Spencer's Pathology of the Lung [6 ed.] 0521509955, 9780521509954

Table of contents :
Cover
Contents
Chapter 1: The normal lung: histology, embryology, development, aging and function
Introduction
Development
Airway and airspace development
Vascular development
Factors regulating lung development
Effect of sex on lung growth
Post-natal lung development
Tracheal growth
Lung
Stem/progenitor cells in the lung
Normal organization
Airways
The trachea
The primary bronchi
The lungs
Blood supply
Lymphatics and lymph nodes
Nerve supply
Pleura
Histology, ultrastructure and function
Trachea, bronchi and bronchioles
Respiratory epithelium
Ciliated cells
Basal cells
Non-ciliated secretory cells
Club cells (Clara cells)
Goblet cells and serous cells
Neuroendocrine cells
Other cells
Brush cells
Intermediate cells
Basement membrane
Smooth muscle
Submucosal glands
Bronchus-associated lymphoid tissue (BALT)
Acini and alveoli
Type I pneumocytes
Type II pneumocytes
Pulmonary interstitium
Pulmonary vasculature
Pleura
Aging
Structural changes
Functional changes
References
Chapter 2: Lung specimen handling and practical considerations
Introduction
Cytology specimen processing
Small specimen histological processing
Large surgical pathology specimen processing
Lung wedge biopsies
Segmentectomy, simple lobectomy, bronchoplastic resection, pneumonectomy, and autopsy lungs
Evaluation of the large specimen in known or suspected cases of primary lung carcinoma
Large pleural biopsy
Ancillary studies
Artifacts in biopsy
Atelectasis/overinflation
Intra-alveolar hemorrhage
Crush artifact
Sponge artifact
Bubble artifact
Incidental lesions
Large airways
Ossification of tracheobronchial cartilage
Oncocytic metaplasia of bronchial submucosal glands
Bronchial submucosal elastosis and adipose tissue
Parenchymal
Scar
Apical cap
Metaplastic bone and dystrophic ossification
Smooth muscle hyperplasia
Schaumann bodies
Asteroid bodies
Calcium oxalate crystals
Mallorys hyaline-like material in type II pneumocytes
Corpora amylacea
Blue bodies
Senile amyloid
Vascular
Megakaryocytes
Pigmentation of elastic fibers
Microthrombi
Bone marrow emboli
Others
Hamazaki-Wesenberg bodies
Entrapped pleura
Applications and misapplications of the lung biopsy
Efficacy of the transbronchial biopsy
High utility
Probably diagnostic
Possibly diagnostic
Unreliable
Problems with the biopsy
Issues of tissue
Problems in interpretation
References
Chapter 3 Congenital abnormalities and pediatric lung diseases, including neoplasms
Introduction
Congenital malformations
Trachea
Abnormality of tracheal length
Tracheal agenesis
Tracheal stenosis
Intrinsic tracheal stenosis
Diffuse stenosis
Funnel-shaped tracheal stenosis
Segmental stenosis
Extrinsic stenosis
Tracheomalacia
Tracheoesophageal fistula
Tracheobronchiomegaly (Mounier-Kuhn syndrome)
Other causes of tracheal obstruction
Bronchus
Bronchial atresia
Bronchial stenosis
Bronchomalacia
Abnormal bronchial origin and branching
Bronchial isomerism syndromes
Broncho-biliary fistula
Lung
Pulmonary agenesis
Lobar agenesis and other lesser congenital pulmonary anomalies
Pulmonary malformations including cysts
Bronchogenic cysts
Congenital adenomatoid malformation
Subtypes (Table 6)
Large cyst type (Stocker type 1)
Small cyst type (Stocker type 2)
Solid adenomatoid malformation (Stocker type 3)
Peripheral lung cyst (Stocker type 4)
Acinar dysplasia
Pulmonary sequestration and related lesions
Extra-lobar sequestration
Intra-lobar sequestration
Isolated systemic arterial supply
Bronchopulmonary foregut malformation
Polyalveolar lobe
Congenital lobar emphysema
Pulmonary hyperplasia
Congenital pulmonary lymphangiectasis
Enteric (enterogenous) cysts
Alveolar capillary dysplasia
Abnormal and ectopic tissues in the lungs
Pulmonary hypoplasia
Inadequate thoracic volume
Impairment of fetal breathing
Oligohydramnios
Primary or idiopathic
Diaphragmatic eventration and hernia
Diaphragmatic hernia
Primary ciliary dyskinesia
Cystic fibrosis
Introduction
Clinical features
Genetics and the basic defect
Pathophysiology of the airways and the basic defect
Infection and inflammation
Histopathology and histopathogenesis of lung disease
a1-Antitrypsin deficiency
Perinatal pathology
Hyaline membrane disease
Pulmonary surfactant metabolic dysfunction disorders
Bronchopulmonary dysplasia
Pathology
Old BPD
Exudative and early reparative phase
Subacute fibroproliferative stage
Chronic fibroproliferative stage
New BPD
Pathogenesis and outcomes
Wilson-Mikity syndrome
Air leaks: pulmonary interstitial emphysema and pneumothorax
Complications of other ventilatory techniques
High-frequency jet ventilation
Extra-corporeal membrane oxygenation
Pulmonary hemorrhage
Thrombosis and embolism
Meconium aspiration syndrome
Persistent pulmonary hypertension of the newborn
Diffuse interstitial lung disease in children
Pulmonary interstitial glycogenosis
Neuroendocrine cell hyperplasia of infancy
Infection
Ascending infection
Neonatal bacterial pneumonia
Viral infection
Cytomegalovirus
Herpes simplex virus
Respiratory syncytial virus
Metapneumovirus
Other viruses
Pediatric lung tumors
Introduction
Pleuropulmonary blastoma
Clinical and radiographic features
Macroscopic pathology
Histopathology
Pathogenesis
Treatment and prognosis
Genetics
Differential diagnosis
Rhabdomyosarcoma
Ewing sarcoma
Synovial sarcoma
Congenital peribronchial myofibroblastic tumor
Myofibromatosis
Inflammatory myofibroblastic tumor
Disseminated juvenile xanthogranuloma
Other tumors
References
Chapter 4 Pulmonary bacterial infections
Background
Epidemiology
Clinical manifestations
Routes of injury
Radiological manifestations
Pathogenesis and normal host defense
Management
Prognosis and natural history
Microbiological work-up
Histological features of bacteria that cause pneumonia
Common bacterial causes of pneumonia
Anaerobic bacteria
Clostridium spp.
Clostridium perfringens
Clostridium septicum
Peptostreptococcus
Chlamydiaceae
Chlamydia trachomatis
Chlamydophila pneumoniae
Chlamydophila psittaci (ornithosis or psittacosis)
Enterobacteriaceae
Escherichia coli
Klebsiella pneumoniae
Proteus mirabilis
Serratia marcescens
Other Gram-negative bacteria
Haemophilus influenzae
Legionella spp
Legionella micdadei (Pittsburgh pneumonia agent)
Legionella pneumophila (Legionnaires disease)
Pseudomonas aeruginosa
Mycoplasmas
Mycoplasma pneumoniae
Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium and Ureaplasma spp
Gram-positive bacteria
Staphylococcus spp.
Staphylococcus aureus
Staphylococcus spp. (coagulase-negative)
Botryomycosis
Streptococcaceae
Enterococcus spp.
Streptococcus agalactiae
Streptococcus pneumoniae (pneumococcal pneumonia)
Streptococcus pyogenes
Streptococcus viridans group
Uncommon bacterial causes
Acinetobacter spp.
Actinomyces and Nocardia spp.
Actinomyces israelii
Nocardia asteroides
Bacillus spp.
Bacillus anthracis (anthrax)
Bacillus cereus
Bacillus sphaericus
Bartonella spp. (bacillary angiomatosis and bacillary peliosis)
Brucella spp.
Burkholderia spp.
Burkholderia pseudomallei (Melioidosis)
Burkholderia cepacia
Chromobacterium violaceum
Francisella tularensis (tularemia)
Leptospira spp.
Micrococcus spp
Moraxella (Branhamella) catarrhalis
Neisseria spp
Pasteurella spp.
Rhodococcus equi (malakoplakia)
Salmonella spp.
Treponema pallidum (syphilis)
Tropheryma whipplei (Whipple disease)
Yersinia pestis (plague)
Rickettsiales
Anaplasma phagocytophilum (anaplasmosis)
Coxiella burnetii (Q fever)
Ehrlichia chaffeensis (monocytotropic ehrlichiosis)
Rickettsia conorii
Rickettsia rickettsii (Rocky Mountain spotted fever)
Orientia tsutsugamushi (scrub typhus)
References
Chapter 5 Pulmonary viral infections
Introduction
Patterns of respiratory viral disease
Epidemiology
Seasonality
Radiographic appearances
Pathophysiology
Pulmonary defenses
Patterns of lung injury due to viral infection
Tracheobronchitis/bronchiolitis
Diffuse alveolar damage
Individual viruses
Influenzavirus
Severe acute respiratory syndrome
Respiratory syncytial virus
Parainfluenza viruses
Measles
Adenovirus
Cytomegalovirus
Herpesvirus
Varicella zoster virus
Epstein-Barr virus
Human herpesvirus-6
Hantavirus pulmonary syndrome
Human immunodeficiency virus-1
Chapter 6 Pulmonary mycobacterial infections
Introduction
Epidemiology
Microbiological diagnosis
Clinical and laboratory manifestations
Radiological manifestations
Pathogenesis
Pathology
Macroscopy
Histopathology
Clinicopathologic classification
Primary pulmonary tuberculosis
Secondary pulmonary tuberculosis
Environmental mycobacteriosis
Clinicopathological correlation
Differential diagnosis
Sarcoidosis
Fungal infection
Other infections/infestations
Wegener granulomatosis
Churg-Strauss syndrome
Bronchocentric granulomatosis
Rheumatoid nodule
Foreign-body-type cells from aspiration
Granulomatous inflammation in lymph nodes draining cancer
Clinical diagnosis
Clinical management
Prognosis and natural history
References
Chapter 7: Pulmonary mycotic infections
Introduction
General epidemiology
Pulmonary aspergillosis
Introduction
Organisms
Epidemiology
Genetics
Pathogenesis
Different forms of aspergillosis
Allergic forms of pulmonary aspergillosis
Aspergilloma
Introduction
Clinical features
Pathology
Diagnosis
Chronic pulmonary aspergillosis
Introduction
Clinical features
Pathology
Diagnosis
Angioinvasive-disseminated aspergillosis
Introduction
Clinical features
Pathology
Diagnosis
Granulomatous (tuberculoid) pulmonary aspergillosis
Tracheobronchial aspergillosis
Other forms of pulmonary aspergillosis
Clinical laboratory diagnosis of Aspergillus
Culture
Antibody tests
Biomarker detection
Galactomannan
beta-d-Glucan detection
Molecular diagnostics
Differential diagnosis
Treatment and prognosis
Pulmonary mucormycosis
Introduction
Organisms
Epidemiology
Clinical features
Pathology
Diagnosis
Differential diagnosis
Treatment and prognosis
Pulmonary scedosporiosis
Introduction
Organism
Epidemiology
Clinical features
Pathology
Diagnosis
Treatment and prognosis
Pulmonary fusariosis
Introduction
Organism
Epidemiology
Clinical features
Pathology
Diagnosis
Treatment and prognosis
Pulmonary histoplasmosis
Introduction
Organism
Epidemiology
Pathogenesis
Clinical features
Pathology
Diagnosis
Culture
Serological tests
Antigen detection tests
Differential diagnosis
Treatment
Pulmonary cryptococcosis
Introduction
Organism
Epidemiology
Clinical features
Pathology
Diagnosis
Culture
Serological tests
Antigen detection tests
Differential diagnosis
Treatment and prognosis
Pulmonary blastomycosis (North American blastomycosis)
Introduction
Organism
Epidemiology
Clinical features
Pathology
Diagnosis
Culture
Serological tests
Antigen detection tests
Differential diagnosis
Treatment and prognosis
Pulmonary coccidioidomycosis
Introduction
Organism
Epidemiology
Clinical and pathological features
Primary pulmonary infection
Chronic pulmonary infection
Coccidioidoma
Progressive pulmonary disease
Disseminated coccidioidomycosis
Diagnosis
Culture
Skin tests
Serological tests
Antigen detection tests
Differential diagnosis
Treatment and prognosis
Pulmonary paracoccidioidomycosis (South American blastomycosis)
Introduction
Organism
Epidemiology
Clinical and pathological features
Acute and subacute pulmonary disease
Progressive pulmonary disease
Diagnosis
Culture
Serological tests
Antigen detection tests
Differential diagnosis
Treatment
Pulmonary candidiasis
Introduction
Organism
Epidemiology and pathogenesis
Clinical features
Endobronchial spread
Hematogenous spread
Pathology
Diagnosis
Non-culture-based methods
Antibody detection
Antigen detection
(1,3)-beta-d-Glucan detection
Molecular methods
Differential diagnosis
Prognosis and treatment
Pneumocystis jirovecii infections
Introduction
Organism
Epidemiology
Clinical features
Pathology
Classic (typical) Pneumocystis pneumonia
Atypical features of Pneumocystis jirovecii pneumonia
Extrapulmonary Pneumocystis jirovecii infection
Diagnosis
Serological tests
beta-d-Glucan detection
Molecular diagnostics
Differential diagnosis
Treatment and prognosis
Pulmonary sporotrichosis
Introduction
Organism
Epidemiology
Clinical features
Pathology
Diagnosis
Culture
Serological tests
Differential diagnosis
Treatment and prognosis
Rare pulmonary fungal infections
Adiaspiromycosis
Malassezia spp.
Phaeohyphomycoses
References
Chapter 8: Pulmonary parasitic infections
Introduction
Principles of parasitic infection
Diagnosis of parasitic lung disease
Protozoal infections
Leishmaniasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Treatment
Amebiasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Treatment
Free-living amebic infections
Trypanosomiasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
African trypanosomiasis
Malaria
Introduction
Epidemiology
Organism
Clinical features
Pathology
Pathogenesis
Diagnosis
Differential diagnosis
Treatment and drug toxicity
Babesiosis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis and differential diagnosis
Toxoplasmosis
Introduction
Epidemiology
Organism
Clinical findings
Pathology
Diagnosis
Differential diagnosis
Treatment
Microsporidiosis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Differential diagnosis
Treatment
Cryptosporidiosis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Treatment
Very rare protozoal lung infections
Trichomonas
Lophomonas
Cyclospora
Balantidium
Helminths
Pulmonary eosinophilia syndromes
Filarial infections
Lymphatic filariases and tropical pulmonary eosinophilia syndrome
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Pathogenesis
Dirofilariasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Treatment
Soil-transmitted helminth infections
Ascariasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Treatment
Hookworm infection
Strongyloidiasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Differential diagnosis
Diagnosis
Treatment
Visceral larva migrans
Introduction
Toxocariasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Uncommon nematode lung infections
Enterobiasis
Anisakiasis
Capillariasis
Angiostrongyliasis
Gnathostomiasis
Mammomonogamiasis
Loiasis
Onchocerciasis
Subcutaneous dirofilarasis
Trematode infections
Schistosomiasis
Introduction
Epidemiology
Organism
Clinical features
Acute schistosomiasis (Katayama syndrome)
Chronic schistosomiasis
Pathology
Diagnosis
Treatment
Paragonimiasis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Treatment
Other uncommon food-borne trematode infections of lung and pleura
Fascioliasis
Alaria
Cestode lung infections
Hydatid cyst - echinococcosis
Introduction
Echinococcus granulosus hydatid lung disease
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Differential diagnosis
Echinococcus multilocularis hydatid lung disease
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Treatment of hydatid disease
Cysticercosis
Introduction
Epidemiology
Organism
Clinical features
Pathology
Diagnosis
Sparganosis
Pentastome infections
Armillifer pentastomiasis
Linguatulosis
References
Chapter 9 Acute lung injury
Introduction
Etiology of ALI/ARDS
Epidemiology
Radiological findings in ALI/ARDS
Pathological features of acute lung injury
Exudative phase
Proliferative phase
Acute fibrinous organizing pneumonia
Samples submitted to pathology departments from patients with suspected ALI/ARDS
Pathogenesis of ALI
Exudative phase
Inflammatory cells
Neutrophils
Macrophages
Platelets
Endothelial cells
Alveolar epithelial cells
Coagulation system
Fibroproliferative phase
Organization and fibrosis versus resolution
Management of ALI/ARDS
Summary
References
Chapter 10 Interstitial lung diseases
Introduction
History of classification
Epidemiology
Genetics
Idiopathic pulmonary fibrosis and usual interstitial pneumonia
Etiology
Clinical presentation
Radiological findings
Pathology
Differential diagnosis
Pathogenesis
Prognosis and natural history
Nonspecific interstitial pneumonia
Clinical presentation
Radiological findings
Pathology
Differential diagnosis
Pathogenesis
Prognosis and natural history
Diffuse alveolar damage and acute interstitial pneumonia
Clinical presentation
Radiological findings
Pathology
Variants
Acute fibrinous organizing pneumonia (AFOP)
Regional alveolar damage
Differential diagnosis
Pathogenesis
Treatment and prognosis
Acute exacerbation of idiopathic pulmonary fibrosis
Clinical presentation
Radiological findings
Pathology
Differential diagnosis
Pathogenesis
Prognosis and natural history
Smoking-related interstitial lung disease
Respiratory bronchiolitis/respiratory bronchiolitis-interstitial lung disease
Clinical presentation
Radiological findings
Pathology
Differential diagnosis
Prognosis
Desquamative interstitial pneumonia
Clinical presentation
Radiological findings
Pathology
Differential diagnosis
Pathogenesis
Prognosis and natural history
Provisional and recently described entities
Pulmonary hypertension and interstitial lung disease
Pulmonary calcification and ossification
Pulmonary calcification
Pulmonary ossification
Pulmonary alveolar microlithiasis
Pulmonary alveolar proteinosis
Etiology
Clinical features
Radiological findings
Pathology
Differential diagnosis
References
Chapter 11 Metabolic and inherited connective tissue disorders involving the lung
Introduction
Lysosomal storage diseases
Gaucher disease
Introduction
Epidemiology
Genetics
Clinical manifestations
Pulmonary radiology
Pathology
Cytology
Electron microscopy
Laboratory findings
Pathophysiology
Differential diagnosis
Prognosis and natural history
Niemann-Pick disease, types A and B
Introduction
Epidemiology
Genetics
Clinical manifestations
Pulmonary radiology
Pathology
Cytology
Electron microscopy
Laboratory findings
Pathophysiology
Differential diagnosis
Prognosis and natural history
Niemann-Pick disease, type C
Introduction
Epidemiology
Genetics
Clinical manifestations
Pulmonary radiology
Pathology
Electron microscopy
Laboratory findings
Pathophysiology
Treatment and prognosis
Fabry disease
Introduction
Epidemiology
Genetics
Clinical manifestations
Pulmonary radiology
Pathology
Cytology
Electron microscopy
Laboratory findings
Pathophysiology
Prognosis and natural history
Hermansky-Pudlak syndrome
Introduction
Epidemiology
Genetics
Clinical manifestations
Pulmonary radiology
Pathology
Cytology
Electron microscopy
Pathophysiology
Differential diagnosis
Prognosis and natural history
Mucopolysaccharidoses
Introduction
Epidemiology
Genetics
Clinical manifestations
Pulmonary radiology
Pathology
Electron microscopy
Laboratory findings
Pathophysiology
Prognosis and outcome
GM1 gangliosidosis
Introduction
Clinical manifestations
Pathology
Electron microscopy
Prognosis and natural history
Krabbe disease (globoid leukodystrophy)
Introduction
Clinical manifestations
Pulmonary radiology
Pathology
Electron microscopy
Prognosis and treatment
Pompe disease (glycogen storage disease type II)
Introduction
Genetics
Clinical features
Pulmonary radiology
Pathology
Electron microscopy
Prognosis and treatment
Farber disease
Epidemiology
Genetics
Clinical features
Pathology
Electron microscopy
Laboratory findings
Pathophysiology
Treatment and prognosis
Aminoaciduria
Lysinuric protein intolerance
Introduction
Epidemiology
Genetics
Clinical manifestations
Pulmonary radiology
Pathology
Electron microscopy
Laboratory findings
Pathophysiology
Treatment and prognosis
Inherited connective tissue disorders
Marfan syndrome
Epidemiology
Genetics
Clinical features
Pulmonary radiology
Pathology
Electron microscopy
Pathophysiology
Prognosis and treatment
Ehlers-Danlos syndromes
Epidemiology
Genetics
Clinical features
Pulmonary radiology
Pathology
Laboratory findings
Pathophysiology
Prognosis and treatment
References
Chapter 12 Hypersensitivity pneumonitis
Introduction
Clinical features
Classification
Incidence and prevalence
Acute exacerbations of chronic HP
Clinical testing
Pulmonary function testing and pathophysiology
Immunological testing
Bronchoalveolar lavage
Inhalational challenge
Skin testing
Radiological findings
Introduction
Acute HP
Subacute HP
Chronic HP
Etiology and pathogenesis
Inciting agents
Immunological background
Effect of smoking on HP
Pathological findings
Role of the surgical lung biopsy in HP
Acute HP
Subacute HP
Bronchiolitis
Interstitial pneumonitis
Granulomas
Airspace organization (intraluminal fibrosis)
Chronic HP
Granulomas
Bronchiolitis, interstitial pneumonitis
Fibrosis
Vasculopathy
Chronic HP with acute exacerbation
Special forms of HP
Hot tub lung
Japanese summer-type hypersensitivity pneumonitis (SHP)
Isocyanate-related HP
HP-like syndrome in occupational exposure to metal-working fluids
Differential diagnosis of HP
Usual interstitial pneumonia/idiopathic pulmonary fibrosis
Nonspecific interstitial pneumonia
Lymphoid interstitial pneumonia
Organizing pneumonia and cryptogenic organizing pneumonia
Centrilobular/bronchiolocentric interstitial pneumonias
Granulomatous disorders to be distinguished from HP
Sarcoidosis
Drug-related interstitial lung disease
Cholesterol granuloma
Pneumocystis pneumonia
Aspiration bronchiolitis/pneumonia
Treatment, prognosis and survivial
References
Chapter 13 Sarcoidosis
Introduction
Epidemiology
Genetic associations and etiological considerations
Clinical manifestations
Radiographic findings
Macroscopic pathology
Histopathology
Inclusions
Cytology
Electron microscopy
Diagnosis of sarcoidosis
Laboratory findings
Immunopathogenesis and clinical associations
Differential diagnosis
Natural history, pathophysiology, prognosis and treatment
Sarcoidosis variants
Nodular sarcoidosis
Necrotizing sarcoid granulomatosis
References
Chapter 14 Occupational lung disease
Introduction
Asbestos
Introduction and occupational risk
Background levels
Non-occupational risk and reference population levels
Regulatory activity and exposure standards
Deposition and clearance of asbestos fibers
Asbestos bodies and non-asbestos ferruginous bodies
Asbestosis
Epidemiology
Mechanisms and pathogenesis of asbestos-related fibrosis
Clinical features
Radiology
Macroscopic pathology
Histopathology
Differential diagnosis
Grading of asbestosis
Prognosis
Asbestos and lung cancer
Methods of tissue analysis
Silicotic lung disease
Introduction
Mineralogy
Occupations causing exposure to silica and pathogenesis
Clinical features
Radiological features
Pathological features
Associated immune dysfunction
Future trends
Mixed-dust pneumoconiosis and mixed pneumoconiosis
Respiratory diseases resulting from coal and coalmine dust
Pathogenesis
Coal workers pneumoconiosis (CWP)
Clinical features
Radiology
Pathology
Complicated CWP and PMF
Pathology
Differential diagnosis
Diffuse interstitial fibrosis
Emphysema and chronic bronchitis, and carcinoma of the lung
Pneumoconioses associated with non-asbestos silicates (silicatoses)
Talcosis (talc pneumoconiosis)
Kaolin pneumoconiosis
Mica pneumoconiosis
Zeolites
Vermiculite
Diseases from metals
Beryllium
Hard-metal (cobalt) lung disease (tungsten carbide pneumoconiosis)
Siderotic lung disease: iron, hematite miners lung
Silicon carbide pneumoconiosis
Rare earth pneumoconiosis
Dental technicians pneumoconiosis
Benign pneumoconioses/nuisance dusts
Titanium pneumoconiosis
Stannosis
Barium pneumoconiosis (baritosis)
Lung diseases caused by metal fumes
Welders pneumoconiosis (WP)
Aluminum lung disease
Cadmium pneumonitis
Mercury pneumonitis
Lung disease caused by non-asbestos mineral fibers
Man-made fibers, mineral and synthetic
Nylon flock workers lung
Miscellaneous fibers and agents: carbon, non-asbestos ferruginous bodies and diacetyl carbon
Popcorn workers/food flavorers lung disease
References
Chapter 15 Eosinophilic lung disease
Introduction
Asthma
Introduction
Epidemiology and genetics
Clinical manifestations
Radiological findings
Macroscopic pathology
Histopathology
Airway lumen
Airway epithelium
Basement membrane
Inflammatory infiltrate
Vessels
Mucous glands
Wall thickness
Cytology of asthma
Laboratory findings
Prognosis and natural history
Non-asthmatic eosinophilic bronchitis
Introduction
Clinical manifestations
Histopathology
Prognosis and natural history
Chronic eosinophilic pneumonia
Introduction
Clinical manifestations
Radiographic findings
Histopathology
Differential diagnosis
Natural history and prognosis
Allergic bronchopulmonary fungal disease
Introduction
Clinical manifestations
Radiographic findings
Histopathology
Bronchiectasis and mucoid impaction of bronchi
Bronchocentric granulomatosis (BCG)
Natural history and prognosis
Acute eosinophilic pneumonia (AEP)
Introduction
Clinical manifestations
Radiographic manifestations
Histopathology
Laboratory findings
Differential diagnosis
Prognosis and natural history
References
Chapter 16 Drug- and therapy-induced lung injury
Introduction
Pathogenesis of drug-induced pulmonary disease
Histological patterns associated with drug toxicity
Diffuse alveolar damage
Chronic interstitial pneumonitis/nonspecific interstitial pneumonia
Organizing pneumonia
Eosinophilic pneumonia
Pulmonary edema
Pulmonary hemorrhage/vasculitis
Pulmonary hypertension/veno-occlusive disease
Granulomatous inflammation
Pleural disease
Pulmonary drug toxicity associated with selected agents
Amiodarone
Methotrexate
Busulfan
Bleomycin
Nitrofurantoin
Rapamycin and rapamycin analogs
Monoclonal antibodies and targeted inhibitors
Monoclonal antibodies
Tyrosine kinase inhibitors
Radiation
Radiation pneumonitis
Chemotherapeutic and radiation effects on pulmonary carcinomas
Radiofrequency ablation
Transfusion-related acute lung injury
Pulmonary pathology associated with illicit drug use
Cocaine
Marijuana and related substances
Heroin
Manifestations of intravenous drug abuse
Other miscellaneous drugs
References
Chapter 17 Chronic obstructive pulmonary disease and diseases of the airways
Introduction
Chronic obstructive pulmonary disease in general
Introduction
Epidemiology of COPD
Genetics
Clinical manifestations
Radiographic findings
Pathophysiology
Pathology of COPD
Large airways
Small airways
Pathogenesis of bronch(iol)itis
Course and prognosis
Treatment
Chronic bronchitis in COPD
Definition
Epidemiology
Pathophysiology
Pathology
Emphysema in COPD
Definition
Classification
Estimation of severity
Emphysema related to alpha-1-antitrypsin deficiency
Pathogenesis of emphysema
Emphysema as a consequence of proteolytic/antiproteolytic imbalance and oxidant damage
Emphysema as a result of disruption of the lungs homeostatic maintenance and repair system
Emphysema as an immunological process
Pulmonary vascular disease in COPD
Systemic effects of COPD
Right ventricular changes
Panniculitis
Small vessel vasculitis
Liver abnormalities
Osteoporosis
Body mass wasting and skeletal muscle dysfunction
Diaphragmatic dysfunction
Systemic inflammation
Other forms of emphysema (Table 4)
Non-destructive emphysema (hyperinflation)
Development-related airspace enlargement
The ``senile´´ lung
Starvation
Localized giant bullous emphysema: Birt-Hogg-Dubé syndrome
Placental transmogrification
Bronchiectasis
Definition
Classification
Clinical
Gross anatomic
Microscopic anatomic
Quasi-etiological (Table 5)
Epidemiology
Clinical
Radiology
Pathology
Pathophysiology
Course and prognosis
Therapy
Special entities
Aspiration
Middle lobe syndrome
Broncholithiasis
Plastic bronchitis
Tracheobronchopathia osteochondroplastica
Small airway diseases/bronchiolitis
Definition and history
Clinical features
Classification
Acute bronchiolitis
Obliterative bronchiolitis
Proliferative bronchiolitis
Constrictive bronchiolitis
Constrictive bronchiolitis related to neuroendocrine hyperplasia
Constrictive bronchiolitis related to ingestion of toxic agents
Diseases of the bronchioles with or without adjacent parenchymal disease
Smokers bronchiolitis with or without adjacent interstitial lung disease
Diffuse panbronchiolitis
Follicular bronchiolitis
Eosinophilic bronchiolitis
Granulomatous bronchiolitis
Mineral dust disease
Peribronchiolar metaplasia (Lambertosis)
Pathophysiology
Treatment and prognosis
References
Chapter 18 Pulmonary vascular pathology
Introduction
Classification of pulmonary hypertension
Handling of tissue specimens
Pulmonary vascular anatomy and histology
Dual blood supply
Pulmonary arteries
Supernumerary arteries
Capillaries
Pulmonary veins
Bronchial circulation
Lymphatics
Vascular remodeling during development and aging
Radiological findings in pulmonary hypertension
Genetics of pulmonary hypertension
Pathogenesis of pulmonary hypertension
Introduction
Bone morphogenic protein receptor-2 (BMPR2)
Serotonin (5-hydroxytryptamine, 5HT) and appetite suppressants
Inflammation
Endothelium and thrombosis
Hypoxia
Hypoxia and pulmonary vascular tone
Hypoxia and vascular remodeling
Histopathological patterns of hypertensive pulmonary vascular disease
Introduction
Plexogenic arteriopathy
Epidemiology and risk factors
Histopathology of plexogenic arteriopathy (Table 5)
Plexogenic arteriopathy: differential diagnosis
Effects of therapy
Thrombotic arteriopathy and chronic thromboembolic pulmonary hypertension (CTEPH)
Epidemiology, risk factors
Histopathology of thrombotic arteriopathy (Table 7)
Pulmonary infarcts
Differential diagnosis
Non-thrombotic causes of pulmonary artery embolism
Hypoxic arteriopathy
Epidemiology and causal factors
Histopathology of hypoxic arteriopathy (Table 9)
Congestive vasculopathy
Epidemiology, causal factors
Histopathology of congestive vasculopathy (Table 11)
Pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis
Introduction
Epidemiology and clinical features
Radiological features of PVOD/PCH
Histopathology (Table 13)
Differential diagnosis
Congenital heart disease with hypo- or hyperperfusion
Cardiac malformations with pulmonary hypoflow
Cardiac malformations with pulmonary hyperflow
Pulmonary vasculopathy in pulmonary hypertension associated with connective tissue diseases
Introduction
Systemic sclerosis
Systemic lupus erythematosus (SLE)
Pulmonary hypertension in interstitial lung diseases
Pulmonary hypertension in vasculitis
Acknowledgements
References
Chapter 19 Pulmonary vasculitis and pulmonary hemorrhage syndromes
Introduction
ANCA-associated diseases
Wegener granulomatosis
Introduction
Epidemiology
Genetics
Clinical manifestations
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Laboratory findings
Pathogenesis
Differential diagnosis
Prognosis and natural history
Microscopic polyangiitis
Introduction
Epidemiology
Genetics
Clinical manifestations
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Laboratory findings
Pathogenesis
Differential diagnosis
Prognosis and natural history
Churg-Strauss syndrome
Introduction
Epidemiology
Genetics
Clinical manifestations
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Laboratory findings
Pathogenesis
Differential diagnosis
Prognosis and natural history
Goodpasture syndrome
Introduction
Epidemiology
Genetics
Clinical manifestations
Radiographic findings
Macro- and histopathology
Cytology
Ultrastructural findings
Laboratory findings
Pathogenesis
Differential diagnosis
Prognosis and natural history
Diffuse pulmonary hemorrhage syndromes and capillaritis
Polyarteritis nodosa
Introduction
Epidemiology
Clinical manifestations
Radiographic findings
Macroscopic pathology
Histopathology
Immunohistochemistry
Electron microscopy
Laboratory findings
Pathogenesis
Differential diagnosis
Prognosis and natural history
Giant cell arteritis
Introduction
Epidemiology
Genetics
Clinical manifestations
Histopathology
Laboratory findings
Pathogenesis
Differential diagnosis
Natural history and prognosis
Takayasu arteritis
Introduction
Epidemiology
Genetics
Clinical manifestations
Radiographic findings
Macroscopic pathology
Histopathology
Laboratory findings
Pathogenesis
Differential diagnosis
Prognosis and natural history
Behçet syndrome
Introduction
Epidemiology
Genetics
Clinical manifestations
Radiographic findings
Macroscopic pathology
Histopathology
Laboratory findings
Pathogenesis
Differential diagnosis
Prognosis and natural history
References
Chapter 20 The pathology of lung transplantation
Introduction
The pathologists role in lung transplantation
Methods for monitoring the pulmonary allograft
Transbronchial biopsy
Bronchoalveolar lavage
Open lung biopsy (OLB) or video-assisted lung biopsy (VATS)
A temporal approach to lung transplant pathology
Perioperative and early post-transplant period (up to 1 month)
Primary graft dysfunction / ischemic-reperfusion injury
Hyperacute rejection
Vascular and airway anastomotic complications
Infection in the early post-transplant period
Intermediate period after transplantation (1 month - 1 year)
Acute cellular rejection
Grade AX (ungradeable specimen)
Grade A0 (no evidence of rejection / NER)
Grade A1 (minimal acute rejection)
Grade A2 (mild acute rejection)
Grade A3 (moderate acute rejection)
Grade A4 (severe acute rejection)
Morphological mimics and pitfalls in the diagnosis and grading of acute rejection
Issues related to the diagnosis and classification of acute rejection
Airway inflammation without scarring
Antibody-mediated rejection
Infection
Viral infections including cytomegalovirus (CMV)
Common community-acquired viral infections
Fungal infections
Post-transplant lymphoproliferative disorder and other EBV-related disorders
1. Early lesions including plasmacytic hyperplasia (PH) and infectious-mononucleosis (IM)-like PTLD
2. Polymorphous PTLD
3. Monomorphic PTLD
4. Classic Hodgkin lymphoma-type PTLD
Other EBV-associated neoplasms
Other pulmonary complications in lung transplant recipients
Late period after transplantation ( 12 months)
Obliterative bronchiolitis and bronchiolitis obliterans syndrome
Chronic vascular rejection
Recurrent disease in the allograft
Future directions
References
Chapter 21 The lungs in connective tissue disease
Introduction to connective tissue diseases
Secondary complications in connective tissue disease
Acute exacerbation of connective tissue diseases
Idiopathic interstitial pneumonias as a presenting manifestation of connective tissue disease
Genetics of connective tissue diseases
Rheumatoid arthritis
Pleuritis
Rheumatoid nodules
Interstitial lung disease
UIP pattern in rheumatoid arthritis
NSIP pattern in rheumatoid arthritis
Organizing pneumonia pattern in rheumatoid arthritis
Lymphocytic interstitial pneumonia pattern in rheumatoid arthritis
Diffuse alveolar damage pattern in rheumatoid arthritis
Airway disease
Obliterative bronchiolitis pattern (constrictive bronchiolitis) in rheumatoid arthritis
Follicular bronchiolitis / lymphoid hyperplasia in rheumatoid arthritis
Chronic bronchiolitis
Bronchiectasis
Vascular disease
Primary pulmonary hypertension in rheumatoid arthritis
Diffuse alveolar hemorrhage/capillaritis in rheumatoid arthritis
Other rare pulmonary manifestations of rheumatoid arthritis
Systemic lupus erythematosus
Pleuritis
Acute lupus pneumonitis
Involvement of pulmonary vasculature
Diffuse alveolar hemorrhage
Pulmonary hypertension
Interstitial lung disease
Shrinking lung syndrome
Drug reactions
Antiphospholipid antibody syndrome
Scleroderma
Interstitial lung disease in scleroderma
NSIP and UIP patterns
Other patterns of parenchymal involvement
Pulmonary hypertension in scleroderma
Lung cancer in scleroderma
Sjögren syndrome
Interstitial lung disease
NSIP and LIP patterns
Other patterns of parenchymal involvement
Airway disease
Tracheobronchial sicca (xerotrachea)
Chronic bronchiolitis
Pulmonary arterial hypertension
Marginal zone B-cell lymphoma and nodular lymphoid hyperplasia
Polymyositis/dermatomyositis
Interstitial lung disease
Other rare pulmonary manifestations
Mixed connective tissue disease
Interstitial lung disease
Pulmonary hypertension
Other pulmonary manifestations
Undifferentiated connective tissue disease
Interstitial lung disease
Ankylosing spondylitis
Relapsing polychondritis
Behçet syndrome
Pyoderma gangrenosum
Sweet syndrome
Pulmonary involvement by inflammatory bowel disease
References
Chapter 22 Benign epithelial neoplasms and tumor-like proliferations of the lung
Introduction
Bronchial inflammatory polyps
Introduction
Classification, cell of origin, pathogenesis and etiology
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Bronchial papillomas
Introduction
Squamous cell papillomas
Classification, cell of origin, pathogenesis and etiology
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Glandular and mixed squamous cell and glandular papillomas
Multifocal micronodular pneumocyte hyperplasia
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Papillary adenoma
Introduction
Classification, cell of origin and etiology
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Alveolar adenoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Sclerosing hemangioma
Introduction
Classification, cell of origin, pathogenesis and etiology
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pulmonary hyalinizing granuloma
Introduction
Clinical details, including epidemiology and etiology
Genetics
Radiological details
Macroscopic pathology
Histopathology
Cytopathology
Immunohistochemistry
Electron microscopy
Pathogenesis
Clinicopathological correlation
Differential diagnoses
Prognostic factors and natural history
Pulmonary endometriosis
Introduction
Clinical details, including epidemiology and etiology
Genetics
Radiographic details
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Pathogenesis
Clinicopathological correlation
Differential diagnosis
Prognostic factors and natural history
References
Chapter 23 Pulmonary pre-invasive disease
Introduction
Bronchial carcinogenesis
Etiology of SD/CIS
Morphologically recognizable pre-invasive bronchial lesions
Mucous cell (goblet cell) hyperplasia
Basal cell hyperplasia
Squamous metaplasia
Squamous dysplasia and carcinoma in situ
Macroscopic features of SD/CIS
Microscopic features of SD/CIS
Exfoliative cytology and the diagnosis of SD/CIS
Problems in the histological assessment of SD/CIS
Differential diagnosis of SD/CIS
Molecular biology of bronchial pre-invasive lesions
Hyperproliferation
p53 and related proteins
P16 (ink4a)-CyclinD1-CDK4-RB pathway
Tyrosine kinase (TK) signaling pathways
Angiogenesis and related factors
Cell immortalization
Transcription factors and other intracellular effectors
Other tumor suppressor genes
Human papilloma virus, cell adhesion molecules and other assorted factors
Studies of genomic instability and other global expression data
Predicting the progression of bronchial pre-invasive disease
Bronchial pre-invasive lesions: comment and conclusion
Carcinogenesis in peripheral lung epithelium
Morphologically recognizable pre-invasive bronchioloalveolar lesions
Atypical adenomatous hyperplasia
Macroscopic features of AAH
Microscopic features of AAH
Issues in the diagnosis of AAH
Differential diagnosis of AAH
Prevalence of AAH
Etiology and other associations of AAH
Molecular biology of peripheral lung adenocarcinogenesis
Cell morphometry and cytofluorimetry
Hyperproliferation
p53 and related proteins
P16 (ink4a)-CyclinD1-CDK4-RB pathway
Tyrosine kinase (TK) signaling pathways
Angiogenesis and related factors
Cell immortalization
Transcription factors and other intracellular effectors
Other tumor suppressor genes
Lineage markers, cell adhesion molecules, matrix metalloproteinases and other factors
Studies of clonality, genomic instability and other global expression data
The clinical relevance and prognosis of AAH
Peripheral lung adenocarcinogenesis: comment and conclusions
Pulmonary pre-invasive disease: final comments
References
Chapter 24 Epidemiological and clinical aspects of lung cancer
Introduction
Epidemiology
Incidence and mortality
Gender
Age
Race
Histology
Etiologies
Tobacco
Radon
Environmental pollution
Occupational factors
Infections
Human papilloma virus
Epstein-Barr virus
Human immunodeficiency virus
Bacterial infections
Fungal infections
Lung injury
Diet
Genetic susceptibility
Familial lung cancer
Single gene polymorphism studies and lung cancer risk
Genome-wide studies and lung cancer risk
Genome-wide linkage studies and familial lung cancer
Lung cancer screening
Clinical manifestations
Radiographic tools
Methods of diagnosis
Lung cancer treatment and prognosis
Acknowledgements
References
Chapter 25 Lung cancer staging
Introduction
The TNM classification
T classification
N classification
M classification
Optional descriptors
Specific situations
Overall survival
Conclusion
References
Chapter 26Immunohistochemistry in the diagnosis of pulmonary tumors
Introduction
TTF-1
Role of TTF-1 in embryology and non-neoplastic conditions
TTF-1 expression in lung tumors
TTF-1 expression in non-pulmonary tumors
Prognostic value of TTF-1 in lung carcinoma
Comparison of monoclonal antibodies and practical considerations
Napsin
Surfactant proteins
Neuroendocrine markers
p16 and p63
Cytokeratins
Other markers
Problems of differential diagnosis
Small cell carcinoma versus lymphoid infiltrates
Small cell carcinoma versus small cell variant of squamous carcinoma
Squamous cell carcinoma versus adenocarcinoma
Differential diagnosis of clear cell carcinoma
Adenocarcinoma of the lung versus metastatic adenocarcinoma of the colorectum
Adenocarcinoma of the lung versus metastatic adenocarcinoma of the breast
Diagnosis of metastatic melanoma
References
Chapter 27 Adenocarcinoma of the lung
Introduction
Classification and cell of origin
Genetics
Special epidemiological features
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Epidermal growth factor receptor (EGFR) signaling pathway
Kirsten-rat derived retroviruses causing sarcoma (KRAS) mutations
Echinoderm microtubule-associated protein-like 4 anaplastic lymphoma receptor tyrosine kinase (EML4-ALK) translocation
BRAF mutation
STK/LKB1
Other mutations
Copy number alterations
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Adenocarcinoma variants
Invasive mucinous adenocarcinoma
Colloid adenocarcinoma
Fetal adenocarcinoma
Enteric adenocarcinoma
References
Chapter 28 Squamous cell carcinoma of the lung
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Squamous cell carcinoma variants
Papillary SCC
Clear cell SCC
Small cell SCC
Basaloid SCC
References
Chapter 29 Large cell carcinoma and adenosquamous carcinoma of the lung
Large cell carcinoma
Large cell carcinomas, not otherwise specified
Introduction
Cell of origin
Special clinical findings
Radiographic findings
Macroscopy pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Genetic and molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Large cell carcinoma variants
Basaloid carcinoma
Prognosis
Lymphoepithelioma-like carcinoma
Prognosis
Clear cell carcinoma
Large cell carcinoma with rhabdoid phenotype
Adenosquamous carcinoma
Prognosis and treatment
References
Chapter 30 Salivary gland neoplasms of the lung
Introduction
Mucoepidermoid carcinoma
Classification and cell of origin
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings and genetics
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Adenoid cystic carcinoma
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Epithelial-myoepithelial carcinoma
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Acinic cell carcinoma
Classification and cell of origin
Genetics and molecular findings
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pleomorphic adenoma
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pneumocytic adenomyoepithelioma
Macroscopic pathology
Histopathology
Immunohistochemistry
Electron microscopy
Differential diagnosis
Clinicopathological correlation
Prognosis and natural history
Mucous gland adenoma
Definition
Clinical features
Macroscopic pathology
Radiographic findings
Histopathology
Immunohistochemistry
Differential diagnosis
Clinicopathological correlation
Prognosis and natural history
Oncocytoma
References
Chapter 31 Neuroendocrine tumors and other neuroendocrine proliferations of the lung
Introduction
Classification schemes
Clinical features
Typical carcinoid tumor
Atypical carcinoid tumor
Large cell neuroendocrine carcinoma
Small cell carcinoma
Radiographic findings and macroscopic pathology
Carcinoid tumor
Large cell neuroendocrine carcinoma
Small cell carcinoma
Histopathology and cytopathology
Typical carcinoid tumor
Atypical carcinoid tumor
Large cell neuroendocrine carcinoma
Small cell carcinoma
Immunohistochemistry
Neuroendocrine antigens
Other antigens
Electron microscopy
Typical carcinoid
Atypical carcinoid
Large cell neuroendocrine carcinoma
Small cell carcinoma
Genetics
Carcinoid tumors
Large cell neuroendocrine and small cell carcinomas
Differential diagnosis
Distinguishing neuroendocrine from non-neuroendocrine pulmonary tumors and tumors metastatic to the lungs
Distinguishing pulmonary neuroendocrine tumors from each other
Prognosis and natural history
Typical carcinoids
Atypical carcinoids
Large cell neuroendocrine carcinoma
Small cell carcinoma
Other pulmonary tumors displaying neuroendocrine differentiation
Non-small cell carcinomas with neuroendocrine differentiation
Pulmonary blastoma
Primitive neuroectodermal tumor
Desmoplastic small round cell tumor
Carcinomas with rhabdoid phenotype
Paraganglioma
Proliferative lesions of pulmonary neuroendocrine cells
Reactive proliferation of pulmonary neuroendocrine cells
Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH)
Clinical and radiological features
Pathology
Reactive proliferation of pulmonary neuroendocrine cells and DIPNECH
Tumorlets and carcinoid tumors
Peritumoral proliferation of pulmonary neuroendocrine cells
References
Chapter 32 Sarcomatoid carcinomas and variants
Introduction
Pleomorphic carcinoma and spindle cell carcinoma
Definition
Clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Differential diagnosis
Prognosis and natural history
Giant cell carcinoma
Definition
Clinical features
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Differential diagnosis
Prognosis and natural history
Carcinosarcoma
Definition
Clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Differential diagnosis
Prognosis and natural history
Pulmonary blastoma
Historical overview
Definition and cell of origin
Clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Differential diagnosis
Prognosis and natural history
Chapter 33 Mesenchymal and miscellaneous neoplasms
Introduction
Pulmonary hamartoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Minute pulmonary meningothelial-like nodules
Introduction
Clinical features
Classification and cell of origin
Genetics
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary meningioma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary lipoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary liposarcoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary extraosseous osteoma
Pulmonary osteogenic sarcoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pulmonary chondromas
Introduction
Classification and cell of origin
Genetics
Radiographic findings
Special clinical features
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Differential diagnosis
Prognosis and natural history
Primary pulmonary chondrosarcoma
Neurogenic tumors
Granular cell tumor
Introduction
Classification and cell of origin
Genetics
Radiographic findings
Special clinical features
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pulmonary rhabdomyosarcomas
Primary pulmonary leiomyoma/leiomyosarcoma
Pulmonary vein sarcoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Benign metastasizing leiomyoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary glomus tumors
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Lymphangioleiomyomatosis
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pulmonary angiomyolipoma
Clear cell (sugar) tumor (PEComa)
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Diffuse pulmonary lymphangiomatosis
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Arteriovenous malformations
Introduction
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Hemangiomas
Pulmonary epithelioid hemangioendothelioma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Kaposi sarcoma
Introduction
Classification and cell of origin
Special clinical features
Genetics
Radiographic findings
Macroscopic findings
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary angiosarcoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Fibrohistiocytic neoplasms
Pulmonary giant cell tumor
Inflammatory myofibroblastic tumor
Introduction
Etiology
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pulmonary artery sarcomas
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary synovial sarcoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Intra-pulmonary thymomas
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Pulmonary teratoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
Primary pulmonary malignant melanoma
Introduction
Classification and cell of origin
Genetics
Special clinical features
Radiographic findings
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Clinicopathological correlation
Differential diagnosis
Prognosis and natural history
References
Chapter 34 Pulmonary lymphoproliferative diseases
Introduction
Lymphoid tissue in the normal lung
Intra-pulmonary lymph nodes
Bronchus-associated lymphoid tissue
Benign lymphoid proliferations
Lymphoid proliferation in interstitial lung disease
Rare benign pulmonary lymphoid proliferations
Follicular bronchiolitis
Clinical features
Pathology
Lymphoid interstitial pneumonia
Clinical features
Pathology
Differential diagnosis
Pulmonary nodular lymphoid hyperplasia
Clinical features
Pathology
Differential diagnosis
Castleman disease
Multicentric Castleman disease
Clinical features
Pathology
Primary pulmonary B-cell lymphomas
Concept and histogenesis
Marginal zone lymphoma of mucosa-associated lymphoid tissue (MALT-type)
Clinical features
Pathology
Immunohistochemistry
Molecular pathology
Differential diagnosis
Treatment
Diffuse large B-cell lymphoma
Clinical features
Pathological features
Immunohistochemistry and molecular pathology
Other primary pulmonary B-cell lymphomas
Lymphomatoid granulomatosis
Clinical features
Pathology
Immunohistochemistry
Differential diagnosis
Treatment
Intravascular large B-cell (angiotropic) lymphoma
Clinical features
Pathology
Immunohistochemistry and molecular pathology
Differential diagnosis
Plasma cell disorders
Amyloidosis
Pulmonary amyloidosis in systemic amyloidosis
Primary (localized) pulmonary amyloidosis
Immunohistochemistry and molecular pathology
Light chain deposition disease
Tumor-associated amyloid
Multiple myeloma
Plasmacytoma
Clinical features
Pathology
Immunohistochemistry and differential diagnosis
Treatment
Hodgkin lymphoma
Primary pulmonary T-cell lymphomas
Anaplastic large cell lymphoma
Other primary pulmonary T-cell lymphomas
Pulmonary lymphomas in immunodeficient patients
Age-related EBV-associated B-cell lymphoproliferative disorders
Pulmonary lymphoma in congenital immunodeficiency
HIV/AIDS-related pulmonary lymphoma
Post-transplant immunoproliferative disorders in the lung
Pulmonary lymphoma associated with cytotoxic drugs
Secondary pulmonary changes in systemic lymphoma
Secondary malignant lymphoma
Clinical features
Specific lymphoma types
B-cell lymphomas
T-cell lymphomas
Hodgkin lymphoma
Pulmonary complications of leukemia
Systemic mast cell disease
Myeloid metaplasia in the lung
Lung biopsy in leukemic patients
Histiocytic disorders
Langerhans cell histiocytosis
Clinical features
Radiology
Diagnosis
Pathology
Histogenesis
Differential diagnosis
Multisystem and malignant Langerhans cell disease
Other tumors of histiocytes and dendritic cells
Erdheim-Chester disease
Clinical features
Pathology
Rosai-Dorfman disease
Pleural lymphomas
Primary effusion lymphoma
Clinical features
Pathology
Immunohistochemistry and molecular pathology
Differential diagnosis
Pyothorax-associated lymphoma
Clinical features
Pathology
Immunohistochemistry and molecular pathology
Differential diagnosis
Secondary pleural lymphoma
References
Chapter 35: Metastases involving the lungs
Introduction
Route of spread for metastases to reach the lung
Incidence
Clinical features
Patterns of pulmonary metastases
Parenchymal nodules
Pulmonary lymphangitic carcinomatosis
Endobronchial metastases
Tumor emboli
Unusual patterns of metastatic spread
Lepidic
Interstitial and/or intra-alveolar spread
Intrathoracic nodal spread
Specific organ system metastases
Head and neck
Thyroid
Breast
Liver
Pancreas
Colon and rectum
Kidney
Ovary
Uterus
Gestational trophoblastic diseases
Prostate
Testis
Neuroendocrine carcinomas
Sarcomas
Malignant melanoma
Lymphomas and leukemias
References
Chapter 36 Diseases of the pleura
Introduction
Normal pleural fluid volume and cell content
Mesothelial cell function
Pleural effusion
Physiology
Clinical features
Transudates and exudates
Pleural transudates
Congestive cardiac failure
Hepatic cirrhosis
Peritoneal dialysis
Urinothorax
Nephrotic syndrome
Atelectasis
Pleural exudates
Infective pleural effusions
Eosinophilic pleural effusions
Radiology of pleural effusions
Pathophysiology of pleural effusion
Treatment
Pneumothorax
Epidemiology
Primary spontaneous pneumothorax (PSP)
Pathogenesis
Secondary spontaneous pneumothorax (SSP)
Genetics
Clinical features of pneumothorax
Pathology
Pathophysiology
Differential diagnosis
Pulmonary Langerhans cell histiocytosis
The pleura in cystic fibrosis
Eosinophilic vasculitis
Pleural fibrosis, idiopathic pleuritis and other changes
Dressler syndrome (post-cardiac injury syndrome)
IgG4-related sclerosing disease
Apical cap
Pathogenesis
Differential diagnosis
Amyloid
Granulomatous pleural disease
Parasitic infections
The pleura in connective tissue diseases
Rheumatoid disease
Systemic lupus erythematosus (SLE)
Other collagen diseases
Drug-induced pleural disease
Pleural endometriosis
Chylothorax
Calcifying fibrous tumor of the pleura
Clinical features
Pathology
Differential diagnosis
Prognosis
Solitary fibrous tumor of the pleura
Introduction
Cell of origin
Genetics
Clinical features
Radiology
Macroscopic pathology
Histopathology
Cytology
Immunohistochemistry
Electron microscopy
Molecular findings
Differential diagnosis
Clinicopathological correlation
Prognosis, natural history and therapy
Benign asbestos-related pleural disease
Introduction
Pleural plaques
Factors affecting plaque production
Epidemiology
Etiology
Non-asbestos causes of pleural plaques
Pleural plaques and smoking
Routes by which asbestos fibers reach the pleura
Genetics
Lag period between asbestos exposure and plaque development
Clinical features
Radiology
Pathology
Macroscopic appearance
Histopathology
Pathological differential diagnosis
Diffuse pleural fibrosis
Mesothelioma
Desmoid tumor
Risk of development of pleuro-pulmonary malignancy in the presence of pleural plaques
Lung cancer
Mesothelioma
Benign asbestos-related pleural effusion
Definition and epidemiology
Etiology
Lag period between asbestos exposure and development of an asbestos-related effusion
Genetics
Clinical manifestations
Pathology
Differential diagnosis
Prognosis and natural history
Diffuse pleural fibrosis (DPF) (termed pachypleuritis in Europe)384
Incidence
Genetics
Etiology
Clinical manifestations
Lag period between asbestos exposure and development of DPF
Radiographic findings
Pathology
Macroscopic
Histology
Asbestos fiber counts in DPF
Differential diagnosis
Progression and prognosis
Risk of development of pleuropulmonary malignancy in cases of DPF
Pathophysiology
Folded lung (shrinking pleuritis with rounded atelectasis)
Clinical manifestations
Etiology
Radiology
Pathology
Cytology
Macroscopic pathology
Histopathology
Asbestos count in rounded atelectasis (RA)
Differential diagnosis
Infection
Pleural plaques
Prognosis
Atypical mesothelial hyperplasia (AMH)
Atypical mesothelial hyperplasia versus so-called in situ malignant mesothelioma and mesothelioma with minimal invasion
Ancillary techniques in reactive mesothelial hyperplasia versus neoplastic processes
Malignant pleural mesothelioma (MPM)
Introduction
Anatomical sites
Epidemiology
Asbestos fiber type and threshold for developing mesothelioma
Types of exposure to asbestos
Occupational exposure to asbestos
Paraoccupational mesothelioma (non-work-related mesotheliomas), including mesothelioma in women
Exposure to asbestos in buildings (outside Turkey)
Environmental mesothelioma
``Familial mesothelioma´´
Mesothelioma in children
Occupational and other risk factors for peritoneal mesothelioma
Non-asbestos causes of mesothelioma
Erionite - discussed above
Fluoro-edenite
Thorotrast
Ionizing radiation
Man-made mineral fibers
Simian virus 40
Latent period from exposure to asbestos to development of mesothelioma
Mechanisms of asbestos-induced oncogenesis
Cell signaling by asbestos in MPM, including reactive oxygen species
Apoptotic control in MPM
MicroRNA (MiRNA)
Tumor suppressor genes
Epidermal growth factor receptor (EGFR) and MPM
PI3K/AKT pathway in MPM
Growth factors and MPM
Matrix metalloproteinases (MMPs) and MPM
Chromosomal changes in pleural mesothelioma
Clinical features of malignant pleural mesothelioma
Radiology
Staging of malignant pleural mesothelioma
Primary, non-pleural, mesotheliomas
Peritoneal mesothelioma (MPeritM)
Pericardial mesothelioma
Mesothelioma of the ovary
Mesothelioma of the tunica vaginalis
Testicular mesothelioma
Primary hepatic mesothelioma
Cystic tumor (``mesothelioma´´) of the atrioventricular node
Cytology
Macroscopic pathology
Diffuse mesothelioma
Localized mesotheliomas
Synchronous bilateral pleural tumors
Spread of pleural mesothelioma
Intra-pulmonary spread
Distant spread
Peritoneal mesothelioma (MPeritM)
Examination of lungs from a pleural mesothelioma
Histology of pleural and peritoneal mesotheliomas
Epithelioid mesothelioma
Sarcomatoid mesothelioma
Desmoplastic mesothelioma
Biphasic or mixed mesothelioma
Histological subtypes of epithelioid mesothelioma (Table 12)
Deciduoid peritoneal mesothelioma
Microcystic mesothelioma (adenomatoid)
Mucin-positive mesotheliomas
Lipid-rich diffuse MPM
Clear cell mesothelioma
Pleomorphic (giant cell) mesothelioma
Lepidic pattern (in lung)
Small cell mesothelioma
Histological variants of sarcomatoid mesothelioma
Lymphohistiocytoid mesothelioma
Reproducibility of histopathological diagnosis of mesothelioma vs. adenocarcinoma
Electron microscopic features of mesotheliomas
Epithelioid mesothelioma
Sarcomatoid mesothelioma
Biphasic mesothelioma
Special stains in mesothelioma
Histochemistry
Mucins
Immunohistochemistry
The ability of immunohistochemistry to distinguish between epithelioid mesothelioma (EpM) and pulmonary adenocarcinoma (PAC)
Carcinoma markers
Carcinoembryonic antigen (CEA)
Glycoproteins Ber-EP4 and B72.3
MOC-31
E-cadherins
Thyroid transcription factor-1 (TTF-1)
Blood group antigens (Lewisy)
Intermediate filaments and cytokeratins (CK)
Cytokeratin-negative mesotheliomas
Mesothelioma markers
Calretinin
Wilms´ tumor gene product (WT1)
Cytokeratin 5/6
D2-40
Podoplanin
N- and E-cadherin
HBME-1
Miscellaneous immunohistochemical markers
GLUT1
Newer antibodies
An immunohistochemical algorithm for differentiating between mesothelioma and adenocarcinoma
The role of immunohistochemistry in the diagnosis of biphasic and sarcomatoid mesothelioma
Cytokeratins
CK5/6
Calretinin
HBME-1, thrombomodulin, N-cadherin and WT-1
Separation of benign from malignant pleural disease
The role of immunohistochemistry in differentiating between benign and malignant mesothelial proliferations
p53
Epithelial membrane antigen
Desmin
Bcl-2 and p-170
Argyrophil nucleolar organizer regions (AgNOR)
Other stains
Well-differentiated papillary mesothelioma (WDPM)
Clinical features
Pathology
Electron microscopy
Differential diagnosis
Prognosis
Differential diagnosis of pleural mesothelioma
Pleural epithelioid tumors
Carcinoma of lung
Primary squamous carcinoma of the pleura
Carcinoma, metastatic from an extrapulmonary site
Thymic tumors
Serous papillary carcinoma of the ovary
Adenomatoid tumor of the pleura
Malignant melanoma
Epithelioid hemangioendothelioma
Pleural-based lymphoma
Alveolar rhabdomyosarcoma
Biphasic tumors
Sarcomatoid carcinoma and carcinosarcoma
Synovial sarcoma
Sarcomatoid tumors
Sarcomatoid carcinoma
Pleural spindle cell sarcomas
Solitary fibrous tumor of the pleura
Desmoplastic round cell tumor (DRSCT)
Peripheral nerve sheath tumors
Ganglioneuroma and other neural neoplasms
Primitive neuroectodermal tumor (PNET)/Ewing sarcoma
Leiomyoma, smooth muscle tumor of undetermined malignant potential and leiomyosarcoma
Desmoid tumor
Inflammatory myofibroblastic tumors (IMT)
Other tumors
Differential diagnosis of peritoneal mesothelioma in women
Peritoneal mesothelial proliferations
Peritoneal mesothelial hyperplasia
Peritoneal inclusion cysts
Multiloculated peritoneal inclusion cysts (``benign cystic mesothelioma´´) (MPIC)
Well-differentiated papillary mesothelioma (WDPM)
Adenomatoid tumor
Peritoneal serous lesions
Endosalpingiosis
Peritoneal serous borderline tumors
Low-grade peritoneal serous carcinomas
High-grade peritoneal serous carcinoma
Primary peritoneal carcinoma
Role of immunocytochemistry in distinguishing mesothelioma from ovarian and peritoneal serous tumors
Pathophysiology of malignant pleural mesothelioma
Prognosis in malignant pleural mesothelioma
Other biological prognostic factors
Prognosis after extrapleural pneumonectomy for pleural mesothelioma
References
Index

Citation preview

Spencer’s Pathology of the Lung Sixth Edition

Spencer’s Pathology of the Lung Sixth Edition Edited by

Philip Hasleton, MD FRCPath Professor of Pathology, University of Manchester, UK, and Visiting Professor of Pathology, Hebrew University, Haddasah Medical School, Jerusalem, Israel

and

Douglas B. Flieder, MD Professor of Pathology, Fox Chase Cancer Center, Philadelphia, PA, USA

CAMBRIDGE UNIVERSITY PRESS Cambridge, New York, Melbourne, Madrid, Cape Town, Singapore, São Paulo, Delhi, Mexico City

Cambridge University Press, United VRG The Edinburgh Building, Cambridge CB2 8RU, UK Published in the United States of America by Cambridge University Press, New York www.cambridge.org Information on this title: www.cambridge.org/9780521509954 Sixth edition © Cambridge University Press 2013 Legacy material by Herbert Spencer © The Estate of the late Herbert Spencer 2013 First, Second, Third, Fourth and Fifth editions © The McGraw-Hill Companies Inc. 1962, 1968, 1977, 1985, 1996 This publication is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. Sixth edition first published 2013 Printed and bound in the United Kingdom by the MPG Books Group A catalogue record for this publication is available from the British Library Library of Congress Cataloging-in-Publication Data Spencer’s pathology of the lung. – 6th ed. / edited by Philip Hasleton, Douglas B. Flieder. p. ; cm. Pathology of the lung Includes bibliographical references and index. ISBN 978-0-521-50995-4 (set : Hardback) – ISBN 978-1-107-02433-5 (volume 1 : Hardback) – ISBN 978-1-107-02434-2 (volume 2 : Hardback) I. Hasleton, P. S. II. Flieder, Douglas B. III. Spencer, United VRG, Herbert, 1915– Pathology of the lung. IV. Title: Pathology of the lung. [DNLM: 1. Lung Diseases–pathology. 2. Lung–pathology. 3. Lung Diseases–diagnosis. WF 600] 616.20 407–dc23 2011046214 Two volume set ISBN 978-0-521-50995-4 Hardback Volume 1 ISBN 978-1-107-02433-5 Hardback Volume 2 ISBN 978-1-107-02434-2 Hardback Cambridge University Press has no responsibility for the persistence or accuracy of URLs for external or third-party internet websites referred to in this publication, and does not guarantee that any content on such websites is, or will remain, accurate or appropriate. All material contained within the CD-ROM is protected by copyright and other intellectual property laws. The customer acquires only the right to use the CD-ROM and does not acquire any other rights, express or implied, unless these are stated explicitly in a separate licence. Every effort has been made in preparing this book to provide accurate and up-to-date information which is in accord with accepted standards and practice at the time of publication. Although case histories are drawn from actual cases, every effort has been made to disguise the identities of the individuals involved. Nevertheless, the authors, editors and publishers can make no warranties that the information contained herein is totally free from error, not least because clinical standards are constantly changing through research and regulation. The authors, editors and publishers therefore disclaim all liability for direct or consequential damages resulting from the use of material contained in this book. Readers are strongly advised to pay careful attention to information provided by the manufacturer of any drugs or equipment that they plan to use.

PSH To my wife for her infinite patience and understanding and to my children and grandchildren, whom I hope to now see more often. DBF To my wonderful parents Sally and Bill and truly exceptional wife Rita. For my terrific children Anna and Jonny.

Contents List of contributors ix Foreword to the First Edition xiv Preface to the Sixth Edition xv Acknowledgements xvi

Volume 1 1.

The normal lung: histology, embryology, development, aging and function 1 Neil Sahasrabudhe, John R. Gosney and Philip Hasleton

2.

Lung specimen handling and practical considerations 41 Leslie Anne Litzky and Anthony Gal

3.

Congenital abnormalities and pediatric lung diseases, including neoplasms 66 Stephen J. Gould, A.K. Webb and Anna Kelsey

4.

Pulmonary bacterial infections 146 Mark Woodhead, Mary Klassen-Fischer, Ronald C. Neafie, Ann-Marie Nelson, Jeffrey R. Galvin and Teri J. Franks

11. Metabolic and inherited connective tissue disorders involving the lung 409 Gail Amir and Annick Raas-Rothschild 12. Hypersensitivity pneumonitis John C. English

439

13. Sarcoidosis 475 Douglas B. Flieder, Abraham Sanders and Michael N. Koss 14. Occupational lung disease 512 Thomas Sporn and Victor L. Roggli 15. Eosinophilic lung disease 563 Henry D. Tazelaar, Joanne L. Wright and Jay H. Ryu 16. Drug- and therapy-induced lung injury Mary Beth Beasley and Glenn A. Rudner

585

5.

Pulmonary viral infections 182 Richard L. Kradin and Jay Fishman

17. Chronic obstructive pulmonary disease and diseases of the airways 605 Wim Timens, Hannie Sietsma and Joanne L. Wright

6.

Pulmonary mycobacterial infections 206 Luciane Dreher Irion and Mark Woodhead

18. Pulmonary vascular pathology 661 Katrien Grünberg and Wolter J. Mooi

7.

Pulmonary mycotic infections 226 Said Khayyata, Caroline B. Moore, Malcolm D. Richardson, Philip Hasleton and Carol Farver

8.

Pulmonary parasitic infections 288 Sebastian Lucas, Philip Hasleton, Ann-Marie Nelson and Ronald C. Neafie

19. Pulmonary vasculitis and pulmonary hemorrhage syndromes 711 Eugene J. Mark, Rex Neal Smith, John H. Stone, Douglas B. Flieder, Amita Sharma and Osamu Matsubara

9.

Acute lung injury 342 William A.H. Wallace, A. John Simpson and Nik Hirani

10. Interstitial lung diseases 366 Andrew G. Nicholson and Alexandra J. Rice

20. The pathology of lung transplantation Gerald J. Berry and Helen M. Doran

767

21. The lungs in connective tissue disease 804 Donald G. Guinee Jr. and William D. Travis

vii

Contents Volume 2 22. Benign epithelial neoplasms and tumor-like proliferations of the lung 847 Douglas B. Flieder 23. Pulmonary pre-invasive disease Keith M. Kerr

889

24. Epidemiological and clinical aspects of lung cancer 945 Erik Thunnissen, Michael Unger and Douglas B. Flieder 25. Lung cancer staging 1004 Eric Lim and Peter Goldstraw 26. Immunohistochemistry in the diagnosis of pulmonary tumors 1015 Paul William Bishop 27. Adenocarcinoma of the lung 1043 Douglas B. Flieder, Alain C. Borczuk and Masayuki Noguchi 28. Squamous cell carcinoma of the lung 1093 Douglas B. Flieder and Elisabeth Brambilla 29. Large cell carcinoma and adenosquamous carcinoma of the lung 1114 Sylvie Lantuejoul, Michèle Fior-Gozlan, Gilbert R. Ferretti and Denis Moro-Sibilot

viii

30. Salivary gland neoplasms of the lung 1127 Sanja Dacic, Sebastian Gilbert, Iclal Ocak and Joan Lacomis 31. Neuroendocrine tumors and other neuroendocrine proliferations of the lung 1151 John R. Gosney 32. Sarcomatoid carcinomas and variants 1186 Yukio Nakatani, Kenzo Hiroshima and Eugene J. Mark 33. Mesenchymal and miscellaneous neoplasms Timothy C. Allen, Philip T. Cagle and Douglas B. Flieder 34. Pulmonary lymphoproliferative diseases Bruce J. Addis

1224

1316

35. Metastases involving the lungs 1375 Bruno Murer, Marco Chilosi, Philip Hasleton and Douglas B. Flieder 36. Diseases of the pleura 1408 Philip Hasleton, Francoise Galateau-Salle, Juliet King, Giulio Rossi, Sylvie Lantuejoul, Rebecca Preston, Durgesh N. Rana and Godfrey Wilson

Index

1565

Contributors

Bruce J. Addis, MBBS DCP FRCPath Consultant Histopathologist, Southampton University Hospitals NHS Trust, Southampton, UK Timothy C. Allen, MD JD Professor of Pathology, Chair, Department of Pathology, The University of Texas Health Science Center at Tyler, Tyler, TX, USA Gail Amir, MB ChB Department of Pathology, Hadassah Medical Center/Hebrew University, Jerusalem, Israel Mary Beth Beasley, MD Associate Professor of Pathology, Mt. Sinai Medical Center, New York, NY, USA Gerald J. Berry, MD Professor of Pathology, Co-Director of Surgical Pathology, Stanford University Medical Center, Stanford, CA, USA Paul William Bishop, BA MB BCh FRCPath Department of Pathology, University Hospital of South Manchester, Manchester, UK Alain C. Borczuk, MD Department of Pathology, Columbia University Medical Center, New York, NY, USA Elisabeth Brambilla, MD Departement d’Anatomie Cytologie Pathologique, Centre Hospitalier Universitaire, Grenoble, France Philip T. Cagle, MD Professor of Pathology and Laboratory Medicine, Weill Medical College of Cornell University,

New York, NY, and The Methodist Hospital, Houston, TX, USA Marco Chilosi, MD Professor of Pathology, Department of Pathology, University of Verona, Verona, Italy Sanja Dacic, MD PhD Associate Professor of Pathology, Department of Pathology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Helen M. Doran, FRCPath Consultant Histopathologist, Department of Histopathology, Clinical Sciences Building, Wythenshawe Hospital, Manchester, UK John C. English, MD FRCPC Vancouver General Hospital, Pathology & Laboratory Medicine, Vancouver, BC, Canada Carol Farver, MD Section Director, Surgical Pathology, Director, Pulmonary Pathology, Pathology and Laboratory Medicine Institute, Cleveland Clinic, Cleveland, OH, USA Gilbert R. Ferretti, MD PhD Department of Radiology, CHU Michallon, Joseph Fourier University, INSERM U 823, Grenoble, France

ix

List of contributors

Michèle Fior-Gozlan, MD Department of Pathology, CHU Michallon, Joseph Fourier University, INSERM U 823, Grenoble, France Jay Fishman, MD Director, Infectious Disease Transplantation Service, Infectious Disease Unit, Department of Medicine, Boston, MA, and Associate Professor, Harvard Medical School, Boston, MA, USA

Stephen J. Gould Consultant Pediatric Pathologist, John Radcliffe Hospital, Headington, Oxford, UK Katrien Grünberg, MD PhD VU Medical Center, Department of Pathology, Amsterdam, The Netherlands

Douglas B. Flieder, MD Professor of Pathology, Department of Pathology, Fox Chase Cancer Center, Philadelphia, PA, USA

Donald G. Guinee Jr., MD Department of Pathology, Virginia Mason Medical Center, Seattle, WA, USA

Teri J. Franks, MD Pulmonary and Mediastinal Pathology, The Joint Pathology Center, Silver Spring, MD, USA

Philip Hasleton, MD FRCPath Professor of Pathology, University of Manchester, UK, and Visiting Professor of Pathology, Hebrew University, Haddasah Medical School, Jerusalem, Israel

Anthony Gal, MD Department of Pathology and Laboratory, Emory University School of Medicine, Atlanta, GA, USA Francoise Galateau-Salle Department of Pathology, MesoPath Center, University of Caen, Caen, France Jeffrey R. Galvin, MD Chief, Thoracic Radiology, American Institute for Radiologic Pathology, Silver Spring, MD, USA, and Professor, Diagnostic Radiology and Pulmonary/Critical Care Medicine, University of Maryland School of Medicine, Baltimore, MD, USA Sebastian Gilbert, MD Assistant Professor of Surgery, Department of Surgery, Division of Thoracic Surgery, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Peter Goldstraw, MB FRCS Imperial College and the Academic Division of Thoracic Surgery, The Royal Brompton Hospital, London, UK

x

John R. Gosney, MD FRCPath Department of Histopathology, Royal Liverpool Hospital, Liverpool, UK

Nik Hirani, PhD MRCP Senior Clinical Lecturer and Honorary Consultant, Respiratory Medicine Unit, College of Medicine and Veterinary Medicine, University of Edinburgh, and Royal Infirmary of Edinburgh, Edinburgh, UK Kenzo Hiroshima, MD Department of Pathology, Tokyo Women’s Medical University Yachiyo Medical Center, Yachiyo-shi, Chiba, Japan Luciane Dreher Irion, MSc PhD FRCPath Consultant Histopathologist, Central Manchester University Hospitals NHS Foundation Trust, and Visiting Lecturer, University of Manchester, Manchester, UK Anna Kelsey, MRCS LRCP FRCPath Department of Diagnostic Histopathology, Royal Manchester Children’s Hospital, Manchester, UK Keith M. Kerr, FRCPath FRCPEd Consultant Pathologist and Hon Professor of Pulmonary Pathology,

List of contributors

Department of Pathology, University of Aberdeen School of Medicine, Aberdeen Royal Infirmary, Aberdeen, UK

Leslie Anne Litzky, MD Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA, USA

Said Khayyata, MD Assistant Professor of Pathology, William Beaumont Hospital-Troy Campus, Oakland University William Beaumont School of Medicine, Troy, MI, USA

Sebastian Lucas, FRCP FRCPath Department of Histopathology, King’s College London School of Medicine, St. Thomas’ Hospital, London, UK

Juliet King, BM DM FRCS Cth Consultant Thoracic Surgeon Guys and St. Thomas NHS Foundation Trust, London, UK

Eugene J. Mark, MD Professor of Pathology, Harvard Medical School, and Pathologist, Massachusetts General Hospital, Boston, MA, USA

Mary Klassen-Fischer, MD Infectious Disease Pathology, The Joint Pathology Center, Silver Spring, MD, USA Michael N. Koss, MD Department of Pathology, Keck School of Medicine of the University of Southern California, Los Angeles, CA, USA Richard L. Kradin, MD MS MLA DTMH Director, Infectious Disease Surgical Pathology, Departments of Pathology and Medicine (Pulmonary and Critical Care), Massachusetts General Hospital, and Associate Professor, Harvard Medical School, Boston, MA, USA Joan Lacomis, MD Associate Professor of Radiology, Department of Radiology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Sylvie Lantuejoul, MD PhD Department of Pathology, CHU Michallon, Joseph Fourier University, INSERM U 823, Grenoble, France Eric Lim, MD Msc FRCS Imperial College and the Academic Division of Thoracic Surgery, The Royal Brompton Hospital, London, UK

Osamu Matsubara, MD PhD Professor and Chair of Department of Pathology, National Defense Medical College, Tokorozawa, Saitama, Japan Wolter J. Mooi, MD PhD VU Medical Center, Department of Pathology, Amsterdam, The Netherlands Caroline B. Moore, PhD MSB Principal Clinical Scientist Mycology Reference Centre, Manchester Academic Health Science Centre, University Hospital of South Manchester, Manchester, UK Denis Moro-Sibilot, MD Department of Respiratory Medicine, CHU Michallon, Joseph Fourier University, INSERM U 823, Grenoble, France Bruno Murer, MD Surgical Pathology Unit, Department of Clinical Pathology, Ospedale dell’Angelo, Mestre-Venice, Italy Yukio Nakatani, MD Department of Diagnostic Pathology, Graduate School of Medicine, Chiba University, Chiba, Japan Ronald C. Neafie, MS Formerly Chief, Parasitic Diseases Pathology Branch,

xi

List of contributors

Division of Infectious and Tropical Diseases Pathology, Department of Environmental and Infectious Disease Sciences, Armed Forces Institute of Pathology, Washington DC, USA Ann-Marie Nelson, MD Infectious Disease and AIDS Pathology, The Joint Pathology Center, Silver Spring, MD, USA Andrew G. Nicholson, FRCPath Professor of Respiratory Pathology, Imperial College London, and Consultant Histopathologist, Royal Brompton Hospital, Sydney Street, London, UK Masayuki Noguchi, MD Department of Pathology, Institute of Basic Medical Sciences, University of Tsukuba, Tsukuba-shi, Ibaraki, Japan Iclal Ocak, MD Assistant Professor of Radiology, Department of Radiology, University of Pittsburgh Medical Center, Pittsburgh, PA, USA Rebecca Preston MBBS BSc MRCP FRCP Department of Radiology, Guy’s and St Thomas’ NHS Foundation Trust, London, UK Annick Raas-Rothschild, MD Department of Human Genetics, Hadassah Medical Center/Hebrew University, Jerusalem, Israel Durgesh N. Rana MBBS FRCPath Manchester Cytology Centre, Manchester Royal Infirmary, Manchester, UK Alexandra J. Rice, FRCPath Consultant Histopathologist, Royal Brompton Hospital, Sydney Street, London, UK Malcolm D. Richardson, PhD FSB FRCPath Professor of Medical Mycology, Mycology Reference Centre, Manchester Academic Health Science Centre,

xii

University Hospital of South Manchester, Manchester, UK Victor L. Roggli, MD Professor of Pathology, Department of Pathology, Duke University Medical Center, Durham, NC, USA Giulio Rossi, MD PhD Section of Pathologic Anatomy Azienda Ospedaliero-Universitaria Policlinico of Modena Modena, Italy Glenn A. Rudner, MD Assistant Clinical Professor of Pathology, Mt. Sinai Medical Center, New York, NY, USA Jay H. Ryu, MD Division of Pulmonary and Critical Care Medicine, Mayo Clinic Rochester, Rochester, MN, USA Neil Sahasrabudhe, MB ChB FRCPath Department of Histopathology, Royal Blackburn Hospital, Blackburn, UK Abraham Sanders, MD Department of Pulmonary Medicine, New York-Presbyterian Hospital, Sanford A. Weil Medical College of Cornell University, New York, NY, USA Amita Sharma, MD Instructor of Radiology, Harvard Medical School, and Assistant Radiologist, Division of Thoracic Imaging and Intervention, Massachusetts General Hospital, Boston, MA, USA Hannie Sietsma, MD PhD Department of Pathology, Martini Hospital Groningen, Groningen, The Netherlands A. John Simpson MD PhD FRCPE Professor of Respiratory Medicine, Newcastle University, Newcastle-upon-Tyne, UK Rex Neal Smith, MD Associate Professor of Pathology, Harvard Medial School, and

List of contributors

Associate Pathologist, Massachusetts General Hospital, Boston, MA, USA Thomas Sporn, MD Associate Professor, Department of Pathology, Duke University Medical Center, Durham, NC, USA John H. Stone, MD Associate Professor of Medicine, Harvard Medical School, and Director, Clinical Rheumatology, Massachusetts General Hospital, Boston, MA, USA Henry D. Tazelaar, MD Chair, Department of Laboratory Medicine and Pathology, Professor of Pathology, College of Medicine, Mayo Clinic, Mayo Clinic Arizona, Scottsdale, AZ, USA Erik Thunnissen, MD PhD Department of Pathology 1E15, VU Medical Center, Amsterdam, The Netherlands Wim Timens, MD PhD Professor of Pathology, Department of Pathology, University Medical Center Groningen, Groningen, The Netherlands William D. Travis, MD Attending Thoracic Pathologist, Department of Pathology,

Memorial Sloan Kettering Cancer Center, New York, NY, USA Michael Unger, MD Professor of Medicine Director, Pulmonary Endoscopy and High-Risk Lung Cancer Program, Fox Chase Cancer Center, Philadelphia, PA, USA William A.H. Wallace, MB ChB (Hon) PhD FRCPE FRCPath Consultant Pathologist and Honorary Reader in Pathology, College of Medicine and Veterinary Medicine, University of Edinburgh and Royal Infirmary of Edinburgh, Edinburgh, UK A.K. Webb, MBBS FRCP Manchester Adult Cystic Fibrosis Centre, Wythenshawe Hospital, Manchester, UK Godfrey Wilson, MB BCh BAO FRCPath Consultant Gynecological Histopathologist Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK Mark Woodhead, BSc DM FRCP Consultant in General & Respiratory Medicine, and Honorary Clinical Professor, University of Manchester, Central Manchester University Hospitals NHS Foundation Trust, Manchester, UK Joanne L. Wright, MD Professor, University of British Columbia, Department of Pathology, St Paul’s Hospital, Vancouver, BC, Canada

xiii

Foreword to the First Edition

At first glance the lungs may seem uncomplicated, but many wise men have gone astray in their labyrinths. When apparently “simplified” as in emphysema, they have remained refractory to analysis. Disease commonly results in a profound but variable revision of their architecture. Their tumors form a bewildering array and some exert profound metabolic effects unsuspected until recently. Blood comes to the lungs from both sides of the heart, in a proportion that may deviate considerably from the norm under particular conditions. The vessels reflect alterations in hemodynamics, and when themselves changed, they can profoundly affect the work of the heart. The pulmonary capillaries, lying as a filter astride the venous outflow of all other organs, must often suffer the consequences. With each breath, also, the innermost recesses of the respiratory tract are brought very much into contact with a sometimes hostile external environment. The lungs are thus vulnerable from all sides. That we are not more often disabled we owe to their marvelous capacity to recover from injury and to their large reserve. A man’s medical history and the traces of his habits and his trade are often inscribed upon the lungs – for him who can read. Not since the monumental contribution of Fischer in the

xiv

Handbook of Henke-Lubarsch have the lungs been so thoroughly or so well read, and the reading so well recorded as in this volume. Recent years have witnessed the identification, and even the introduction, of many new agents of pulmonary disease. Many other conditions such as “eosinophilic granuloma”, while still of unknown etiology, have been defined in anatomical terms. Cardiopulmonary disease in the broadest sense is now much better understood than it was twenty years ago. The intelligent use of the cardiac catheter in man and in many ingenious experiments in animals and the development of cardiac surgery have greatly broadened our comprehension of this subject. Although the current exponential increase in knowledge indicates how much there is yet to learn, the time is surely ripe for a sound and comprehensive statement of what is now known. Doctor Spencer has supplied this need admirably, and with a fine sense of history. Only a rare concurrence of meticulous scholarship and discernment could have enabled the condensation of so much information into so little space. This work will long be of interest and value to all students of disease. Averill A. Liebow 1962

Preface to the Sixth Edition

Medicine has drastically changed since Dr. Liebow introduced the first edition of this textbook 50 years ago. Otto Lubarsch and Friedrich Henke are unknown today to most practicing pathologists and “eosinophilic granuloma” is no longer of unknown etiology. Technological advances in radiology, immunohistochemical developments and molecular discoveries along with an ever-expanding therapeutic armamentarium have led to new clinicopathological disease classifications that have greatly impacted chest medicine. Our understanding and classification of interstitial lung diseases and, for example, the five categories of “angiitis and granulomatoses” have greatly evolved beyond mid-twentieth-century beliefs, and today a diagnosis of “non-small-cell carcinoma” is almost universally frowned upon, especially by chest oncologists! Specialized pulmonary hospitals were once rare, but now multidisciplinary disease teams in such institutions and other centers around the world effectively treat patients with respiratory diseases. Simply put, progress, although seemingly slow and uncertain, alters medical knowledge and patient care almost daily. In this light, any attempt to produce an up-to-date educational and clinically useful tome is fraught with obstacles, not least of which is the threat of immediate obsolescence. However, to shirk from such a task would be in the best sense a disservice to those interested in updated concepts, and in the worst, nothing less than intellectual cowardice. This fiftieth anniversary edition demonstrates that the current generation of pulmonary pathologists accepts the task to update this veritable classic pulmonary pathology textbook.

It is interesting to note that the first edition comprised 23 chapters while this sixth edition contains 36 chapters. The current two-volume set includes focused discussions of clinical and radiographic presentations along with pathophysiology and clinicopathological correlations. Genetic and molecular findings are included for many diseases, but do not monopolize discussions. These expositions are invaluable for the pathologist working in a multidisciplinary care setting. Given the international nature of medicine, experts in particular topics from around the world were recruited to contribute their time and knowledge. It was astounding how many of the contributors fondly remembered learning lung pathology from prior editions of this textbook and it is our hope that this tradition continues. While some suggest that the days of multi-authored textbooks are cumbersome relics of the past, we believe this format offers bona fide expert discussions in one source. Since medical knowledge is no longer constrained to libraries and printed materials, or even personal computers, an electronic version of the textbook allows for the instant access many of us now need. In closing, we hope, as Averill Liebow wrote in 1962, that “this work will long be of interest and value to all students of disease”. Philip Hasleton, MD FRCPath Douglas B. Flieder, MD August 2012

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Acknowledgements

This transatlantic collaboration has many individuals to thank. We would like to thank all the authors who gave of their valuable time and accepted, when necessary, our suggestions. The expert medical library staff at Manchester Royal Infirmary under Rene Banerjee and the Fox Chase Cancer Center Talbot Library under Beth Lewis were incredibly helpful at locating articles and providing informatics support. UnitedColette Curry, Helen Carruthers and the Medical Illustration Department at Wythenshawe Hospital as well as Joe Hurley and Karen Trush at Fox Chase Cancer Center cheerfully provided skilled help at creating and cleaning figures and tables. Jean Schofield gave much time to the project. Lori Schaffert worked tirelessly as DF’s assistant and if not for her, we shudder to think what fate would have befallen this project. Special thanks are extended to Kristen Edwards at Fox Chase Cancer Center and Klaus Irion at the Liverpool Heart and Chest Hospital for sharing radiographic images, Martin

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Selig at Massachusetts General Hospital for contributing electron micrographs, and Aletta Frazier at the American Institute for Radiologic Pathology and University of Maryland School of Medicine for her wonderful medical illustrations. Many other colleagues also contributed images. We would also like to thank the many professionals at or affiliated with Cambridge University Press. United VRG. Nick Dunton and Nisha Doshi were helpful throughout the difficult process of writing and editing this large manuscript while production editor Caroline Mowatt and copy editor Roger Borthwick focused on producing the highest-quality textbook possible. Last but not least, DF would also like to thank Arthur Patchefsky at Fox Chase Cancer Center for his encouragement and generous help, and Maggie Flieder, whose company on long nightly walks provided many needed breaths of fresh air.

Chapter

1

The normal lung: histology, embryology, development, aging and function Neil Sahasrabudhe, John R. Gosney and Philip Hasleton

Introduction

Table 1 Stages of lung growth

Knowledge of normal lung anatomy and function is important for the interpretation of lung biopsies and resections. An understanding of different cell structures and functions allows for a greater appreciation of disease states. In addition basic pulmonary embryology explains congenital pulmonary defects. At the other extreme of life, knowledge of how the lung ages is important, not only for consideration of other diseases, such as hiatus hernias with co-existent aspiration, but also because of the world’s increasing elderly population.

Stage

Time

Main events

Embryonic

0–7 weeks

Formation of trachea, right and left main bronchi, segmental bronchi, and vasculogenesis around airway buds

Pseudoglandular

7–17 weeks

Differentiation of epithelial cells, formation of conduction airways and terminal bronchioles, formation of pulmonary arteries and veins

Canalicular

17–27 weeks

Formation of respiratory bronchioles, alveolar ducts and primitive alveoli, differentiation of type I and type II pneumocytes and formation of alveolar capillary barrier

Saccular

27–36 weeks

Increment in gas exchange areas, further differentiation of type I and type II cells

Alveolar

36 weeks – 2 years Up to 18–22 years

Septation and multiplication of alveoli Enlargement of terminal bronchioles and alveoli

Development The key events of pulmonary embryogenesis and postnatal development are discussed in this chapter. For a more detailed account, the reader is referred to two monographs1,2 and several review articles.3–7 The events of human lung growth are divided into five continuous stages, based on anatomic and histological characteristics.8 These are the embryonic, pseudoglandular, canalicular, saccular and alveolar stages (Table 1) (Figures 1 and 2). Airway and vascular development are closely related. The conducting airways are formed in the embryonic and pseudoglandular stages, while gas exchange units characterized by vascularization and reduction of mesenchyme are formed in the canalicular, saccular and alveolar stages.6

Airway and airspace development The lung first appears as a diverticulum or bud from the ventral wall of the foregut 22 to 26 days after fertilization. This bud grows caudally, with the epithelial cells from the foregut endoderm invading the surrounding mesenchyme to form the trachea (Figure 3). During the fourth week of gestation, the caudal end of the trachea divides into two bronchial buds, each proceeding to form the right and left main bronchi. By 32 to 35 days, the lobar bronchial buds form and up to 10 days later, the segmental and subsegmental bronchial buds develop.1 Further dichotomous branching continues and by 14 weeks 70% of the total airway is formed.9 At the end of the

Reprinted from Joshi S, Kotecha S. Lung growth and development. Early Hum Dev 2007;83:789–794. With permission from Elsevier.

pseudoglandular stage (17 weeks), the development of conducting airways up to the terminal bronchioles is complete.10 Human lung contains undifferentiated lung stem cells, nested in niches in the distal airways. These cells are selfrenewing, clonogenic, and multipotent in vitro. After injection into damaged mouse lung in vivo, human lung stem cells form human bronchioles, alveoli and pulmonary vessels integrated

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

1

(a)

(b)

(c)

(d)

(e)

(f)

Figure 1. (a–c) Developing lung at 14 weeks. Very immature lung at the late pseudoglandular stage. Developing airways are lined by cuboidal undifferentiated cells containing glycogen. Interstitium is wide and relatively poorly vascularized but new vessels are rapidly formed by vasculogenesis within the mesenchyme. (d–f) Fetal lung at 20 weeks. Canalicular stage lung with more elongated developing airways. Interstitium is reduced; vascularization is increased and some capillaries are starting to push into the cuboidal epithelium visible mainly at the airway branch points. (Images provided by Dr Stephen Gould, Oxford, UK.)

2

(a)

(b)

(c)

(d)

(e)

(f)

Figure 2. Stages in lung development. (a–c) Lung at 26 weeks. Late canalicular/early saccular stage, with increasingly close contact between the capillaries and the airway lumen. The blood-gas barriers are approaching the thinness found in the adult. Hence it is around this stage that premature infant survival may be possible. Cuboidal type II pneumocytes which, at least ultrastructurally, are producing surfactant are still very visible but type I pneumocytes are also differentiating over the capillaries. (d–f) The lung at 38 weeks. The lung is relatively mature and alveolarization is progressing quickly. By this stage, some 30–50% of the alveoli present in the adult have been formed. (Images provided by Dr Stephen Gould, Oxford, UK.)

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Chapter 1: The normal lung: histology, embryology, development, aging and function

(a)

(b)

(c) Esophagus

Mesenchyme

Trachea Pleural cavity

structurally and functionally with the damaged organ. The formation of a chimeric lung was confirmed by detection of human transcripts for epithelial and vascular genes. In addition, the self-renewal and long-term proliferation of human lung stem cells was shown in serial-transplantation assays.11 A population of endogenous or lung-resident (LR) mesenchymal stromal cells (MSCs) in human adult lungs has been identified in transplanted allograft bronchoalveolar lavage (BAL).12 Lung-resident mesenchymal stromal cells (LR-MSCs) were characterized as fibroblast-like clonal cells that differentiated into adipocytes, chondrocytes and osteocytes in vitro. They expressed mesenchymal markers, vimentin and prolyl-4-hydroxylase, and stem cell markers CD73, CD90, and CD105. Hematopoietic markers, CD14, CD34, and CD45, were not detected. More than 95% of the lungs’ MSCs were donor-derived. Using sex-mismatched lung transplant donor-recipient pairs, the data suggested a population of self-renewing LR-MSCs. The same authors13 demonstrated that lavage LR-MSCs were increased within 3 months, as well as more than 2 years after lung transplantation. Greater numbers of LR-MSCs were found in association with a recent diagnosis of BOS (bronchiolitis obliterans syndrome), a histological diagnosis of bronchiolitis obliterans (BO), or histopathological organizing pneumonia (OP). During this pseudoglandular stage, the epithelial and mesenchymal cells differentiate to form cartilage, submucosal glands, bronchial smooth muscle and the different epithelial cells. At this time, the airways are lined by tall columnar cells proximally and cuboidal cells distally. Ciliated, non-ciliated, basal, and goblet cells are also present. The respiratory bronchioles, alveolar ducts, and primitive alveoli form during the canalicular stage. Two important events occur in this stage, namely differentiation of pulmonary epithelium into type I and type II pneumocytes and formation of the alveolar capillary barrier with a marked increase in the vasculogenesis of the primitive interstitium. Surfactant protein is detectable by 24 weeks of intrauterine life.4 During the saccular stage, further enlargement and dilatation of the primitive alveoli into saccules occurs, with thinning

4

Figure 3. Diagram showing (a) the growth of the trachea and esophagus from primitive buds, (b) into the main bronchi surrounded by mesenchyme and (c) further differentiation of bronchi. Adapted from Sadler TW. Langman’s Medical Embryology, 6th ed. Williams and Wilkins, Baltimore, 1990.

of the airway walls due to reduction in the mesenchyme. This process continues into the alveolar stage, with development of true alveoli by formation of interalveolar walls (called secondary septa). This leads to an extensive increase in surface area. Alveolar multiplication then continues for at least 2 years, into postnatal life.14 Radial count estimation correlates better with total gestational age, crown-rump length, body weight, and fixed lung volume than any other morphometric parameter assessed. The radial count method provides a reliable index of lung growth in intrauterine and early postnatal development. The radial count method of Emery and Mithal was applied to the lungs of 37 infants of gestational age 19–42 weeks.15 There was a progressive increase in complexity of the terminal lung units throughout gestation. In intrauterine and early post-natal groups radial counts correlated very closely with the total gestational age (gestational age plus survival time after birth) of the child.

Vascular development The airways and vessels develop simultaneously, with the airways acting as a template for the development of pulmonary blood vessels. The pulmonary vasculature develops via two separate processes, angiogenesis and vasculogenesis. Angiogenesis is the sprouting of new vessels from existing ones and vasculogenesis is the de novo formation of vessels from the mesenchymal endothelial precursor cells.3 The proximal pulmonary arteries are thought to grow by the process of angiogenesis, originating from the sixth aortic arch, while peripheral blood vessels develop from vasculogenesis.5,16 The initial development sequence begins by formation of the heart at the end of the third week. The heart expels blood into the paired cranial ventral aortae, which are connected to the dorsal aortae by six aortic arches. The main pulmonary artery and the left and right main pulmonary arteries arise from the sixth aortic arch (Figure 4). The pulmonary arteries then continue to grow by angiogenesis around the airway buds from 4 to 16 weeks. The pre-acinar pulmonary veins also develop during this period.

Chapter 1: The normal lung: histology, embryology, development, aging and function

Right

Left

1

2

3

AT AS RLP

4 6 PA

Figure 4. Brachial arch arteries connecting the ventral aortic sac (AS) with the right and left dorsal aortae (LDA and RDA) in a 5 mm embryo. On the left, the dorsal and ventral sprouts of the sixth (pulmonary) arch have nearly met (RLP). On the right side, the arch is complete. From the ventral sprouts, plexiform vessels (PA) pass to the lung bud. Adapted from Congdon ED. Transformation of the aortic arch system during the development of the human embryo. Contribution to Embryology (Carnegie Institute) Washington 1922; 14:47.

LDA

RDA

A

The capillaries form by vasculogenesis. The process begins with the formation of spaces in the primitive mesenchyme, which harbor groups of progenitor endothelial cells called angioblasts. These cells differentiate and form endothelial cells. The endothelial cells fuse to form capillary channels and tubes. The newly formed tubes then connect to the existing vessels.5,17 During the alveolar stage of lung development, the secondary septa in the alveoli contain a double capillary network, with intervening supportive central connective tissue. During the postnatal period, the double capillary network gradually merges as a single capillary layer, termed microvascular maturation.18

Factors regulating lung development The development and growth of the lung is influenced by many factors including fetal lung fluid secretion and fetal breathing movements, environmental factors, such as tobacco smoke and pollutants including ozone and particulate matters, and various growth and transcriptional factors including hormones (Table 2).4 The major factor responsible for the pulmonary vascular development is vascular endothelial growth factor (VEGF). Vascular endothelial growth factor is a pluripotent growth and permeability factor; many different lung cells produce VEGF and also respond to it. Besides its critical role in fetal lung development, it also serves as a maintenance factor

Table 2 Possible role of some growth and transcriptional factors in lung growth

Transcriptional and growth factors

Possible role in lung growth and development

FOXA1, FOXA2, GATA4 and GATA6

Formation and maintenance of foregut

Tbx4

Localization of bud site

Fibroblast growth factors (FGFs)

Localization of organs derived from foregut Induction of budding and branching (FGF10) Alveolization Type II cell differentiation and induction of Surfactant protein C (FGF 2, FGF 7)

Sonic hedgehog

Suppresses FGF 10 expression and prevents branching events at sites where branching is stereotypically determined not to take place

Bone morphogenic protein 4

Formation and control of dorsal and ventral branches

HOX genes

Defines overall three-dimensional orientation

Epidermal growth factor

Airway proliferation, differentiation and branching

Platelet-derived growth factor

Alveolization

Retinoic acid

Induction of FGF 10 and endodermal differentiation

Transforming growth factor

Lung repair after pulmonary insult and matrix production Inhibits cell proliferation

Insulin-like growth factor

Airway proliferation

Vascular endothelial growth factor

Vasculogenesis, angiogenesis and lymphangiogenesis

Granulocyte-macrophage colony-stimulating factor

Macrophage differentiation

Reprinted from Joshi S, Kotecha S. Lung growth and development. Early Hum Dev 2007;83:789–94. With permission from Elsevier.

during adult life. In addition to the physiological functions of this protein, there is increasing evidence VEGF also plays a role in several acute and chronic lung diseases, such as acute lung injury, severe pulmonary hypertension, and emphysema (see Chapters 9, 17, and 18).19 Several genes play a role in the very early development of the trachea and primary lung buds. One of the most important is fibroblast growth factor (FGF). A full account of the growth factor signaling is given in an excellent chapter, which forms the basis of this section.20 FGF 1, 2, 9, 10, and 18 play overlapping but distinct roles in the developing lung.

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Chapter 1: The normal lung: histology, embryology, development, aging and function

FGF 10 has been linked with mesenchymal-epithelial interaction, especially during branching. It is expressed focally in the distal mesenchyme, adjacent to stereotypically determined branching sites. It is hypothesized that FGF 10 governs the directional outgrowth of lung buds during branching by triggering chemotaxis and proliferation of the adjacent epithelium. This causes unidirectional growth of the primary lung buds. The chemotactic response of the lung endoderm involves the coordinated movement of the entire epithelial tip, with all its cells, towards an FGF 10 source. An equally important factor in determining the specificity of the FGF signaling response may be the presence or absence of key downstream intermediate genes. One such example is tyrosine protein phosphatase Shp2, present in embryonic lung branch tips. This gene is essential for FGF transduction. In addition FGF 10 controls the differentiation of epithelium by inducing surfactant protein C (SP-C) expression and upregulating expression of bone morphogenetic protein 4 (BMP4), a regulator of lung epithelial differentiation. Transforming growth factor-p signaling, mediated by transforming growth factor-p type II receptor, plays distinct roles in developing mouse lung epithelium. The integrated functions of this receptor are very important in embryonic lung branching morphogenesis and development of alveoli in the post-natal lung. The developmental immaturity of lung structure and function, resulting from loss-of-function mutations in transforming growth factor-b signaling pathway components, may contribute to early post-natal respiratory problems, such as bronchopulmonary dysplasia (see Chapter 3). It may also increase the susceptibility to respiratory diseases later in life, including emphysema.21 Overexpression of TGFb1 in the developing fetal monkey lung causes severe progressive pulmonary and pleural fibrosis, as well as pulmonary hypoplasia.22 TGFb1 overexpression triggered mesenchymal cell proliferation that appeared to continue after the overexpression of exogenous TGFb1 was discontinued. Recessive mutations in latent transforming growth factor-b binding protein 4 (LTBP4) gene leading to disrupted pulmonary, gastrointestinal, urinary, musculoskeletal, craniofacial, and dermal development have been described.23 Patients have severe respiratory distress, with cystic and atelectatic changes in the lungs, complicated by tracheomalacia and diaphragmatic hernia. Respiratory failure is the usual cause of death. Impaired synthesis and deficient deposition of LTBP4 into the extracellular matrix appears responsible for defective elastic fiber assembly in all tissues affected by the disease. Abrogation of TGF-p type II receptor (TpRII) in mouse lung epithelium causes retardation of post-natal lung alveolarization, with markedly decreased numbers of type I alveolar epithelial cells. No abnormalities in prenatal lung development are observed. In contrast, blockade of TpRII in mesodermderived tissues, including lung mesenchyme, results in mildly abnormal lung branching and reduced cell proliferation after mid-gestation. This is accompanied by multiple defects in other organs, including diaphragmatic hernia.

6

Effect of sex on lung growth Transcriptomic analyses by Kho et al. provide interesting findings regarding lung development.24 They showed a major influence of surfactant-associated genes, even in the early phase of lung development. At the canalicular and saccular periods, more mature lung development has been described in the female fetus than in the male.25 The synthesis of surfactant in the fetal lung is sexually dimorphic.26 Female neonates are more responsive to hormonal accelerators of surfactant production. The influence of sex on the expression profiles has been studied in mice but only in a narrow gestational age range.27 Such studies may help in the understanding of why the male lung is disadvantaged at birth.

Post-natal lung development The early years of life are extremely important in lung development. This is the time when many chronic respiratory diseases appear to have their origins. For example, bronchial asthma arises from interactions between genetic predisposition and infection, while allergy-induced airway inflammation leads to airway remodeling as early as the first 3 years of life.28 In cystic fibrosis lung disease appears to start soon after birth with pulmonary inflammation leading to functional change within the first few months of life29 and structural damage.30 Extremely low gestational age newborns are at increased risk of chronic lung disease (bronchopulmonary dysplasia) and developmental delay (see Chapter 3).31 In addition, low birth weight and lower weight gain in early childhood are associated with modest reductions in adult lung function across a broad range of measures including lung volumes and diffusing capacity. These findings are independent of a number of potential confounding factors, including low socioeconomic status and maternal smoking.32 Weight gain between birth and the age of 3 years is also associated with higher values for static lung volumes in the fully adjusted analyses, which is consistent with this being the main stage of alveolar development. These findings partially correlate with the morphological results that all the alveoli are present by the age of 2 years but further lung growth continues to the age of 8.32

Tracheal growth Wailoo and Emery studied 452 children with apparently normal tracheas ranging from 28 weeks’ gestation to 14 years.33 In the neonatal period the trachea is funnel-shaped with the upper end wider than the lower. It becomes cylindrical with increasing age. The ratio of cartilage to muscle remains constant throughout childhood. The trachea appears to grow at a greater rate in relation to crown-rump length from 1 month to 4 years of age than in utero or around puberty.

Lung At birth a complete set of airways are present but the most peripheral are relatively short. The formation of alveoli starts in late uterine life but most of these air sacs (more than 85%)

Chapter 1: The normal lung: histology, embryology, development, aging and function

are formed after birth.34 The alveoli are formed from tissue ridges on the existing primary septa. This produces a large number of small buds along the primary septa. Myofibroblasts, elastic fibers and collagen fibrils are the probable driving forces for septation. Inside the pre-existing septa platelet-derived growth factor (PDGF) receptor-positive smooth muscle precursors proliferate and move to locations where the new septa are to be formed. Alveolarization does not occur in PDGF-Adeficient mice.6 Alveolarization of the acinus primarily occurs between birth and 2 years; significant but slower growth is seen up to 8 years. The ratio of pulmonary diffusion to alveolar volume remains constant in the first 2 years of life. This is consistent with lung growth in this age group occurring because of an increase in the number of alveoli rather than an expansion of the same number of alveoli.35 These findings are in keeping with morphometric data on number of alveoli per unit volume and mean linear intercept in the post-natal period.36 After this time, values plateau, suggesting alveolarization is complete. Radial counts correlate well with the chronological age of the child.37 Pulmonary diffusing capacity and alveolar volume, measured by DLCO and alveolar volume, increase with age and somatic size among infants and toddlers. Sex differences are primarily related to somatic size. There are no sex differences when pulmonary diffusing capacity is related to alveolar volume. However, in a morphometric study, individual lung units, alveolar dimensions, and number of alveoli per unit area and volume did not differ between boys and girls, but boys had bigger lungs than girls for the same stature. This resulted in a larger total number of alveoli and a larger alveolar surface area in boys than in girls for a given age and stature. Boys may have more respiratory units than girls.36

Stem/progenitor cells in the lung

This is a “hot” area for research, as stem cells offer new tools for the investigation of pathogenetic and developmental pathways. It is likely that many stem cell populations exist in the lung with distinct lineage potential. The ability to purify and functionally assay these populations requires consistent use of well-defined protocols for isolation, culturing, and functional assays.38 This area is still in its infancy and, while continuously “offering” new possible treatments, has yet to provide a proven therapeutic return. An unanswered question is whether adult lung epithelial stem cells are a distinct population or whether some multipotential embryonic progenitors persist into adult life. Evidence suggests that in liver and pancreas, the embryonic cells that build tissue are different from those that repair and maintain it.39 In the lung evidence suggests lung embryonic progenitors and adult stem cells are separate, although lineagelinked, populations.40 The pools of epithelial stem/progenitor cells are widely distributed over the alveolar surface.41 They are located in

the basal layer of the upper airways, within or near neuroendocrine cell rests, at the bronchoalveolar junctions, as well as within the alveolar epithelial surface.42 The most important of these are the alveolar epithelial cells, which have a large surface area. Either alveolar epithelial cells contain a subpopulation of progenitor cells or most alveolar epithelial cells can reactivate into a progenitor-like state in response to injury. Another subset of Club (Clara) cells has been identified, based on their location at the bronchiolar-alveolar junction. They co-express secretoglobin 1a1 (Scgb1a1, also known as CC10 or CCSP), and an alveolar type II cell marker surfactant protein C (SftpC or SpC). These cells proliferate following lung injury. Based on their in vitro differentiation potential, it has been proposed that they are bronchioalveolar stem cells (BASCs) that give rise to both bronchiolar and alveolar cells in vivo.43

Normal organization Airways For convenience of anatomical description, the airways are divided into the upper and lower respiratory systems. The upper respiratory system comprises the nasal cavity, paranasal sinuses, and pharynx, while the larynx, trachea, bronchi, and bronchioles are the lower respiratory tract. The nose and paranasal sinuses act as a first line of defense against bacteria and inhaled particles, through the filtering function of the nasal hairs, as well as the irregular structure of the nasal bones and mucosa. In addition these structures warm and humidify the inspired air. Thus in a patient with a tracheostomy, this first line of defense is bypassed. The mechanics of the upper airways, important in obstructive and sleep apnea, CheyneStokes respiration, and the obesity hypoventilation syndrome, are often neglected by the histopathologist. An excellent review article is available.44 The trachea divides into the right and left primary extrapulmonary or mainstem bronchi. Each of these then gives rise to secondary (lobar) bronchi, which supply the lung lobes. These further ramify into tertiary (segmental) bronchi, which supply the segments of each lobe. The bronchi branch progressively into bronchioles. The smallest are terminal bronchioles that constitute the most distal component of the conducting part of the airways. The terminal bronchioles give rise to the acinus, which is the part of the respiratory system involved in gas exchange and comprises respiratory bronchioles, alveolar ducts, alveolar sacs, and the alveoli.

The trachea The trachea begins anterior to the sixth cervical vertebral body, where it is attached to the inferior portion of the cricoid cartilage of the larynx. It ends in the mediastinum at the level of the fifth thoracic vertebral body, where it branches to form the right and left primary bronchi. The total length and diameter are approximately 11 cm and 2.5 cm, respectively. The trachea is formed of 15–20 C-shaped cartilage rings, the

7

Chapter 1: The normal lung: histology, embryology, development, aging and function

open sides facing posteriorly, where the trachealis muscle completes the circumference. These rings protect the trachea from frontal injury and also prevent collapse during the negative intrathoracic pressure associated with respiration.

The primary bronchi The trachea branches into the right and left primary bronchi, which are separated by an internal ridge called the carina. The structure of these primary bronchi is similar to that of the trachea, being formed of C-shaped cartilage rings. The right primary bronchus is larger in diameter than the left. It closely follows the general direction of the trachea, whereas the left diverges at a greater angle, especially in females. For years it was thought that such anatomy resulted in greater right lung aspiration but recent studies call this into question.45 Each primary bronchus enters the corresponding lung at the pulmonary hilum or root, a groove along the medial surface of each lung, which also provides entry to pulmonary arteries and veins, nerves and lymphatics. All these structures at the root of the lung are surrounded by connective tissue. The relationship of the pulmonary artery, mainstem bronchus, and pulmonary veins is well defined and constant.46

The lungs Each lung lies in its corresponding pleural cavity. The apex of each lung extends above the first rib, while the concave base rests on the superior surface of the diaphragm. In general, the right lung is wider than the left, due to the projection of the heart towards the left side. Conversely, the left lung is longer than the right as the dome of the diaphragm is higher on the right side, because of the underlying liver.47 The right lung is heavier than the left, weighing approximately 700 g in adult men and 500 g in adult women as compared to 600 g for the left lung in men and 450 g in women.47 There is a wide variation in autopsy weights, in part due to differences in the degree of terminal pulmonary edema and congestion from one individual to another. In a study of the organ weights in 684 adult Caucasian forensic autopsies,48 the mean lung weight
standard deviation was 663
239 g for the right lung in males, 583
216 g for the left lung in males, 546
207 g for the right lung in females and 467
174 g for the left lung in females. Such organ weight tables need regular updating, as the normal values of organ weight change with time, secondary to genetic and environmental factors.48 Lung weight tends to diminish slightly in the elderly, probably due to alveolar enlargement (see Chapter 17). The lungs are normally divided into lobes that are separated by fissures. Interlobar fissures are deep depressions that extend from the outer lung surface towards the center. The visceral pleura also dips into the fissure, making the lung surfaces lying within the fissures smooth and thus allowing free movement of individual lobes.47 The right lung is divided into upper, middle and lower lobes, with the horizontal/minor fissure separating the upper and middle lobes. The oblique fissure separates the upper and

8

Figure 5. Macroscopic image of the azygous lobe.

middle lobes from the lower lobe. The horizontal fissure is usually less well developed than the oblique. The left lung is divided into upper and lower lobes, separated by the oblique fissure. A rudimentary structure, the lingula, is present on the left and is considered the equivalent of the middle lobe. It is, however, part of the left upper lobe, rather than a separate lobe, and appears antero-inferiorly as a small tongue-like projection. Variations in the anatomy of fissures are common and include accessory fissures, fissures in abnormal locations and incomplete or absent lobar fissures. Such structural anomalies have no functional significance but may cause radiological confusion. The prevalence of these abnormalities varies between studies.49–52 A well-known anomaly is the azygous lobe, present in 1% of normal individuals. This is caused by extrinsic compression of the lung by an aberrant azygous vein in the right upper thorax, resulting in a depression (fissure) from the top to the bottom of the right upper lobe (Figure 5). It does not reflect any underlying segmental division of the bronchi. Within the lung, the primary bronchi divide to form secondary (lobar) bronchi. One secondary bronchus goes to each lobe, so the right lung has three secondary bronchi and the left lung has two secondary bronchi. The right primary bronchus gives rise to the right upper lobe bronchus and continues as the bronchus intermedius. It then divides into the right middle lobe bronchus and right lower lobe bronchus. The left primary (main) bronchus is longer than the right and divides into the left upper lobe bronchus and left lower lobe bronchus. The secondary bronchi branch to form tertiary (segmental) bronchi (Figure 6). Each tertiary bronchus supplies air to a single bronchopulmonary segment. Bronchopulmonary segments are considered the anatomic units of the lung, each possessing its own bronchus, and pulmonary arterial, venous and lymphatic systems. They are constant in their topographic anatomy (Figures 7, 8 and 9).47 Each segment is surrounded by connective tissue septa, which are continuous with the pleural surface. The segmental bronchus traverses down the center of the segment, accompanied

Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 6. The segmental bronchi and the principal groups of lymph nodes related to the hilar region of the bronchi. 1. Right main bronchus. 2. Right epiarterial bronchus. 3. Right upper apical segmental bronchus. 4. Right upper posterior segmental bronchus. 5. Right upper anterior segmental bronchus. 6. Right middle lobar bronchus dividing into lateral and medial divisions. 7. Right lower superior (apical) segmental bronchus. 8. Right lower lobe anterior basal segmental bronchus. 9. Right lower lobe lateral basal segmental bronchus. 10. Right lower lobe posterior basal segmental bronchus. 11. Left main bronchus. 12. Lingular bronchus dividing into superior and inferior lingular segmental branches. 13. Left upper lobe apical segmental bronchus. 14. Left upper lobe posterior segmental bronchus (usually 13 and 14 arise by a common bronchus from the left upper lobe bronchus). 15. Left upper lobe anterior segmental bronchus. 16. Left lower lobe superior (apical) segmental bronchus. 17. Left lower lobe anterio-medial basal segmental bronchus. 18. Left lower lobe lateral basal segmental bronchus. 19. Left lower lobe posterior-basal segmental bronchus. A. Right superior bronchial nodes. B. Inferior tracheobronchial nodes. C. Left superior bronchial nodes. D. Nodes around the root of the middle lobe bronchus. E. Lingular nodes.

by a corresponding branch of the pulmonary artery. The draining veins, on the other hand, run in the intersegmental planes. The right lung has ten bronchopulmonary segments, while the left has eight. From the trachea to the terminal bronchioles, the airways have a purely conducting function. Each tertiary bronchus branches several times and ultimately forms the non-respiratory or membranous bronchioles, which differ from bronchi by the absence of cartilage and seromucinous glands in their walls. Terminal bronchioles form the most distal branches of the nonrespiratory bronchioles. The terminal bronchioles are 0.7–1 mm in diameter tubes and correspond on average to the 16th dichotomous division of the airway tree.53 Terminal bronchioles are the smallest airways completely lined by bronchial epithelial cells and the last of the alveoli-free conducting airways. Bronchoscopy is a widely performed procedure for assessment of the airways with many indications. Some of these

Figure 7. Anatomy of the bronchopulmonary segment. The draining pulmonary vein circulates in the intersegmental plane, marking the boundaries of the segment. (Reprinted from Ugalde P, Camargo J, Deslauriers J. Lobes, fissures and bronchopulmonary segments. Thorac Surg Clin 2007;17:587–99, with permission from Elsevier.)

require knowledge of airway dimensions to optimize the interventions or assess disease progression. Bronchoscopists may rely on pre-procedure imaging, usually with computed tomography (CT), or on derivation of quantitative information directly from bronchoscopic images. Recently, anatomical optical coherence tomography has been described as an emerging technique for measuring airway dimensions during bronchoscopy.54 The acinus is distal to the terminal bronchiole and its components have an increasing number of alveoli in their walls, where most of the gas exchange occurs. An acinus is usually defined as the lung tissue supplied by a single terminal bronchiole and is composed of respiratory bronchioles, alveolar ducts, and alveolar sacs with their alveoli.55,56 Each terminal bronchiole divides into two to five respiratory bronchioles, which progressively have more alveoli in their walls.57 A respiratory bronchiole in turn divides into two or three alveolar ducts. Each alveolar duct usually ends in four alveolar sacs, which are common chambers connected to multiple individual alveoli. In a stereological analysis of six adult human lungs, the mean number of alveoli was 480 million (range 274–790 million) and was closely related to the total lung volume.58 The alveolar surface area in adults is approximately 70–80 m2.56 Stereology is the technique of quantitative characterization of irregular 3D objects based on measurements

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Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 8. Topographic anatomy and numerical designation of the ten right bronchopulmonary segments. (Reprinted from Ugalde P, Camargo J, Deslauriers J. Lobes, fissures and bronchopulmonary segments. Thorac Surg Clin 2007;17:587–99, with permission from Elsevier.)

Figure 9. Topographic anatomy and numerical designation of the eight left bronchopulmonary segments. (Reprinted from Ugalde P, Camargo J, Deslauriers J. Lobes, fissures and bronchopulmonary segments. Thorac Surg Clin 2007;17:587–99, with permission from Elsevier.)

made on 2D sections. This technique acts as a bridge between the understanding of lung structure and function in various studies on the lung.59,60 The connective tissues from the lung hila extend progressively into the lung parenchyma in the form of fibrous partitions or trabeculae. The smallest unit separated by the trabeculae is called the pulmonary lobule or the secondary lobule of Miller (Figure 10).61 Each pulmonary lobule consists of three to five acini.62 The trabeculae of the subpleural lobules

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(at the periphery of the lung) are continuous with the connective tissue of the visceral pleura.

Blood supply The lungs have a dual circulation. The low-pressure, highvolume pulmonary system carries deoxygenated blood from the right side of the heart to the lungs for gas exchange with the inspired air in the alveolar spaces. The high-pressure,

Chapter 1: The normal lung: histology, embryology, development, aging and function

low-volume bronchial circulation delivers oxygenated blood to the airways, pleura and walls of the pulmonary arteries and veins (vasa vasora).63 Various communications exist between the pulmonary and bronchial systems.64 Their exact location has long been a topic of discussion, and pre-, post-, and capillary sites have all been demonstrated at post mortem.65 The pulmonary arterial trunk arises and bifurcates into the right and left main trunks at the level of the fourth thoracic vertebral body (Figure 11). The pulmonary arteries enter the hila of the lungs with the main bronchi. They run in parallel with the airways within the lung.5,66 The pulmonary arteries branch synchronously with the airways to ultimately become the pulmonary capillaries in the alveolar walls. Pulmonary venules begin on the venous side of the alveolar capillary bed and join to form pulmonary veins. They travel within the interlobular septa, along a separate course to the arteries and bronchi. The main pulmonary veins lie adjacent to the pulmonary arteries and main bronchi in the lung

Figure 10. Part of a lung fixed with formalin vapor, showing well-delineated Miller’s secondary lobules (arrow). Anthracotic pigment is prominent.

(a)

(b)

hilum. Pulmonary veins carry oxygenated blood to the left side of the heart. The bronchial arteries usually arise from the descending aorta, between the level of the fifth and sixth thoracic vertebrae, and run parallel to the airways.67 On the venous side, most of the bronchial venules and veins join the pulmonary veins through bronchopulmonary venous anastomoses to return their blood to the left atrium. A small proportion of blood from the bronchial system drains to the right side of the heart, via the azygous venous system.

Lymphatics and lymph nodes Two separate networks of lymphatics are present in the adult human lungs – a deep plexus located in the connective tissue surrounding the airway branches and a superficial plexus that forms a visceral pleural network (Figure 12). Lymphatics can remove 1.5 l of fluid per hour, proving an effective clearance mechanism of pulmonary edema. The lymphatic vessels of the deep plexus originate from capillaries at the outer edge of the acinus, with none present in the alveoli. The lymphatics surround the airway branches and coalesce at the hilum.68 They travel caudally along the extrapulmonary bronchi and trachea. Bronchial lymphatics from both lungs usually remain ipsilateral, but some may travel in the contralateral mediastinum, after crossing the trachea.67 Ultimately the lymphatics drain into the venous circulation by joining the internal jugulo-subclavian venous angle.69 On the left side, some lymphatics may join the thoracic duct, which in turn drains into the venous circulation.70 Although most lymphatics follow this pathway, some vessels from the lower lobes cross the diaphragm to terminate in the juxta-aortic lymph nodes of the celiac region.71 The D2–40 immunohistochemical stain highlights lymphatics.72 The superficial lymphatic plexus in the visceral pleura comprises lymphatics, which course over the surface of the entire lung toward the hilum, where they anastomose with the lymphatics from the deep plexus. In up to 25% of lungs, they can drain directly into the mediastinal lymph nodes, without anastomosing at the hilum.73

Figure 11. (a) Pulmonary trunk with some elastin (black). (b) The normal aorta consists almost entirely of elastic tissue (Elastic van Gieson).

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Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 13. A subpleural intrapulmonary lymph node showing a reactive lymphoid follicle.

Figure 12. Lymphatic drainage of the left lung. (Reprinted from Riquet M. Bronchial arteries and lymphatics of the lung. Thorac Surg Clin 2007;17:619–38, with permission from Elsevier.)

Along their course, many lymphatics drain into lymph nodes, variably located within the lung and mediastinum. Nodes can be detected within lung parenchyma, with some of the distal lymph nodes located along segmental bronchi. Although these peribronchial lymph nodes are fairly common, more peripheral intraparenchymal nodes are unusual, but can even be present in a subpleural location.74 They may be detected on CT and removed as a “tumor” (Figure 13). Intrathoracic nodes draining the lung are better known through the “Regional Lymph Node Classification for Lung Cancer Staging”.75 In this classification, the intrapulmonary and mediastinal lymph nodes are divided into 14 numbered stations (Figure 14). Each station includes lymph nodes located within a definite region of the lung or mediastinum (see Chapter 25).

Nerve supply The human airways are innervated via afferent and efferent autonomic nerves. These regulate many aspects of airway function, including pulmonary blood flow, mucous gland secretion, ventilation, airway smooth muscle tone, and the cough reflex.76 Input from airway afferent nerves to the central nervous system is integrated in the brainstem and ultimately leads to sensations and various reflex outputs. There are three major groups of lung sensory receptors, namely C-fibers, rapidly adapting stretch receptors (RARs),

12

and slowly adapting stretch receptors (SARs).77,78 The RARs and SARs are mainly mechanoreceptors, which respond to lung inflation and mechanical stimulation. C-fibers, on the other hand, are relatively insensitive to small mechanical stimulation, but are stimulated by chemicals, pulmonary edema, and fever. Each group of receptors has afferent fibers coursing in the vagus nerve, which terminate centrally in the caudal half of the nucleus of the solitary tract in the medulla. Second-order neurons in the pathways from these receptors emanate from the nucleus of the solitary tract and innervate neurons located in the respiratory centers of the medulla, pons, and spinal cord.79 Besides the vagal afferent nerves, the activities of the respiratory centers are also modified by sensory information from other sources, including chemoreceptors in the carotid and aortic bodies, baroreceptors in the carotid and aortic sinuses, irritating physical stimuli in the nasal cavity or larynx, and other visceral sensations, such as pain or body temperature changes. The efferent innervation of the lung is by sympathetic adrenergic fibers, parasympathetic cholinergic fibers, and the inhibitory and excitatory types of non-adrenergic and noncholinergic fibers.80,81 The preganglionic sympathetic (adrenergic) fibers synapse in the second to fourth thoracic sympathetic ganglia. The preganglionic parasympathetic (cholinergic) fibers relay in the ganglia found in the bronchial arterial wall.

Pleura

The pleural space may at times be only approximately 20 mm wide82 and contains a small amount of clear, colorless fluid with a protein concentration of less than 1.5 g/dl. The amount of pleural fluid in each pleural cavity ranges from 4.1 to 12.7 ml. When expressed as per kilogram of body mass, the total pleural fluid volume is 0.26
0.1 ml/kg.83 The pleural cavity is formed by the parietal and visceral pleura. The parietal pleura covers the inner surface of the thoracic cage, mediastinum, and diaphragm. The visceral pleura coats the lung surfaces, including the interlobar fissures.

Chapter 1: The normal lung: histology, embryology, development, aging and function Figure 14. International Association for the Study of Lung Cancer Nodal Chart with stations and zones. (Image courtesy of A. Frazier, MD, Baltimore, Maryland, USA. Reprinted with permission courtesy of the International Association for the Study of Lung Cancer. Copyright © 2008 Aletta Ann Frazier, MD.)

The visceral pleura is reflected at the hilum, where it becomes the parietal pleura. The visceral pleura consists of a layer of connective tissue, covered by a limiting mesothelium lying on a basal lamina. The blood supplies of the parietal and visceral pleura differ. The parietal pleura is supplied by branches of the intercostal and internal mammary arteries. The visceral pleura is mainly supplied by the bronchial arteries. Mesothelium provides for frictionless movement of the organs in the pleural cavity. Numerous microvilli on the mesothelial cell apical surface, covered by a film of hyaluronic acid-rich glycoprotein combined with the small amount of pleural fluid, are responsible for smooth movement.84 The physiology of pleural fluid formation is considered at the beginning of the pleural chapter (see Chapter 36).

Up to 93% of urban dwellers have 2–30 mm flat or slightly nodular, round to oval, dark parietal pleural patches with smooth surfaces mainly in the lower costal and diaphragmatic zones.85 These “black spots” are thought to result from the incorporation via the pleural stomata of inorganic particles including carbon pigment and asbestos fibers from the pleural space into the main connective tissue layer of the parietal pleura.86,87 These patches could be the initiation point for pathological processes induced by such mineral fibers.

Histology, ultrastructure and function Trachea, bronchi and bronchioles There are approximately 40 types of cell in the human respiratory tract (Table 3).88

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Chapter 1: The normal lung: histology, embryology, development, aging and function Table 3 Resident cells of the respiratory tract

Airway epithelial cells Goblet Ciliated Club (Clara) cells Neuroendocrine (neuroendocrine bodies) Basal Intermediate (or parabasal) cells Serous cell (like Club (Clara) cells) – major secretory cell in rat. Also present in human fetal conducting airway mucosa; some reports in adult humans

Smooth muscle cells Fibroblasts/myofibroblasts Hematopoietic and lymphoid tissue Lymphocytes Plasma cells Bronchial mucosal associated lymphoid tissue Megakaryocytes Macrophages Dendritic cells Langerhans cells

Brush cell

Mast cells

Special type cells with numerous intracytoplasmic membranebound inclusions

Eosinophils

Oncocytes

Basophils

Nonciliated columnar cells – unclassifiable columnar cells Metaplastic cells – squamous cells and Clara-mucous cells, bronchiolar metaplasia Alveolar cells Types I–II pneumocytes Transition between types I and II pneumocytes, consisting of cuboidal nonciliated cells Salivary gland cells (in bronchi)

Neutrophils Pleura Mesothelial cell layer Pleuripotent submesothelial fibroblasts Adipose cells – intrapleural fat Lymphatics (see above) Poorly defined cells Stem cells

Serous cells

Perivascular epithelioid cells (PEC) – precursor for LAM cells

Mucous cells

Pluripotent epithelial stem cell – precursor for lung cancer – especially small cell carcinoma – where no precursor is yet identified

Ductal cells Interstitial connective tissue

Endothelial progenitor cells

Smooth muscle

Mucinous cells in certain paediatric conditions

Cartilage Fibroblasts Myofibroblasts Meningothelioid cells of minute meningothelioid nodules

Reprinted with permission of the American Thoracic Society. Copyright © American Thoracic Society. Franks TJ, Colby TV, Travis WD, et al. Resident cellular components of the human lung: current knowledge and goals for research on cell type and phenotyping and function. Proc Am Thorac Soc 2008;5:763–6. Official journal of the American Thoracic Society, Diane Gern, Publisher.

Adipose tissue Neural cells (intrapulmonary nerves) Blood vessels Arteries/veins Endothelial cells (differences in arterioles versus veins versus capillaries) Smooth muscle cells Fibroblasts/myofibroblasts Pericytes Lymphatics Endothelial cells

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The lower respiratory tract is lined by respiratory epithelium (Figure 15), which undergoes transition from a tall pseudostratified columnar ciliated form in the trachea to a simple cuboidal non-ciliated type in the bronchioles. The constituent cells vary in different parts of the airways. The epithelial sheet of the conducting airways functions in conjunction with other epithelial, mesenchymal, and endothelial cells and the extracellular matrix comprising the bronchial walls.89,90 The respiratory epithelium has many functions. It acts as a barrier (especially with the mucociliary escalator), has a secretory function with mucins and growth factors, bronchoconstrictors, as well as degradative enzymes. For a full discussion of these, the reader is referred to Knight and Holgate.89

Chapter 1: The normal lung: histology, embryology, development, aging and function Figure 16. Bronchusassociated lymphoid tissue – this pattern is seen in a smoker.

Figure 15. Normal respiratory (pseudostratified, ciliated, columnar) bronchial epithelium attached to the basement membrane.

Figure 17. Bronchial smooth muscle forming a band in the subepithelial tissue.

The epithelium rests on a basement membrane, beneath which lies the lamina propria formed of connective tissue and also containing lymphatics, blood vessels, nerves, and the mucosa-associated lymphoid tissue (MALT). This is called BALT (bronchus-associated lymphoid tissue) in the lung (see Chapter 34) (Figure 16). A layer of smooth muscle lies deep to the lamina propria, which becomes progressively prominent as the airway diameter decreases (Figure 17). The submucosa lies beneath the smooth muscle and contains the seromucinous glands (Figures 17 and 18), the number of which decreases progressively from the trachea downwards, being absent distal to the tertiary bronchi. The cartilage lies outside the submucosa and again is absent beyond the tertiary bronchi. The outermost layer, adventitia, is formed of fibroelastic connective tissue.

The trachea and bronchi contain numerous mixed seromucinous glands resting on the cartilage in the anterior parts (Figure 19). Posteriorly, the cartilage is absent and there is a band of smooth muscle beneath the glands. The bronchi are cartilaginous conducting airways, greater than 1 mm in diameter. As the bronchi enter the lung, the cartilage progressively becomes discontinuous and irregular, with an increasing area between the cartilaginous plates. In the posterior part of large bronchi, there are dense bands of elastin running longitudinally in the lamina propria.91 Bronchioles are usually less than 1 mm in diameter and do not have seromucinous glands or cartilage in their walls, making them much thinner than the bronchi (Figure 20). The pseudostratification and height of the epithelium decrease progressively towards the lung periphery and it becomes cuboidal. The concentric layer of muscle, outside which lies the peribronchiolar connective tissue, is very important. Bronchoconstriction is greatest at bronchiolar level and accounts for the dyspnea from infection, especially in children, bronchial asthma and COPD.

Respiratory epithelium The cells comprising the respiratory epithelium include ciliated and non-ciliated cells, basal cells, neuroendocrine cells as well as others, such as intermediate and brush cells. The cellular composition of the epithelium lining the conducting airways differs along the proximal to distal axis. Ciliated cells and goblet cells are present from the trachea to the terminal bronchioles, but are more numerous proximally. Ciliated cells are most frequent lining the bronchus. The basal and neuroendocrine cells predominate in the trachea and bronchi, and are rare in bronchioles. Conversely, Club [Clara] cells and serous cells are primarily seen in the bronchioles. Intermediate and brush cells are only identified ultrastructurally.

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Chapter 1: The normal lung: histology, embryology, development, aging and function

(a)

(b)

Figure 18. (a) Seromucinous glands in bronchial wall; (b) mucus extruded from a mucous gland.

Figure 19. Histological section from a bronchus showing respiratory epithelium, submucosal glands and cartilage. Smooth muscle is inconspicuous.

Ciliated cells Ciliated cells are columnar or cuboidal epithelial cells. Bronchial ciliated cells are roughly columnar and measure approximately 20 mm in length and 7 mm. in width and taper at the base.92 Each cell has approximately 250 cilia on the luminal surface (Figure 21). Although ciliated cells are present up to the respiratory bronchioles, their number, height, and cilial length decrease in the more distal airways, implying mucociliary clearance rates are slower in more peripheral airways.93 Each cilium measures about 6 mm in length. Ultrastructurally, the ciliated cell contains electron-lucent cytoplasm. The nucleus is basal, and above it there is a welldeveloped Golgi apparatus. The upper part of the cell is rich in mitochondria; these lie just below the basal bodies, which anchor the ciliary axoneme into the cell cytoplasm. The cilium

16

(or ciliary shaft) is a cytoplasmic extension from the surface of the cell and is covered by the same plasmalemma. Interspersed among the cilia are microvilli. Transverse sections of the ciliary shaft reveal an axial filament complex consisting of nine peripheral doublets of microtubules connected to two central microtubules by radial spokes (9þ2 arrangement) (Figure 22). Each doublet has inner and outer dynein arms, the site of ATPase activity and essential for ciliary movement.94 Cilia maintain a constant upward flow of respiratory secretions to the mouth. Particles larger than 5 µm are trapped in the upper airways and are cleared by ciliary movement, sneezing, and/or coughing. Thus the ciliated cells form an important component of the defense system of the respiratory tract.95 The host defenses and composition of the fluid in the mucociliary escalator are considered in Chapter 4. Ciliary function can be assessed in nasal brush biopsy samples. With the advent of high-speed digital video microscopy, the ciliary beat pattern and frequency can be precisely assessed.96 Basal cells Basal cells are small pyramidal cells with scanty cytoplasm. They are attached to the basement membrane and do not reach the luminal surface. Basal cells predominate in bronchi, and are rare in bronchioles.92,97 There is a direct correlation between the thickness of the epithelium and the number of basal cells. The basal cell has a sparse electron-dense cytoplasm that contains bundles of low molecular weight cytokeratin.98 Basal cells are the only cell types firmly attached to the basement membrane by hemidesmosomes. Integrins (a6b4) facilitate adhesion. Two types of basal cells are recognized.99 One is found mainly in the large airways and is characterized by abundant intermediate filaments, well-developed hemidesmosomes and anchoring fibrils. These cells, type A basal cells, demonstrate CK14 positivity. The second type, type B, contain few

Chapter 1: The normal lung: histology, embryology, development, aging and function

(a)

(b)

Figure 20. (a) Low-magnification view of a peripheral small bronchiole in normal lung parenchyma and no inflammation. (b) Normal bronchiole. The bronchiolar mucosa is low columnar. The cells with clear cytoplasm are neuroendocrine cells, which are probably increased in number due to the chronic inflammation.

Figure 21. Ultrastructure of a bronchiole showing cilia, some cut in transverse section.

intermediate filaments, poorly developed hemidesmosomes and anchoring fibrils, and are CK14 negative. Basal cells have at least two major functions. They serve as a multipotent progenitor cell population of the bronchial airways, capable of renewing the bronchial epithelium. They also have a major role in anchoring the pseudostratified tracheobronchial epithelium to the basement membrane.100–105 Basal cells are thought to secrete a number of bioactive molecules, including neutral endopeptidase, 15-lipoxygenase products and cytokines.89 Non-ciliated secretory cells Club cells (Clara cells) –– Max Clara first described a non-ciliated, non-mucous cell in the human respiratory epithelium of the peripheral conducting airways in 1937.106 It has been

suggested this eponym be replaced by the term “Club cell”, as the tissue obtained for the description was from an individual executed by the Nazi “justice system”.107,108 Club cells are found mainly in the terminal and respiratory bronchioles and are thought to be virtually absent in the bronchi.109 Club-like cells were detected in neuroepithelial bodies of the airway lining in the 1980s, intensifying interest in the cell and its function.110 Club cells are defined by their distinctive PAS-positive, diastase-resistant cytoplasmic granules. Apical cytoplasm protrudes above the level of the adjacent ciliated cells (Figures 23 and 24). Ultrastructurally, they are characterized by cytoplasmic ovoid electron-dense osmiophilic granules and abundant smooth endoplasmic reticula.111 The former are immunolocalized with Club cell 10-kilodalton protein (CC10), an inhibitor of phospholipase A2.112–116 The apical mitochondria are unusual as they have no cristae and show an abundant pale matrix Club cells may act as progenitor cells of the bronchioles and are critical in epithelial renewal following injury. This progenitor function in bronchi is controversial, with conflicting opinions.112,117,118 Club cells also have important secretory functions, including contribution to the mucus pool and extracellular lining fluid and surfactant production.119 In addition to their secretory role, Club cells are believed to metabolize xenobiotic compounds via P450 mono-oxygenases, as well as oxidant gases.120 They may also produce specific antiproteases, such as secretory leukocyte protease inhibitor.121 Specific Clara cell protein (CC10, identical to CC16 or uteroglobin) may play a role as a clinical biomarker of lung disease.122–124 Recent studies suggest the presence of two subsets of bronchiolar Club cells based on naphthalene sensitivity – the more numerous, classic non-ciliated secretory Club cells

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Chapter 1: The normal lung: histology, embryology, development, aging and function

(a)

(b)

Outer dynein arm Inner dynein arm Spoke Spoke head Nexin link Central sheath

Figures 22. (a) Diagram of a cross section of a cilium; (b) ultrastructure of a cilium. Figure 23. Club (Clara) cell with apical cytoplasmic snouts (arrows). Nuclei are not apparent.

and a rarer subpopulation with properties of tissue-specific stem cells (see section above on stem/progenitor cells in the lung).125–128 Goblet cells and serous cells –– Goblet (or mucous) cells are interspersed between the ciliated cells and are more numerous in the bronchi (especially proximally) with fewer numbers in the bronchioles. Such cells stain intensely with diastase/Alcian blue pH 2.5/PAS (periodic acid Schiff), indicating they contain acid mucopolysaccharides. Goblet cells can also proliferate and differentiate into ciliated cells.129 Goblet cells contain distinctive round or oval cytoplasmic granules filled with mucin. After standard chemical fixation with glutaraldehyde, transmission electron microscopy reveals that the apical parts of the cells are expanded by a coalescence of these electron-lucent granules. Their cytoplasm contains abundant endoplasmic reticulum in addition to a welldeveloped Golgi apparatus. Small microvilli are found on the surface of these cells. Mucous granules are discharged from the cell, often with intact limiting membranes as well as some other cell components (Figure 18b). These products pass through pores in the luminal surface.

18

Together with the bronchial submucosal glands, goblet cells secrete high molecular weight mucus glycoproteins (mucins). These help in entrapment and transportation of inhaled irritants, particles, and microorganisms.130 Production of the correct amount of mucus and its viscoelasticity are important for efficient mucociliary clearance. The sialic acid content of the glycoprotein probably determines the viscoelastic profile and hence the relative ease of transport across cilia. Mucins are tightly packed in the intracellular granules of the goblet cell. The number of goblet cells increases in response to acute and chronic insults, such as inhalation of sulfur dioxide, smoking, or persistent infection.131,132 This change is often accompanied by basement membrane thickening. Mucous cell hyperplasia and metaplasia are common in chronic bronchitis and asthma and contribute to productive cough but not to the same extent as the mucous gland hyperplasia (Figure 25) (see Chapter 17).133 Serous cells are rare in the bronchi and are primarily present in bronchioles.134 Morphologically, they are identical to serous cells in the minor salivary glands. In response to adverse conditions, these cells may be able to transform to mucus cells. Ultrastructurally their granule content is electron-dense, rather than electron-lucent.134 The cystic fibrosis transmembrane conductance regulator is localized on serous cells. This regulator controls the transport of sphingosine-1-phosphate (S1P) across the plasma membrane of the cell.135 S1P stimulates cell growth and prevents apoptosis. Other sphingolipid metabolites, including ceramide and sphingosine, arrest cell growth and promote apoptosis.136 Serous cells also secrete a rich mixture of proteins that have antimicrobial, antiprotease, antioxidant, and anti-inflammatory functions. Human b-defensin 2 (hbD-2) is diffusely expressed throughout epithelia of many organs, including the lung, where it is found in the surface epithelia and serous cells of the submucosal glands. The fully processed peptide has

Chapter 1: The normal lung: histology, embryology, development, aging and function

(a)

(b)

Figure 25. Bronchus with mucous cell hyperplasia and thickened basement membrane.

broad antibacterial activity against many organisms. It is saltsensitive and synergistic with lysozyme and lactoferrin. The b-defensin family on mucosal surfaces probably contributes to normal host defense.137 The major antimicrobial polypeptides in the secretions from the serous cells include lysozyme, lactoferrin, epithelial defensins, and secretory leukoprotease inhibitor.138 This liquid is also essential for flushing gland secretions, including mucins, from the gland ducts. The liquid hydrates airway surfaces and supports mucociliary transport.139 Neuroendocrine cells –– Pulmonary neuroendocrine cells (PNCs) constitute the pulmonary component of the diffuse neuroendocrine system (DNS), a system of widely dispersed amine and peptide-secreting regulatory cells. These act in concert with systemic neural and endocrine control systems to

Figure 24. (a) Scanning electron microscopy of Club (Clara) cells; (b) transmission electron micrograph showing a Club (Clara) cell with an apical snout.

Figure 26. Three solitary neuroendocrine cells, immunolabeled for chromogranin, in a terminal bronchiole of a normal adult human lung. Their equal spacing within the respiratory epithelium is characteristic.

regulate physiological processes by a mode of local secretion known as paracrine.140–144 In healthy adult human lungs, PNCs are found from the trachea to the alveoli, but are most numerous in bronchi and bronchioles.145–147 Most human PNCs are solitary (Figure 26). It is unlikely the highly organized, innervated, corpuscular aggregates of PNCs, known as neuroepithelial bodies (NEBs) (Figure 27),148 which are frequent in lower species,149 are ever found in human lungs. It is probable PNC clusters develop in human lungs only when pulmonary tissues are growing and differentiating. Typically this is in fetal lungs and in lungs in which injury has led to the need for regeneration and repair (Figure 28).142 Solitary human PNCs are columnar, triangular or “bottleshaped” with a broad base abutting the basement membrane (Figure 29).149,150 Some have dendritic cytoplasmic extensions

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Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 27. A neuroepithelial body, immunolabeled for neuron-specific enolase, in an alveolar duct of a neonatal rabbit lung. The highly organized arrangement of its component cells and the “cap” of non-neuroendocrine epithelial cells are typical features and are not seen in clusters of human pulmonary neuroendocrine cells.

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Figure 28. A cluster of neuroendocrine cells, immunolabeled for chromogranin, in a human lung affected by bronchopneumonia.

Figure 29. A neuroendocrine cell, immunolabeled for chromogranin, in a human bronchus. The basal concentration of granules and the delicate cytoplasmic process extending towards the lumen are well shown.

Figure 30. Two clusters of neuroendocrine cells in human fetal lung identified by in situ hybridization for mRNA encoding gastrin-releasing peptide.

at their bases that run along the basement membrane away from the cell and some have a long, apical, finger-like process that winds its way between adjacent epithelial cells to reach the airway lumen. In human lungs, clusters of these cells appear to comprise closely apposed solitary cells without the highly organized “organoid” structure of true NEBs. The functions of PNCs are debated. Evidence for a chemoreceptive role for NEBs in lower species, such as the rabbit, is overwhelming.143 There is no evidence for such a function in human lungs; not only are NEBs probably absent, but studies linking increased numbers of human PNCs to hypoxia per se are unconvincing. Studies of PNCs during fetal pulmonary development and in human lungs affected by pulmonary disease show this system of cells almost certainly has a central role

in regulating the development, repair and regeneration of human pulmonary tissues.142,144,151–153 Evidence for a central role for PNCs in human fetal pulmonary growth is strong. Numbers of GRP (gastrin releasing peptide)-containing PNCs, as well as levels of GRP itself, its mRNA (Figure 30), and its receptor, are markedly elevated during the canalicular period of pulmonary development.154–156 GRP is powerfully trophic to human bronchial epithelial cells in vitro.157 It stimulates fetal pulmonary growth and maturation in vivo and in organ culture in the murine and rhesus monkey fetus.158–160 The only other situation in which PNCs are as prevalent as in fetal lungs is when they proliferate in injured lungs (Figure 31).142,152 As described in Chapter 31, the stereotypical

Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 31. Early proliferation of neuroendocrine cells, immunolabeled for chromogranin, in a small bronchus of a human lung affected by bronchopneumonia. Occasional solitary cells are identifiable, but most are replaced by small clusters.

hormone (ACTH) appear.142,152,162,163 The nature of this proliferative process and its relationship to diffuse pulmonary NE hyperplasia (DIPNECH)164 and neuroendocrine neoplasms are discussed in detail in Chapter 31. PNCs have often been referred to as “clear cells” because of their lucent cytoplasm.165 However, this is an inconstant feature and they are difficult to detect in hematoxylin and eosin-stained sections. Early studies trusted unreliable histochemical techniques. It was not until the advent of more specific and reliable immunochemical markers of neuroendocrine differentiation that the characteristics of PNCs became precisely defined.142 Of these antigens (Chapter 31), neuronspecific enolase (NSE), protein gene product (PGP) 9.5, and chromogranin have been most widely used in the identification of PNCs. The range of peptides and amines found in the PNCs of different species is enormous,166 but those established by repeated demonstration in human lungs are GRP, the mammalian analog of amphibian bombesin,167 calcitonin (CT),168 calcitonin gene-related peptide (CGRP),169 and the amine serotonin (5-hydroxytryptamine; 5HT).170 In healthy human lungs, about two-thirds of PNCs contain GRP and most of the remainder CT.145 Detection of chromogranin reflects the presence of neurosecretory (dense core) vesicles (see below) and, in that sense, as with detecting neuroendocrine differentiation in pulmonary tumors,171 is a particularly specific marker reflecting the functional attributes of these cells. The ultrastructural hallmark of PNCs, in common with all cells of the DNS, is the neurosecretory (dense core) vesicle (Figure 34).149 These are spherical structures, varying in size from about 100 to 180 nm, and characterized by an electrondense core separated from a limiting membrane by a clear halo. Otherwise the ultrastructure of PNCs is not particularly unique.149,172 Their nuclei are spherical or ovoid, basal or

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(b)

pattern of this proliferation involves early formation of a monolayer at the base of the bronchial or bronchiolar epithelium followed by irregular aggregation of cells. These cells expand and elevate the mucosa (Figure 32) and should the injurious stimulus persist, tumorlets develop.161 These 2–3 mm aggregates of PNCs invade across the basement membrane into the adjacent parenchyma (Figure 33). This proliferation is accompanied in its early stages by a shift in the predominant secretory product of PNCs from GRP to CT (calcitonin) and, in its later stages, aberrant peptides, like adrenocorticotrophic

Figure 32. (a) Larger aggregates of chromogranin-containing neuroendocrine cells in a terminal bronchiole of a human lung affected by bronchopneumonia; (b) Neuroendocrine cells surrounding the entire bronchiole.

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Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 33. The proliferating neuroendocrine cells in the small airway in this lung affected by bronchiectasis have been immunolabeled for gastrin-releasing peptide. They have breached the basement membrane, locally invading the adjacent parenchyma to form a tumorlet. It is at this stage that a ubiquitous physiological response of the pulmonary neuroendocrine cell system to pulmonary injury becomes disordered and pathological (see Chapter 31).

Figure 34. Neurosecretory (dense core) vesicles in a human pulmonary neuroendocrine cell. Between 100 and 180 nm in diameter, these organelles store the amine and peptide hormones synthesized by and secreted from neuroendocrine cells and also contain proteins, such as the chromogranins.

suprabasal, and sometimes indented. The Golgi apparatus is supranuclear, both smooth and rough endoplasmic reticulum are seen, and free ribosomes are plentiful. Moderate numbers of mitochondria are present. Intermediate filaments are often abundant and characteristically sheaved. Microvilli are present on the apical surface and desmosomes, tight junctions and junctional complexes bind PNCs to their neighbors. Other cells Brush cells –– Brush cells, named for their apical tuft of stiff microvilli, are only distinguished from other epithelial cells immunohistochemically and ultrastructurally (Figure 35). They have also been termed tuft cells and type III pneumocytes (in the alveoli). Brush cells can be identified at light microscopic level by immunostaining with antibodies against villin and fimbrin.173 The cell may be columnar or flask-like, and its most salient feature is the broad, squat microvilli (0.5–1 mm in length and 150–180 nm in width) on its surface that extend into the alveolar space, either perpendicularly or parallel to the alveolar wall. Approximately 120 to 140 microvilli may be found on each cell. The microvilli contain filaments that stretch into the cytoplasm, forming a long root-like structure. There are also glycogen and numerous vesicles in the apical cytoplasm. Occasional parallel arrays of smooth endoplasmic reticulum have also been identified toward the base of the cell. The cell may lie adjacent to either a type I or a type II cell. When adjacent to a type I cell, the alveolar surface of the brush cell is covered by a flange of cytoplasm from the type I that reaches to the base of the microvilli.174 In a study of their distribution, Chang et al. reported that the number of brush cells in the rat was greatest at the first

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Figure 35 Human bronchiolar brush cell with surface microvilli. (Reproduced by kind permission of Professor R. Brody, Raleigh, Durham, USA.)

bifurcation of the alveolar ducts, where they constituted 10% of the epithelial volume.175 They constituted 3% of the tracheal epithelial volume, 1.4% of terminal bronchioles, and 1.8% of the distal alveolar region. Few brush cells were identified in the lobar bronchi, suggesting a differential localization of brush cells along the airways. Brush cells are reliably distinguished from the other epithelial cells at an ultrastructural level by the presence of an apical tuft of stiff blunt microvilli (120–140/cell). They have extremely long microvillar rootlets that stretch into the underlying cytoplasm and may project down to the perinuclear space.174,176 These cells are pear-shaped, with a wide base and narrow apex. Other features include the presence of glycogen and numerous vesicles in the apical cytoplasm.174

Chapter 1: The normal lung: histology, embryology, development, aging and function

Their function is obscure, although a chemoreceptor function has been suggested.174,176,177 Acetylcholine (ACh), a classic transmitter of parasympathetic nerve fibers in the airways, is synthesized by airway surface epithelial cells, including neuroendocrine and brush cells.178 Thus, these cells appear to comprise a diffuse chemosensory system that covers large areas (probably the whole digestive and respiratory apparatuses) with analogies to chemosensory systems described in aquatic vertebrates. Intermediate cells –– These spindle-shaped cells may be a transitional stage of development of basal cells to ciliated epithelial cells but are not well characterized.179

Basement membrane The epithelial cells rest on a basement membrane, which can be demonstrated with a PAS diastase stain. The basement membrane provides mechanical support for cells, acts as a semipermeable barrier between tissue compartments, and regulates cellular migration and differentiation.180 Several structural components are identified in basement membranes, including collagens, glycoproteins, and proteoglycans, although the proportion of these varies between the ultrastructurally defined layers.181 Ultrastructurally, the airway basement membrane (or basal lamina) consists of three layers, the lamina lucida (electronlucent), lamina densa (electron-dense), and lamina reticularis (fine fibrillary collagen and not technically part of the basement membrane). The combination of lamina lucida and lamina densa forms the basal lamina and both these layers are synthesized by the epithelial cells.182 Howat et al. reported oval-shaped pores in the basement membrane of bronchial epithelium.182 They proposed that these pores allowed the cells from the lamina propria to cross into the epithelium without damaging the basement membrane.

Smooth muscle Outside the lamina propria is an almost circular layer of muscle, interrupted only by collagen and bronchial gland ducts. Smooth muscle may be seen as a thin layer when relaxed or as a thicker inner and outer layer when constricted. In larger bronchi, the smooth muscle connects the tips of the cartilage plates. Thus contraction approximates the cartilage plates, causing a reduction in both diameter and length of the bronchi. Airway smooth muscle plays a pivotal role in modulating bronchomotor tone, but it may have an important role in airway inflammation and remodeling, particularly in chronic diseases.183–185 A review of airway smooth muscle, including ultrastructure, electrophysiology, mechanical properties, and pathophysiology is presented by Stephens.186

Submucosal glands Submucosal glands are present in the trachea and bronchi, but are absent in the bronchioles. They lie outer to the smooth muscle and inside or between the cartilage, and have

Figure 36. Oncocytic metaplasia in seromucinous glands.

a gland density of approximately one gland per mm2.187 These are tubuloacinar glands with secretory acini composed of mucous, serous, and mixed acinar units. Intercalated ducts arise from the acini and converge to form excretory ducts, which are continuous with the airway surface. The intercalated ducts are lined by a layer of cuboidal cells, while the excretory ducts show a pseudostratified epithelium of predominantly ciliated columnar cells with intermingled goblet cells and basal cells.188 Mucous cells have large, apical mucin-containing granules, which push the nucleus and cytoplasm to the basal portion of the cells. The serous cells are pyramidal with basal nuclei.189 The apices are filled with numerous electron-dense secretory granules. Normal glands are about 60% serous and 40% mucous by volume, with the serous cells more distally located in the acini.190 The basal surfaces are surrounded by myoepithelial cells, which facilitate emptying of luminal contents by virtue of their contractile properties.191 Oncocytic change is often noted in bronchial gland collecting ducts. These cuboidal cells have eosinophilic cytoplasm due to abundant mitochondria (Figure 36). While metabolically active, they have no recognized functional significance and may represent a form of epithelial cell degeneration.192,193 They are seen in older individuals and in smokers. In one study they were found in 30/33 patients’ bronchial glands at necropsy. Found with similar frequency in the main, upper, and lower lobe bronchi, the cells were not increased in number in chronic bronchitics, questioning the role of smoking.192 Their relationship to bronchial oncocytomas is unknown (see Chapters 2 and 30). Submucosal glands produce a mucin-rich secretion for the conducting airways in response to neurohormonal stimuli.194 Mucous cells of the submucosal glands account for about 95% of the airway mucin production, the remainder being provided by the goblet cells of the surface epithelium. Gland mucus is critical in the airway defense mechanism, as it traps microbes, inhibits their replication, and clears them from the airways.195

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Chapter 1: The normal lung: histology, embryology, development, aging and function

Bronchus-associated lymphoid tissue (BALT) Airway-associated lymphoid tissue is part of the diffuse mucosa-associated lymphoid tissue (MALT) and in the lung is referred to as bronchus-associated lymphoid tissue (BALT). It is well developed in certain species, such as rats and rabbits, in which the overlying respiratory epithelium is attenuated and flattened (called “lymphoepithelium”) and is devoid of ciliated cells. This specialized epithelium is thought to allow passage of soluble and particulate antigenic material from the airways into the underlying lymphoid follicle, where it can be processed.196 Bronchus-associated lymphoid tissue is more frequent in the fetal lung,197 in children and adolescents.198 In the normal adult lung, BALT is absent or scanty and is seen mainly as organized subepithelial aggregations of lymphocytes, which are predominantly present at the points of bifurcation of the proximal airways.197,199–202 It is more common in smokers than non-smokers.199 In pathological states, lymphoid follicles with germinal centers are often found, which are not restricted to the upper airways and have recently been termed inducible BALT by some authors.203,204 Immunophenotypically, the lymphoid tissue of BALT is composed of both B and T lymphocytes, and when wellformed, has B-cell-rich follicles, B cell follicular mantle and marginal zones, and a T-cell-rich interfollicular region. A follicular dendritic cell network is also present and polyclonal plasma cells are identified in the perifollicular tissue.205 Subepithelial lymphoid tissue, present along the airways, is usually considered BALT.206 Besides this subepithelial BALT, smaller, isolated aggregations of lymphoid cells are observed in the airway wall and may play a role in antigen uptake and cell-mediated immune responses.207 Unlike BALT, these isolated aggregates are seen beneath the smooth muscle layer and close to the mucous glands but away from the luminal epithelial surface.

Acini and alveoli Terminal bronchioles constitute the most distal part of the conducting portion of the respiratory tract. The acinus lies beyond the terminal bronchiole and comprises respiratory bronchioles, alveolar ducts, alveolar sacs, and alveoli, which are all involved in gas exchange. The respiratory bronchiole is the first order of bronchiole to bear alveoli, the number of which progressively increases with subsequent branches. The respiratory bronchioles are lined by ciliated cuboidal cells and non-ciliated Club cells, although the number of ciliated cells progressively diminishes distally. Respiratory bronchioles divide into alveolar ducts, which end in alveolar sacs. Numerous alveoli open into both these structures. Each alveolus is cup-shaped with an opening on one side. The inner surface of the alveolus is covered by surfactant, a phospholipid synthesized by the type II pneumocytes and Club cells. Each alveolus is lined by an epithelium, which is inconspicuous by light microscopy in a normal lung (Figure 37). It is

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Figure 37. Normal alveoli are present with the center of the image being occupied by an alveolar duct.

formed of type I–III pneumocytes with an underlying basement membrane. (Type III pneumocytes, so-called brush cells, are discussed above.) The alveolar septum or wall lies between the epithelium of two adjacent alveoli and consists of numerous capillaries surrounded by elastin and collagen fibers and a variable number of cells (see below).

Type I pneumocytes Ninety-three to 96% of the alveolar lining is covered by type I or membranous pneumocytes. Type I cells develop from a yet unidentified fetal progenitor cell(s) and are present at birth.208 Most studies of type I cell turnover in the injured mature rodent lung suggest these cells in the adult are derived from type II cells during alveolar repair.209 It is not known how this cell type is repopulated in the normal human lung. It is also unknown if the in vitro reversibility of type II to type I transition occurs in mammalian lung.210 Type I pneumocytes are 40 mm wide specialized squamous cells with a dense flattened nucleus and abundant cytoplasm, which extends a long way from the nucleus. These cells have no regenerative capacity. They form part of the extremely thin gaseous diffusion barrier between alveolar air and blood. This barrier consists of the attenuated cytoplasm of a type I pneumocyte, the fused basement membrane of the type I pneumocyte and capillary, and the thin cytoplasm of the capillary endothelial cell (Figure 38). In the regions of gas exchange, this cell measures only 0.1 to 0.2 mm thick. The edges of adjacent type I pneumocytes are tightly bound together, providing an intact epithelial barrier. The cytoplasm of type I pneumocytes contains a few mitochondria, a small amount of smooth endoplasmic reticulum, and an occasional lysozyme. Micropinocytic vesicles (caveoli), which probably play a major role in transport of solutes through the cell, are seen in the plasmalemma. Adjacent cells are bound together by tight junctions. This tight barrier helps

Chapter 1: The normal lung: histology, embryology, development, aging and function

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Figure 38. (a) Transmission electron micrograph of human alveolar wall. At the top there is the thin attenuated cytoplasm of a type I cell with basement membrane beneath. No capillary is identified in this image. At the bottom of the image are type II cells. (b) higher magnification of two type I cells, showing an intercellular junction, with a rarely identified nucleus. There is underlying basement membrane with endothelium beneath.

to restrict the movement of ions and water. Type I cell deep cytoplasmic extensions pass through the alveolar wall interstitium between capillaries to reach and cover parts of the opposing alveolar surface.211 This anatomical configuration of one cell covering two surfaces, with the nucleus on only one side, makes it impossible for such a cell to undergo mitosis. It also provides a firm anchor for the cell. The cytoplasm of the type I pneumocyte also covers a large area of the type II pneumocyte alveolar surface. Thus only a small proportion of the type II pneumocyte surface is exposed to the alveolar space. The major function of type I pneumocytes is to allow gas exchange and fluid transport.212 Type I cells express a large number of proteins including T1-a, aquaporin 5 (AQP-5), functional ion channels,213 caveolins, adenosine receptors,214 and multidrug-resistance genes.215 Sodium transport is closely regulated to maintain an appropriate fluid layer on the alveolar surface. Lung epithelium has enormous flexibility to alter the magnitude of salt and water transport. Amiloride-sensitive epithelial sodium channels (ENaC) play an important role. In lung, ENaC is regulated by many transmitter and hormonal agents.216 Cultured rat type I cells express highly selective and non-selective cation channels, the cystic fibrosis transmembrane conductance regulator, and cyclic nucleotide gated channels. mRNA and protein for these transporters are present in various degrees in this cell population.214

Type II pneumocytes Type II pneumocytes account for 60–65% of the total alveolar cells, but cover only 4–7% of the alveolar surface. These cuboidal cells, with a diameter of 8–10 mm, have large, central,

round nuclei with dispersed chromatin, prominent nucleoli and plentiful eosinophilic, vacuolated cytoplasm. The latter is due to phospholipid-filled vesicles. Type II pneumocytes are not distributed uniformly but are located at alveolar wall branch points, where thick elastic fibers are concentrated.217 In the adult rodent lung, type II cells cycle every 28–35 days, and this slow mitotic rate is also believed to occur in humans.218 The free surface of type II pneumocytes features numerous microvilli. The cytoplasm contains numerous mitochondria, rough endoplasmic reticulum, and many lysosomes. The characteristic feature is the presence of osmiophilic lamellar bodies, responsible for surfactant production. The lamellar bodies are membrane-bound structures and the lamellae are mainly composed of phospholipids (Figure 39). The main function of type II pneumocytes is to produce and secrete surfactant. They also proliferate to restore the epithelium after lung injury and differentiate into type I pneumocytes.219 This view of the type II cell as a totipotential cell in the alveolus is challenged in the normal human lung, where it is not known how this cell is repopulated.215 Other functions of type II cells include regulation of fluid balance by conducting solutes and fluids across the transmembrane channels (aquaporins),220 as well as secretion of alpha-1-antitrypsin221 and various cytokines.222 Pulmonary surfactant is composed of approximately 90% lipid and 10% protein, the latter consisting of the four surfactant-associated proteins, surfactant protein (SP) A, SP-B, SP-C, and SP-D, and a large number of other, mostly serum-derived proteins.223 The majority of the surfactant lipids are phospholipids, the most abundant being phosphatidylcholine. Surfactant reduces

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Chapter 1: The normal lung: histology, embryology, development, aging and function

(a)

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the surface tension within the alveoli, preventing alveolar collapse and facilitating gas exchange. It acts in a similar way to a patch of oil on a road, so water aggregates into globules which are removed by lymphatics. Surfactant also has the important function of keeping airways open by maintaining bronchiolar patency during normal and forced respiration. When surfactant is absent, as in bronchopulmonary dysplasia, or lost, as in acute respiratory distress syndrome, small airways and alveolar walls collapse (see Chapter 3).224,225 Surfactant proteins SP-A and SP-D are also known as pulmonary collectins, so-called “pattern recognition molecules” that bind via their carbohydrate recognition domains to oligosaccharides on the surfaces of microorganisms.226 They play an important role in the innate immunity of the lung via viral neutralization and clearance of bacteria, fungi, and necrotic cells, as well as downregulation of allergic reactions and resolution of inflammation.227 The ability of SP-A and SP-D to modulate both pro- and anti-inflammatory responses may be a consequence of whether or not the SP attaches to a pathogen or apoptotic cell.228 Several receptors, SP-R210, CD91/calreticulin, SIRP, and toll-like receptors, mediate these immunological functions. By binding SIRP alpha or calreticulin/CD91, SP-A and SP-D act as dual-function surveillance molecules to suppress or enhance inflammation. They can activate or deactivate inflammatory responses by alveolar macrophages. SP-A and SP-D are also implicated in modulation of the adaptive immune response.229 SP-A and SP-D bind to dendritic cells in a calcium-dependent manner. SP-D also enhances dendritic cell antigen presentation, whereas SP-A inhibits dendritic cell maturation.230,231 These two surfactant moieties also inhibit T cell proliferation, in part by inhibiting calcium signaling.232 Of note, SP-B and SP-C and one or more surfactant lipid constituents appear to share similar immunoregulatory properties with SP-A and SP-D.233 Surfactant composition and pool size is controlled by several physiological processes, including secretion, re-uptake, and degradation by type II cells and macrophages.234 All

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Figure 39. (a) Low-power magnification of a type II cell with a prominent nucleus and microvilli but lamellar bodies are ill-defined. (b) Surface of type II cell with lamellar bodies. Microvilli are present.

Figure 40. Scanning electron micrograph of an alveolus with prominent type II cells with microvilli and a pore of Kohn at the bottom of the image.

surfactant proteins contribute to the internalization of surfactant subtypes by type II epithelial cells.235,236 P63 functions as a receptor for SP-A on type II cells.237 For a fuller discussion of surfactant receptors, the reader is referred to reports by Wright and co-workers.226,238 Several collateral respiratory pathways are described, yet are only identifiable by light microscopy in diseased lung. Pores of Kohn are approximately 8 mm interalveolar communications (Figure 40). Each alveolus has between 13 and 21 such pores.239 Direct communications that are also present between non-respiratory bronchioles and adjacent alveoli are termed Lambert’s canals.240 These pathways of collateral ventilation are not important in the healthy individual but become significant when the airways are diseased. Obstructed bronchial segment(s), an increase in lung volume, increased surface tension, hypocapnia, decreased pulmonary blood flow, and pulmonary edema may affect collateral pathways.241

Chapter 1: The normal lung: histology, embryology, development, aging and function

Pulmonary interstitium The interstitial space is the part of the septal wall lying between the alveolar epithelial and capillary endothelial basement membranes and is normally inconspicuous. The interstitial space is formed by extracellular matrix and a variable number of cells. Pulmonary gas exchange critically depends upon the hydration state and the thinness of the interstitial tissue layer within the alveolar-capillary barrier. The entire lung is invested with a continuous connective tissue framework from the hilum to the visceral pleura and involves the interstitial space, which contains mainly collagen and elastin fibers. These fibers appear to have connections with those of pulmonary arteries. This means that any force exerted on the parenchyma is distributed throughout the organ.242 The extracellular matrix is composed of proteoglycans, which form a gelatinous and hydrated substance, embedding these fibrous proteins.243 The various types of cells seen in the interstitial space include alveolar macrophages, capillary pericytes, myofibroblasts, fibroblasts, mast cells, dendritic cells, lymphocytes, and granulocytes. The basement membrane is produced by alveolar epithelial and endothelial cells. Where the alveolar epithelium covers capillaries, the two basement membranes fuse to form a homogeneous structure. The portion of each membrane subjacent to both the alveolar and capillary endothelium is less dense and is termed the lamina lucida.244 These zones are continuous with a dense zone in each membrane, termed the lamina densa. Sirianni et al. documented apertures in the basal laminae of types 1 and II pneumocytes and endothelial cells.245 Alveolar wall fibroblasts purportedly link pneumocytes with endothelium through these apertures; such linkage provides directional information to migrating leukocytes. In the interstitium, fluid freely moving within the fibrous extracellular matrix equilibrates with water interacting with hyaluronic acid and proteoglycans. The interstitium establishes and maintains adequate interstitial tissue fluid volume by providing a stiff three-dimensional fibrous scaffold. It functions as an efficient safety factor to oppose fluid filtration into the tissue and prevent tissue fluid accumulation. Disturbances of the deposition and/or turnover of the matrix and/or of its three-dimensional architecture and composition may evolve into pulmonary fibrosis.246 The reader is reminded the epithelium and interstitium function as one unit. In pulmonary edema and lung injury, this space becomes thicker, thus impeding gaseous exchange. In acute lung injury, if edema does not resolve, interstitial pulmonary fibrosis follows. Alveolar macrophages, derived from blood monocytes, are an important component of the lung defense mechanism against inhaled particulate materials, microorganisms, and environmental toxins which escape the upper airway defense system.247 These cells are seen both in the interstitium and lying free in the alveolar space. Alveolar macrophages vary in size from 12 to 40 mm in diameter and have a lobulated nucleus. They contain abundant lipid droplets, mitochondria

Figure 41. Smoker’s macrophages showing granular, brown cytoplasm. It does not glisten like hemosiderin. A Perls’ stain would show these smoker’s macrophages are faintly positive, whereas hemosiderin shows strongly positive globules.

and electron-dense secondary lysosomes. The cytoplasm usually contains abundant particulate matter, a large proportion of which is black, due to carbon, often called anthracotic pigment. Macrophages containing phagocytosed material from cigarette smoke are often seen in the alveoli of smokers. These “smoker’s macrophages” have granular brown cytoplasm (Figure 41). Macrophages sequestered in the interstitium are removed by lymphatics to the draining lymph nodes. Those in alveoli mostly migrate into the upper airways, where they are removed by the mucociliary system. Besides their phagocytic role, alveolar macrophages are involved in the regulation of adaptive immune response and inflammation, including antigen presentation and the production of reactive oxygen and nitrogen species as well as metalloproteinases.248–250 Mast cells are potent inflammatory cells containing several vasoactive substances, including histamine and 5-hydroxytryptamine. They measure 10 to 15 µm in diameter and typically contain 600 to 800 nm membrane-bound intracytoplasmic granules with intra-granular inclusions of various forms. In addition, rare long surface filiform microvilli are seen.251 They are normally present both in the alveolar walls252 and in the cartilaginous and membranous airways253 in varying numbers (Figure 42). There has been renewed interest in the association between mast cells and asthma.254 There has also been a recent interest in pulmonary dendritic cells (DCs) due to their possible role in the pathogenesis of inflammatory lung disorders.255–257 Dendritic cells are professional antigen-presenting cells of hematopoietic origin. They form an immunological synapse by meeting naive lymphocytes, directing the proliferation of antigen-specific T cells and, thus, orchestrating the adaptive immune response.258,259 Their distribution in normal lung has not been well documented but the cells appear to reside in bronchiolar epithelium

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Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 42. Mast cell in an edematous interstitium. Mast cells are often associated with edematous processes.

and adventitia. Flow cytometry delineates three subsets of human dendritic cells in the lung, namely myeloid dendritic cells (mDCs) types 1 and 2, and plasmacytoid dendritic cells (pDCs).255,260 One study identified pDCs in small airways of human lungs and highlighted their presence in lymphoid follicles. This finding is compatible with the expression of lymphoid-homing chemokine receptors CXCR3 and CXCR4 on these cells.261 Plasmacytoid dendritic cells represent a unique population of professional antigen-presenting cells with a plasma cell-like morphology and a unique surface receptor phenotype. Through Toll-like receptors (TLRs)-7 and -9, pDCs are capable of producing large amounts of type I interferons in response to viruses and nucleic acid-containing complexes from the host.262,263 In addition, through upregulating inducible T cell co-stimulator ligand, pDCs can generate regulatory T cells.264 The long, protruding dendritic processes of mDCs endow them with the unique capacity to sample antigens in the airway via DEC205 (CD205), macrophage mannose receptors (MMR), and other cell surface molecules that mediate receptor-mediated endocytosis and antigen processing in deep lysosomes or peripheral endosomes.256,265 Using specific receptors, mDCs sense for danger signals while sampling their environment for antigens. Myeloid dendritic cells process the antigen, and present it on major histocompatibility class II and I molecules. They integrate this information with the sensed danger signals by upregulating co-stimulatory molecules and producing specific cytokines. Only a few lymphocytes are normally found in the alveolar interstitium, as they become progressively less in number distal to the bronchioles. Neutrophils and eosinophils are normally present in small numbers in the walls of the conducting airways but rarely in the alveolar interstitium. Scattered megakaryocytes are also often seen in the interstitium, but more often in the alveolar capillaries and are of no diagnostic significance (Figure 43). The megakaryocytes may be intact with multilobed, hyperchromatic nuclei and

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Figure 43. A megakaryocyte (arrow), which is commonly seen in the normal lung.

abundant cytoplasm, but are more often seen as naked or semi-naked hyperchromatic nuclei.266 Often prominent in acute lung injury, megakaryocytes can be confused with circulating malignant cells.

Pulmonary vasculature As already discussed, pulmonary arteries return deoxygenated blood to the lungs, whereas bronchial arteries supply oxygenated blood to maintain the pulmonary tissues.267 In addition, the pulmonary circulation is a platelet factory. Megakaryocytes derived from the bone marrow are fragmented into platelets in the pulmonary circulation. As are all systemic arteries, the pulmonary arteries are composed of three layers, the intima, media, and the adventitia. However, the pulmonary circulation is a low-pressure system (with the normal mean pressure of approximately 10 mmHg) and the structure of the blood vessels somewhat differs from other systemic arteries. Pulmonary artery wall thickness is considerably less than its luminal diameter. There are three categories of pulmonary arterial vessels.268 The largest are the elastic pulmonary arteries, which have multiple concentric elastic lamellae, down to a diameter of about 500 mm. Their branches are the so-called “muscular pulmonary arteries”, which have a thin media of circularly oriented smooth muscle sandwiched between inner and outer elastic laminae (Figure 44). These range from 80 to 500 mm in diameter. Pulmonary arterioles are less than 80 mm in diameter (see Chapter 18). The pulmonary arteries run alongside the airways and the diameters of the pulmonary artery and the accompanying airway are roughly equal in cross section. The elastic pulmonary arteries are thin-walled, as the media contains predominantly elastic fiber lamellae and relatively little smooth muscle. In adults, elastic fiber lamellae are less compact and more irregular and fragmented as compared to infants. Elastic tissue

Chapter 1: The normal lung: histology, embryology, development, aging and function

(a)

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Figure 44. (a) Muscular pulmonary artery, which superficially appears thick-walled. The true media consists of the outer circular muscle. Inside the circular muscle is intimal fibrosis and elastosis. (b) The media is shown well here with an Elastic van Gieson stain. Pulmonary vessels should never be assessed for their medial thickness on H&E sections alone.

Figure 45. Muscular pulmonary artery, which becomes progressively smaller, with eventual loss of the media and fusion of the elastic layers.

remains relatively prominent in the pulmonary arteries until they become the muscular pulmonary arteries, which roughly corresponds to the point where bronchi become bronchioles. Internal and external elastic laminae are present in the muscular pulmonary arteries and extend to the arterioles. As the arterioles get smaller, the two elastic laminae appear fused (Figure 45). This is the result of progressive attenuation of the medial smooth muscle, up to a point where a single fragmented elastic lamina is all that separates the intima from the adventitia.269 The medial thickness of a muscular pulmonary artery is roughly 5% the external diameter of the artery. This generalization aids in the recognition of arterial abnormalities.

The adventitia has been neglected by histopathologists. A rapidly emerging concept is that the vascular adventitia acts as a biological processing center for the retrieval, integration, storage, and release of key regulators of vessel wall function. In response to stress or injury, resident adventitial cells can be activated and reprogrammed to exhibit different functional and structural behaviors. Functions include proliferation, differentiation, upregulation of contractile and extracellular matrix proteins, release of factors directly affecting medial smooth muscle cell tone and growth, as well as stimulating recruitment of inflammatory and progenitor cells to the vessel wall. Each of these changes in fibroblast phenotype directly or indirectly modulates changes in overall vascular function and structure.270 Under certain conditions, the adventitial compartment may be considered the principal injury-sensing tissue of the vessel wall. In response to vascular stresses, such as overdistension and hypoxia, the adventitial fibroblast is activated and undergoes phenotypic changes. It can differentiate into a myofibroblast. Some believe the adventitia is the seat of vascular progenitor cells, capable of differentiating into endothelium or smooth muscle cells.271,272 The terminal branches of the pulmonary arterioles ramify in the alveolar septa to form a widely interconnected capillary basket, which is mainly involved in gas exchange. The capillaries are 7 to 10 mm in diameter and the partial pressure of the alveolar gases controls the amount of blood flowing into them. Pulmonary capillaries are lined by continuous endothelial cells of a non-fenestrated type.63 Besides gas exchange, the pulmonary capillaries perform other functions including production of angiotensin-converting enzyme, deactivation of chemicals such as serotonin, adenosine, synthesis of various substances including fibronectin, heparan sulfate, interleukin 1,

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Chapter 1: The normal lung: histology, embryology, development, aging and function

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Figure 46. (a) Pulmonary vein is patulous with a thin wall. (b) The irregular muscular layer and prominent elastic are apparent (Elastic van Gieson).

(b)

tissue plasminogen activator, endothelin 1, and detoxification of some drugs. Pulmonary capillary endothelial cells, which are also termed microvascular endothelial cells,267 show a higher constitutive expression of adhesion molecules compared to systemic capillaries. These may contribute to the sequestration of a large intravascular pool of neutrophils in the lung.273 Pulmonary endothelial cells are barely detectable by light microscopy. Perhaps one notes nuclei embracing vessel lumens. These cells contain large numbers of caveolae and show long cytoplasmic extensions, which encircle much of the vessel. The cytoplasm contains mitochondria, free ribosomes, rough endoplasmic reticulum, and Weibel-Palade bodies. Endothelial cells also contain microtubules and microfilaments. Pericytes (Roget cells) are closely applied to the outer walls of alveolar capillaries and probably have a contractile function. Their branched cytoplasmic processes are partially embedded in endothelial basement membrane and contain fine filaments similar to those seen in smooth muscle cells. Pulmonary veins have relatively scanty smooth muscle in their media and possess only a single (outer) elastic lamina (Figure 46). The veins often appear indistinct in tissue sections. They are located in interlobar connective tissue septa, as opposed to the pulmonary arteries, which lie with airways within pulmonary lobules.

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Figure 47. (a) Bronchial artery with internal and external circular muscle layers and a prominent adventitia; (b) bronchial artery with no longitudinal muscle, a thick media, an internal elastic layer but a very thin outer elastic, if present at all (Elastic van Gieson).

(b)

The bronchial arteries run parallel to the airways within the bronchovascular sheath. The largest-diameter bronchial arteries can be identified in the airway adventitia (Figure 47). Bronchial arteries have a thicker media than pulmonary arteries. Bronchial arteries can also be distinguished from pulmonary arteries by a single thick internal elastic lamina and a poorly formed, if at all visible, outer elastica. Some have prominent intimal longitudinal muscle, which is not present in the newborn but develops by 3 months, due to stretch.274

Pleura

The pleura is formed of three main components – the mesothelial layer, the basal lamina, and the connective tissue layer (Figure 48). The visceral pleura also contains a welldefined elastic lamina in the submesothelial connective tissue, which is an important layer, since the presence of tumor below or above it contributes to the staging of pulmonary epithelial tumors.275,276 The elastic lamina may be several layers thick rather than a single layer (Figure 49). The mesothelial layer is formed of a single layer of mesothelial cells. These cells may be either flat or cuboidal depending upon their metabolic activity and site.277,278 In general, the parietal pleura consists of a large number of flat cells whereas the visceral pleura has predominantly cuboidal

Chapter 1: The normal lung: histology, embryology, development, aging and function

cells. Mesothelial cells have round nuclei, often with nucleoli, and a moderate amount of cytoplasm (Figure 50). Ultrastructurally, mesothelial cells are characterized by long forked microvilli measuring up to 3 µm in length and 0.1 µm in diameter. There is a higher density of microvilli in mesothelial cells on the visceral pleural surface than on the parietal pleura. The microvilli help trap hyaluronic acid, which decreases the friction between the lung and chest wall. Mesothelial cell cytoplasm is rich in mitochondria, and contains many intermediate filaments. Parietal pleural stomata, ranging from 2 to 8 µm, connect directly with the lymphatics. The basal lamina has wide superficial electron-lucent and thin electron-dense parts.279 It consists of a three-dimensional network of irregular strands, composed of at least five mesothelial-produced substances, namely collagen IV, laminin, heparan sulfate proteoglycan, entactin, and fibronectin.280 The connective tissue layer consists of collagen, lymphatics from the superficial plexus, capillaries, nerves, and a few bundles of smooth muscle. The visceral pleura also contains elastic fibers. Several minor alterations are commonly seen in the pleura. Black spots are composed of mixed dust particles surrounded by collagen, fibroblasts, macrophages, and lymphocytes. Figure 48. Pleura with a thin, discontinuous elastic layer, fibrosis and no identifiable mesothelial cells.

Reactive mesothelial hyperplasia may overlie black spots.87 Black spots are thought to be similar to the “milky spots” described in the peritoneum,279 and to the “Kampmeier foci”, first described by Kampmeier in 1928 in the caudal part of the mediastinal pleura (Figure 51).86,281 Focal pleural and subpleural fibrosis, accompanied by subpleural emphysematous change, is very common in smokers’ lungs (Figure 49). The mesothelial layer of the parietal pleura is not continuous, but ultrastructurally shows 2 and 8 mm gaps, so-called lymphatic stomata (see Chapter 36). These provide for direct communication between the pleural cavity and the lymphatic system for the transport of extravasated lung fluid and particulate matter. These stomata were initially described in the peritoneum, and also noted in the costal, mediastinal, and diaphragmatic parietal pleura.282–285

Aging Aging is a process associated with an inability to maintain and repair somatic cells.286 Aging occurs following cellular senescence, which is a genetic program of essentially irreversible cell cycle arrest that blocks the cell’s response to proliferative stimuli and growth factors.287 Several mechanisms have been proposed to cause cellular senescence including DNA damage, chromatin instability, progressive telomere shortening, overexpression of oncoproteins, and a variety of stress signals, including oxidative damage.288 The lung matures by the age of 20–25 years and thereafter aging is associated with a decline in function.289 Aging leads to a number of structural and functional changes in the respiratory tract.

Structural changes Structural changes are a result of either degenerative changes or compensatory mechanisms. A variety of changes are seen in the trachea, bronchi, alveoli, and blood vessels. The trachea and bronchi may show tracheobronchial cartilage ossification (Figure 52), oncocytic metaplasia of the bronchial glands, fatty metaplasia of the bronchial submucosa and a reduction in the (a)

(b)

Figure 49. (a) Pleura with an “almost” single elastic layer. (b) Another case with more fibrosis in the pleura and several layers of elastic tissue. (Elastic van Gieson.)

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Chapter 1: The normal lung: histology, embryology, development, aging and function

Figure 50. A layer of hyperplastic mesothelial cells. It is only when these cells undergo this change that they are visible.

Figure 52. Calcification of bronchial cartilage.

diameter of small bronchioles. Alveoli often demonstrate a homogeneous increase in distal airspace (alveolar ducts and alveoli) cross-sectional diameter due to lung matrix remodeling. There is also a decrease in the elastin and an increase in collagen content. This is associated with a loss of gas exchange surface area and number of capillaries per alveolus.290,291 The media of the pulmonary trunk and elastic pulmonary arteries show progressive loss of elastic tissue and increased amounts of fibrous tissue and acid mucopolysaccharide ground substance.292 In addition, the elastic pulmonary arteries feature atherosclerotic plaques, while intimal fibrosis commonly occurs in the muscular pulmonary arteries, arterioles and pulmonary veins.293 The arterioles may also show hyalinization of the medial layer. Senile pulmonary amyloid (SSA) most often occurs as an incidental finding in individuals above the age of 80 years.294 It results from the pathological deposition of unmutated (wildtype) transthyretin (TTR) molecules.295 Though most commonly affecting the heart, scattered or diffuse pulmonary interstitial deposition has been reported (see Chapter 34).

32

Figure 51. A Kampmeier focus in the visceral pleura.

Functional changes The decrease in the lung elastic recoil results in increased lung compliance. In addition, chest wall and thoracic spine deformities from osteoporosis and stiffening of the rib cage from calcification of the ribs impair chest wall and respiratory compliance. These changes place the diaphragm at a disadvantage. Decreased muscle strength probably due to diaphragmatic muscle atrophy and age-related changes in fast-twitch fibers can impair cough.289 Muscle atrophy also leads to decreased transdiaphragmatic pressure.296 With aging, there is also a tendency for the small airways to close more readily at a given lung volume, leading to a decrease in expiratory flow rates, gas trapping and an increase in residual volume at the expense of vital capacity.290 A decrease in mucociliary clearance efficiency, due to cilia having a lower beat frequency, and increased microtubular abnormalities are also seen.297 Additionally, a decline in the immune response of the lung, due to various alterations in the number and functions of T- and B-lymphocytes, occurs.95 There are also an increased number of neutrophils and a decreased number of macrophages in lungs of older individuals along with an increase in IgA and IgM, in bronchoalveolar fluid. The ratio of CD4þ/CD8þ lymphocytes increases with age in this fluid, suggesting the presence of primed T cells from repeated antigenic stimuli of the lower respiratory tract mucosa.298 These changes make the elderly more susceptible to respiratory tract infections. In addition to these purely pulmonary changes, other organs must be considered. Cardiac, esophageal, and cerebral diseases, especially stroke and dementia, and increased body weight with concomitant diabetes mellitus and rheumatological disorders, are just a few of the changes that impact respiratory function with age. Aspiration is a problem with increasing age. Silent aspiration is common in the elderly, and has been linked to chronic bronchiolar inflammation.299,300

Chapter 1: The normal lung: histology, embryology, development, aging and function

Poor dentition, pathogenic bacteria in the mouth, and a significant decrease in oropharyngeal clearance are also frequent changes associated with aging.301 The problems are increased

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Chapter

2

Lung specimen handling and practical considerations Leslie Anne Litzky and Anthony Gal

Introduction The current practice of thoracic pathology extends beyond the routine pathological assessment of a pulmonary specimen and requires teamwork with a multidisciplinary approach. All pathologists – whether practicing in smaller community settings or in a larger academic centers – should facilitate an interactive discussion with their clinical and radiology colleagues. This should occur frequently, as technological advances are made, and as important evidence-based studies are added to the literature. This discussion has utility in all phases of the diagnostic evaluation – before, during, and after the specimen has been submitted to the laboratory. Reluctance on the part of the pathologist to engage in these discussions is often a disservice to the individual patient and an impediment to improving the overall quality of medical care and knowledge. Prior to sampling, the issue might center on what constitutes appropriate pathological sampling and in what context. Is an open lung biopsy necessary in a patient with idiopathic pulmonary fibrosis (IPF)? Given the current trend toward personalized chemotherapy requiring numerous molecular tests, will the amount of material in a needle biopsy be sufficient for diagnosis? Are multiple molecular tests in patients with advanced-stage nonsmall cell lung cancer needed and if so which ones? Under what circumstances is a cytological diagnosis of mesothelioma reliable and acceptable? During pathological evaluation, clinical and radiographic correlations are absolutely essential. Is the pathological sampling truly representative of the radiographic abnormality? Do the pathological changes account for the patient’s symptoms and suspected disease? How can additional ancillary tests, such as immunohistochemistry, help to refine the diagnosis? As a corollary, why is it important for the clinician and patient to wait beyond the usual turnaround time for the final diagnosis? Following the final diagnosis, the discussion might focus on the options for further pathological sampling in the case of a non-diagnostic sampling, as well as the appropriate interval for radiographic follow-up and repeat sampling in stable or progressive disease. With an informed awareness of the clinical context in which the pathological sampling has been obtained, the

pathologist must confront the issues of proper specimen processing. The elements required for the structural integrity of the lung are unique among the body organs. In vivo, inhaled exogenous and endogenously produced gases, extra- and intrathoracic pressures, the structure of the airways, as well as the pulmonary circulatory volume all combine to produce the lung’s specific anatomic configuration. These elements are disrupted and artifacts introduced when lung tissue is removed for pathology. Every pulmonary pathologist must be aware of these potential artifacts, since they may compromise the gross or microscopic evaluation. It is also particularly useful – whether for clinical practice, research, or education – to have a working familiarity with the techniques to compensate for such artifacts. This chapter provides an initial overview of specimen processing, followed by a summary of various artifacts or incidental findings seen on microscopy. It concludes with a general discussion of the indications and efficacies of the most common sampling techniques.

Cytology specimen processing Cytology specimens include sputum cytology, bronchial brushings, bronchial washings, bronchoalveolar lavage (BAL), radiologically guided fine needle aspiration (FNA), lymph node aspirates, and pleural fluid. The availability of liquid-based cytology preparations has improved the morphological assessment of cytology specimens, as well as enabling special studies on such material. The processing of cytology specimens will vary greatly depending on the site of acquisition and the proximity to the pathology laboratory. Where the clinical site is close to the laboratory, there is the opportunity to send the specimen fresh. This allows the laboratory more flexibility to effectively triage the sample, if warned in advance of receipt. In general, BAL or pleural fluids can be transported to the laboratory at room temperature, if processing occurs in less than 1 hour.1 If there are processing delays but fixation is not indicated, specimens should be refrigerated. Aliquots for microbiology culture are submitted separately. Cytological preparations can be made for an initial evaluation, while the remainder of the sample is refrigerated overnight. If there is a need for additional studies,

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 2: Lung specimen handling and practical considerations

such as flow cytometry, then the specimen can be sent for ancillary testing. On-site cytological evaluation, where available, may prove helpful by allowing for additional needle passes that can be triaged for ancillary testing. Cytological specimens obtained off-site must be placed directly into fixative for transport to the cytology laboratory. In this instance, it is important to consider the differential diagnosis prior to specimen acquisition. Other complementary evaluations, such as microbiology cultures or flow cytometry that will require special transport media, should be anticipated. Planning prior to the procedure may spare the patient an additional procedure, which is often the consequence of a “non-diagnostic” sample. As discussed below, concurrent cytology and biopsy specimens, obtained either by bronchoscopy or by interventional radiology, often provide complementary information and should be correlated with each other. When there is sufficient cytological material, a “cell block” can be prepared by centrifugation of the specimen, fixation of the pellet, and then standard processing to produce a paraffin block.2 Special studies, such as immunohistochemistry or molecular analysis, can then be performed on the cell block in a similar manner to small biopsies. In some circumstances, there may be even more material in the cell block for these studies and less background stromal cells or inflammation. This may help to improve the diagnostic yield for molecular analysis.

Small specimen histological processing Small histological specimens include wide-needle (18-gauge) core biopsies, as well as biopsies obtained by fiberoptic or rigid bronchoscopy. Transbronchial and endobronchial biopsy specimens usually measure 1–2 mm in greatest dimension, while percutaneous needle biopsies can measure up to 1–2 cm in length but less than 1 mm in diameter. Rigid bronchoscopy specimens can be substantially larger. There are at least four fundamental pathological objectives in the evaluation of these small specimens. The first is not to lose any small fragments in processing – either at the procedure or especially in the laboratory. The second is to avoid drying or other fixation artifacts – again either at the time of the procedure or in the pathology laboratory. The third is to minimize tissue handling, which in itself will also reduce the possibility of specimen loss or mix-up and reduce trauma to the specimen. The fourth objective is to maximize tissue examination for standard diagnostic evaluation and for ancillary testing. In most circumstances, all of the tissue obtained should be submitted for standard light microscopy.3 Specific instances where a small additional piece of tissue might be set aside include fresh tissue for immunofluorescence in alveolar hemorrhage, samples for electron microscopy in suspected cases of ciliary dysmotility, separate samples for microbiology, and fresh tissue for molecular studies. Practice patterns may vary from institution to institution and between countries. These small specimens are usually

42

collected in a dedicated bronchoscopy suite or operating room, by interventional radiology and occasionally at the bedside (e.g., closed pleural biopsies). Preparations prior to and at the time of small specimen collection are essential. Ideally, these procedures should be discussed, documented in a procedure manual, and reviewed regularly with the involved clinicians, radiologists, and pathologists, as well as their technical and administrative staff. Prior to the procedure, clinicians (and their trainees) should be encouraged to contact the pathology laboratory with any questions regarding specimen collection in unusual clinical circumstances. For patient safety, it may be the practice to place these samples into saline first in the bronchoscopy or radiology suite. However, prolonged immersion in saline should be avoided. All appropriate fixatives and transport or culture media should be available on site to minimize specimen degradation during transport. Specimens obtained for special studies, such as electron microscopy, should be directly placed into the appropriate fixative or medium. At the conclusion of the procedure, specimens in saline for routine processing should be quickly transferred into fixative by the clinician performing the procedure. Gently shaking the specimen in fixative in the specimen container helps expand the alveolar spaces and avoid atelectasis.1,4 Following rapid transport to the pathology laboratory, these small specimens must be identified, carefully handled, and described. The specimens should be accessioned and processed in a manner specialized for small samples. If clinically urgent, these small specimens can be rapidly processed using a 4-h tissue processing cycle, without any significant sacrifice in morphological detail. However, immunohistochemical results may be affected. The small fragments should be retrieved from the formalin-filled transport container and wrapped in moist lens paper, prior to placing them in a cassette. The specimen should not be placed between sponges, since this can cause atelectasis and “sponge artifact”.3,4 Pathologists view step sections as essential for proper histological evaluation. The utility of such sections in the diagnosis of diffuse lung disease, as well as neoplasms, has been validated.5 Pathology laboratories should have established protocols for step sectioning and retaining interval levels for ancillary tests. In small biopsies, where the specimen may be cut through, it is often useful to establish a priority order for the special stains. Such protocols increase the probability that key stains will be done, where there is sufficient tissue to validate a diagnosis. It cannot be emphasized enough that the proper processing of small specimens requires careful correlation of the pathological findings with the clinical and radiographic presentations. The clinician, radiologist, and pathologist need to collectively consider whether the sample is representative of the disease process and distribution. Unfortunately, this does not occur consistently in many practice settings. There are a number of factors that contribute to this. One is the hectic pace of clinical medicine and a large pile of unreported slides by the side of the microscope. Another may be the physical

Chapter 2: Lung specimen handling and practical considerations

distance between the clinical site, surgical or radiological facilities, and the pathology laboratory. A third factor is the unfortunate and rather antiquated notion that providing clinical and radiographic information “biases” the pathologist. This attitude leads to a lack of clinical information at the time of tissue processing and may, in some instances, compromise a complete and accurate pathological evaluation. A fourth factor is reluctance on the part of the pathologist to take the initiative in obtaining or verifying clinical and radiographic information. Finally it is not uncommon to obtain a clinical history from one, often junior, clinician, and a different one from the more senior member of the team. It is ironic in this age of cell phones, e-mail, portable electronic devices, electronic medical records and even telepathology (not to mention simple telephones, message machines and walking down a corridor), that our struggles with “communication” still profoundly affect medical care. All pathologists recognize the importance of correlating the gross appearance of specimens with histology. In small specimens, the gross appearance is represented not only by the endoscopic findings but by the radiology, often available on the pathologist’s computer. Therefore, the radiologist should be viewed as an essential co-partner in diagnosis. The pathologist can avoid overinterpretation of nonspecific findings or missing more subtle changes, if a radiographic back-check is performed prior to final diagnosis. Often, communicating directly with the radiologist, rather than through the pulmonologist, brings additional insights. At a minimum, the pathologist should understand whether the radiographic abnormality falls into one of five general categories:6 mass lesion; diffuse bronchial or interstitial disease; patchy disease that can be targeted (for example, alveolar lipoproteinosis); patchy disease that can be difficult to target (for example, pulmonary Langerhans’ cell histiocytosis); and diseases that will probably require a larger sample of tissue, particularly if lobular architecture is important (for example, usual interstitial pneumonia). Communicating directly with the clinician for a clinical back-check is no less important. In addition to ascertaining whether the observed pathology correlates with the patient’s presumed disease, other pertinent history, such as tobacco use, which may cause nonspecific background changes, is also significant. Whether or not the patient is immunocompromised should be considered essential information. General information about pulmonary function tests is useful, particularly in non-neoplastic disease. No discussion of small specimens is complete without some consideration of the current challenge of trying to do more with less tissue. It is difficult to ensure adequacy for standard diagnostic evaluation, while preserving the small amount of remaining material for ancillary molecular testing. There is an ever increasing demand for this type of tissuebased testing. In recent years there has been an explosive growth in the number of proposed molecular markers that might predict prognosis or response to therapy. This is despite the fact that the number of appropriately powered marker

validation studies is still limited. The pathologist is often put in the awkward position of damping this enthusiasm for molecular testing generated by clinicians and “internet-savvy” patients alike. Future technological improvements may minimize the amount of tissue needed for analysis. At the present the pathologist must be able to explain the practical constraints of exhausting the tissue block – either for current studies or future applications – and to sort out the diagnostic priorities in tissue testing.

Large surgical pathology specimen processing Lung wedge biopsies

In addition to standard thoracotomy, the development and general availability of video-assisted thoracoscopic surgery (VATS) has greatly increased the number of wedge biopsies that require pathology evaluation. It is important to remember that wedge biopsies contain only a peripheral portion of pulmonary parenchyma and visceral pleura. There is no sampling of the larger proximal airways, vessels, or lymph nodes. The histopathological findings in these more proximal structures may lead to dramatic but entirely nonspecific changes in the distal parenchyma contained in the wedge biopsy. Again, clinical and radiological correlation is essential for accurate interpretation. Usually, patients will have undergone a prior bronchoscopy to exclude an endobronchial abnormality, but this assumption should be confirmed by the pathologist. Radiographic correlation will provide detailed information about the mediastinum, such as lymphadenopathy, and the parenchymal distribution of the pathological changes. Wedge biopsies are generally undertaken for three indications – diagnosis of diffuse parenchymal disease, diagnosis/and or treatment of a localized parenchymal process, and diagnosis and/or treatment of a mass lesion. Regardless of the indication, the thoracic surgeon must be included in the interactive multidisciplinary discussion to optimize specimen processing and patient care. The operating room is rarely the appropriate setting for a thoughtful discussion of basic thoracic surgical principles, although occasionally, for the benefit of the patient, some pressured verbal exchanges might take place. It is better to come to a general agreement beforehand about proper specimen processing in more relaxed circumstances. At such a time, the standard error rate for frozen sections and the clinical implications of requesting a frozen section, in a case where no preoperative diagnosis has been made, might also be an essential item for discussion. In open wedge biopsies for diffuse parenchymal disease, the average size for each wedge biopsy sample is in the range of 4 cm.7 The choice of biopsy side (right versus left lung) should be related to the radiographic findings or the patient’s prior surgical history. In many instances it is influenced by the surgeon’s training. Cardiac surgeons tend to preferentially sample the left lung – which is acceptable if the surgeon understands the potential pitfalls. Both the lingula and the right middle lobe, which are both temptingly accessible, may

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Chapter 2: Lung specimen handling and practical considerations

have nonspecific increases in fibrosis, inflammation, and vascular thickening.8 For these reasons, it is recommended these sites should not be biopsied. The scarred tip of any lobe should be avoided. Surgeons should try to biopsy the deeper portions of the lung, if it is technically feasible.3 Multiple samples should be taken to include areas of radiographically active disease (such as ground-glass opacities). Areas of honeycomb lung should not be oversampled, as the advancing edge of disease is more likely to provide insights into the underlying etiology. The routine practice of sampling multiple lobes – with the previous caveats in mind – usually provides the pathologist with satisfactory material for evaluation (leaving only the problem of those unclassifiable interstitial pneumonias!).9 If the indication for the surgical procedure is a nodular density, the surgeon should be encouraged to remove the nodular density in its entirety rather than sampling a small piece from the edge. If frozen section is necessary, excision of the entire lesion allows the pathologist more flexibility in selecting a representative sample, thus improving frozen section accuracy. All biopsies suggestive of infection should be routinely cultured for bacteria, fungi, and acid-fast bacilli. Viral cultures can also be sent in special circumstances. Even in the instance of a lesion highly suspicious for malignancy, it is not uncommon to find co-existent infection. These cultures can be initiated by the surgeon in the operating room. In some cases the pathologist can assume the responsibility for obtaining fresh tissue for culture, such as a possible tuberculous lesion. Either practice suffices, provided that the protocol is understood and agreed by both the clinician and pathologist. This avoids a failure of communication resulting in no cultures at all. In all instances, wedge biopsies should be received fresh, as soon as possible after excision for pathological evaluation. The request for a frozen section on a lung wedge biopsy should include the clinical and radiographic findings. While it is a good practice to regard all tissues as potentially infectious, pathologists should insist that a specimen highly suspicious for infection be specifically designated, so that special cryostat precautions can be taken. It should be the standard practice to evaluate all lung frozens in a special Category II biological cabinet to avoid the erroneous clinical diagnosis of cavitating squamous cell carcinoma being confused with tuberculosis. Instances in which frozen sections are recommended include lung disease in immunocompromised patients, acute onset of lung injury, strong suspicion for infection, nodular infiltrates, and nodules or masses without a preoperative diagnosis.3 In the acutely ill patient, frozen section may identify an etiology for which specific therapy can be rapidly instituted. Frozen sections also allow for the pre-ordering of special stains for microorganisms. Doing so improves the diagnostic turnaround time in these critically ill patients. Frozen sections can be used to assess the need for other diagnostic techniques, such as immunofluorescence in alveolar hemorrhage syndromes, flow cytometry and B-5 fixation for lymphoid lesions, and samples for pneumoconiosis analysis.3

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If the surgeon and radiologist are experienced, frozen sections are probably not necessary for the interstitial pneumonias including usual interstitial pneumonia, desquamative interstitial pneumonia/respiratory bronchiolitis interstitial lung disease, nonspecific interstitial pneumonia, chronic hypersensitivity pneumonia, as well as diffuse bronchial and vascular diseases. Intact open biopsies for these indications allow for optimal specimen processing, which prevents artifactual alveolar collapse. Even for these indications, a surgeon will often request a frozen section to confirm that “diagnostic” tissue has been obtained and it is reasonable to accommodate such a request. If the frozen section shows only honeycomb lung, the surgeon should sample the lung in less advanced areas of disease. Whatever the indication, unfixed tissue must be handled gently and with sharp scalpels or razor blades to reduce artifacts. Upon receipt of the wedge biopsy, the pathologist should at a minimum understand whether the biopsy is for diffuse and probably non-neoplastic lung disease or if the wedge excision is for a nodule that is possibly malignant. The procedures for handling these two different clinical indications differ. In wedge excision for a possible malignant nodule, the visceral pleura overlying the lesion should be inked prior to sectioning. This practice provides the surgical pathologist with a microscopic landmark during his/her evaluation of pleural invasion. The wedge excision will also typically include one or two staple lines. If this wedge is superseded by a completion lobectomy, as is the current standard recommendation for a primary lung carcinoma, these staple lines become irrelevant. If the wedge is intended to be the definitive surgery – as in inflammatory lesions, metastases, or primary lung tumors in patients for whom completion lobectomy is contraindicated – these staple lines must be evaluated as important surgical margins. It is not usually necessary to disrupt these stapled margins at the time of frozen section, if the reason for this procedure is focused on diagnosis. The wedge biopsy can be sectioned in one of two ways – either by traditional “bread loafing” or by a simple bisection of the wedge from the visceral pleura towards the staple line. In either instance, the wedge should be carefully assessed to ensure there is a gross abnormality that corresponds to the radiographically identified nodule or palpated abnormality. This sectioning should minimize sampling error at the time of frozen section. It should allow for sampling in an area away from the affected visceral pleura, which will avoid disruption artifacts. Some surgeons incise into the specimen prior to receipt in the pathology laboratory. They should be instructed, if possible, to incise away from the visceral pleura overlying the mass. This will avoid disrupting the visceral pleura in an area that may impact accurate tumor staging. Some surgeons or their assistants are even willing to apply ink before their incision. Once a preliminary diagnosis has been rendered, any fresh tissue required for ancillary studies is taken and the specimen

Chapter 2: Lung specimen handling and practical considerations

can be fixed. After an appropriate fixation interval, subsequent processing depends on whether the patient underwent additional surgery. If the wedge is intended as definitive surgery, then the staple lines should also be inked and the lesion in relationship to the stapled parenchymal margin should be submitted for permanent section evaluation. As discussed in the subsequent section on lobectomies, all potential adenocarcinomas 2 cm or less should be submitted in their entirety. An alternative approach for all wedge resections is for the surgeon to gently distend the lung tissue with formalin, using a finegauge needle and formalin. It is then placed in an adequate amount of formalin and covered with a thin sheet of tissue. This ensures the specimen is properly fixed, which can be important, especially over a weekend or holiday period. Different strategies have been described for processing wedge biopsies for non-neoplastic diseases. Individual experimentation with these techniques should be encouraged and the results personally evaluated for their suitability in a specific practice setting. Whatever the technique, surgical lung biopsies for non-neoplastic disease are typically a once in a patient’s lifetime procedure and should be optimally and entirely processed.1 Katzenstein describes a straightforward approach of carefully removing the staple line by cutting along the lung surface adjacent to the staples with small scissors.3 A sharp scalpel or razor blade is then used to serially section the wedge specimen perpendicular to the long axis using a gentle sawing motion. In this method, the sectioning should always begin at the lung margin and proceed toward the pleural surface. This is because the pleural surface is firmer than the underlying lung. This strategy will produce less atelectasis. If a wedge biopsy is received intact and no fresh tissue needs to be saved, others have described techniques for distending the specimen using a small-gauge needle and formalin to expand the alveolar spaces and eliminate alveolar collapse.10 Multiple formalin injections can be made at the staple line. This is best accomplished with a 5 ml or smaller syringe. Infusion should be performed slowly and overdistension avoided. The tissue is then covered with tissue or gauze to ensure the pleural surface does not dry out. Once fixed for an appropriate interval, the wedge should be entirely processed to maximize the evaluation of a possibly patchy disease process. An alternative technique for distension, which can be easily modified for the frozen section and has interesting applications for research, is to use diluted cryomatrix (TissueTek® OCT compound) medium (Figure 1).11 This technique requires a slightly larger-gauge needle than those used to inject formalin. This practice facilitates cryosectioning and prevents atelectasis. Although both distension techniques may create artifacts, such as overexpansion of airspaces and dilatation of lymphatics, they are not obstacles to accurate evaluation. Pathologists are most delighted when they receive an intact specimen from a surgeon that allows them optimal control over gross evaluation, processing, fixation, and microscopic examination. Clinical practice is rarely so perfect and there are a number of strategies and techniques that can be used to

Figure 1. Preventing atelectasis during frozen sectioning with cryomatrix. During the frozen section technique, the installation of cryomatrix via a syringe can be used to inflate a small piece of lung and prevent atelectasis. Left panel without cryomatrix, right panel with cryomatrix.

rescue a specimen that is not intact. Whether or not a specimen is received intact prior to fixation is largely dependent on the setting. Common reasons include prior frozen section, prior sampling for culture and other studies, such as immunofluorescence, fresh tissue for research protocols/tissue banking and molecular assays, and the inquisitive surgeon. Vacuum chamber formalin fixation is another simple technique which reduces artifactual atelectasis in a previously cut fresh lung.12 This method requires an inexpensive counter-top vacuum chamber and access to suction. The previously cut wedge specimen and the thawed frozen section are placed into an open cup of formalin in the vacuum chamber. The chamber is closed and the vacuum is turned on at a gentle setting. As air is removed, formalin fills the tissue. After several hours, depending on the size of the specimen, the tissue is ready for further processing. Whatever processing approach one adopts, artifactual atelectasis should be avoided as much as possible, since the microscopic evaluation of appropriately expanded lung sections allows for significantly better pathological evaluation and trainee instruction. As will be discussed in the subsequent section, artifactual atelectasis is commonly overinterpreted and can result in major diagnostic error.4

Segmentectomy, simple lobectomy, bronchoplastic resection, pneumonectomy, and autopsy lungs The pathological evaluation of these large specimens requires some fundamental knowledge of surgical techniques and

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Chapter 2: Lung specimen handling and practical considerations

Figure 3. Use of probes. A sharp knife is used to section the lung, using the bronchial probes to establish a place of sectioning

Figure 2. Sleeve lobectomy. The right main bronchus and bronchus intermedius are divided and the right upper lobe is removed. There are two bronchial resection margins, proximal (main stem) and distal (bronchus intermedius) that must be submitted for pathological evaluation. Image courtesy of L. Kaiser, MD, Philadelphia, Pennsylvania, USA. Reused from Larry R. Kaiser: Atlas of General Thoracic Surgery, Mosby-Year Book, Inc. St. Louis, Missouri, 1997, part IV, Pulmonary resections, chapter 21, Sleeve lobectomy, Figure 21–2, p. 96, with permission from Elsevier Ltd.

their indications. A segmentectomy is defined as an anatomic surgical resection, including a small segmental bronchus and artery. The segmental bronchus and artery must be identified on the specimen and sampled as surgical resection margins. Simple lobectomy is the commonest surgical procedure performed for stage I lung cancer and in some specific instances of non-neoplastic disease. Bronchoplastic resections are seen in larger specialized thoracic centers. Many bronchoplastic procedures can be performed depending on the location of the lesion, but the basic principle is to spare lung parenchyma by resecting only a portion of the major airways and re-anastomosing the proximal and distal ends of the remaining airway (Figure 2). In this instance, there are two bronchial margins that must be pathologically evaluated. Pneumonectomies are performed on a select group of patient with malignant disease, as well as for some specific cases of non-neoplastic disease. Explants from lung transplant recipients, as well as autopsy specimens, are also included in this category. It is best to have these larger specimens brought directly to the laboratory in a fresh state during working hours. Specimens can be temporarily stored in a dedicated refrigerator during the night and processed the next morning.

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Processing of these large specimens for non-neoplastic disease or suspected lung cancer will be discussed here. Specific issues related to lung cancer staging are covered in Chapter 25. Whenever a lobe or whole lung is removed, there is artifactual lung collapse that makes histological evaluation difficult. Unless bronchial or vascular anomalies need to be evaluated in an unfixed specimen, formalin inflation through the bronchus or trachea is recommended. This can be accomplished via gravity drainage, which uses a plastic tube attached to a formalin tank 2–3 feet above the specimen, or with a large formalin-filled syringe. The plastic tube or syringe is wedged into the airway and the specimen is filled with formalin until maximally expanded. It is usually unnecessary to clamp the airway to retain formalin. In a centrally obstructing lesion, it may be necessary to use a syringe and needle for localized formalin distension of the distal parenchyma or previously incised areas. The lung should then fix for at least 1–2 hours, while overnight is optimal. One should always consider the need for special studies while the specimen is still fresh. If fresh tissue is required for microbiology or other purposes, small pieces of peripheral lung can be obtained, without significantly affecting formalin distension. A fixed lung is firm and easy to slice thinly for optimal gross examination. The two techniques most popular for sectioning are Liebow’s bronchial probe technique and the “slab” method. Both methods require a very sharp knife. Extra trimming may be required to completely open the vessels and smaller airways. Since it is virtually impossible to cut slices thinner than 0.5 cm, one must palpate slices in order to identify and localize radiographic or unsuspected lesions. In the Liebow bronchial probe method (Figure 3), probes are passed down the airways and used as a guide for slicing. This technique is excellent for demonstrating a tumor’s relationship to the airways or distal parenchymal disease (Figure 4). The resulting cuts are in neither a sagittal nor a coronal plane but

Chapter 2: Lung specimen handling and practical considerations Figure 5. Slab section. This slab section of lung from a patient with cystic fibrosis vividly demonstrates bronchiectasis and purulent mucous plugging.

Figure 4. Bronchial probe. This section of lower lobe, cut using the bronchial probe method and trimmed, demonstrates bronchiectasis.

this is of little consequence for presentation and sampling purposes.13 The slab method is most frequently used for whole lungs at autopsy. The main stem bronchus is trimmed and then the hilar surface of the lung is laid down on the cutting board. The lung is serially sliced in an established plane with as thin and even slices as possible. The anteriorto-posterior, apical-to-inferior dimension demonstrates a maximum amount of lung and is excellent for appreciating regional variations (upper vs. lower lobe predominant disease, hilar or peripheral process, etc.) (Figure 5). Other specific orientations, such as sagittal slicing, are used to demonstrate and evaluate emphysema and other diffuse pulmonary diseases. Although some pathologists may have a strong personal preference by habit or training for “breadloafing”, there are some compelling reasons to utilize one of the two above-mentioned techniques. These techniques allow for the rational orientation of the lung and facilitate more accurate gross descriptions. These types of orientation help to avoid common trainee problems, such as confusing a post-obstructive pneumonia with the tumor mass, missing airway lesions, and confusing lymph node metastases with primary tumors. This orientation also allows for a greater appreciation of the disease geography, as with centrilobular emphysema and pulmonary fibrosis. Lungs fixed and cut in these manners are also excellent for educational demonstrations or photographs. The effect of an outstanding gross photograph cannot be overestimated – in terms of both its didactic value and its simple ability to delight and impress the trainee or clinician. Although there is much that can be done in these days of digital imagery to rescue a suboptimal photograph, adherence to a few fundamental photography guidelines will usually suffice to produce a quality image. As with other types of organs, the image is best when taken in an anatomically correct orientation. When the section of lung does not lie flat, it is a good idea to use modeling clay or some other material to prop

up the specimen and maintain it in a uniform plane. If the specimen is being photographed in a fresh state, excess blood should be blotted away. A paper towel soaked in alcohol can be briefly applied to the surface in order to reduce the sheen from a fresh specimen and can be similarly applied to a formalinfixed specimen to brighten the colors. There are alternatives to formalin fixation or sectioning that may be specifically indicated, particularly for research. Computed tomogram (CT) correlation studies may require a transverse or axial plane of sectioning. Diluted cryomatrix inflation of the whole lung combined with rapid freezing has also been used for RNA analysis and other specialized techniques requiring snap-frozen tissue.14 There have been a number of specialized techniques devised and described in pulmonary research. Some of these techniques were summarized in previous editions of this book, as well as in other general reference publications.15 Radio-opaque material injection into vessels or airways is one such process. Advanced radiological techniques such as micro-CT are converging, at least in an experimental setting, with the detailed structural evaluation that was once only possible through more precise methods of inflation for volumetric lung studies (Figure 6). Whether these technological advances will be incorporated into future clinical practice and how they will impact on histopathology remain unknown.

Evaluation of the large specimen in known or suspected cases of primary lung carcinoma The diagnosis of malignancy is now often confirmed prior to resection. In such cases there is usually information about the primary tumor and other abnormalities, such as potential satellite lesions – at least to the radiologist and surgeon. Many

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Chapter 2: Lung specimen handling and practical considerations

Figure 6. MicroCT. Volume-rendered micro-CT image of emphysematous lung tissue showing large regions of tissue destruction. The specimen was obtained from the explanted lung of a transplant recipient with severe chronic obstructive pulmonary disease. The tissue was inflated, frozen in liquid nitrogen vapor, fixed in 1% glutaraldehyde solution, post-fixed in 1% osmium tetroxide, and critical-point dried. Micro-CT scanning was performed on the dry tissue using an eXplore Locus-SP micro-CT scanner (GE Healthcare) with 16 µm isotropic resolution. Volume rendering was performed using OsiriX software (www.osirix-viewer.com). (Image courtesy of A. Wright, PhD, Philadelphia, Pennsylvania, USA.)

of these additional lesions will be designated as ill-defined ground-glass opacities, which may be difficult to palpate or to detect grossly. It is prudent to know as much about the radiological abnormalities as possible, prior to gross evaluation. Radiology has its limitations – particularly in lesions under 0.5 cm – and there is still no substitute for a careful and independent gross pathology examination. Chapter 25 addresses the lung cancer staging system but there are a few fundamental points that should be made in the context of specimen handling. It is particularly important that pathologists and their trainees understand the importance of accurate tumor measurement and its potential impact on adjuvant therapeutic decisions and overall prognosis. For over a decade, the TNM classification for non-small cell lung cancer (6th edition) was based on the revision of the International Staging System published by Mountain in 1997.16 In this staging system, stratification of T1 from T2 tumors incorporated both a size cutoff (⩽ 3 cm or > 3 cm), as well as the descriptors of visceral pleural invasion, hilar atelectasis, and obstructive pneumonitis. The 2009 TNM revision (7th edition) has expanded the number of tumor size descriptors to include 2, 3, 5 and 7 cm cutoffs.17 In the 2009 revision, T1 tumors are subclassified into T1a (⩽ 2 cm) and T1b (> 2 and ⩽ 3 cm). T2 tumors are subclassified into T2a (> 3 and ⩽ 5 cm), T2b (> 5 and ⩽ 7 cm) and T3 (> 7 cm).18 The 2009 revision has retained the non-sizebased T2 criteria, although some controversy does exist as to whether these criteria should be linked to size criteria, rather than remaining as independent criteria.19,20 The issue of fresh versus fixed lung tumor measurement is addressed in the 7th edition TNM classification.21 Measurement

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of the tumor in the unfixed specimen is advocated, on the basis of one study which looked at tumor size pre- and postfixation.22 The study, which excluded any tumors with visceral pleural invasion, main bronchus involvement, or associated with atelectasis or obstructive pneumonia, reported that 20% of tumors > 3 cm shrank by an average of > 1 cm with a 10% stage shift from stage Ib to Ia. In addition, it was noted that not all lung tumors showed shrinkage after formalin fixation or even a constant fixation rate. The pathological criteria and use of elastic stains to evaluate visceral pleural invasion was recently reviewed on behalf of the International Staging Committee.23 One way to approach the evaluation of the visceral pleura is to carefully ink the visceral pleura overlying the lung lesion prior to sectioning. Such a practice ensures that one can discern the true visceral pleural surface from subpleural fibrosis or incompletely cut histological sections. In many peripheral lung tumors, visceral pleural puckering is obvious and ink should be applied generously to this area. This ink helps identify the visceral pleural surface in histological sections. If a frozen section is requested (or the surgeon feels it necessary to cut into the specimen for some other reason), the lesion should be sampled in an area away from the most obviously affected visceral pleura to avoid disruption artifacts. In the 7th edition of the TNM, visceral pleural invasion (VPI) is defined as invasion beyond the elastic layer, including invasion to the visceral pleural surface (see Chapter 25). Elastic stains are recommended when the level of pleural invasion is not clear on H&E evaluation. Pre-ordering an elastin stain on sections most suspicious for visceral pleural invasion will improve turnaround time and result in more accurate staging. Although not a formal part of the lung cancer staging system, thoracic pathology experts also recommend that all known or potential adenocarcinomas 2 cm or less be submitted for pathological evaluation in their entirety. This allows complete morphological evaluation and identification of tumor subtypes, which may carry therapeutic and prognostic significance. In the American Joint Committee on Cancer/International Union for Cancer Control (AJCC/UICC) Lung Cancer Staging system, thoracic lymph nodes are divided into N1, N2, and N3 nodes. N2 and N3 nodes are mediastinal lymph nodes that should be separately identified and designated by the surgeon (see Chapter 25). N1 nodes include peribronchial and hilar lymph nodes attached to lobectomy specimens. The pathologist must carefully examine the resection specimen for all N1 nodes. These N1 nodes are more easily identified in a specimen with a peripheral lung tumor but can be quite difficult to identify in a more central tumor. Often, particularly with an inexperienced trainee, a metastasis to contiguous N1 lymph nodes is overlooked macroscopically. Cutting the lung by the bronchial probe method allows a better appreciation of the pathology (Figure 7). The carbon pigment present in most of these nodes further facilitates lymph node recognition. This type of sectioning also highlights the secondary pathologies of post-obstructive pneumonia, bronchiectasis, and

Chapter 2: Lung specimen handling and practical considerations

representative samples. Many surgeons are reluctant to biopsy the lung in such circumstances, if it can be identified amidst the dense fibrosis, for fear of a prolonged air leak. However, deeper “interface” biopsies of the chest wall can be taken with minimal morbidity. “Interface” biopsies should be encouraged, as they are often critical in demonstrating tumor infiltration into fat and muscle. The size of pleural biopsies varies greatly. Smaller biopsies should be submitted in toto but sampling of larger decortication or pleurectomy specimens requires knowledge of the clinical history and an informed gross examination. Larger pleural specimens should be carefully examined for peripheral adherent lung and skeletal muscle. Sections from these areas should be submitted for the reasons given above. Benign pleural fibrosis appears uniform throughout. It is rubbery and any areas of firmness or nodularity should be sampled. The number of sections should correlate with the index of suspicion for malignancy.

Ancillary studies Figure 7. Dissection by probe method. It is easy to appreciate both the main tumor mass and the gross involvement of lymph nodes when the specimen is cut along the airway.

atelectasis. The distance from the bronchial or medial resection margin is also more easily noted.

Large pleural biopsy As with interstitial lung disease, VATS has greatly influenced the approach to and management of pleural disease. Depending on the indication, larger specimens may be sent to the pathology laboratory for processing. Receipt of these larger samples is typically viewed with relief by both general and expert pathologists. They both understand that with pleural biopsies, larger biopsies usually result in a higher level of diagnostic confidence. This is particularly true when a thoughtful surgeon, an attentive pulmonologist, and an astute radiologist contribute to the evaluation. Benign and malignant pleural diseases are covered in depth in Chapter 36 and therefore only a few basic principles of processing are warranted. When the pleural space is open and there is diffuse nodular disease, the surgeon will have a wider range of options for biopsy sites. It is preferable to include at least one wedge biopsy of the lung that will help to define the visceral pleural pathology in relationship to the underlying lung. In addition multiple biopsies from the parietal pleura, chest wall, mediastinum, and diaphragm should be taken. When the pleural space is partially or completely obliterated, the surgeon will face greater technical difficulty in assessing the pleural disease (diffuse pleural fibrosis versus malignancy) and obtaining

The appropriate use of ancillary studies must be grounded in a well-formulated differential diagnosis and correlated with morphology. The clinician and the radiologist contribute greatly by providing a complete and accurate history, which helps the diagnostic work-up and avoids unnecessary tests. In some areas, such as interstitial pulmonary fibrosis, a combined diagnostic approach in a joint meeting is the only rational way to achieve the correct diagnosis. A general overview is provided here; more specific indications for these ancillary tests are covered in subsequent chapters. Despite a great deal of technological innovation and enthusiasm for molecular testing, histochemical stains remain a mainstay in the diagnosis of neoplastic and non-neoplastic lung disease. Histochemical stains have the great virtues of rapid turnaround time, low cost, technical simplicity, and applicability to standard cytology and histology samples. Histochemical stains can be divided into those routinely performed to detect microorganisms (Grocott methenamine silver, Ziehl-Neelsen, Gram), those staining matrix substances (Masson trichrome, Movat pentachrome, elastic van Giesen, and Congo red), those that demonstrate intracellular mucin (mucicarmine, periodic acid-Schiff), and those detecting other substances, such as iron or calcium. Immunohistochemistry has become an integral part of tumor diagnosis and aids in the diagnosis of certain nonneoplastic diseases, such as Langerhans’ cell histiocytosis. Immunohistochemistry has also been applied to the diagnosis of mycobacterial, fungal, viral, and bacterial infections. Immunohistochemistry in pulmonary pathology has been extended into prognostic markers and therapeutic response predictors. The technique is based on a primary antigen-antibody reaction and a secondary antibody-enzyme complex, which interacts with a chromogen for a microscopically visible color reaction. Immunohistochemistry can be performed on either frozen or

49

Chapter 2: Lung specimen handling and practical considerations

formalin-fixed tissue, depending on the antibody. It is good practice not to use a tissue block that contains previously frozen material for immunohistochemistry, if other tissue blocks are available. Unfortunately, there are very few antibodies which approach 100% sensitivity and specificity. As immunohistochemical experts emphasize, it is diagnostically irrelevant to speak of overall sensitivity and specificity for a particular antibody. Rather it is more appropriate to speak of relative sensitivity and specificity within a particular differential diagnosis. This requires clinical interaction and morphological expertise in generating a differential diagnosis, in addition to critical assessment of the immunohistochemical results with appropriate controls (see Chapter 26). Immunohistochemistry is often used in the work-up of lung and pleural tumors, either to better characterize the primary or to exclude metastatic disease. It is vital to understand the staining patterns of the normal lung and pleura, particularly in a small or distorted specimen. For example, cytokeratin antibodies will stain benign bronchial and alveolar epithelia, as well as reactive mesothelial cells, which can be entrapped within tumors and lead to a false positive interpretation. Staining with a panel of antibodies to support one’s diagnostic impression within a differential diagnosis can enhance diagnostic accuracy. The histopathologist must be aware of the staining for a particular monoclonal antibody in tumors in the differential diagnosis. Thus calretinin, while positive in some epithelioid mesotheliomas, may stain the nuclei of some sarcomatoid carcinomas (see Chapter 36). In individual cases, immunohistochemical analysis remains an exercise in probabilities and may not be sufficient for certain clinical circumstances. New markers are often introduced with initial published reports of high sensitivities and specificities. After additional studies and incorporation into daily practice, more exceptions appear. There is no substitute for a good clinical history, thorough physical examination, high-quality radiology, and pathology evaluation based on routine hematoxylin and eosin-stained sections. Electron microscopy (EM) has ceded much ground over the past 25 years as the number of antibodies suitable for immunohistochemistry on routine formalin-fixed paraffin embedded tissue dramatically increased. Electron microscopy’s role in tumor differential diagnosis has been greatly diminished, even for diagnosing malignant mesothelioma. The diagnosis of epithelioid mesothelioma is satisfactorily confirmed with an immunohistochemical panel in most cases. Electron microscopy, if available, can be reserved for the occasional instances in which the immunohistochemical profile is equivocal.24 Electron microscopy is occasionally useful in establishing the correct diagnosis of well to moderately differentiated epithelioid tumors, when immunohistochemical results are equivocal.25 It is reasonable, if the tumor sample is large enough, to set aside a small sample for EM. Prompt fixation in a recommended fixative (glutaraldehyde or methanol-free

50

formaldehyde) is preferred, although commercial formalin may be used if the fixation is rapid.13 Although material can be retrieved from a paraffin block, the preservation is often suboptimal, depending on the fixation interval and the ultrastructural features to be identified. Special care should be taken in obtaining the sample. A new sharp blade should be used and cut with a to-and-fro motion, rather than by exerting downward pressure.13 In non-neoplastic lung disease, electron microscopy remains essential for the evaluation of cilia in ciliary dyskinesia syndromes.1 In this instance, rapid fixation is once again essential and should be available on site at the time of bronchoscopy. Other selected uses in non-neoplastic disease include the demonstration of electron-dense deposits. It is an exciting but unsettling time in pulmonary pathology. New molecular techniques for the evaluation of neoplastic and infectious diseases have been introduced into the pathology laboratory, although the degree to which these techniques have been incorporated into clinical practice varies worldwide. In some centers, molecular testing is not only being used in diagnosis, but also to address prognosis and to assess therapeutic targets. The potential applications for immunohistochemistry and in situ hybridization have been similarly expanded. The evaluation of epidermal growth factor receptor (EGFR) abnormalities in patients with pulmonary adenocarcinoma is a typical example. At present, there is relatively little consensus as to how these tumors should be routinely tested for EGFR and whether the results of immunohistochemistry, mutation analysis or fluorescent in situ hybridization correlate with each other or are independent predictive variables. As the authors of one recent study examining these issues concluded, there is a need to develop more evidencebased or consensus standardization guidelines for the performance and interpretation of EGFR tests in routine clinical practice.26 Similar conclusions can be drawn from any recent survey of the literature, which is now replete with proposed molecular markers in pulmonary neoplasia. Fewer studies address the cost-benefit analysis associated with new assay validation and implementation into the clinical laboratory. Another issue that must be addressed within the clinical laboratory and in collaboration with clinical colleagues is what to test. The new molecular technologies can be applied to a bewildering array of different fresh biological samples, including tissue, aspirates, effusion fluid, and bronchial specimens. There are some advantages to using cytological material over histological sections to perform molecular assays in terms of DNA and RNA preservation. Although not an insurmountable obstacle for some assays, formalin fixation and paraffin embedding can impair nucleic acid retrieval. It is also more difficult to control the prevailing environmental conditions of paraffin block storage and histological processing. When adopting a new molecular assay, the clinical pathology laboratory faces the additional challenge of deciding who should perform and interpret the test.27 Whether molecular profiling

Chapter 2: Lung specimen handling and practical considerations

will improve tumor subclassification and differential diagnosis in a cost-efficient and reproducible manner remains to be seen. The same issues pertain to infectious diseases, where the menu of potential studies includes immunohistochemistry, in situ hybridization, and PCR-based assays.

Artifacts in biopsy Under ideal circumstances, a properly obtained representative sample of lung should provide useful diagnostic information. Unfortunately, there are several potential artifacts that may occur in transbronchial biopsies (TBBx) or in open lung biopsies, which can distort lung tissue and potentially lead to misleading diagnoses (Table 1).1,3,4,8,28,29 The artifacts may occur during tissue procurement, while being transported to the laboratory, or in the tissue processing.4

Table 1 Artifacts in lung biopsies3,4,7,8,28–30

Bronchoscopy

Laboratory

Atelectasis

Atelectasis

Hemorrhage

Sponge

Pinch

Bubble

Crush

Poor-quality histology

Dried or poorly fixed Biopsy of pleura

Figure 8. Atlectasis. Prominent areas of atelectasis are frequently present in transbronchial and open lung biopsies. In this tranbronchial biopsy, the sharp interface of normal lung adjacent to collapsed lung is helpful in identifying atelectasis, as opposed to a fibrosing disorder.

Atelectasis/overinflation Atelectasis is a frequent finding in all types of lung biopsies and could lead to mistakes in diagnosis.4,7,30 This commonly occurs in TBBx due to tissue compression. The changes are most prominent in the central portions of the biopsy specimen (Figure 8). Not infrequently, atelectasis in TBBx may be misinterpreted as fibrosis. This can be avoided through proper fixation and careful comparison with other areas in the biopsy. If not certain, a trichrome stain may clearly show delicate thin alveolar septa, as opposed to definitive interstitial fibrosis. In open lung biopsies, various distension techniques have been proposed to prevent artifactual atelectasis. Overinflation of lung tissues could occur by direct instillation of cryomatrix into a non-formalin-fixed lung. As a consequence, dilated alveoli could be misdiagnosed as emphysema or lymphangiectasia.

Intra-alveolar hemorrhage Intra-alveolar hemorrhage is a frequent finding and is usually an artifact resulting from the biopsy-inducing filling of the alveolar spaces with fresh blood (Figure 9).7 If coarse hemosiderin-laden macrophages are present, this suggests that bleeding occurred in the past (Figure 10). An iron stain highlights hemosiderin in macrophages as clumpy, dark-blue material. Caution is advised since the finely pigmented golden brown and carbonaceous intracytoplasmic material seen in macrophages from cigarette smokers can resemble hemosiderin. It is

Figure 9. Artifactual intra-alveolar hemorrhage. Commonly seen in transbronchial and open lung biopsies, intra-alveolar hemorrhage results from the filling of the alveolar spaces with fresh blood during the biopsy procedure.

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Chapter 2: Lung specimen handling and practical considerations

Figure 10. Real intra-alveolar hemorrhage. The intra-alveolar hemorrhage is associated with hemosiderin-laden macrophages, which indicates that there had been past episodes of bleeding.

Figure 11. Iron-laden intra-alveolar macrophages. The coarse brown granules of hemosiderin in intra-alveolar macrophages in the left panel should be distinguished from the finely pigmented golden brown and carbonaceous intra-cytoplasmic material seen in macrophages from cigarette smokers in the right panel.

Crush artifact Crush artifact is frequently seen in the setting of small cell carcinoma, but may be seen in benign and malignant lymphoproliferative disorders.31 Careful correlation with cytology specimens and examination of other areas of the biopsy, especially any fragments outside the main biopsy, may help to establish a diagnosis (Figure 12).32 If the crush artifact is so severe that interpretation is impossible, the pathologist must concede to this reality and inform the clinician, rather than risk a dangerous overinterpretation.

Sponge artifact The use of commercially available foam embedding sponges in tissue cassettes can lead to artefactual clefts in lung parenchyma (Figure 13).4 The irregularly shaped, variably sized, triangular spaces are present in lung parenchyma without granuloma formation or polarizable foreign material. This could be misinterpreted as foreign-body reaction. Figure 12. Crush artifact. Biopsy forceps can crush any tumor beyond histological recognition but small cell carcinoma and lymphoid lesions are prone to distortion. Other areas of this particular tumor demonstrated obvious small cell carcinoma morphology.

only faintly Perls’-positive and does not glisten, like hemosiderin (Figure 11). In some cases of recent massive alveolar hemorrhage, hemosiderin-laden macrophages may be absent, rendering clinical correlation a necessity.

52

Bubble artifact This is a common finding in lung biopsies due to crushed or collapsed airspaces and has been referred to as “pseudolipoid pneumonia”.4,29 It consists of irregularly shaped, roundto-slightly oval spaces within the lung parenchyma, without evidence of a foreign-body giant cell, fibrinous material, or an inflammatory response (Figure 14). An associated

Chapter 2: Lung specimen handling and practical considerations Figure 13. Sponge artifact. The use of biopsy foam pads in tissue cassettes causes tissue artifacts. The center panel illustrates “barb-like” prongs of the foam material, viewed microscopically. In the tissues, triangular, irregular shapes are due to intrusions of the foam material in the right panel.

Table 2 Incidental findings in lung specimens

Large airways Ossification of bronchial cartilage Oncocytic metaplasia of bronchial submucosal glands Bronchial submucosal elastosis Alveolar parenchyma Scar Apical cap Foci of smooth muscle hyperplasia Metaplastic bone/dystrophic ossification Corpora amylacea Blue body Schaumann body Asteroid body Calcium oxalate crystal Hamazaki-Wesenberg bodies Mallory’s hyaline-like material in type II pneumocytes

Figure 14. Bubble artifact. This consists of irregularly shaped round-to-slightly oval spaces within the lung parenchyma, without evidence of a foreign-body giant cell, fibrinous material, or an inflammatory response.

foreign-body giant cell reaction warrants consideration of a lipoid pneumonia.

Incidental lesions A large number of nonspecific lesions may often be identified in all types of lung specimens. They can occur in various locations, including the airways, lung parenchyma, vasculature, or other sites (Table 2). While some are of no pathological consequence,

Vascular structures Megakaryocyte Small emboli Iron encrustation of elastic fibers Bone marrow emboli Other Entrapped pleura

they can be associated with certain diseases or need to be distinguished from other similar-appearing lesions.

Large airways

Ossification of tracheobronchial cartilage With aging, calcification and ossification of cartilage are common in large airways and are not usually of pathological consequence.

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Chapter 2: Lung specimen handling and practical considerations

Figure 16. Bronchial submucosal elastosis. This benign fibroelastotic process may distort overlying respiratory epithelium. Also note the thickened basement membrane.

Figure 15. Oncocytic metaplasia. Samples from older individuals often demonstrate oncocytic change in seromucinous acini.

Bone marrow cellular elements may be readily identified in the ossified cartilage. In lung allograft patients prominent ossification, calcification, and fibrovascular ingrowth of bronchial cartilage have been noted, possibly related to ischemic change.33

Oncocytic metaplasia of bronchial submucosal glands The bronchial submucosal glands may show pronounced oncocytic changes with advanced age. There is abundant eosinophilic cytoplasm filling the glands (Figure 15).34 This should be distinguished from bronchial oncocytoma and oncocytic carcinoid tumors (see Chapters 30 and 31).

Bronchial submucosal elastosis and adipose tissue Increased submucosal elastosis is another finding in the airways from elderly patients (Figure 16). Some asthmatic patients may show changes in the extracellular matrix that lead to elastosis and elastic fiber fragmentation in large airways.35 On rare occasions, foci of adipose tissue may be present in the bronchial lamina propria in elderly patients.

Parenchymal Scar

Scarification in the lung is fairly common and increases with aging. A localized pulmonary scar is usually found in the periphery of the lung with essentially unremarkable lung underneath the zone of fibrosis. A healed infection is the most common of all etiologies. This usually occurs in the lung tips and in the lingula.8 Microscopically, there is considerable dense eosinophilic collagen deposition with patchy chronic inflammation and possibly entrapped carbonaceous material or hemosiderin. Bronchiolar

54

metaplasia of lung parenchyma may be present and should not be mistaken for the true honeycombing fibrosis, as seen in interstitial pneumonias. It may be difficult in limited samples to separate localized scarring from a diffuse interstitial pneumonitis. Careful scrutiny of subjacent lung parenchyma is essential as well as clinical and radiographic correlations.

Apical cap Apical caps are increasingly noted in resection specimens, particularly those from the apices of the upper lobes in elderly patients.36 The etiology is most likely related to chronic ischemia and possibly infection.36 Grossly there are white, irregular scarred areas with induration (Figure 17a). At low magnification, they show a pyramidal shape associated with overlying pleural adhesions, and/or hyaline pleural plaques (Figure 17b). Senile emphysematous changes often accompany the apical cap. There is dense basophilic fibrosis with replacement of the pulmonary parenchyma with abundant eosinophilic collagen. Prominent stromal elastosis may be present in some cases and this is best appreciated on elastic and trichrome stains.

Metaplastic bone and dystrophic ossification Localized discrete foci of bone or calcification may be seen incidentally in large airways, fibrous scars, or old healed granulomas (Figure 18). When found incidentally, they have no pathological significance. More diffuse or extensive forms of pulmonary ossification, i.e., nodular/dendriform pulmonary ossification, are discussed elsewhere (see Chapter 10).

Smooth muscle hyperplasia Incidental discrete foci of interstitial smooth muscle may be seen, usually in women (Figure 19). An etiology is not recognized. These lesions are small, typically measuring less than 1 mm. They should be differentiated from other smooth muscle proliferations in the lung such as “benign metastasizing leiomyoma”, primary pulmonary leiomyoma/leiomyosarcoma, and lymphangioleiomyomatosis (see Chapter 33). More advanced smooth

Chapter 2: Lung specimen handling and practical considerations

(a)

(b)

Figure 17. Apical fibrous cap. (a) An irregular subpleural tan semisolid mass is worrisome for malignancy. (b) Lung parenchyma is distorted and overrun by richly vascular fibroelastotic tissue with scattered lymphoid aggregates.

Figure 18. Pulmonary ossification. Areas of metaplastic ossification are commonly seen in lung biopsies.

Figure 19. Smooth muscle hyperplasia. Small foci of smooth muscle hyperplasia predominantly involve the pulmonary interstitium.

muscle hyperplasia is commonly seen in the setting of the diffuse chronic fibrosing interstitial pneumonias, such as usual interstitial pneumonia and desquamative interstitial pneumonia.37

concentric lamellar bodies may or may not be birefringent and are derived from lysosomes. They stain positively with histochemical stains for calcium and iron.

Schaumann bodies Basophilic calcified bodies, termed Schaumann or conchoid bodies, commonly occur in sarcoidosis and in other granulomatous disorders (Figure 20) (see Chapter 13).38,39 These

Asteroid bodies Asteroid bodies represent distinctive spiculated or star-shaped structures that typically occur within multinucleated giant cells (Figure 20) (see Chapter 13).38,39 They should not be mistaken for fungi or other exogenous materials.

55

Chapter 2: Lung specimen handling and practical considerations Figure 20. Sarcoidal inclusions. Basophilic calcified bodies, termed Schaumann bodies, are seen in left panel. The central panel shows a distinctive spiculated or star-shaped structure, known as an asteroid body. Pale, irregularly shaped or block-like structures signify calcium oxylate crystals in the right panel. H&E.

and ultrastructural features with hepatic Mallory’s hyaline in alcoholic steatohepatitis. This was first described in asbestosis, but is a nonspecific finding in diverse non-neoplastic lung disorders.44

Corpora amylacea Scattered corpora amylacea are common in alveoli from older individuals.45,46 The etiology is uncertain. Some authors postulate that the bodies form by sequential aggregation, fusion, and coalescence of degenerated alveolar macrophages,46 while others suggest a localized reaction to foreign material.45,47 These round to slightly oval eosinophilic structures exhibit fine concentric circumferential lines (Figure 22). Corpora amylacea have no particular diagnostic importance, except that principal differential diagnostic considerations include distinction from inhaled or aspirated exogenous material, fungal forms, or pulmonary alveolar microlithiasis. Figure 21. Mallory’s hyaline-like material. Type II pneumocytes in diseased lung may contain deeply eosinophilic cytoplasmic material, identical to that commonly seen in alcoholic steatohepatitis. Image courtesy of W. Travis, MD, New York, New York, USA.

Calcium oxalate crystals These crystals, composed of calcium oxalate, are birefringent, typically block-like or irregular in configuration (as opposed to the needle-like appearance of silicates), and can occur independently of Schaumann bodies (Figure 20) (see Chapter 13).38,40,41 They can also be found in other diseases, although less frequently, such as infectious granulomatous disorders. These inclusions could falsely lead the pathologist to conclude the granulomas are of a foreign body type. Often the granulomas are associated with birefringent crystals within giant cells and macrophages. One can find poorly stained, lucent materials within or surrounded by giant cells in over two-thirds of open lung bodies in sarcoidosis.42 These inclusions are believed to be a byproduct of macrophage or giant cell metabolism, rather than exogenous material that produces the granulomata.

56

Blue bodies Blue bodies are intra-alveolar laminated basophilic concretions of an uncertain etiology.48 The round, blue-grey, slightly laminated structures are found in alveolar spaces and coexist with pigmented alveolar macrophages (Figure 23). By histochemical stains, iron and calcium may be identified. Electron microscopic studies have demonstrated calcium carbonate and other inorganic substances.48 Blue bodies are most often seen in the setting of desquamative interstitial pneumonia, chronic eosinophilic pneumonia, and occupational exposure to environmental dusts.48–50

Senile amyloid Focal deposits of amyloid occur commonly in the lung and other organs in elderly patients as a frequent incidental finding without evidence of other forms of amyloidosis.51 This is best appreciated in the lung at post-mortem examination and there is a strong correlation with advanced age.

Mallory’s hyaline-like material in type II pneumocytes

Vascular

Intracytoplasmic hyaline material, resembling Mallory’s alcoholic hyaline, may be present in type II pneumocytes (Figure 21).43,44 Pulmonary hyaline shares similar histochemical

Circulating megakaryocytes are occasionally seen in small blood vessels in open lung biopsies.52 The lung is a “platelet

Megakaryocytes

Chapter 2: Lung specimen handling and practical considerations

(a)

(b)

Figure 22. Corpora amylacea. (a) These round to slightly oval eosinophilic-to-basophilic structures are commonly seen in lung biopsies. (b) They measure up to 200 μm and are composed of glycoproteins. Although they are periodic acid-Schiff positive, one should not confuse them with fungi.

Figure 23. Blue bodies. The round, blue-gray, slightly laminated structures are found in alveolar spaces and coexist with pigmented alveolar macrophages.

Figure 24. Megakaryocyte. These bone marrow-derived cells travel through the pulmonary circulation and are prominent in acute inflammatory conditions. They should not be mistaken for tumor, trophoblast or virally infected cells.

factory”. Megakaryocytes are normally released from the marrow and in the pulmonary circulation they are buffeted against the vascular walls. Here the platelets are liberated, leaving only the bare nuclei visible on histopathology. The

megakaryocytes are most frequently seen in patients with acute lung injury or underlying systemic disorders affecting the lung.52–55 These irregularly shaped, hyperchromatic structures are located in the interstitial capillaries (Figure 24). While they

57

Chapter 2: Lung specimen handling and practical considerations

Figure 25. Iron encrustation. Intravascular prominent pigmentation of brownblack irregular material signifies previous episodes of pulmonary hemorrhage.

Figure 26. Intravascular thrombosis in diffuse alveolar damage. Microthrombi in the setting of diffuse alveolar damage results from vascular remodeling.

represent a nonspecific finding, care should be taken to not mistake them for viral inclusions, malignancy, or trophoblastic cells.

Pigmentation of elastic fibers In certain conditions, there may be prominent pigmentation deposition in pulmonary vascular walls. This consists of iron and calcium salts within the elastica of pulmonary arteries and veins. This has been termed “iron encrustation”, “mineral pulmonary elastosis”, or “endogenous pneumoconiosis”.56,57 Microscopically, there is deposition of brown-black irregular material within the elastica laminae associated with giant cells and it is better appreciated in histochemical stains for iron and calcium (Figure 25). This may be seen in the setting of longstanding alveolar hemorrhagic disorders, pulmonary venoocclusive disease, and in any condition with a raised left atrial pressure. In the Western world mitral stenosis is the most common cause. It must not be confused with the basophilia seen in the vessels associated with a small cell carcinoma.

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Figure 27. Bone marrow embolus. Commonly found at autopsy from individuals with sickle cell disease or blunt trauma including post-cardiac resuscitation, vascular spaces feature hematopoetic elements within marrow adipose tissue.

Microthrombi

Bone marrow emboli

Small microthrombi in the small blood vessels may or may not be of pathological consequence. They are common in acute lung injury, such as in the organizing phase of diffuse alveolar damage (Figure 26).55,58 They are best regarded as a pulmonary form of disseminated intravascular coagulation (DIC). Diffuse microthrombi can suggest a pulmonary microvascular disorder, overwhelming infection, DIC, or tumor-related thrombotic pulmonary microangiopathy.59

At autopsy, bone marrow emboli in the pulmonary microvasculature are most frequently related to compression of ribs and sternum during cardiopulmonary resuscitation.60,61 Rarely, they may be seen in the setting of cardiothoracic surgery following rib retraction or sternal disruption. This consists of bone marrow elements and adipose tissue in small arterioles (Figure 27). In contrast, fat emboli arising from the bone marrow elements are more typically seen in certain diseases, such as in sickle cell anemia and other hemoglobinopathies,

Chapter 2: Lung specimen handling and practical considerations Figure 28. Hamazaki-Wesenberg bodies. In the left panel, in H&E stain, these round, ovoid or spindle-shaped, yellow-brown structures are seen in subcapsular sinuses in lymph nodes. They may appear black in AFB stain in the right panel.

and following blunt trauma, including post-cardiac resuscitation, and long bone fractures.62,63

Others

Hamazaki-Wesenberg bodies In patients with sarcoidosis, the lymph nodes may reveal distinctive small yellow-brown bodies in the subcapsular sinus, known as Hamazaki-Wesenberg bodies (see Chapter 13).64–66 Similar structures have been seen in other sites and in other disorders, but they are most frequently encountered in sarcoid. These round, ovoid, or spindle-shaped structures measure 1–15 μm and are not birefringent (Figure 28). They stain black in the Ziehl-Neelsen, methenamine silver or Fontana-Masson stains and red with the periodic acid-Schiff stain. Because these bodies can closely resemble budding yeasts, their recognition is important to exclude an incorrect diagnosis of disseminated fungal infection. They probably represent lipofuscin and ultrastructurally show large lysosomes with protein, glycoprotein, and iron.

Entrapped pleura Portions of entrapped visceral pleura may be seen in TBBx and pose diagnostic difficulties.67 Microscopically, this consists of thin strips or clusters of bland, low cuboidal epithelial cells

with accompanying subjacent fibrovascular stroma or fat (Figure 29). They may be embedded within the lung parenchyma or loosely occasionally associated with fibrin or blood and when nodular could be potentially mistaken for a neoplasm.68 Immunohistochemical stains for WT-1 and calretinin establish the mesothelial nature of the cells. Recognition of this finding is important, as it may indicate that the biopsy caused a pneumothorax. The bronchoscopist should be immediately notified.

Applications and misapplications of the lung biopsy As a semi-invasive or invasive procedure, the lung biopsy is widely recognized as an invaluable tool for the diagnosis and management of diverse pulmonary disorders. In the past century, many technical advances have allowed surgeons, pulmonologists, and interventional radiologists to acquire lung tissue for accurate diagnoses. Accordingly, the transbronchial lung biopsy (TBBx), endobronchial or bronchial biopsy, transthoracic needle biopsy, open lung biopsy (OLBx), and videoassisted thoracoscopic surgery (VATS) biopsy are the current diagnostic procedures for obtaining samples for histopathological analysis.30,69 While surgical lung biopsies remain the most sensitive and specific test available for many patients with

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Chapter 2: Lung specimen handling and practical considerations

lung diseases, they should be utilized when a confident diagnosis cannot be gleaned from available clinical and radiographic data. While the various types of lung biopsies can yield useful information in certain clinical contexts, there are a number of potential problems which hinder proper interpretation of lung biopsies. A first-hand knowledge of the indications and limitations of lung biopsies is necessary for proper patient care and diagnosis.

Efficacy of the transbronchial biopsy Transbronchial biopsy is often the first step in obtaining tissue in diverse clinical situations with the anticipation of arriving at a definitive diagnosis and avoiding the use of a more invasive biopsy procedure. Although the TBBx can be a highly effective tool for the diagnosis of certain lung diseases, its role is quite limited and the outcome of the TBBx is highly disease- and situation-dependent (Table 1).30,70–72

High utility Transbronchial biopsy can be diagnostic in relatively few circumstances (Table 3).6,28,70,73,74 In primary lung carcinomas, TBBx is particularly efficacious; the addition of complementary cytological specimens, such as bronchial brushings and washings, increases the overall yield of fiberoptic bronchoscopy.34,71 Endobronchial ultrasound (EBUS) directed biopsy, which is often performed at the time of bronchoscopy, is particularly helpful for procuring tissue from enlarged lymph

Table 3 Reliability of transbronchial biopsy6,28,30,70,73,74

High utility Lung cancer and some metastases Sarcoidosis Opportunistic infections in immunocompromised patients Rejection in lung transplantation recipients Probably diagnostic Alveolar hemorrhage Capillaritis in anti-neutrophil cytoplasmic antibody-related disorders Goodpasture syndrome Lupus pneumonitis Alveolar proteinosis Lymphangioleiomyomatosis Unusual tumors Possibly diagnostic Wegener granulomatosis Eosinophilic pneumonia Lymphoid interstitial pneumonia Obliterative bronchiolitis Drug toxicity Unreliable Usual interstitial pneumonia Desquamative interstitial pneumonia Respiratory bronchiolitis-associated interstitial lung disease Nonspecific interstitial pneumonia Hypersensitivity pneumonitis Pulmonary Langerhans cell histiocytosis Figure 29. Entrapped pleura. The left panel shows small pieces of entrapped visceral pleura in a transbronchial biopsy. At higher magnification, the right panel shows strips of low cuboidal epithelial cells with accompanying subjacent fibrovascular stroma and fat.

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Chapter 2: Lung specimen handling and practical considerations

nodes and lung parenchyma and aids in lung cancer staging.75,76 Metastatic tumors can also be detected by TBBx, particularly in cases of lymphangitic carcinomatosis. In non-neoplastic diseases, such as in sarcoidosis, TBBx and/or blind bronchial biopsies, either by rigid or flexible bronchoscopes, are frequently useful (see Chapter 13). For certain infections and disorders in immunocompromised patients, fiberoptic bronchoscopy is particularly effective; bronchoalveolar lavage and brushings serve as the primary diagnostic modalities and TBBx is supplemental.77,78 Finally, in lung transplant recipients, the diagnosis of acute allograft rejection can be accurately rendered, but other entities unique to this patient population are more difficult to assess by TBBx.79,80

Probably diagnostic In certain situations, TBBx may be diagnostic, particularly if the pathology fits with the clinical and radiographic presentations. The histological finding of diffuse alveolar hemorrhage with or without necrotizing capillaritis could suggest a diagnosis of Wegener granulomatosis, other anti-neutrophil cytoplasmic antibody-related lung diseases, Goodpasture syndrome, or lupus pneumonitis. Fresh hemorrhage is frequently present in TBBx and may represent an artifact rather than a specific disease process. A few rare disorders, such as pulmonary alveolar proteinosis or lymphangioleiomyomatosis, could potentially be diagnosed by TBBx, given their characteristic histology and judicious use of special stains, including immunohistochemistry. Other less common tumors, often occurring in the central portions of the lung, might be diagnosed on TBBx. Special caution is needed to separate carcinoid tumor from small cell carcinoma in TBBx samples.

Possibly diagnostic It is improbable that the TBBx will be effective in many nonneoplastic disorders and a diagnosis is often the result of serendipity. For example, in Wegener granulomatosis, it is essential to find both the necrotizing granulomas and vasculitis; however, to find these changes in a small biopsy would be exceptional. Inflammatory changes in blood vessels adjacent to necrotizing granulomas, due to various infections, could be mistaken for a true vasculitic disorder.81 Therefore, extreme caution should be exercised in making a diagnosis of Wegener granulomatosis on TBBx. Various disorders characterized by pulmonary eosinophilia, such as acute, chronic, or drug-induced eosinophilic pneumonias, could be deduced by the presence of tissue eosinophilia. Some lymphoproliferative disorders, such as lymphoid interstitial pneumonia, Hodgkin lymphoma, or high-grade lymphoma, could be potentially diagnosed on TBBx, if other disorders are properly excluded. However, these instances represent exceptions rather than the rule. In small airways disorders, such as respiratory bronchiolitis, constrictive bronchiolitis, and cryptogenic organizing pneumonia, TBBx may not be able to sample sufficient tissue to render a certain diagnosis. Finally, in environmental,

drug-induced lung disease, or radiation injury, TBBx may show nonspecific histological features that overlap with other disorders.

Unreliable Transbronchial biopsy cannot be relied on to make an accurate diagnosis in the setting of diffuse interstitial lung disease, as the histological changes are seldom specific and may be misleading.1,8,70,74,82–84 There is an increased tendency to perform TBBx as a first step in evaluating patients with a suspected interstitial lung disease. In certain situations in which the CT findings are specific and the histological features suggest a fibrosing interstitial lung disorder, accurate classification is nevertheless impossible. Increasing numbers of patients present with atypical radiographic or clinical findings and this predicates the use of a more invasive biopsy procedure for accurate diagnosis. The limited sample size, the inability to evaluate various regions of the lung, and potential artifacts render the TBBx of little use for the diagnosis of many interstitial lung disorders (see Chapter 10).

Problems with the biopsy Issues of tissue

Histologically, the lung responds in a limited way to a variety of insults. In other words, the morphological changes are seldom specific and can be due to a variety of causes, irrespective of etiology. A pattern approach is frequently used, particularly for non-neoplastic lung diseases, and several of these major patterns form a useful framework for diagnosis. In the setting of many interstitial lung diseases, the pathological findings overlap with other disease processes and a small biopsy sample, such as TBBx, may not render sufficient tissue to look for specific histological findings.1,31,83 Another major problem with TBBx is that certain low-power architectural features, essential for establishing the diagnosis of particular interstitial lung diseases, cannot be accurately assessed in samples with a limited size.1,31,84 It is frequently challenging to separate specific from nonspecific histological changes in lung biopsies. In particular, the presence of a nonspecific feature in TBBx may or may not be relevant to diagnosis.85 For example, a chronic interstitial lymphoid infiltrate could be a “nonspecific” finding or represent features of lymphoproliferative disorder, chronic hypersensitivity pneumonia, cellular nonspecific interstitial pneumonia, or drug toxicity.1,28 In TBBx specimens, it is common to find alveolar hemorrhage, edema, fibrosis, or peribronchiolar inflammation. It is often impossible to determine whether any of these changes have any diagnostic significance. Controversy exists regarding the methods and indications for OLBx or VATS lung biopsy. The sample size and the number of sites biopsied by the surgeon varies considerably; some prefer to biopsy a single site, whereas others will sample several different areas. Ideally, the surgeon should procure

61

Chapter 2: Lung specimen handling and practical considerations

tissue from several different areas, showing active disease, normal-appearing lung (if present), and transitional areas.9 It may be difficult to determine whether a biopsy specimen is representative of an actual disease process. For example, if the surgeon only samples the lingula, the histopathological changes could reflect local histological alterations and not true diffuse disease (see above).8,82,84,86 In addition, a biopsy taken from an area of densely fibrotic or honeycomb lung is seldom informative, as this is the final common pathway of a large number of pulmonary disorders.83 When the pathologist encounters honeycombing fibrosis as the sole pathological finding, one should not use the term “end-stage lung”, unless there is radiographic evidence of extensive diffuse bilateral disease.

Problems in interpretation “Inconsistencies of opinion, arising from changes of circumstances, are often justifiable.” Daniel Webster (1782–1852). Histological evaluation and interpretation of lung biopsies are frequently problematic. This may be due to the pathologist’s level of experience and confidence, as well as the presence or absence of clinical, radiographic, and other pertinent information. While proper interpretation of the lung biopsy is

References 1. Travis WD, Colby T, Koss MN, et al. Handling and analysis of bronchoalveolar lavage and lung biopsy specimens with approach to patterns of lung injury. In Atlas of Non Tumor Pathology, Fascicle 2. American Registry of Pathology, 2002. 2. Andrews TD, Wallace WA. Diagnosis and staging of lung and pleural malignancy – an overview of tissue sampling techniques and the implications for pathological assessment. Clin Oncol (R Coll Radiol) 2009;21(6):451–63. 3. Katzenstein AL. Chapter 1. Handling and interpretation of lung biopsies. In Katzenstein’s and Askin’s Surgical Pathology of Non-Neoplastic Lung Disease, 4th ed. Philadelphia: W.B. Saunders, 2006. 4. Kendall DM, Gal AA. Interpretation of tissue artifacts in transbronchial lung biopsy specimens. Ann Diagn Pathol 2003;7(1):20–4. 5. Nagata N, Hirano H, Takayama K, Miyagawa Y, Shigematsu N. Step section preparation of transbronchial lung biopsy. Significance in the diagnosis of diffuse lung disease. Chest 1991;100(4):959–62. 6. Wall CP, Gaensler EA, Carrington CB, Hayes JA. Comparison of transbronchial

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critical for appropriate patient management, variance of opinion commonly occurs in the interpretation of lung biopsies. Most lung tumors can be readily classified as either “small cell carcinoma” or “non-small cell carcinoma” (see Chapter 27). In smaller biopsies, such as TBBx or needle biopsies, further classification into subgroups of non-small cell lung cancer (i.e., squamous cell carcinoma, adenocarcinoma, or large cell carcinoma) may not always be possible.87–90 The pulmonary neuroendocrine tumors are especially noted for difficulties in classification, in both TBBx and even larger specimens.91–93 There is significant interobserver variability in the interpretation of lung biopsies from diffuse interstitial lung disorders.31,94–96 Many general surgical pathologists, as well as experienced lung pathologists, have difficulties in classification and diagnosis. While there is very good agreement for the diagnosis of sarcoidosis, lesser degrees of certainty are present for cryptogenic organizing pneumonia or diffuse alveolar damage, and poor concordance in diagnosis between usual interstitial pneumonia and nonspecific interstitial pneumonia is reported.97 A dynamic multidisciplinary approach by the addition of clinical and radiographic information is critical to assigning a proper diagnosis and classification of idiopathic interstitial pneumonias.

and open biopsies in chronic infiltrative lung diseases. Am Rev Respir Dis 1981;123(3):280–5. 7. Kadokura M, Colby TV, Myers JL, et al. Pathologic comparison of video-assisted thoracic surgical lung biopsy with traditional open lung biopsy. J Thorac Cardiovasc Surg 1995;109(3):494–8.

13. Corrin B, Nicholson AG, eds. Pathology of the Lungs, 2nd ed. Churchill Livingstone Elsevier, 2006. 14. Wang IM, Stepaniants S, Boie Y, et al. Gene expression profiling in patients with chronic obstructive pulmonary disease and lung cancer. Am J Respir Crit Care Med 2008;177(4):402–11. 15. Whimster WF. Techniques in pulmonary pathology. In Hasleton PS, ed. Spencer’s Pathology of the Lung, 5th ed. New York: McGraw-Hill, 1996.

8. Gaensler EA, Carrington CB. Open biopsy for chronic diffuse infiltrative lung disease: clinical, roentgenographic, and physiological correlations in 502 patients. Ann Thorac Surg 1980;30(5):411–26.

16. Mountain CF. Revisions in the International System for Staging Lung Cancer. Chest 1997;111(6):1710–7.

9. Halkos ME, Gal AA, Kerendi F, Miller DL, Miller JI, Jr. Role of thoracic surgeons in the diagnosis of idiopathic interstitial lung disease. Ann Thorac Surg 2005;79(6):2172–9.

17. Goldstraw P, ed. IASLC International Staging Committee. Chapter 25. Lung. In Edge SB, Byrd D, Compton CC, et al. eds. AJCC Cancer Staging Manual. New York: Springer, 2010.

10. Churg A. An inflation procedure for open lung biopsies. Am J Surg Pathol 1983;7(1):69–71. 11. Gianoulis M, Chan N, Wright JL. Inflation of lung biopsies for frozen section. Mod Pathol 1988;1(5):357–8. 12. van Kuppevelt TH, Robbesom AA, Versteeg EM, et al. Restoration by vacuum inflation of original alveolar dimensions in small human lung specimens. Eur Respir J 2000;15(4): 771–7.

18. Goldstraw P, Crowley J, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol 2007;2(8): 706–14. 19. Ou SH, Zell JA, Ziogas A, AntonCulver H. Prognostic significance of the non-size-based AJCC T2 descriptors: visceral pleura invasion, hilar

Chapter 2: Lung specimen handling and practical considerations

atelectasis, or obstructive pneumonitis in stage IB non-small cell lung cancer is dependent on tumor size. Chest 2008;133(3):662–9. 20. Milroy R. Staging of lung cancer. Chest 2008;133(3):593–5. 21. Travis WD. Reporting lung cancer pathology specimens. Impact of the anticipated 7th Edition TNM classification based on recommendations of the IASLC Staging Committee. Histopathology 2009;54(1):3–11. 22. Hsu PK, Huang HC, Hsieh CC, et al. Effect of formalin fixation on tumor size determination in stage I non-small cell lung cancer. Ann Thorac Surg 2007;84(6):1825–9. 23. Travis WD, Brambilla E, Rami-Porta R, et al. Visceral pleural invasion: pathologic criteria and use of elastic stains: proposal for the 7th edition of the TNM classification for lung cancer. J Thorac Oncol 2008;3(12):1384–90. 24. Husain AN, Colby TV, Ordonez NG, et al. Guidelines for pathologic diagnosis of malignant mesothelioma: a consensus statement from the International Mesothelioma Interest Group. Arch Pathol Lab Med 2009; 133(8):1317–31. 25. Ordonez NG. The diagnostic utility of immunohistochemistry in distinguishing between epithelioid mesotheliomas and squamous carcinomas of the lung: a comparative study. Mod Pathol 2006;19(3):417–28. 26. Gupta R, Dastane AM, Forozan F, et al. Evaluation of EGFR abnormalities in patients with pulmonary adenocarcinoma: the need to test neoplasms with more than one method. Mod Pathol 2009;22(1):128–33. 27. Schmitt FC, Longatto-Filho A, Valent A, Vielh P. Molecular techniques in cytopathology practice. J Clin Pathol 2008;61(3):258–67. 28. Katzenstein AL. Chapter 17. Transbronchial biopsy. In Katzenstein’s and Askin’s Surgical Pathology of Non-Neoplastic Lung Disease, 4th ed. Philadelphia: W.B. Saunders, 2006. 29. Colby TV, Yousem SA. Pulmonary histology for the surgical pathologist. Am J Surg Pathol 1988;12(3):223–39. 30. Gal AA. Use and abuse of lung biopsy. Adv Anat Pathol 2005; 12(4):195–202.

31. Nicholson SA, Beasley MB, Brambilla E, et al. Small cell lung carcinoma (SCLC): a clinicopathologic study of 100 cases with surgical specimens. Am J Surg Pathol 2002;26(9):1184–97. 32. Davenport RD. Diagnostic value of crush artifact in cytologic specimens. Occurrence in small cell carcinoma of the lung. Acta Cytol 1990;34(4):502–4. 33. Yousem SA, Dauber JH, Griffith BP. Bronchial cartilage alterations in lung transplantation. Chest 1990; 98(5):1121–4. 34. Matsuba K, Takizawa T, Thurlbeck WM. Oncocytes in human bronchial mucous glands. Thorax 1972; 27(2):181–4. 35. Mauad T, Xavier AC, Saldiva PH, Dolhnikoff M. Elastosis and fragmentation of fibers of the elastic system in fatal asthma. Am J Respir Crit Care Med 1999;160(3):968–75. 36. Yousem SA. Pulmonary apical cap: a distinctive but poorly recognized lesion in pulmonary surgical pathology. Am J Surg Pathol 2001;25(5):679–83. 37. Ovenfors CO, Dahlgren SE, Ripe E, Ost A. Muscular hyperplasia of the lung: a clinical, radiographic, and histopathologic study. AJR Am J Roentgenol 1980;135(4):703–12. 38. Ma Y, Gal A, Koss MN. The pathology of pulmonary sarcoidosis: update. Semin Diagn Pathol 2007;24(3):150–61. 39. Kirkpatrick CJ, Curry A, Bisset DL. Light- and electron-microscopic studies on multinucleated giant cells in sarcoid granuloma: new aspects of asteroid and Schaumann bodies. Ultrastruct Pathol 1988;12(6):581–97. 40. Reid JD, Andersen ME. Calcium oxalate in sarcoid granulomas. With particular reference to the small ovoid body and a note on the finding of dolomite. Am J Clin Pathol 1988;90(5):545–58. 41. Visscher D, Churg A, Katzenstein AL. Significance of crystalline inclusions in lung granulomas. Mod Pathol 1988; 1(6):415–9. 42. Gal AA, Koss MN. The pathology of sarcoidosis. Curr Opin Pulm Med 2002;8(5):445–51. 43. Kuhn C, 3rd, Kuo TT. Cytoplasmic hyalin in asbestosis. A reaction of injured alveolar epithelium. Arch Pathol 1973;95(3):190–4. 44. Warnock ML, Press M, Churg A. Further observations on cytoplasmic

hyaline in the lung. Hum Pathol 1980;11(1):59–65. 45. Hollander DH, Hutchins GM. Central spherules in pulmonary corpora amylacea. Arch Pathol Lab Med 1978;102(12):629–30. 46. Dobashi M, Yuda F, Narabayashi M, et al. Histopathological study of corpora amylacea pulmonum. Histol Histopathol 1989;4(2):153–65. 47. Yamanouchi H, Yoshinouchi T, Watanabe R, Fujita J, Takahara J, Ohtsuki Y. Immunohistochemical study of a patient with diffuse pulmonary corpora amylacea detected by open lung biopsy. Intern Med 1999;38(11):900–3. 48. Koss MN, Johnson FB, Hochholzer L. Pulmonary blue bodies. Hum Pathol 1981;12(3):258–66. 49. Gardiner IT, Uff JS. “Blue bodies” in a case of cryptogenic fibrosing alveolitis (desquamative type) an ultra-structural study. Thorax 1978; 33(6):806–13. 50. Kung IT, Hsu C, Chan SC, Leung BS, Ng DW. Frequency of “blue bodies” in pulmonary cytology specimens. Diagn Cytopathol 1987;3(4):284–6. 51. Kunze WP. Senile pulmonary amyloidosis. Pathol Res Pract 1979;164(4):413–22. 52. Soares FA. Increased numbers of pulmonary megakaryocytes in patients with arterial pulmonary tumour embolism and with lung metastases seen at necropsy. J Clin Pathol 1992; 45(2):140–2. 53. Sharma GK, Talbot IC. Pulmonary megakaryocytes: “missing link” between cardiovascular and respiratory disease? J Clin Pathol 1986;39(9):969–76. 54. Mandal RV, Mark EJ, Kradin RL. Megakaryocytes and platelet homeostasis in diffuse alveolar damage. Exp Mol Pathol 2007; 83(3):327–31. 55. Hasleton PS. Adult respiratory distress syndrome – a review. Histopathology 1983;7(3):307–32. 56. Pai U, McMahon J, Tomashefski JF, Jr. Mineralizing pulmonary elastosis in chronic cardiac failure. “Endogenous pneumoconiosis” revisited. Am J Clin Pathol 1994;101(1):22–8. 57. Lendrum AC. Pulmonary haemosiderosis of cardiac origin. J Pathol Bacteriol 1950;62(4):555–61.

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58. Tomashefski JF, Jr. Pulmonary pathology of acute respiratory distress syndrome. Clin Chest Med 2000; 21(3):435–66. 59. Pinckard JK, Wick MR. Tumor-related thrombotic pulmonary microangiopathy: review of pathologic findings and pathophysiologic mechanisms. Ann Diagn Pathol 2000; 4(3):154–7. 60. Hashimoto Y, Moriya F, Furumiya J. Forensic aspects of complications resulting from cardiopulmonary resuscitation. Leg Med (Tokyo) 2007;9:94–9.

71. Jones AM, Hanson IM, Armstrong GR, O’Driscoll BR. Value and accuracy of cytology in addition to histology in the diagnosis of lung cancer at flexible bronchoscopy. Respir Med 2001; 95(5):374–8. 72. Danel C. [Usefulness of transbronchial and surgical biopsies for the management of interstitial lung disease.] Rev Pneumol Clin 2005; 61(3):149–57.

lung biopsy indicated in every patient? Am J Respir Crit Care Med 1995; 151(3 Pt 1):909–14. 83. Gal AA, Staton GW, Jr. Current concepts in the classification of interstitial lung disease. Am J Clin Pathol 2005;123 Suppl:S67–81. 84. Dacic S, Yousem SA. Idiopathic pulmonary fibrosis: histologic classification of idiopathic chronic interstitial pneumonias. Am J Resp Cell Mol Biol 2003;29:S5–S9.

73. Shure D. Transbronchial biopsy and needle aspiration. Chest 1989; 95(5):1130–8.

85. Wilson RK, Fechner RE, Greenberg SD, Estrada R, Stevens PM. Clinical implications of a “nonspecific” transbronchial biopsy. Am J Med 1978;65(2):252–6.

62. Graham JK, Mosunjac M, Hanzlick RL, Mosunjac M. Sickle cell lung disease and sudden death: a retrospective/ prospective study of 21 autopsy cases and literature review. Am J Forensic Med Pathol 2007;28(2):168–72.

74. Miller RR, Evans KG. Lung biopsy. In Thurlbeck WM, Churg AM, eds. Pathology of the Lung, 2nd ed. New York: Thieme, 1995.

86. Newman SL, Michel RP, Wang NS. Lingular lung biopsy: is it representative? Am Rev Respir Dis 1985;132(5):1084–6.

75. Annema JT, Rabe KF. State of the art lecture: EUS and EBUS in pulmonary medicine. Endoscopy 2006;38 Suppl 1: S118–22.

63. Husebye EE, Lyberg T, Roise O. Bone marrow fat in the circulation: clinical entities and pathophysiological mechanisms. Injury 2006;37 Suppl IV:S8–18.

76. Colt HG, Murgu SD. Interventional bronchoscopy from bench to bedside: new techniques for early lung cancer detection. Clin Chest Med 2010;31(1):29–37.

87. Burnett RA, Howatson SR, Lang S, et al. Observer variability in histopathological reporting of non-small cell lung carcinoma on bronchial biopsy specimens. J Clin Pathol 1996;49(2):130–3.

64. Sieracki JC, Fisher ER. The ceroid nature of the so-called “HamazakiWesenberg bodies”. Am J Clin Pathol 1973;59(2):248–53.

77. Rano A, Agusti C, Jimenez P, et al. Pulmonary infiltrates in non-HIV immunocompromised patients: a diagnostic approach using non-invasive and bronchoscopic procedures. Thorax 2001;56(5):379–87.

61. Dzieciol J, Kemona A, Gorska M, et al. Widespread myocardial and pulmonary bone marrow embolism following cardiac massage. Forensic Sci Int 1992;56(2):195–9.

65. Ro JY, Luna MA, Mackay B, Ramos O. Yellow-brown (Hamazaki-Wesenberg) bodies mimicking fungal yeasts. Arch Pathol Lab Med 1987;111(6):555–9. 66. Senba M, Kawai K. Nature of yellow-brown bodies. Histochemical and ultrastructural studies on the brown pigment. Zentralbl Allg Pathol 1989;135:351–5. 67. Bejarano PA, Garcia MT, Ganjei-Azar P. Mesothelial cells in transbronchial biopsies: a rare complication with a potential for a diagnostic pitfall. Am J Surg Pathol 2007;31(6):914–8. 68. Chan JK, Loo KT, Yau BK, Lam SY. Nodular histiocytic/mesothelial hyperplasia: a lesion potentially mistaken for a neoplasm in transbronchial biopsy. Am J Surg Pathol 1997;21(6):658–63. 69. Gibbs AR, Seal RM. ACP Broadsheet 123: January 1990. Examination of lung specimens. J Clin Pathol 1990; 43(1):68–72.

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70. Churg A. Transbronchial biopsy: nothing to fear. Am J Surg Pathol 2001;25(6):820–2.

78. Narayanswami G, Salzman SH. Bronchoscopy in the human immunodeficiency virus-infected patient. Semin Respir Infect 2003; 18(2):80–6. 79. Glanville AR. The role of bronchoscopic surveillance monitoring in the care of lung transplant recipients. Semin Respir Crit Care Med 2006; 27(5):480–91. 80. Stewart S, Fishbein MC, Snell GI, et al. Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant 2007;26(12):1229–42.

88. Chuang MT, Marchevsky A, Teirstein AS, Kirschner PA, Kleinerman J. Diagnosis of lung cancer by fibreoptic bronchoscopy: problems in the histological classification of non-small cell carcinomas. Thorax 1984; 39(3):175–8. 89. Cataluna JJ, Perpina M, Greses JV, et al. Cell type accuracy of bronchial biopsy specimens in primary lung cancer. Chest 1996;109(5):1199–203. 90. Thomas JS, Lamb D, Ashcroft T, et al. How reliable is the diagnosis of lung cancer using small biopsy specimens? Report of a UKCCCR Lung Cancer Working Party. Thorax 1993; 48(11):1135–9. 91. Pelosi G, Rodriguez J, Viale G, Rosai J. Typical and atypical pulmonary carcinoid tumor overdiagnosed as small-cell carcinoma on biopsy specimens: a major pitfall in the management of lung cancer patients. Am J Surg Pathol 2005;29(2):179–87.

81. Ulbright TM, Katzenstein AL. Solitary necrotizing granulomas of the lung: differentiating features and etiology. Am J Surg Pathol 1980;4(1):13–28.

92. Marchevsky AM, Chuang MT, Teirstein AS, Nieburgs HE, Kleinerman J. Problems in the diagnosis of small cell carcinoma of the lungs by fiberoptic bronchoscopy. Cancer Detect Prev 1984;7(4):253–60.

82. Raghu G. Interstitial lung disease: a diagnostic approach. Are CT scan and

93. Travis WD, Gal AA, Colby TV, et al. Reproducibility of neuroendocrine lung

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tumor classification. Hum Pathol 1998;29(3):272–9. 94. Bouros D, Wells AU, Nicholson AG, et al. Histopathologic subsets of fibrosing alveolitis in patients with systemic sclerosis and their relationship to outcome. Am J Respir Crit Care Med 2002;165(12):1581–6.

95. Hunninghake GW, Zimmerman MB, Schwartz DA, et al. Utility of a lung biopsy for the diagnosis of idiopathic pulmonary fibrosis. Am J Respir Crit Care Med 2001;164(2):193–6. 96. Katzenstein AL, Zisman DA, Litzky LA, Nguyen BT, Kotloff RM. Usual interstitial pneumonia: histologic

study of biopsy and explant specimens. Am J Surg Pathol 2002; 26(12):1567–77. 97. Nicholson AG, Addis BJ, Bharucha H, et al. Inter-observer variation between pathologists in diffuse parenchymal lung disease. Thorax 2004;59(6):500–5.

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Chapter

3

Congenital abnormalities and pediatric lung diseases, including neoplasms Stephen J. Gould, A.K. Webb and Anna Kelsey

Introduction This chapter will discuss lung disorders that present throughout the childhood years, including infancy and the perinatal period. Some structural abnormalities are now detected earlier with recent advances in prenatal ultrasound. Areas considered include congenital malformations and disease processes associated with the transition from an intrauterine to an extrauterine existence, particularly when this occurs preterm. Cystic fibrosis (CF) and other inheritable conditions that typically present first in the pediatric age range will also will be covered. The pediatric aspects of conditions such as infection and tumors are also included but entities typically encountered in adults that rarely affect children will only be cross-referenced.

Congenital malformations Congenital malformations are morphological defects in part of an organ, a complete organ or a larger region of the body, due to an intrinsically abnormal developmental process.1 A morphological defect of the same structures due to the extrinsic breakdown of, or interference with, an originally normal developmental process is termed a disruption. These may be difficult to distinguish, especially if the disruption has occurred early in gestation. Some malformations are presumed multifactorial, due to interactions between genetic factors and environmental agents; others are purely environmental. It may be difficult to be sure where, in this etiological spectrum, some malformations should be placed. To understand defects in lung development it is essential to remember the embryology outlined in Chapter 1. Bronchopulmonary malformations are uncommon. The spectrum and frequency with which any abnormality is encountered varies depending on the nature of the clinical practice. Routine prenatal diagnostic scanning recognizes many silent anomalies. Their existence is detected on scan, usually midtrimester, and they subsequently disappear or at least are asymptomatic.2,3 There is debate as to the management of these “silent” lesions;4 some authorities believe they should be removed only if symptomatic. Others recommend intervention even if they remain clinically silent. Consequently, it is

extremely difficult to determine the incidence of many abnormalities with any degree of precision. However, in the Oxford region of Great Britain between 2000 and 2007, the rate of thoracic anomalies (excluding cardiac), including those lesions diagnosed by prenatal diagnostic ultrasound, was 0.5/1000 live births.5

Trachea The normal trachea has up to 22 transversely arranged C-shaped cartilagenous rings, which open posteriorly. They extend from the lower border of the larynx to the carina. There may be a significant number of incomplete or Y-shaped rings in “normal” tracheas. Demonstrating minor defects in the cartilaginous skeleton of the trachea may require the use of clearing techniques.6,7

Abnormality of tracheal length A short trachea may result from a reduction in the number of tracheal cartilages, usually from a mean of 17 to less than 15. This finding is commonly associated with a congenitally short neck. The Klippel-Feil syndrome, short neck associated with a low occipital hairline, decreased neck mobility, and often cervical vertebral fusion, is the most common cause.8 Other conditions include a wide spectrum of skeletal dysplasias, hypoplastic left heart syndrome, some chromosomal disorders, and neural tube defects.9 The main risk from the short trachea is from intubation of a single bronchus10 but late presentation with wheezing has been reported.11 Radiologically, a short trachea may be suspected and diagnosed from the position of the carina in relation to the vertebral column (normally T4–T5). Too many tracheal rings may be a component of some variants of tracheal stenosis (see below).

Tracheal agenesis Tracheal agenesis is a very rare malformation with approximately only 150 cases reported since its first description in 1900.12 Its incidence has been estimated as 1:50 000 births, with a slight male preponderance.13

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 1 Agenesis of the trachea

Table 2 Classification of tracheal stenosis

Type

Abnormality

Proportion

I

A

Total pulmonary agenesis

B

Main bronchi arise from esophagus

10%

Intrinsic A. Diffuse generalized hypoplasia (30%) 1. Diffuse posterior cartilage ring fusion 2. Solid cartilaginous sleeve

C

Fused main bronchi with tracheoesophageal fistula

56%

B. Funnel-like tapering stenosis (20%)

D

Tracheoesophageal fistula with atretic strand between larynx and distal trachea

10%

E

Agenesis of the upper trachea with direct tracheoesophageal communication

5%

F

Blind bronchial bifurcation with no esophageal communication

5%

G

Short segment tracheal agenesis

5%

8%

C. Segmental stenosis (50%) II

Extrinsic

Figure 1. Tracheal agenesis with communication between fused bronchi and esophagus. (Image courtesy of Dr Phil Cox, Birmingham, UK.)

Modified from.16

Tracheal agenesis is rarely diagnosed prenatally. There may be maternal polyhydramnios with an ultrasound scan detecting features suggestive of a high airway obstruction, including fetal hydrops, ascites, lung hyperechogenicity, and a flattened diaphragm. Laryngeal atresia is a more common specific cause of this presentation but a specific diagnosis of tracheal agenesis has been reported by magnetic resonance imaging (MRI) scan.14 More commonly, presentation is at birth with severe respiratory distress, cyanosis, poor lung expansion or sometimes no respiratory effort and bradycardia. Bag and mask ventilation may be helpful if there is an esophageal or broncho-tracheal fistula. Intubation is impossible, at which point an upper airway obstruction is usually identified. Tracheal agenesis has been the subject of two classification systems. The first and simpler by Floyd et al.15 recognizes three main types whereas that of Faro et al.16 recognizes seven (Table 1). The most critical factor in the success of initial ventilation relies on the presence of a fistula between the lower respiratory tract and the esophagus (Figure 1). Communication between fused bronchi and the esophagus is the commonest fistula. The embryology of this region at the critical time, after 22 days post-ovulation, is unclear and so the cause of the malformation is uncertain. Traditionally, agenesis was explained by failure of the tracheo-esophageal septum to form properly. More recent evidence suggests the lower respiratory tract starts as an outpouching of the pharynx. As each foregut tube and the trachea develop, the intervening mesenchyme forms the septum. Normal bronchial branching does not occur until the elongated tracheal tube grows into the appropriate bronchial mesenchyme. Delayed budding of the respiratory tract from the primitive foregut may lead to the lung bud growing from the esophagus directly into bronchial mesenchyme. This probably stimulates immediate formation of the lung lobes, without tracheal development.17 Tracheal agenesis rarely occurs in isolation; associated malformations are identified in 94% of cases.17 Patterns of abnormalities overlap with those seen in association with tracheo-esophageal fistula such as the VACTERL spectrum

(a non-random association of Vertebral anomaly, Anal atresia, Cardiac, Tracheo-Esophageal fistula with atresia, Renal anomalies, and Limb abnormality (often radial)). Not surprisingly, tracheal agenesis cases also show other laryngeal anomalies and lung lobation defects. Associated malformations include complex congenital cardiac abnormalities, isolated ventricular septal defects, vertebral anomalies and more caudal defects, including renal dysplasia and anal atresia. Tracheal agenesis appears to be a sporadic defect with a low recurrence risk in future births. Tracheal agenesis is almost uniformly lethal, usually at birth or in the post-natal period. Immediate survival depends on whether adequate ventilation can be established via an esophageal fistula. Reconstructive surgery has generally met with very limited success,12 with infants, at best, living for a few weeks after surgery. There have been occasional reports of long-term survival.18,19

Tracheal stenosis Tracheal stenosis can be due to an intrinsic abnormality of the trachea or an extrinsic abnormality, causing a deformation. The latter is more common. Intrinsic stenosis is rare, with a rate estimated by one Canadian group as 1:64 500 live births. Although a simplification of the various patterns that can occur, Cantrell and Guild20 classified tracheal stenosis on a morphological basis (Table 2). Anton-Pacheco21 suggests a

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

(b)

Figure 2. (a) Posterior view of trachea with diffuse tracheal stenosis although the presence of a reduced tracheal lumen is not obvious from this view. No pars membranacea is visible and stenosis is associated with complete tracheal rings. (b) In transverse section the trachea has a greater resemblance to “funnel-shaped” diffuse tracheal stenosis. The mucosa of the upper trachea is severely ulcerated from attempted intubation. A complete ring is clearly visible in the upper right hand corner and other blocks show cartilage posteriorly. The lumen is reduced to 2 mm diameter at its narrowest. All blocks are oriented in the same direction (arrow points anteriorly).

clinical approach. The clinical presentation is variable. Severe stenosis presents in the neonatal period with stridor or respiratory distress.22 In the older infant, recurrent pneumonia may be more prominent, while activity-induced wheezing, dyspnea and intercostal retraction may be observed.23 Milder forms of stenosis may be all but asymptomatic or have wheezing and features which may mimic asthma in late childhood or adolescence.

Intrinsic tracheal stenosis Intrinsic stenosis is subdivided into three broad categories, namely diffuse, funnel-shaped, and segmental. Diffuse stenosis Diffuse tracheal stenosis has two slightly differing appearances. If there is a diffuse abnormality, the trachea is narrowed for most or its entire length (Figure 2). The luminal diameter in affected newborns is often as small as 1–3 mm. The abnormality commences immediately below the cricoid and is limited to the trachea. The major bronchi are not involved. The major underlying pathology is posterior fusion of the cartilage ring.20 Abnormalities associated with diffuse stenosis include Fallot’s tetralogy, agenesis of one lung and diaphragmatic hernia.24 In the second variant of diffuse tracheal stenosis, the trachea is typically described as a solid or complete cartilaginous sleeve, although Davis et al.25 show this is a slight oversimplification. Rather than a solid tube, they argue that a rudimentary pars membranacea may be present posteriorly and the trachea may also show other abnormalities and

68

deformity. This abnormality can involve the bronchial tree and include an unusually long right main bronchus or a short left main bronchus, together with pulmonary “pseudoisomerism” (see below).8 It is not clear whether the functional deficits in ventilation associated with this abnormality result from the tracheal stenosis or are due to the other tracheal features, such as an abnormal length, rigidity, and/or angulation. Almost invariably, the diffuse cartilaginous sleeve variant of tracheal stenosis is reported in children with craniosynostosis syndromes, such as Crouzon disease, Apert and Pfeiffer syndromes26,27 in which abnormalities in fibroblast growth factor receptor genes are a feature.28 These associations suggest this diffuse form of tracheal stenosis, like the other mesenchymal defects present in the syndromes, represents a fundamental mesenchymal defect in which normally discrete structures fuse.25 Funnel-shaped tracheal stenosis In this subtype, the trachea immediately below the larynx is normal but there is a gradual reduction in luminal diameter to a minimum directly above the carina.20 Again, the main tracheal ring abnormality is a reduction in, or absence of, the pars membranacea. Absence leads to complete fusion of the tracheal cartilages. The lower trachea is typically stenotic with a complete tracheal ring.8,29,30 Although it might be thought the stenosis is due to extrinsic compression, there appears to be a primary abnormality of the trachea. This particular anomaly has been studied and subclassified in more detail by Wells et al.31 Funnel-shaped

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

stenosis is rarely present in the absence of anomalies elsewhere; unilateral pulmonary agenesis is a particularly well-described association.24,32 Funnel-shaped tracheal stenosis is also often associated with a “sling” left pulmonary artery, in which there is an anomalous origin of the left pulmonary artery from the posterior aspect of the right pulmonary artery. The left pulmonary artery then reaches the hilum of the left lung by passing over the right main bronchus and between the trachea and the esophagus. Sling left pulmonary artery may be associated with bronchial “pseudoisomerism”. In this condition there is an abnormally short left main bronchus and long right main bronchus, so they are of nearly equal length.33 Unlike true isomerisms, where the further bronchial subdivisions are symmetric, in “pseudoisomerism” bronchial subdivisions are within the normal range. Segmental stenosis This is the commonest form of tracheal stenosis, often comprising a narrowing of some three to five cartilage rings in the lower third of the trachea.23 Landing and Dixon8 described individual complete cartilaginous rings, the so-called “Napkin-ring” cartilages, which were evenly distributed in the sub-cricoid, mid-trachea and supra-tracheal areas. Mid-tracheal stenosis was described by Wells et al.34 in association with Down syndrome. In these cases the ring cartilages may even be arranged as helical segments. Stenosis is not always a pure cartilage-related anomaly and has been described secondary to muscular hypertrophy.35 Le Bret et al.36 described a localized stenosis, in which soft tissue was a significant component of the anomaly together with disrupted incomplete cartilage rings. Associations with a wide variety of other extra-bronchopulmonary abnormalities are described, including tracheo-esophageal fistulae and vascular slings.

Extrinsic stenosis In neonates and infants, the most common cause of extrinsic tracheal compression is an abnormally located or unusually large blood vessel. The clinical presentation is similar to intrinsic stenosis and includes stridor, recurrent chest infections, cough, respiratory distress during feeding, episodic respiratory distress, and cyanosis. Landing and Dixon8 provide the relative frequencies of these anomalies (Table 3). As noted above, there is a relationship between the severity of symptoms and the degree of stenosis – the earlier and more severe the symptoms, the longer and more critical the stenosis. A similar relationship exists with prognosis. Operative interventions include resection and anastomosis, cartilage patches and slide tracheoplasty,37 the latter being used more frequently for the longer segment stenoses. Herrera and colleagues23 reviewed the outcome of surgery from a number of studies. After slide tracheoplasty, the caliber of the airway is often doubled, but it is still smaller than the rest of the trachea and the usual diameter for an age-matched tracheal diameter.

Table 3 Vascular anomalies associated with extrinsic tracheal stenosis

Anomaly Vascular ring:

Frequency Double aortic arch Right aortic arch with left ligamentum arteriosum

47% 20%

Anomalous innominate artery (arising further left)

11%

Anomalous left carotid artery (arising further right)

4%

Sling (retrotracheal) left pulmonary artery

3%

Right aortic arch with aberrant left subclavian artery

1%

Modified from.8

Tracheal growth continues, albeit slowly. The airway may remain noisy because of the irregularity of the airway and further intervention may be needed for granulation tissue accumulation.23 Age and associated abnormalities affect mortality. Stenosis requiring surgery in the presence of an associated cardiac anomaly in infants less than 1 month of age carries a particularly high risk. Mortality can approach 100%. The risk reduces with age, to approximately 70% after the neonatal period and to less than 20% by the age of 2 years in cases where no cardiac anomaly is present. There is also a role for conservative non-operative management in the presence of minimal symptoms.38

Tracheomalacia Tracheomalacia implies an inadequacy or softness of the tracheal cartilages and is one of the commoner tracheal abnormalities.39 The incidence is difficult to estimate due to variations in definition and techniques used in diagnosis. Incidences range between 1:1445 infants40 and 1:2600.41 Males appear to be more commonly affected than females.42 Tracheomalacia may be isolated but can coexist with malacia of the bronchus, more commonly the left bronchus, or the larynx. Presentation may be in the neonatal period but is often slightly later, in early infancy. Expiratory stridor with a cough, sometimes described as barking, is the most common clinical manifestation. Inspiratory stridor may occur if the proximal extra-thoracic trachea is involved.43 Computed tomograms (CT) and MRI may be helpful initial diagnostic investigations but many believe bronchoscopy remains the more sensitive diagnostic tool. Tracheomalacia can be divided into primary and secondary forms and further subdivided into segmental or diffuse. It is also diagnosed in normal infants, especially in the context of prematurity. There are many associations, including mucopolysaccharidoses, chromosomal defects, such as trisomies 21 and 9, and some skeletal dysplasias.43 It could be argued that true “malacia” only occurs in association with some of the skeletal dysplasia syndromes.6 An abnormality of tracheal form and cartilaginous deficiency is also well documented in association with tracheo-esophageal fistulae.44 While this

69

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

intervention may be indicated, especially in the presence of life-threatening events. Tracheostomy or, more rarely, resection and/or reconstruction are established interventions.43 Deaths directly attributable to tracheomalacia are uncommon. Many infants outgrow their symptoms because of gradually increasing tracheal diameters. There is some evidence that, rarely, new areas of malacia develop.52

Tracheoesophageal fistula

Figure 3. Tracheomalacia. This trachea, from a 24 week gestation baby with other anomalies, is flat with soft, floppy cartilage. The “inward” collapse posteriorly of the pars membranacea is visible.

might be considered secondary, some have argued this represents a primary cartilaginous defect.43 Acquired or secondary tracheomalacia may occur when there is damage to the cartilage from traumatic intubation or as a complication of mechanical ventilation.45 Collapse of a structurally normal trachea may be associated with excessive expiratory effort, as in asthma. Primary tracheomalacia should only be considered after external compression and localized intratracheal airway obstruction are excluded.8 External compression/ secondary tracheomalacia is often associated with congenital cardiac abnormalities in as many as 58% of cases.24 There may be few overt macroscopic abnormalities in tracheomalacia apart from a visible increase in the pars membranacea and, if severe, a flattened or mis-shapened trachea (Figure 3). Spontaneous improvement in mild variants over an 18–24 month period suggests the abnormally thin or floppy cartilage may represent a form of “tracheal immaturity”.46 This theory probably applies to the tracheas of preterm infants. In most cases, tracheal samples demonstrate a wider breadth of changes. There is a reduction in the ratio of cartilage to soft tissue and the amount of longitudinal muscle, which normally lies inside transversely oriented fibers posteriorly, is reduced.47,48 The ratio of cartilage to soft tissue should be at least 4.5:1, a ratio that remains fairly constant throughout childhood.49,50 In the lower trachea of a normal infant, however, at the trigone, the posterior membrane is relatively large and the tracheal profile may be “flattened” in the antero-posterior diameter.51 This feature is not present in the adult. The significance of this profile in recurrent respiratory symptoms is uncertain. Prognosis is generally good for most cases of tracheomalacia and many infants, especially when symptoms are very mild, can be treated conservatively. Stenting may be affected by continuous positive airways pressure (CPAP). Surgical

70

Tracheoesophageal fistula (T-O fistula) with esophageal atresia is one of the more commonly encountered thoracic abnormalities. Incidences range from 1:800 to 1:5000 births.53,54 It may cause maternal polyhydramnios or, in the neonate, excessive secretions or choking, usually in association with feeding. With a prenatal ultrasound, absence of the gastric bubble associated with polyhydramnios may suggest tracheo-esophageal fistula. From 10% to 30% of cases are detected in this manner.55,56 Both these ultrasound signs, however, may be associated with a wide range of other diagnoses.55 Tracheoesophageal fistula is a sporadic malformation with a very high rate of associated malformations. Up to 70% of affected low-birth-weight infants have additional malformations.57 The main association of T-O fistula is seen in the VACTERL syndrome (see above). The embryological basis of T-O fistula and esophageal atresia is uncertain but it is intimately associated with the failure of normal division of the primitive foregut into an anterior respiratory component and a dorsal intestinal component. Broadly, there are two postulated patterns of normal embryogenesis.58 The first is that an anterior bud from the primitive foregut rapidly grows caudally and divides into the lung buds (“tap and water theory”). The second postulates that primitive foregut, once formed, is split into two components, either by a septum which grows rostrally between the ventral and dorsal aspects of the primitive tube, or, alternatively, there is inward collapse of the lateral walls of the primitive foregut tube, which fuse to form a ventral and dorsal tube.58 How abnormalities of either of the above processes account fully for the commonly found malformations is unclear. There is good evidence that disturbance of the normal patterning genes intimately associated with the normal development of this region, such as sonic hedgehog, is involved. Interestingly, while both theories can help to explain the fistulae, at present, neither accounts well for the usual association of esophageal atresia, which may be a subsequent event.58 Tracheoesophageal fistulas are classified into five main subtypes (Figure 4a). A short atretic esophagus, with the lower esophagus arising from the lower trachea, is the commonest variant,59 accounting for over 80% of cases (Figure 4b). Esophageal atresia with no fistula, tracheo-esophageal fistula with no atresia, esophageal atresia with a fistula between a proximal atretic esophagus and trachea and esophageal atresia with tracheal fistulae between both proximal and distal segments of the esophagus consititute the less common subtypes.

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

86%

(b)

8%

4.2%

1.4%

0.8%

Figure 4. (a) Diagram illustrating the main subtypes of tracheoesophageal fistula. (b) Tracheoesophageal fistula. Opened lower esophagus is in continuity with the lower trachea.

Additional exceedingly rare variants exist and include a broncho-esophageal fistula. Tracheomalacia is also commonly seen in these patients. However, tracheal development is usually normal in cases of esophageal atresia without fistula formation (see above).60 These other abnormalities frequently determine patient outcome. In term infants, survival from isolated T-O fistula approaches 95%.61 Particular risk factors are low birthweight and prematurity. There has been a steady improvement and, even when both these risk factors are present, survival in these infants exceeds 50%.61,62 Short- to medium-term morbidity from complications such as dysphagia (45%), respiratory infections (29%), and gastro-esophageal reflux (48%) is more problematic.63

Tracheobronchiomegaly (Mounier-Kuhn syndrome) Tracheobronchiomegaly is an extremely rare condition characterized by an enlarged trachea and/or main bronchi, more than three standard deviations greater than the normal diameter.64,65 Saccular bulging of the intercartilaginous membranes amounting to tracheal diverticulae may be seen. These posterior outpouchings can cause a flaccid wall. Although childhood cases have been reported, presentation tends not to be until the fourth or fifth decade, when patients present with chronic cough and repeated respiratory tract infections. Diagnosis is primarily by CT scan or bronchoscopy.66 The pathogenesis is unclear and a relatively late presentation raises the possibility that this is an acquired condition. An association with inheritable conditions, such as Ehlers-Danlos syndrome, cutis laxa and skeletal dysplasia,65,67,68 suggests that an elastic tissue defect may be responsible for the anatomic manifestation. The occurrence in siblings suggests the condition may be inherited in an autosomal recessive manner, supporting a genetic basis.69 Prognosis is variable. Affected individuals may develop bronchiectasis, emphysema, and ultimately respiratory failure. Therapy is aimed at clearing secretions, although stenting may be helpful in the advanced stages.70 Lung transplantation is an option but is not without complications.71

Other causes of tracheal obstruction A variety of masses and intraluminal obstructions have been recorded, particularly in the newborn. These include mucus retention cysts72 and fibrous webs.73 Perhaps not strictly a congenital lesion, tracheobronchopathia osteoplastica represents an overgrowth, into the lumen, of tracheal cartilage, possibly with ossification. These nodules typically involve the anterior and lateral walls while sparing the posterior membranous wall.74 It is often asymptomatic and found as an incidental finding usually at autopsy (> 90%), so the true incidence is difficult to assess. Presentation is usually relatively benign with, most commonly, hoarseness and chronic cough but hemoptysis and asthma-like symptoms have also been reported. Rarely, it can cause significant central airway obstruction.74 While it is more a condition of the adult presenting after the age of 50, it has been reported in children (see Chapter 17).75 Familial occurrence in a mother and daughter has been reported.76

Bronchus

Bronchial atresia Bronchial atresia is usually considered a rare abnormality and it is often an isolated lesion.77–79 It may be underdiagnosed, partly because it is very difficult to demonstrate pathologically80 but also because it may be asymptomatic. In one study, the atresia was an incidental radiographic finding in up to 58% of cases.78 Isolated bronchial atresia more commonly presents in the second decade, sometimes with dyspnea. In the younger age group, recurrent cough and fever with infection is a more likely clinical scenario.80 Radiologically, the atresia may be associated with infiltrates or distal emphysema. In a recent relatively large series,80 the right lower lobe was more commonly affected than the right and left upper lobes; the right middle lobe was the least affected. The atresia usually affects a segmental bronchus but subsegmental and lobar bronchi can also be involved. Distal lung usually contains a bronchial mucocele along with other features of obstruction, including a lipid pneumonia or hyperinflated emphysematous lung.80

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

The bronchus may be obstructed by circumferential or eccentric luminal fibrosis or the lumen may be occluded by hyperplastic but otherwise normal-appearing bronchial mucosa (Figure 5). Bronchial atresias have been reported from early gestation and most are congenital.81,82 More recently, bronchial atresia has been increasingly recognized as a fundamental component of many adenomatoid and other pulmonary malformations.83,84 This suggests atresia may be a commoner anomaly than previously thought. The manifestations of bronchial atresia are therefore protean. It may be the precise timing of the occurrence of the atresia and any associated abnormalities that determines the clinical and pathological presentation.

Bronchial stenosis Bronchial stenosis may be an isolated lesion but is frequently part of a more generalized segment of abnormality involving the trachea. As in tracheal stenosis, bronchial stenosis can be intrinsic or due to extrinsic compression. Acquired stenosis,

Figure 5. Transverse section across bronchial atresia. Two lumina are seen, one representing proximal lumen, the other, distal lumen (arrowheads).

(a)

72

(b)

due to post-traumatic fibrosis, may also occur.85 Compared with extrinsic bronchial stenosis, congenital intrinsic stenosis is extremely rare.21,86 One case report noted the stenosis was associated with anomalous cartilage segmentation and secondary squamous metaplasia.86 Extrinsic stenosis is most commonly associated with congenital heart disease and there may be an overlap with bronchomalacia.87 Compression may occur when pulmonary arteries, possibly enlarged secondary to pulmonary hypertension, compress the left main bronchus or the left upper lobe bronchus (Figure 6). Stenosis has also been recorded in association with an abnormally placed descending aorta (Figure 7) or displaced aortic arch.88,89 The left main bronchus may be compressed in conditions associated with an enlarged left atrium. Other causes include bronchogenic cyst.90

Bronchomalacia Bronchomalacia is similar to, and often coexists with, tracheomalacia.42 This bronchial cartilage abnormality can be considered a form of bronchial stenosis, since it leads to airway collapse during respiration. In infants with wheezing and/or other symptoms, the mild form may be incorrectly attributed to other forms of reactive airway disease.41,91 The commonest cause of bronchomalacia is acquired damage to the cartilage from mechanical ventilation. In congenital disease, there may be a widespread cartilaginous deficiency that may lead to childhood bronchiectasis92,93 but segmental bronchomalacia is also described.94,95 The pathology of bronchomalacia is poorly described. Gupta et al.94 attribute an intrapulmonary segment of bronchomalacia to separated and flattened cartilage plates. Recalling that the extrapulmonary main bronchus should have horseshoe-shaped cartilage similar to the trachea, MacMahon and Ruggieri95 illustrate an abnormal main bronchus composed of isolated cartilaginous islands, rather than a well-formed plate. Bronchomalacia is frequently associated with other anomalies, when it is sometimes designated secondary, as opposed to primary isolated malacia. In addition to malacia elsewhere

Figure 6. (a) Heart has been lifted upward to reveal the left main pulmonary artery crossing the main bronchus. (b) The isolated left main bronchus showing the focus of stenosis caused by the pulmonary artery. (Image courtesy of Dr Mike Ashworth, London, UK.)

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 7. Bronchial compression from descending aorta.

(in up to 50% of cases), bronchomalacia may be associated with other anomalies including cardiovascular malformations, the VACTERL association with tracheoesophageal fistula94,96,97 and various skeletal dysplasias.8 There is also an association with gastroesophageal reflux. A significant genetic component is suggested by reports of familial isolated bronchomalacia98 and its occurrence in twins.99 The lung distal to the malacic focus may demonstrate congenital lobar emphysema. Treatment and outcome vary according to the severity and associated abnormalities. In one series, the majority with isolated malacia of the left main bronchus grew out of their condition but some had persistent exercise limitation.91 More severe malacia requires more active therapy, including long-term ventilatory support,100 bronchopexy or stenting.97,101,102

Abnormal bronchial origin and branching Abnormal bronchial origin and branching describes a wide range of anomalies. Most are minor but these anomalies may be associated with significant complications. Since most are asymptomatic, a true incidence is unknown. McLaughlin et al.103 identified a tracheal bronchus in 2% of children requiring bronchoscopy. A similar proportion was found by Sanchez and colleagues,104 who, in a series of 580 bronchoscopies, found various tracheobronchial anomalies in 9.6% of cases. Seventy percent of those involved the right upper bronchus (see below). Spiral CT scans demonstrate the wide range of anomalies and variation in normal anatomy in aymptomatic patients.105 The cause of bronchial branching anomalies is not clear. Since the major branches of the tracheobronchial tree are formed by 16 weeks gestation, the etiology must have its origins before this time. The main lobar bronchi form around 4–5 weeks gestation (post-ovulation). Three main developmental events may account for the final pattern of bronchial distribution; disruption to any one or all three mechanisms might lead to excess or aberrantly distributed bronchi.105,106 The reduction theory suggests there is an underlying

symmetric “ground plan” for bronchial development. Modification to this plan results from suppression or even regression of partly developed bronchi. Secondly, developing bronchi may migrate from their initial position. Thirdly, bronchi develop because of the inducting effect of bronchial mesenchyme on foregut endoderm. Abnormal morphogenesis may cause bronchi either to fail to develop or to develop at an abnomal site. Abnormal bronchial branching may be asymptomatic but patients might present with stridor, cough, respiratory distress, or recurrent, usually right-sided pneumonia.103,104,107,108 Occasionally, the anomaly may only manifest itself during other medical interventions, such as intubation.109 Associated conditions include Down syndrome.104 Atwell110 described a variety of abnormalities based on 1200 bronchograms. Additional major bronchi were confined exclusively to the right upper lobe. The anomalies included double right upper lobe bronchi and an origin of the right upper lobe bronchus from the trachea. Fewer cases with a reduction in major bronchi were found and again these exclusively involved the right upper lobes. Minor abnormal branching patterns were more common in this study, notably trifurcation of the left upper lobe bronchus, accounting for 10% of cases. Branching abnormalities are frequently associated with other anomalies, such as the association of a “bridging” bronchus with a sling left pulmonary artery31 and tracheal stenosis. The “bridge” is formed by the bronchus supplying the right lower and/or middle lobes crossing the mediastinum from its origin on the left main bronchus. The lobe associated with the aberrant bronchus may show bronchiectasis104 or chronic inflammation. In one case, the lobe was hypoplastic.103

Bronchial isomerism syndromes Isomerism might be regarded as a curiosity, in which the abnormal bronchial branching leads to two similar lungs, that is, two bilobed “left” lungs or two trilobed “right” lungs. In most cases, pulmonary isomerism is associated with multiple other defects, most notably cardiac and splenic, and the pulmonary abnormality is of relatively little functional significance. These syndromes appear to reflect an inability of the developing embryo to establish early left-right asymmetry. This process is is linked to a variety of chromosomal loci.111–113 Normally the right upper lobe bronchus is eparterial, i.e., it lies above the right pulmonary artery, which passes between the right upper and right middle bronchus. The left main stem bronchus is approximately twice as long as the right and both main branches are hyparterial, i.e., they lie below the pulmonary artery. All organs (thoracic and abdominal) may be complete mirror images, i.e., situs inversus. Occasionally there may be situs ambiguous, in which the left and right main bronchi and major divisions are symmetric. This is usually associated with a complex malformation. In situs ambiguous, or heterotaxy, the lungs may have three lobes with an isomeric “right-sided” eparterial right main bronchus, or isomeric, bilobed “left” lungs with hyparterial

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 4 Summary of main thoracic anomalies associated with major isomerism subtypes

Asplenia

Polysplenia

Lung

Bilateral trilobed with eparterial bronchi

(81–93%)

Bilateral bilobed with hyparterial bronchi

(72–88%)

Superior vena cava

Bilateral

(46–71%)

Bilateral

(33–50%)

Inferior vena cava

Bilateral

(16–28%)

Interrupted with azygos continuation

(58–100%)

Pulmonary veins

Total anomalous to systemic vein

(64–72%)

Anomalous drainage due to abnormal atrial septum

(37–50%)

Congenital heart malformation

AV canal defects

(84–92%)

AV canal defects

(80%)

Absence/hypoplasia of a ventricle

(44–55%)

Absence/hypoplasia of a ventricle

(37%)

Double-outlet right ventricle

(82%)

Double-outlet right ventricle

(17–37%)

Stenosis/ atresia Pulmonary

(88–96%)

Stenosis/atresia Pulmonary Aortic

(42–43%) (17–22%)

Modified from.115

bronchi. There is usually, but not invariably, a parallel symmetry of the atria.114 They are either bilateral morphologically right-sided or bilateral morphologically left-sided. Asplenia is more typically associated with right isomerism while polysplenia with left isomerism (Table 4).115 Many malformation complexes have been described, and Landing and colleagues outlined five of the commoner subgroups of isomerism-related syndromes.8,116 However, there is considerable overlap in the constellation of abnormalities found in any one case. Further, the occurrence of different patterns within families suggests these heterotaxies may be part of a spectrum, rather than distinct entities. Many genetic abnormalities and inheritance patterns have been described, as well as some environmental factors.117 In general, most of these syndromes are of sporadic inheritance but autosomal recessive, dominant and sex-linked patterns of inheritance have been recorded. Overlap in syndromes occurs.

Broncho-biliary fistula This is a rare anomaly, in which the right mainstem bronchus and left hepatic duct are connected by a fistula that passes with the esophagus through the diaphragm.118 It usually presents in childhood with cough, bile-stained sputum, and recurrent chest infections. The clinical picture may suggest cystic fibrosis but chest infections are uncommon in this condition before the age of 3 months. Late presentation in adults has been reported.119 The embryological basis of this condition is uncertain, but it may represent a form of upper gastrointestinal tract duplication.120

Lung

Pulmonary agenesis Agenesis of the lungs describes the unilateral or bilateral absence of the lung parenchyma and vasculature. Cases where

74

Figure 8. Unilateral aplasia of lung. Residual right main bronchial stump (arrow). This abnormality was also associated with a complex congenital heart disease.

the residual main bronchus is present (Figure 8) are designated aplasia by Schneider and Schwalbe.121 Unilateral and bilateral agenesis are rare and the incidence imprecise. Mardini and Nyhan122 suggest a combined incidence of some 0.5–1 per 10 000 babies. Maltz and Nadas,123 reviewing 164 cases of lung agenesis, found an equal sex incidence in bilateral agenesis but a slight preponderance of females with unilateral agenesis (56%). Unilateral agenesis may affect the left and right lungs equally.124

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Features related to unilateral agenesis can be identified by prenatal ultrasound but are not specific. Findings are similar to those of other intrathoracic lesions or congenital diaphragmatic hernia.125 At birth, unilateral agenesis may cause cyanosis, tachypnea and respiratory distress123 as well as features due to mediastinal shift. Presentation may be later in childhood126 or even adulthood127 with nonspecific respiratory symptoms. Where there is a rudimentary bronchus, especially in childhood, the main problem relates to a solitary lung with poor resistance to infection and development of recurrent infections.128 The blind bronchial stump gives rise to “spillage” pneumonia.8 In bilateral agenesis, the laryngo-tracheal bud either develops as far as the larynx or the trachea.129,130 In the latter case, the pulmonary artery joins the aorta by the ductus arteriosus. Bronchial arteries and veins are usually absent. In unilateral agenesis, morphometry indicates that, in the remaining lung, the alveolar number is increased and “compensates” for the alveolar loss from the agenetic lung.131 A wide range of abnormalities are associated with pulmonary agenesis. VACTERL-related anomalies are well described132 and in unilateral agenesis there is evidence that ipsilateral anomalies are more frequent. Cunningham and Mann124 noted associated ipsilateral hemifacial microsomia and abnormalities of the radial bone. An underlying vascular disruption sequence is one possible explanation. Karyotypic study in cases of pulmonary agenesis is usually normal but evidence for genetic causes comes from the familial occurrence.133,134 Agenesis is also associated with a number of conditions and syndromes, which are either autosomally recessively inherited or there are known abnormal gene loci.135

Lobar agenesis and other lesser congenital pulmonary anomalies Abnormalities of major pulmonary fissures may be present in over 2% of fetal autopsies.136 While not of any major functional significance, there is an association with malformations elsewhere. Cases with absence of the right middle lobe8 or left lower lobe associated with stenosis of the left main bronchus have been described. Anomalous bronchial distribution usually causes no disabilities and falls into the province of anatomical variation. Congenital anomalies of pulmonary lobes are varied but include common abnormalities, such as the azygos lobe, cardiac lobe, double-lobed upper lobe of left lung in association with Fallot’s tetralogy, and isomerism of the right lung.137 An azygous lobe is a portion of the right upper lobe, growing medially to the right posterior and common cardinal veins. It may rarely be seen on the left side of the chest. A cardiac lobe occurs when the anterior basal segment of the right lower lobe is separated from the rest of the lobe. A horseshoe lung is an uncommon congenital abnormality caused by the partial fusion of the bases of both lungs behind the pericardial sac (Figure 9). It may be associated with

Figure 9. Horseshoe lung with posterior fusion of the lungs. In this case, there were other abnormalities including esophageal atresia (arrowhead) and tracheoesophageal fistula (arrow).

pulmonary hypoplasia but in the absence of other abnormalities is usually asymptomatic. This abnormality is described in association with wide variations in pulmonary vascular distribution and other anomalies, many of which are within the VACTERL spectrum.138–141 Most cases have been associated with right lung hypoplasia and the scimitar syndrome, a variant of partial pulmonary venous drainage anomaly, but horseshoe lung with left lung hypoplasia is recorded.142,143 The lungs may herniate into the mediastinum144 while herniation into the neck (ectopia) has been described in iniencephalus, Klippel-Feil syndrome and the “cri du chat” (5p-) syndrome138–148 The cause is unknown, although there appears to be a reduction or absence of fascia in this area. An unusual herniation of the lung into an extra-thoracic location, so-called “bag-pipe” lung, has been described in a 16-week-gestation fetus.149 Again, the underlying mechanism is unknown but is probably secondary to an early intrauterine event.

Pulmonary malformations including cysts This section concentrates on the congenital pulmonary malformations that commonly lead to a clinical or radiological diagnosis of pulmonary cystic disease or simply a cyst in

75

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 5 Classification of congenital lung malformation and lung cysts

I.

Congenital lung malformation A. Bronchopulmonary malformation 1. Bronchogenic cysts; (peripheral cysts) 2. Bronchial atresia (i) Isolated (ii) With systemic vascular arterial/venous connection (true intralobar sequestration) (iii) With communication with GI tract (bronchopulmonary foregut malformation) (iv) Systemic vascular connection to normal lung 3. Cystic adenomatoid malformation, large cyst type (i) Isolated (Stocker type 1) (ii) With systemic vascular connection (hybrid lesion) 4. Cystic adenomatoid malformation, small cyst type (Stocker type 2) (i) Isolated (ii) With systemic vascular connection (hybrid lesion) 5. Extralobar sequestration (i) With GI tract communication (bronchopulmonary foregut malformation) – with or without small cyst type adenomatoid malformation (ii) No communication with GI tract – with or without small cyst type adenomatoid malformation 6. Pulmonary hyperplasia and related (i) With laryngeal atresia (ii) Solid adenomatoid malformation (Stocker, type 3) (iii) Polyalveolar lobe 7. Congenital lobar overinflation (emphysema) (i) With intrinsic bronchial abnormality (ii) With extrinsic bronchial compression

II. Other congenital cystic malformation 1. Simple parenchymal cysts 2. Lymphangiomatous cysts 3. Enteric cysts, foregut duplications 4. Mesothelial cysts III. Acquired cysts (perinatal/pediatric) 1. Cystic pleuropulmonary blastoma (type 1) 2. Infection (i) Bacterial (ii) Parasitic 3. Other (i) Encysted pulmonary interstitial emphysema (ii) Inhaled foreign body (iii) Other Modified from83

the fetus or young (Table 5). Other acquired causes are considered elsewhere. Age at presentation is wide but over the past few decades, antenatal diagnosis of pulmonary cysts has become common. There is a considerable temptation on the part of obstetricians and radiologists to determine very specific diagnoses and labels to apply at this early stage. Very often this exercise is

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impossible and there is a very good argument for leaving attempts at specific diagnosis until after birth, when other techniques are available.150 This has no adverse effects, since precise classification of a cyst does not affect antenatal management. If any antenatal maneuver is considered, it is probably on the basis of the secondary effects of the cyst, such as mediastinal displacement or hydrops. Even when cystic lesions are studied pathologically, they do not always fit sharply into traditional diagnostic “boxes”. Clements and Warner151 proposed, a little in the manner of the sequential analysis of cardiac abnormalities, that bronchopulmonary abnormalities could be approached in a similar fashion152 and coined the term “malinosculation”, described as a “congenitally abnormal connection or opening of one or more of the four components of lung tissue”, i.e., the airway, parenchyma, arterial supply, and venous drainage. The classification has a three-step construction. The first describes the abnormality of the bronchopulmonary airway or arterial blood supply or both. Thus the abnormality may be in the bronchopulmonary airway but with a normal pulmonary arterial supply. This is bronchial or bronchopulmonary malinosculation. At the other end of the spectrum is an anomalous arterial supply to an area of lung with a normal bronchopulmonary airway. This is arterial pulmonary malinosculation. There are grades in between, which are termed broncho-arterial malinosculations. The second step in the classification defines the associated anomalies of venous drainage. The final stage is to describe any associated abnormality of the lung parenchyma. This approach emphasizes that many malformations form a spectrum of conditions. This notion has been further emphasized by Langston,83 who prefers a more descriptive approach to bronchopulmonary malformations (i.e., small cyst, large cyst, and solid). She considers many bronchopulmonary cystic lesions as a part of a sequence. Underlying bronchial atresia appears to be a fundamental component of many cystic abnormalities, even though it may be difficult to demonstrate.83,84,153 Others emphasize the spectrum of pathology in these lesions and state that traditional diagnostic labels and definitions may prove inadequate.154–157 As noted above, distinguishing between various cystic lung lesions prenatally is difficult and often of little immediate relevance to management. Postnatal imaging may be undertaken to assess the need for and timing of surgery. Chest X-ray is often used more to assess the extent of the abnormality (Figure 10) rather than to detect it. The presence, for instance, of mediastinal displacement may indicate the patient is a candidate for early surgery. Computed tomography is better at identifying cystic lesions (Figure 11) and indeed other pathologies, such as lobar emphysema. Unless the infant is symptomatic, it is possible to wait a few months before this investigation is undertaken. The CT may identify an aberrant vessel and alter the diagnosis from an adenomatoid malformation to a sequestration but there is an overlap in appearances.157 Computed tomography may help to define the extent of surgery required157 and 3D

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 10. Post-natal X-ray of congenital cystic lung. Multiple “bubbly” cystic lesions in the lower lobe of the right lung (arrow).

reconstruction may assist the surgeon in “mapping” the origin and distribution of the abnormal vessel. Other modalities are of less importance. Magnetic resonance imaging may be associated with too much breathing artifact to help further. In general, angiograms are not contributory, although they may play a role in very specific circumstances where, for instance, embolization of aberrant vessels is contemplated.

Bronchogenic cysts Bronchogenic cysts are congenital cysts most commonly found in the anterior mediastinum or around the hilum.158 The sex incidence is equal at all ages. Presentation may occur across the age spectrum, usually in the first decade, but appearance in adult life is also well recognized.159–161 It may be an incidental radiological finding but bronchial obstruction with stridor or wheezing may be the main signs in the very young.162 In older children and adults, chest pain or symptoms secondary to infection may be more prominent.159 Bronchogenic cysts are more common in the left lung and they have been described in association with other pulmonary abnormalities. They often coexist with other congenital pulmonary defects, such as bronchial atresia.164–166 There is no association with congenital cystic disease in other organs. Despite their label, cysts conforming to the appearance of bronchogenic cysts may be found in a very wide range of sites, including the paratracheal region in the neck,167 esophageal wall,168 pericardium,169 a variety of locations below the diaphragm,170,171 the base of the tongue172 and, even wider afield, subcutaneously.173 Peripheral162,174 or intra-pulmonary bronchogenic cysts have been reported but may be difficult to distinguish from

Figure 11. CT scan defines the cystic lesions, in this case an adenomatoid malformation, better than X-ray.

abscesses or other acquired pathology. Communication with the bronchial tree may predispose to infection174 but Stocker175 believes many of these lesions represent examples of type 1 congenital cystic adenomatoid malformations (see below). Antenatal ultrasound imaging may detect these lesions as fluid or mucus-filled cysts, found either along the trachea or esophagus or in the perihilar region, with a predilection for the carinal area. After birth, they can be demonstrated by chest X-ray as a globular mass, often in the sub-carinal area. More accurate identification can be obtained by a CT or MRI scan (Figure 12). Pathologically, clear diagnostic criteria are required, especially for bronchogenic cysts in unusual locations. The cysts are unilocular and, at least in older patients, may be up to 10 cm in diameter (Figure 13). Communication with the tracheobronchial tree should not exist, but lesions may be adherent to the structures. If a communication is noted, particularly when these are multiple, another pathology such as an abscess should be suspected. The cyst fluid may be clear or, if infected, contain pus and hemorrhage inside a thickened fibrous wall. The cyst lining is respiratory, sometimes with focal squamous metaplasia. This is not specific to bronchogenic cysts, as esophageal cysts may also demonstrate such epithelia. The most reliable criterion is the presence of cartilage in the wall176 but this may require serial sections for its detection (Figure 14). Seromucinous glands and fibromuscular connective tissue are also present in the wall. Bronchogenic cysts are thought to arise as abnormal “late” buds from the primitive foregut. To explain some of the more

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 12. Axial image of a contrast-enhanced CT scan showing a bronchogenic cyst (arrow) in a sub-carinal position. (Image courtesy of Dr Ash Chakraborty, Oxford, UK.)

Figure 13. Bronchogenic cyst. This 3.0 cm cyst is well circumscribed with a fibrous wall.

Figure 14. Wall of bronchogenic cyst showing respiratory epithelial lining with cartilage. Squamous metaplasia may be present and the inflammation is more marked here than in many.

unusual locations, the concept of “migrating bronchogenic rests” is invoked.172 The failure of separation of primitive foregut and notocord may explain the occurrence of intradural bronchogenic cysts.177 Where necessary, removal is often undertaken via thoracoscopy and outcome is excellent in both children and adults.178,179

Congenital adenomatoid malformation

These lesions are traditionally described as congenital “cystic” adenomatoid malformation (CCAM) but more recently as congenital pulmonary adenomatoid malformation (CPAM), this term emphasizing that not all these lesions are cystic. Three subtypes were originally described180 and two further subtypes were added, based on the concept that each subtype reflected an abnormality at a different level in the tracheobronchial tree, i.e., type 0, proximal bronchial anomaly; type 4, peripheral lung.181 While the Stocker classification has widespread currency and is helpful in providing a framework to classify different abnormal subtypes, the basis of this classification83 has been challenged. In many situations, the precise

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typing of these cysts may be an academic exercise, but an awareness of their possible relationship to neoplastic lesions is critical. Langston83 uses a more descriptive approach to the labeling of these abnormalities. Such is beneficial, especially early in gestation or when there are unusual features154,182,183 The prevalence of adenomatoid malformations is difficult to estimate, since lesions can be diagnosed in very different circumstances from early gestation to adult life. Furthermore, ultrasound studies very clearly demonstrate that malformations identified in mid-trimester may shrink and not be readily seen at term. It may occur in 1:11 000 up to 1:35 000 births.184,185 Adenomatoid malformations are generally considered sporadic, and while very occasional chromosomal abnormalities have been described in individual cases, a consistent association with a specific chromosomal anomaly has not been described.4,186 The initial presentation and management of adenomatoid malformation and indeed related pathology, such as sequestration, are often similar. Initial presentation and diagnosis are commonly prenatal, when a specific diagnosis is uncertain. A diaphragmatic hernia may also enter the differential diagnosis, although it is a more critical distinction, as the prognosis and post-natal management is very different. Cystic lung disease may be an incidental ultrasound finding, although larger lesions can cause mediastinal shift and hydrops or ascites caused by venous obstruction. A lung or lobe will show increased echogenicity, with the appearance of a solid and/or cystic chest mass. The size of these malformations may change during the pregnancy and there are well-documented examples of both regression and progression.4,187,188 Whether or not there has been apparent regression prenatally, further postnatal assessment is required to assess their extent and the need for surgery (see below).

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 6 Summary of pathological features of main adenomatoid malformations subtypes

Macroscopy

Microscopy

Comment

Large cyst adenomatoid malformation (type 1)

Well defined; larger cysts 2–7 cm postnatally

Pseudocolumnar ciliated epithelium; thin fibroelastic wall; occasional mucigenic epithelium and/or cartilage

In communication with main bronchial tree. Large cysts may cause mediastinal shift. Rarely, late bronchioloalveolar carcinoma

Small cyst adenomatoid malformation (type 2)

Multicystic; mms to < 2 cm diameter postnatally

Bronchiolar type epithelium; intervening alveoli

Associated with bronchial atresia; changes also found in sequestration

Solid adenomatoid malformation (type 3)

Enlarged but solid lung with no obvious cysts

Uniform gland-like structures which may become slightly distended; scattered bronchioles

Associated with bronchial atresia; may be associated with prenatal mediastinal shift

Peripheral adenomatoid malformation (type 4)

Large peripheral cysts

Flattened, sometimes cuboidal epithelium

Exclude subepithelial immature small cells of cystic pleuropulmonary blastoma

Figure 15. Large cyst adenomatoid malformation in a mid-trimester-gestation baby. The abnormality is confined to the right upper lobe with the readily visible cysts up to 1.5 cm in diameter

A long-term, albeit low, risk of malignancy, such as rhabdomyosarcoma, is well recognized from case reports.189,190 More recently, a link between large cyst (type 1) adenomatoid malformation and adenocarcinoma, bronchioloalveolar subtype, that may present in the second decade has been demonstrated (see below). The distinction between the peripheral cyst type adenomatoid malformation and well-differentiated cystic pleuropulmonary blastoma (CPPB) can be extremely difficult; indeed, so much so, that some question whether all of these adenomatoid malformation subtypes are CPPB (see below).83

Subtypes (Table 6)

Large cyst type (Stocker type 1) As with all pulmonary cysts, these are now commonly diagnosed prenatally. They may present in infancy with respiratory

Figure 16. Large cyst adenomatoid malformation with columnar epithelium on a thin fibromuscular layer. The appearance of the small papillary formations of the epithelium lining the collapsed cyst is typical. Small bars of cartilage (inset) may be present.

distress, secondary to mass effect of a large cyst (Figure 15). Cyst size clearly depends on age at gestation or age at presentation but they may be up to 7 cm in infancy. They are often multilocular with adjacent smaller bronchiolar type cysts merging with more normal lung, so the malformation is poorly delineated. The epithelial lining is usually respiratory (Figure 16), i.e., ciliated columnar, but characteristic islands of mucigenic epithelium may occasionally be found (Figure 17). The wall contains a thin fibromuscular layer, in which occasional cartilage bars may be found. There is communication with the bronchial tree and sometimes with a systemic artery. Complications, including hydrops or pulmonary hypoplasia, are rare. Even at a young age, bronchioloalveolar carcinoma has been reported in association with large cystic

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Figure 17. Mucigenic epithelium is found in some large cyst adenomatoid malformations.

Figure 19. Small cyst adenomatoid malformation are lined by bronchiolar type epithelium. The histological margins of the abnormality are also ill-defined.

Figure 20. Small cyst adenomatoid malformation with rhabdomyoblasts in the interstitium.

malformations. For this reason alone, it has been argued that maintaining the distinction between large and small cyst adenomatoid malformation should continue.157 Evidence is growing that the mucinous cell clusters may be pre-malignant and form a continuum with bronchioloalveolar carcinomas.191–194

intervening alveoli. Cartilage is absent and mucigenic epithelelium is not seen. The anomaly can be very poorly delineated and merge imperceptibly with normal lung. Rarely, striated muscle is present throughout the malformation (Figure 20). Small cyst adenomatoid malformation may be associated with malformations elsewhere, especially in the urinary tract.195 It is likely that this maldevelopment is secondary to airway obstruction, such as bronchial atresia. This may be difficult to demonstrate,84,153,196 especially when the abnormal lung has been resected thorascopically. Adjacent lung often features obstructive processes including mucus retention and/or a lipoid macrophage reaction.

Small cyst type (Stocker type 2) This usually affects only a lobe or part of a lobe but occasionally may affect a whole lung. Prenatally, it may be impossible to distinguish small from large cyst disease by ultrasound. Postnatally, these cysts are smaller and the extent of the lesion less well defined (Figure 18). Cysts range from millimeters up to 2 cm in infants or older individuals.157 Microscopically the cysts have a bronchiolar-like structure and epithelial lining (Figure 19). Cysts may largely replace the normal lung parenchyma or be more scattered, with relatively normal-appearing

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Figure 18. Resected lobe partly affected by small cyst adenomatoid malformation. The margins of the lesion are ill defined.

Solid adenomatoid malformation (Stocker type 3) This is a rarer subtype, in which the lung or lobe of lung is massively increased in size, so it is more likely to be associated

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 21. Solid adenomatoid malformation, diagnosed as cystic lung antenatally and requiring early resection because of respiratory distress. Here, the abnormal lung is more central. Residual pale normal lung is noted at the bottom of the resection.

(a)

Figure 22. The histology shows the more solid, less cystic change typical of this malformation; the inset highlights the “adenomatoid” aspect of the abnormality.

(b)

Figure 23. (a) Peripheral adenomatoid: malformation with multiple thin septae and flattened cuboidal epithelium (inset). (b) The cyst walls of the well differentiated type 1 cystic pleuropulmonary blastomas are similar except small hyperchromatic “blastematous” cells may be present beneath the epithelium. This may be focal.

with mediastinal shift than the other subtypes (Figure 21). Microscopically, the lung appears relatively immature with an overgrowth of immature terminal airspaces.196 The small “cysts” are lined by cuboidal epithelial cells with only very scattered bronchiolar structures (Figure 22). Langston83 views this as very closely related to the enlarged lungs associated with laryngeal atresia, i.e., it is a form of hyperplastic lung (see below). As such this implies an underlying obstructive origin. Peripheral lung cyst (Stocker type 4) The status of this cyst subtype is disputed. These peripheral cysts have been regarded as a form of hamartomatous malformation of the distal acinus181 that may present in early childhood. They may be incidental findings, but have been

described in association with pneumonia, spontaneous pneumothoraces or respiratory distress.197 They comprise thin-walled cysts lined by a simple flattened or cuboidal epithelium (Figure 23a). It is becoming clear that many of these lesions are neoplastic and represent well-differentiated cystic pleuropulmonary blastomas (Figure 23b). Extremely careful evaluation is required before a label of peripheral (type 4) adenomatoid malformation is considered appropriate (see below).198,199,200 Prognosis of adenomatoid malformation is usually good, unless there is bilateral disease or fetal hydrops develops.201,202 In utero death may rarely occur,197 which has prompted prenatal intervention in some cases.203,204 Since between 25 and 40% of cases are diagnosed radiographically, it is controversial whether asymptomatic adenomatoid malformations should be

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Figure 24. Acinar dysplasia in a term gestation baby. There is very abnormal development of the acini beyond the bronchiolar level. Acini are lined by columnar cells. (Image courtesy of Dr Chris Wright, Newcastle, UK.)

resected.205–210 Because of the residual risk of infection and malignancy, there is an increasing consensus156,206 but no uniform move211,212 towards the surgical removal of malformations, before they become symptomatic.

Acinar dysplasia This is a very rare malformation that is incompatible with life and has been the subject of a few case reports. Stocker considers this lesion a type of adenomatoid malformation (type 0).181 Severe maldevelopment of the terminal part of the lung is the morphological finding. In some cases, the macroscopic appearance is that of a severely hypoplastic lung.196,213,214 But in others,215 the lung weights may be normal and the severity of the abnormality is more apparent only at the microscopic level. Cardiac and renal anomalies are associated with some cases215 and it has been reported in siblings.196 Histologically, there is severe disturbance of the normal pulmonary architecture, in which the lung appears to have developed only to the level of bronchioles. Normal acinar development is lacking (Figure 24). Terminal sacs are lined by pseudostratified, bronchial type columnar epithelium. Davidson et al.214 described it as an apparent arrest at the pseudoglandular stage of development. As a bilateral disease, it is uniformly fatal.

Pulmonary sequestration and related lesions Much of the early literature uses the term “accessory lung” or “lobe” to label pulmonary tissue that is separate from the main pulmonary mass but that occurs within the lung or below the diaphragm. Many of these lesions were “true” accessory lungs and connected to parts of the gut, including the esophagus or stomach, by an accessory bronchus. The term “sequestration” was originally introduced by Pryce216 to label these “disconnected” masses with an anomalous

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Figure 25. Left sided, extra-lobar sequestration with arterial communication with the descending aorta. This was associated with diaphragmatic hernia (Image courtesy of Dr Colene Bowker, Oxford, UK.)

pulmonary supply. This term has gradually replaced the notion of “accessory” lobes or lungs. Sequestrations were divided into intralobar (ILS) and extralobar (ELS). Subsequent case reports detailed a wide variety of arterial, venous and bronchial connections. This led to difficulty in incorporating them into the simple concept of ILS or ELS, with or without gastrointestinal tract communication. Gerle et al.217 suggested the term bronchopulmonary foregut malformation to describe those lesions with gastrointestinal tract fistulae. Many sequestrations are now identified initially in utero by prenatal ultrasound and are part of the differential diagnosis of congenital cystic lung lesions. Postnatally chest X-rays may be used to screen patients and to assess the extent of the abnormality, rather than detecting the abnormality. This section will deal with sequestrations and other related lesions associated with an anomalous vascular supply. It is in the context of sequestration, especially the intralobar type, where difficulties with nomenclature often arise. Extra-lobar sequestration Extralobar sequestration (ELS) describes an isolated mass of lung invested in its own pleura with no bronchial communication and an anomalous blood supply (Figure 25). Its isolation as a mass identifies this as a distinct malformation. Conran and Stocker218 described the characteristics of 50 cases of ELS. Excluding those diagnosed prenatally, 61% were diagnosed within the first 3 months of life but one patient was not diagnosed until age 67 years. The male:female ratio was 1:1. Respiratory distress was the commonest

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 26. Axial and 3D surface-rendered reconstructed images show a large vessel arising from the lower thoracic aorta to supply the abnormal segment. (Images courtesy of Dr Ash Chakraborty, Oxford, UK.)

presentation. Lesions were distributed in both hemithoraces, posterior and anterior mediastinum and subdiaphragmatic. The most common location was the left hemithorax (48%). Overall 50% of cases, especially those in the left hemithorax, showed histological features of small cyst-type adenomatoid malformation. Rhabdomyomatous dysgenesis was common. Extralobar sequestration may be associated with other malformations. Besides adenomatoid malformation, ELS has been associated with congenital heart disease, diaphragmatic hernia, and pulmonary hypoplasia, although the latter is strictly a deformation.219–221

Intra-lobar sequestration Intralobar sequestration (ILS) is defined as a lobe or part of a lobe of lung that lies within the visceral pleura, isolated from the normal tracheobronchial tree and associated with an anomalous blood supply. Because the typical description of ILS was of a late childhood or adult condition presenting following multiple episodes of infection, it has been suggested that the majority of ILS are acquired through a combination of inflammation-related obstruction to the airway and reactive hypertrophy of pulmonary ligament arteries.220 This view has been challenged. Langston83 argues that ILS is bronchial atresia associated with a systemic blood supply. Malformations that conform to the strict definition of ILS in children have been recorded in several series.222–224 In addition, there have been a number of reports describing the combination of small cyst adenomatoid malformation with an anomalous systemic blood supply. Often the cystic lung has been detected by prenatal ultrasound scan. Particularly in the pediatric surgical community, these have been described as “hybrid lesions,” i.e., a cross between a form of sequestration and adenomatoid malformation.155,226,227 If one regards adenomatoid malformation as a consequence of bronchial atresia, the adenomatoid change is secondary and part of a sequence. Other features that strongly suggest underlying bronchial atresia include individually dilated bronchi, mucus retention, and simplified large alveoli. These other changes may be more

widespread than the very focal adenomatoid malformation. This is an area where past definitions and concepts may be confusing and simple descriptions of the airway and vascular connections may be better. At the younger end of the age spectrum, ILS and related lesions may be detected by prenatal ultrasound, with features shared with other cystic lung diseases. Occasionally the anomalous vessel will give rise to early cardiac failure, secondary to systemic hypertension. In “later life” and especially in adults, ILS presents more commonly with infection. Fifteen percent of ILS cases remain asymptomatic and are detected on routine radiology as a cystic area or a discrete mass. Bronchography shows ectactic bronchi near the lesion but none entering it.228 Computed tomography may confirm the cystic nature of the mass and demonstrate the systemic feeding vessel (Figure 26). Despite the varied ages of affected individuals and clinical presentations, other characteristics of ILS are similar.224 Fiftyfive percent of cases are left-sided229 and bilateral involvement has been reported. ILS is usually seen in the posterior basal segment of the lower lobe. The lesion receives its blood supply from either the thoracic or upper abdominal aorta, or from an artery arising from one of these portions of the aorta. The venous drainage is via the pulmonary veins of the affected segment or may occasionally join the azygos veins.230 Intralobar sequestration, unlike ELS, is rarely associated with other anomalies. ILS may be resected in the neonatal period, following prenatal detection. Macroscopically, the abnormal segment is not always readily distinguishable from adjacent normal lung unless there is well-developed cystic change (Figure 27). Histologically, inflammation is absent or minimal in these early presenting lesions. Bronchioles may be dilated with evidence of obstruction and mucus retention (Figure 28), sometimes throughout the abnormal segment. If features amounting to small cyst adenomatoid malformation are present, another result of bronchial obstruction, then these are sometimes referred to as “hybrid” lesions. The intervening alveoli may also be affected. Size is variable but their structure is simplified in comparison with normal adjacent lung (Figure 29).

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 27. Intra-lobar sequestration. Resected lung with systemic (clipped) vessel arrowed. On the right the lung cross section shows an ill-defined segment with prominent vasculature. Changes related to bronchial obstruction were present histologically. Cystic lung was diagnosed prenatally.

Figure 28. Mucus retention from bronchial obstruction in a sequestered lobe. Figure 29. Elastic stain of (left) normal lung and (right) sequestered lobe. In the sequested lobe the alveoli are simplified, smaller and there is an increase in elastic tissue.

Although alveolar hypoplasia is typically described,157 larger alveoli may also be seen, which may reflect the underlying obstruction. Resection in older children and adults often follows investigation for repeated infection. The abnormal segment presents as an atelectatic mass of lung or may be replaced entirely by a collection of thin to viscid yellow-white fluid or sometimes gelatinous material-filled cysts. There is pleural fibrosis and adhesions to adjacent structures. The cystic cavities may appear bronchiectatic but bronchiectasis does not have a systemic blood supply and is rarely as localized as an ELS. Because of repeated infections, fibrosis and cystic change tend to be more pronounced in the older patients. The fibrosis and cystic

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change are caused by the accumulation of secretions in the non-communicating bronchi. Histologically, pulmonary parenchyma is replaced by the results of chronic inflammation, largely secondary to infection. The cysts are lined by cuboidal, columnar or rarely squamous epithelium and contain amorphous eosinophilic material, as well as foamy macrophages. Residual bronchi and bronchioles are surrounded by lymphoplasmacytic infiltrates. There is cuboidalization of inflamed alveoli, and thick-walled systemic vessels litter the interstitium. The edge of the lesion may be sharply demarcated from the adjacent normal lung or blend diffusely with it. Acute bronchopneumonia may be seen. Squamous cell carcinoma has been described, but is exceedingly rare.231

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 30. (a) Congenital lobar emphysema. Chest X-ray showing congenital overinflation of the upper lobe of the left lung. The overinflation makes the lung more translucent. It is shifting the mediastinum to the right side. (b) Axial image of a CT scan viewed on lung windows show the darker low attenuation areas of overinflation and decreased vascularity.

Isolated systemic arterial supply Lungs with an anomalous arterial supply are often considered within the realm of sequestrations. These segments typically receive an arterial supply from the lower thoracic aorta, while the tracheobronchial anatomy is normal.216 The lung parenchyma is normal and complications are due to the cardiovascular effects of the aberrant vessel, rather than the increased risk of infection. Heart failure, due to shunting between systemic and pulmonary circulations, may be a presenting feature in infancy222 and hemoptysis in older age groups.232,233 Resection may be necessary but increasingly embolization is being used.234–236

Bronchopulmonary foregut malformation This term was coined by Gerle,217 to describe “sequestered lobes”, encompassing both ELS and ILS, where the abnormal segment communicates with the gastrointestinal tract.237,238 The abnormal pulmonary segment arises either from the esophagus (usually the lower part) or from the stomach; the former is commoner. It is right-sided in 70–80% of cases.238 The sex incidence is equal and most cases present in the first year of life, although adult presentation is recorded. Presentation may be due to chronic cough, recurrent pneumonia or respiratory distress. Associated anomalies are common, overlapping with ELS, and include diaphragmatic hernia, and abnormalities of the VACTERL type.239,240 The macroscopic and microscopic appearances of the abnormal lung segment broadly correspond to whether the segment is extra- or intra-lobar. These lung segments differ from typical sequestrations only in their communication with

the bowel, although they may demonstrate more intense inflammation. The open communication with the gastrointestinal tract leads to widespread honeycomb change.

Polyalveolar lobe

First described by Hislop and Reid,241 polyalveolar lobe is characterized by a significant increase in alveoli numbers in the presence of a normal bronchial tree and vascular pattern. Polyalveolar lobe primarily affects upper lobes, particularly the left upper, and causes complications due to compression of adjacent structures and mediastinal shift. The underlying cause of the abnormality is unclear. It presumably results from a relatively late gestational event, since the bronchial tree is established at 16 weeks of gestation. The relationship to adenomatoid malformation and the hyperplastic lung, seen in laryngeal atresia, is not certain. The increase in the number of distal airspaces is suggestive of underlying airway obstruction as a pathogenetic feature.83,242,243

Congenital lobar emphysema Congenital lobar emphysema is an overdistended segment or lobe of lung due to airway obstruction.153 The lesion can affect an entire lobe or part of a lobe. It may be diagnosed by prenatal ultrasound227,244–247 but might be initially labeled simply as cystic lung disase. It more commonly presents in the neonate or early infancy with life-threatening respiratory distress (Figure 30). The upper lobes are more frequently affected than the lower and males slightly more commonly than females.

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Macroscopically, the cut surface of the resected segment may appear relatively normal, although some distended alveoli may be apparent (Figure 31). Histologically, the lung parenchyma features distended terminal airspaces (Figure 32) and sometimes alveoli are ruptured. Bronchial compression or collapse, usually secondary to an intrinsic process, such as bronchomalacia, is responsible for the lesion.248 Since it may result from external bronchial compression, other congenital abnormalities, particularly congenital heart disease, such as

ventricular septal defects or Tetralogy of Fallot, are a common accompaniment.249,250 Lobar emphysema is usually acquired following secondary bronchial damage in early childhood. Bronchial damage may follow ventilation associated with chronic lung disease; sometimes with polyp formation251 and is reported in neonatal intensive care survivors.156 A form of lobar emphysema or emphysema affecting a whole lung may occur following bronchial damage during childhood viral bronchiolitis, otherwise known as Macleod syndrome (see Chapter 17).156,252

Pulmonary hyperplasia

Figure 31. Lobectomy for congenital lobar emphysema. Cut surface may look normal but foci of distended alveoli are visible. Compressed, normal lung is present above and to the right of the abnormal lung.

Pulmonary hyperplasia has a similarity to lobar emphysema in that both are the consequence of an obstructed airway causing an expanded lung. Pulmonary hyperplasia is usually bilateral and the obstruction is most often due to laryngeal atresia. Tracheal agenesis is also a reported association.253 The obstruction prevents the normal egress of fetal lung liquid, a chloride-rich secretion produced throughout in utero life by the lungs and which only stops at or shortly before delivery. Hyperplastic lungs may present in the antenatal period, when ultrasound can detect fetal ascites.254 The lungs may be hyperechogenic255 and, like the cystic malformations described above, a specific diagnosis may not be possible from the prenatal features. The results of high airway obstruction have been described under the term “congenital high airway obstruction syndrome (CHAOS)”. This is usually fatal at birth but, recently, prenatal detection has led to planned ex utero intrapartum treatment (EXIT). These are procedures in which there is an attempt to secure the airway at delivery. Some success has been reported.255 Figure 32. Normal lung (a) compared with lung resected for congenital lobar emphysema. (b) The emphysematous lung may appear relatively normal except for the overdistended airspaces.

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 7 Lymphangiectasia

1. Primary:

(a) limited to the lungs (b) pulmonary and mediastinal involvement (c) generalized (including intestinal, hepatic and osseous involvement)

2. Secondary:

to obstruction of the pulmonary venous outflow

Figure 33. Typical hyperplastic lung associated with laryngeal atresia in a mid-trimester loss. The heart is obscured and the diaphragm pushed downward and flat. The combined weight of these lungs (50 g) was five times the expected weight for gestation.

Pathologically, the typical macroscopic findings are those of grossly enlarged lungs that fill the chest tightly (Figure 33); there may be rib markings on the pleural surfaces. The diaphragm is pushed downward and is horizontal rather than dome-shaped.242 Pulmonary hyperplasia is often the first clue to the presence of a high airway obstruction.257 Laryngeal atresia may be a feature of Fraser syndrome,256 an autosomal recessive condition that may demonstrate a wide variety of abnormalities including cryptophthalmos, syndactyly, genital abnormalities and renal agenesis. The lung to body weight ratios are high. Histologically, the terminal sacs or alveoli may be distended and the numbers of alveoli appear increased (Figure 34). Sometimes lungs appear more mature than might be expected for gestation. Morphometry has confirmed that there is an increased alveolar surface area for body weight and sometimes for gestation.258 The amount of lung DNA per kg body weight is only marginally raised, suggesting that airway obstruction is not a great stimulus to cell proliferation.

Congenital pulmonary lymphangiectasis Congenital lymphangiectasis is a rare condition. It is commoner in males than females by a ratio of 2:1. It may be familial.259 It is frequently associated with other congenital anomalies, such as asplenia and congenital heart disease, including total anomalous venous drainage, common atrialventricular valve defects, ostium secundum, pulmonary stenosis, ventricular septal defect, mitral atresia, hypoplastic left heart and cor triatrium. Accessory lobes and renal abnormalities, such as a hydroureter, have also been documented.260 The condition has also been reported in association with Noonan syndrome, a rare autosomal dominant genetic disorder characterized by facial dysmorphism, short stature and cardiac anomalies amongst other features,261,263 and congenital icthyosis.262

Figure 34. Hyperplastic lung at 23 weeks. For 23 weeks there are more airspaces distal to the bronchioles than expected, and distension is also prominent.

Lymphangiectasia has been subject to a number of different classifications264,265 and can either be due to a primary developmental abnormality of the lymphatics or secondary to obstruction (Table 7). The latter may occur in association with congenital heart disease, especially abnormalities of pulmonary venous return. Secondly, lymphangiectasia can be confined to the lung or can be generalized. In the latter case, it may be associated with other often syndromic conditions such as Hennekam syndrome, in which there is facial dysmorphism with intestinal lymphangiectasia, lymphedema of the limbs and mental retardation.266 While congenital lymphangiectasia is usually sporadic, autosomal recessive inheritance is recognized in some cases.186 Initial presentation may be antenatally in the second trimester with maternal polyhydramnios and fetal hydrops, associated with a marked pleural effusion.267 There may be a chylothorax.268 Stillbirth may occur, in which case the lymphangiectasia may be an autopsy diagnosis. The disease more commonly presents in the newborn within hours of birth and causes respiratory distress. Macroscopically the lungs are larger than normal, inelastic, lobulated and firm. Multiple, up to 1.5 cm in diameter, cysts are fluctuant on palpation and may be seen through the pleura. They may be mistaken for air cysts due to interstitial emphysema. The cysts may be present around bronchi and interlobular septa. Subpleural lymphatics are easily seen and there is a honeycomb effect with small cysts containing clear

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

(b)

(c) Figure 35. Congenital lymphangiectasia. (a) Lung is lobulated and firm. (Photograph courtesy of Dr Mike Ashworth, London, UK.) (b) Cysts are apparent on the cut surface. (c) The interlobular septum is expanded with dilated thinwalled lymphatics.

fluid (Figure 35a,b). The cysts increase in size towards the hilum. These lymphatics form a network of channels rather than isolated cysts.268 Histologically there are prominent lung septa and dilated lymphatics with thin-walled endothelial-lined spaces and sporadic smooth muscle varying from 15 mm to 1.5 cm in diameter (Figure 35c). These cysts localize to thickened visceral pleura, interlobular spaces and bronchovascular sheaths.269 Cells lining the dilated channels stain with D2–40, confirming their lymphatic nature.270,271 The intrapleural and interlobular lymphatics are usually more prominent than the others and are devoid of valves. The ectatic lymphatics are seen in the early stages of the disease close to pleura and next to pulmonary veins. Lymphocytes may be present and lumina contain eosinophilic material. There is no endothelial overgrowth to suggest a lymphangioma.

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While a rapidly fatal outcome is usual,272 longer survival is not unheard of.273 The outcome may be modified by in utero therapy, such as thoracocentesis to reduce the pressure on the lungs and the likelihood of pulmonary hypoplasia. This is followed by surgery in the immediate neonatal period.267,271

Enteric (enterogenous) cysts These cysts are not pulmonary in origin but are mentioned briefly as they enter the differential diagnosis for bronchogenic cysts. Enterogenous cysts are a form of unilocular gastrointestinal duplication cysts, which may occur anywhere along the gastrointestinal tract. In the thorax they typically occur in the right posterior mediastinum attached to the esophagus. Bronchial connection is rare. These cysts may lie in the mediastinum and can be associated with anomalies of the lower cervical and upper thoracic vertebrae.

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Presentation is often incidental and may occur in adults. Multiple cysts have been reported.274 In the neonate, cough or other respiratory symptoms occur in approximately 75% of cases.275 Histologically, squamous epithelium is typical but gastric fundic epithelium or small intestinal epithelium can also be seen. The wall may contain muscle but not cartilage. Pancreatic islets in the wall have also been reported.276 The gastric epithelium can cause ulceration and cyst rupture. Thorascopic resection is frequently utilized.275 Morbidity is usually limited to damage to local structures.

Alveolar capillary dysplasia Alveolar capillary dysplasia (ACD) refers to a combination of poor capillarization of the terminal sacs or alveoli. The incidence is unknown, although results from one center, where six cases were seen within an 18-year period, suggested it is a relatively common cause of clinically diagnosed idiopathic pulmonary hypertension in the newborn.277 Presentation is mainly in the early neonatal period, although some cases present slightly later, up to 2 months of age.278,279 The clinical picture is that of respiratory distress associated with severe pulmonary hypertension in the presence of a structurally normal heart.280–282 If a cardiac anomaly is present, it is one not normally associated with pulmonary hypertension. Diagnosis can be made on biopsy if it aids in management. Unfortunately diagnosis is often only made at autopsy.283,284 Alveolar capillary dysplasia is associated with other malformations, including congenital heart disease,285 gastrointestinal anomalies such as malrotation286 and adenomatoid malformation.287 While most cases appear to be sporadic, there is evidence of autosomal recessive inheritance in some families.288 Up to 10% of cases may have a familial association.289

Macroscopically the lungs are unremarkable although the effects of pulmonary hypertension may be seen in the right ventricle. Microscopically, the capillary dysplasia is a failure of normal capillary development.290 Superficially, alveolar vascularization may appear normal and congested, and thin-walled vessels may be prominent. However, these vessels are within the interstitium and fail to push into the alveolar walls to form normal very thin blood-air barriers (Figure 36). The lack of normally positioned capillaries is highlighted by vascular immunohistochemical markers, such as CD31, CD34 or Factor VIII (Figure 37).

Figure 36. Alveolar capillary dysplasia in a 34 week gestation infant. Dilated capillaries may be very prominent within the interstitium but few are pushing into the alveoli to form normal blood-air barriers. Maturation appears immature. Figure 37. CD34 staining of normal lung and alveolar capillary dysplasia. Diseased lung (left) features interstitial capillaries in contrast to normal lung of similar gestational age (right).

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 38. (a) Misalignment of the pulmonary veins commonly seen in association with alveolar capillary dysplasia. Pulmonary veins share the same adventia as the arteries. (b) Architecture may be highlighted with an EVG stain.

Misalignment of the pulmonary vein is usually seen in association with ACD. One observes an abnormal vascular bundle adjacent to the bronchial structure containing both pulmonary arteries and veins (Figure 38). Normally the pulmonary artery will show marked medial hypertrophy. These abnormalities are not always uniformly present throughout the lungs. Normal blood-air barriers do not form. It is possible the large capillary-like vessels in the intersitium are immature and fail to differentiate into capillaries. This failure of differentiation indicates they are incapable of forming the smaller normal capillaries that have the capacity to push into the developing alveoli and form normal blood-air barriers.290 At present, ACD is fatal. Alveolar capillary dysplasia is associated with a variety of microdeletions or point mutations in the FOX gene cluster at 16q24.1.289 In one study, point mutations in the FOXF1 gene were associated with bowel malrotation, while microdeletions of FOXF1 were also associated with hypoplastic left heart syndrome. Deletions in all three FOX gene clusters (FOXF1, FOXC2 and FOXL1) demonstrate gastrointestinal atresias and a variety of cardiovascular malformations in addition to ACD.

Abnormal and ectopic tissues in the lungs Scattered striated muscle fibers are well described in association with small cyst adenomatoid malformations. More widespread proliferation, referred to as rhabdomyomatous dysplasia (Figure 39), may be isolated or an incidental finding in association with other abnormalities such as sequestration291 or congenital heart disease.292–294 Ectopic pancreatic295 and adrenal cortical tissue296 have been reported. The latter

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Figure 39. Widespread rhabdomyomatous tissue in a segment of lung.

may develop from early fetal coelomic cells lying lateral to the root of the mesentery. These cells may become transposed above the growing septum transversum and incorporated into the pleura. Heterotopic tissues are usually an incidental finding in the pediatric age group but there is a low risk that they may give rise to tumors later in life.297 Ectopic tissues are occasionally found for reasons other than aberrant development. Pulmonary glial nodules are associated with anencephaly and this may be related to the cerebral disruption and transfer of tissue to the lung, either via blood vessels or ingestion (Figure 40).298,299 Intravascular cerebral tissue, usually cerebellum, may rarely occur as an embolic phenomenon following a difficult or traumatic, usually term,

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 40. Ectopic, probably aspirated, glial tissue in the airway in an anencephalic fetus. Figure 42. Ectopic liver tissue above the right hemidiaphragm.

Figure 41. Cerebellar tissue within pulmonary vasculature. This is an important, if rare, marker of traumatic injury, often at birth.

mortality.302–304 Ten to 20% of neonatal autopsies feature this deformation. The causes of pulmonary hypoplasia cover a broad spectrum of conditions (Table 8) but can be grouped into four main categories.

Inadequate thoracic volume Accounting for 30 to 40% of cases, simple physical restriction of the thoracic space is responsible for poor lung growth. Specific causes include many of the skeletal dysplasias, where there is a small thoracic volume due to poor rib growth. Displaced abdominal organs, e.g., in diaphragmatic hernia, is one of the commonest causes, particularly of unilateral pulmonary hypoplasia. Pleural effusions, e.g., associated with fetal hydrops, form a further important subgroup.

Impairment of fetal breathing delivery.300,301 It should not be mistaken for hamartomatous or heterotopic tissue (Figure 41). The cerebellar tissue in the lung may not be functionally significant compared with emboli into the coronary vessels, which it often accompanies, but it is an important and usually more obvious marker. Ectopic liver tissue, including bile duct epithelium invested by visceral pleura, may rarely be found in the right lower lobe (Figure 42).

Pulmonary hypoplasia Strictly speaking, pulmonary hypoplasia is a deformation rather than a malformation. It is due to physical restrictions on lung growth or pathophysiological events that impede lung growth, occuring potentially late in gestation. It is one of the commoner conditions encountered in perinatal pathology. In both the pre- and post-surfactant eras, pulmonary hypoplasia contributed and contributes significantly to neonatal

This is a difficult group to define and may only account for 5–10% of cases, when it is acting in isolation. Experimental evidence suggests the low-volume, high-frequency breathing normally performed by the fetus in utero is important in lung development.305–307 Interference with fetal breathing may be a factor in some severe cerebral malformations. Rare examples of pulmonary hypoplasia are associated with severe anencephaly. Studies also suggest a possible relationship between poor development of the arcuate nucleus and pulmonary hypoplasia.308,309 It may be acquired if cerebral damage from hypoxia or ischemia affects the respiratory centers. Poor muscular activity, as in some congenital muscular dystrophies, may produce a similar result.

Oligohydramnios Pulmonary hypoplasia associated with maternal oligohydramnios is one of the most common presentations. Oligohydramnios may result from reduced production of fetal urine

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 8. Classification of pulmonary hypoplasia

Mechanism

Subgroup

Examples

Primary or idiopathic

Chromosomal abnormalities Familial

Trisomy 21, 18, 13

Inadequate volume

Skeletal

Skeletal dysplasia thoracic

Impairment of fetal breathing

Diaphragmatic hernia Effusions Cerebral

Skeletal muscle Diaphragm Oligohydramnios

Inadequate production

Excessive loss

Rhesus disease Fetal hydrops Hypoxia/ischemia Malformation – Anencephaly with brain stem involvement Congenital dystrophy Amyoplasia ?Exomphalos Urinary tract anomalies – Renal agenesis – Cystic dysplasia – Urethral obstruction Chronic leakage of liquor amnii

N.B.: some of the specific examples might fit into more than one group; e.g. diaphragmatic hernia might cause pulmonary hypoplasia due to the presence of herniated bowel in the thorax but also the absence of normal breathing movements.

due to renal tract abnormalities (e.g. renal agenesis, cystic dysplasia or urinary tract obstruction), or chronic loss of amniotic fluid from premature rupture of membranes. From the pathogenetic viewpoint, this is one of the more problematic groups. Initial concepts suggested oligohydramnios and the restricted uterine cavity allowed compression of the fetal thorax, but later theories concentrated on the significance of lung liquid in normal lung development.310–312 During development, the lung produces a chloride-rich lung liquid that gradually drains into the amniotic fluid via the upper respiratory tract. Secretion is normally switched off at birth. The relevance of this fluid to normal lung development was suggested by observations, amongst others, of natural experiments, such as is seen in Fraser syndrome.256 When the major abnormality is limited to renal agenesis, then the lungs are likely to be hypoplastic, as expected in the presence of oligohydramnios. However, when, as sometimes may occur, there is laryngeal atresia as well as renal agenesis, despite the oligohydramnios, the lungs are frequently large and “hyperplastic” rather than hypoplastic (see also pulmonary hyperplasia, above). This led to the recognition that distension of the lung generated by normal lung liquid pressure is important for

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normal in utero lung development. This pressure is reduced in the presence of oligohydramnios when there is rapid lung liquid efflux. In Fraser syndrome, however, the pressure is maintained by airway obstruction caused by the laryngeal atresia. This concept has led to attempts to reverse the pulmonary hypoplasia associated with some conditions, most commonly diaphragmatic hernia, by occluding the airway prenatally with plugs.313,314 This has met with some success. In a wide range of animal studies there is good evidence that airway occlusion enhances lung growth and maturation although the latter is not entirely normal.315

Primary or idiopathic This is a diagnosis of exclusion, reserved for instances where pulmonary hypoplasia cannot be attributed to any of the above causes.316,317 Sometimes, in a severely growth-retarded infant, the lungs are particularly affected. Genetic factors are important in some cases and hypoplasia can be found in association with trisomy 13, 18 or 21.303,306 Hypoplasias in twins318 or familial cases319 are two examples of primary hypoplasia. Prenatally, pulmonary hypoplasia is often diagnosed after a known association has been identified. It is often inferred by ultrasound scanning when pathologies, such as lethal skeletal dysplasia or chronic pleural effusions, are recognized. Because of these well-known associations, presentation of pulmonary hypoplasia at birth may be anticipated and indeed is an important aspect in the counselling of parents about prognosis. The infant may be born apparently normally but deteriorates rapidly with severe respiratory distress that responds poorly to resuscitative measures. With ventilation, chest movements may be poor and, even if some response is obtained initially, increasing pressures are required with diminishing results. Death may occur in minutes or hours. Occasionally, less severe cases may permit initially successful ventilation. Respiratory distress syndrome often develops, requiring relatively high pressures to maintain adequate oxygenation. Outcomes vary. Some infants die in the neonatal period and others may progress to chronic lung disease (see below). In these “clinical” cases, the criteria for diagnosis are often presumptive. Pulmonary hypoplasia may be first suspected from the external appearance of the baby. Severe oligohydramnios leads to external compression of the baby and external features associated with Potter syndrome, small nose, low-set ears and micrognathia, may be evident. Pathological diagnosis of pulmonary hypoplasia at autopsy is usually straightforward, although it may be missed.320 In stillbirths and early neonatal deaths, the lungs appear small within the thorax (Figure 43). After removal, the lungs are clearly small, relative to the heart (Figure 44). If the infant is resuscitated successfully for a time, this feature may not be so striking. Careful inspection of the surface may reveal subpleural pulmonary interstitial emphysema. A more objective measure can be used by determining the lung:body weight ratios. Ratios of < 0.015 in fetuses/neonates < 28 weeks gestation or < 0.012 in fetuses/neonates  28 weeks gestation

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 43. Bilateral pulmonary hypoplasia secondary to severe chronic pleural effusions.

are indicative of pulmonary hypoplasia.321,322 These ratios define pulmonary hypoplasia at the clearly pathological severe end of the spectrum, although lesser degrees of pulmonary hypoplasia may still impose some functional impairment.323 One must remember pneumonia or significant neonatal survival tends to increase lung weights. Radial alveolar counts are also useful in establishing the diagnosis. Described by Emery and Mithal,324 this procedure is a measure of the complexity of the acinus and involves counting the number of developing alveolar spaces along a line from the mid-point of a respiratory bronchiole to the nearest pleural or interlobular septum. Morphometric studies indicate hypoplastic lungs from any cause have a reduced radial alveolar count.321 The normal count and range gradually increases from early gestation through childhood.325 Cooney and Thurlbeck306,325 pointed out a number of precautions and allowances that have to be made in applying this technique. These same researchers also326 suggested the most satisfactory evaluation of lung growth was obtained by combining the radial count and the fixed lung volume. They found, in a small number of cases studied, that radial count was a poor predictor of lung maturity in hypoplastic lungs, while the ratio of lung/ body weight was only of use if it was severely depressed. Histologically, the lung is small and there is relatively poor development of the acinus. Wigglesworth defined two main groups of pulmonary hypoplasia: those with poor growth but apparently normal maturation of the epithelial component; and those lungs with impairment of both lung growth and maturation (Figures 45 and 46). This latter form is seen more in pulmonary hypoplasia associated with oligohydramnios.258,327 Immaturity of surfactant development is associated with the oligohydramnios cases.328 Elastin is notably absent from septal crests, especially in those cases with impairment of both lung growth and maturation.329 Assessing the full impact of pulmonary hypoplasia on neonatal mortality and morbidity is problematic, in part

Figure 44. Typical severe pulmonary hypoplasia. With a normal-sized heart, the diaphragmatic surface of the lungs should be close to the heart apex.

Figure 45. Severe pulmonary hypoplasia in a baby of 37 weeks gestation. The lung is very small and immature (inset).

because of the difficulties in clinical diagnosis and the spectrum of disease.323 There are those with severe hypoplasia, and 100% mortality where the etiology can be traced back into early gestation and oligohydramnios (see above). In other parts of the spectrum a mortality of close to 50% is noted in babies with probable pulmonary hypoplasia associated with prolonged membrane rupture from the second trimester.330

Diaphragmatic eventration and hernia Eventration of the diaphragm describes a membrane-covered hernial sac, representing one half of the diaphragm, which

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 46. At appprox. 35 weeks, normal lung (left) compared with hypoplastic lung (right) where growth distal to terminal bronchiole is poor. Maturity, however, is comparable.

perforation site. These hernias are usually small and covered by both pleura and peritoneum. They may be associated with trisomy 18. Presentation may be in the newborn period with respiratory distress and signs of mediastinal shift, or the infant can be asymptomatic and present at a later time. Prognosis of isolated eventration is good, with many repairs now undertaken thoracoscopically.332

Diaphragmatic hernia

Figure 47. Eventration of the diaphragm. Part of the liver protrudes into the right hemithorax covered by a thin membrane partly lifted by the probe.

protrudes into the thorax (Figure 47). It is probably secondary to denervation, atrophy and muscle development failure.258 This entity may be associated with pulmonary hypoplasia331 or extralobar sequestration, especially if the eventration is on the left side. Eventration is often indistinguishable from a muscular deficiency with enlargement of the central tendinous portion of the diaphragm. This is termed a hernia of the foramina of Morgagni and is formed by protrusion of the diaphragm through a muscular defect at the internal mammary artery

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Congenital diaphragmatic hernias (DH) may be central, left or right-sided. Left-sided defects are commonest (85%) with an incidence of one in 2000–3500 deliveries; 5% are bilateral.333 Diaphragmatic hernia is often associated with other congenital abnormalities, particularly those of the central nervous system, gastrointestinal tract, genitourinary tract and heart.334 In one series, nearly 20% of babies had other malformations, likely to be fatal.335 Usually diaphragmatic hernias are sporadic but there is a high incidence of associated chromosomal abnormalities, particularly in cases detected early by fetal ultrasound.336 Familial examples are well recorded.337 Fryns syndrome is an autosomal recessive syndrome with craniofacial and neurological abnormalities, distal limb and nail hypoplasia, genitourinary and cardiac anomalies, particularly ventriculoseptal defect, as well as diaphragmatic aplasia.339 Presentation is now common in the antenatal period. Polyhydramnios may occur in late gestation but earlier diagnosis may follow routine ultrasound when a mass, which may be cystic and sometimes associated with mediastinal shift, is detected. The differential diagnosis includes pulmonary malformations, such as adenomatoid malformation or sequestration. In the neonatal period, presentation may be at birth with

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 48. Typical severe diaphragmatic hernia where the left hemidiaphragm is aplastic. In this case, bowel is the most visible abdominal content in the thoracic cavity causing mediastinal shift.

respiratory distress if pulmonary hypoplasia is severe. Otherwise, herniated bowel filled with air causes lung compression and respiratory distress in infants.333 Late presentation in childhood usually manifests with pulmonary symptoms; however, gastrointestinal symptoms, such as vomiting and pain related to incarcerated bowel, may be protean.340 Pathologically, diaphragmatic aplasia or agenesis, representing the extreme end of the spectrum, is often seen. Rarely, this aplasia may be bilateral. In a left-sided defect, the hemithorax may contain part of the left lobe of liver, spleen, bowel loops and stomach (Figure 48). Because of the bowel displacement, malrotation is common. The associated left lung is usually hypoplastic (Figure 49) and there may be significant right-sided mediastinal displacement, which, if severe, may also cause a degree of right lung hypoplasia. Histology is not especially contributory, although it may allow a comparison between a severely hypoplastic left lung and relatively normal right lung. In right-sided hernias, the defect is often “plugged” by the liver. While this may be in the right hemithorax, the pulmonary hypoplasia may not be so severe. The prognosis of diaphragmatic hernia, despite newer therapeutic modalities, such as nitric oxide and extracorporeal membrane oxygenation (ECMO), remains guarded. Early presentation, a marker of severity and associated malformations equate with mortality rates of 40–50%.335,341 Most deaths occur early before surgery. Long-term morbidity with gastroesophageal reflux may be significant.

Primary ciliary dyskinesia Primary ciliary dyskinesia has taken its place alongside CF and A1AT as one of the “big three” genetic causes of chronic lung disease. The associated condition was first described in 1933 by Kartagener in a group of men with situs inversus, bronchiectasis, chronic rhino-sinusitus and absent frontal sinuses. Some were subsequently found to have immotile cilia. The disorder is due to a primary abnormality of the ciliary

Figure 49. Hypoplastic left lung secondary to severe diaphragmatic hernia. Here, the right lung is relatively normal.

axoneme. Cilia have critical functions in the respiratory tract, sperm motility and the determination of normal lateralization during development. The prevalence, which is probably underestimated, is of the order of 1:15 000–30 000 births.342 Primary ciliary dyskinesia is an autosomal recessive condition with some reports of X-linked disease. The ciliary axoneme contains over 250 proteins, so there are a large number of genes associated with ciliary dysfunction.343 Abnormal genes have been identified, amongst others, on chromosomes 5p15, 7 and 9p13–21. There is increasing potential for genetic testing, with a mutation on one allele being identified in up to 25% of PCD cases.343 Diagnosis is often relatively late because many of the symptoms and signs are common and nonspecific.342 The possibility of PCD should be considered in children with recurrent respiratory and ear infections unresponsive to treatment. Later, infertility with azoospermia may lead to diagnostic testing.344 The main CT features associated with suspected PCD include bronchiectasis, bronchial wall thickening and dilatation, mucus plugging, mottled shadowing, hyperinflation, and dextrocardia.345,346 The lower lung fields are usually involved, with relative sparing of the upper lobes. The right middle lobe and lingula often demonstrate bronchiectasis. Initial investigations usually involve screening procedures. The saccharine test measures nasal mucociliary-clearance time. A small particle of saccharine, approximately 0.5 mm3, is placed on the child’s inferior turbinate. The normal patient usually appreciates the strong sweet taste in less than 30 minutes.347 This test may be inappropriate for children under the age of six because of compliance problems. The presence of a low nasal nitric oxide has also been used as a screening test but may be less useful in children.348

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 51. Abnormal extra tubules in two of these cilia. This sort of abnormality is not necessarily associated with ciliary dykinesia.

Figure 50. Cilia with normal arrangement of 9 + 2 tubules. The central tubules are oriented in a similar direction. The inner and outer dynein arms (arrowheads) are not always easily visible

The diagnostic investigations of choice are an assessment of ciliary beat frequency and beat pattern, together with ultrastructural studies. It is possible to assess nasal respiratory ciliary motility and ultrastructure 16 hours after death and possibly longer.349 Ciliary beat assessment is a non-invasive test, requiring a cytological brush sample from the inferior turbinate and lateral nasal wall mucosa.350 Frequency is measured photometrically, as the strips of epithelium are easily seen by bright-field illumination. It is necessary to quantitate not only ciliary beat frequency, but also beat pattern. In PCD the beat frequency is significantly reduced and the ciliary wave form is grossly abnormal.350,351 Ciliary beat observations are complemented by ultrastructural studies (Figure 50). Up to 10% of cilia from control subjects may have abnormalities, so the mere presence of an ultrastructural anomaly is insufficient for diagnosis.352 The initial defect described in Kartagener syndrome was a lack of dynein arms in the ciliary axoneme.353 Most defects associated with PCD are associated with absence of an outer dynein arm, inner arm or a combination of both.354 Primary ciliary diskinesia may be associated with absence of the inner tubular pair.355 Papon et al., in a study of over 1100 patients investigated for ciliary dyskinesia, found that 65% of those with a significant cilial ultrastructural abnormality had an anomaly of the outer dynein arms (shortening or absence).352 One-third also had an inner dynein arm abnormality, 18% had an isolated abnormality of the inner dynein arm, and 20% featured an abnormality of the central microtubules. The proportions of the abnormalities were broadly similar in adults and children. Cilial orientation may also be important to normal function and a case has been described with normal ciliary structure but random ciliary orientation. The direction of effective

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ciliary stroke in this patient was random and there was a reduction in ciliary beat frequency. Thus, effective mucociliary clearance did not occur.356 Not all cilial abnormalities are associated with PCD. For example, patients with retinitis pigmentosa have abnormal cilia with irregularly arranged outer doublets and a variation in the number from two to 10. Respiratory tract viruses, air pollutants and other chronic respiratory diseases can also cause abnormal cilia. Compound cilia, supernumerary microtubules in the axoneme, and loss of cilia or ciliated cells are nonspecific changes found in either congenital or aquired diseases.357 Heavy smokers may show three types of abnormalities. These include compound cilia with two to 27 axial filament complexes, swollen cilia with an increase in matrix but only a single axial filament, and cilia with no central complexes. Such cilia are considered functionally incompetent.358 Further abnormalities noted in patients, apparently without the immotile cilia syndrome, include intracytoplasmic microtubular doublets and cilia within periciliary sheaths.359 As a guide, structural abnormalities of varying types, found in less than 10% of cells, are unlikely to indicate a primary cilial disorder (Figure 51).360 While PCD represents a generalized ciliary disorder, acquired injury to the ciliated epithelium may be restricted to one site in the body. The nasal mucosa may show secondary changes more frequently, possibly because it is more susceptible to infection and other insults.361 Thus, when a nasal biopsy is inconclusive, bronchial biopsy may be of benefit. If distinguishing primary from secondary changes in the cilia proves persistently difficult, it may be necessary to culture ciliated cells. Secondary damage is rare in these cells.342 Lung disease, such as bronchiectasis, associated with PCD is progressive if not treated. While this may only emerge in late childhood or adolescence, there is evidence that lung damage can be detected even in infancy if considered.362 Critically, there is evidence that appropriate therapy, such as

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

physiotherapy and aggressive treatment of infection, may prevent further deterioration of lung function.363

Table 9 Changes in care for CF patients over the last 25 years

1982

2008

Cystic fibrosis Introduction

Patient characteristics Age 16–31 years Lung and liver disease

Age 16–67 years Multisystem disease

Cystic fibrosis is the most commonly inherited lethal disease. However, a disease previously associated with childhood mortality is now one of adult survival. The gene was isolated in 1989.364–366 This seminal scientific discovery, as yet, has not been translated into therapy; but mean survival is now into the fourth decade of life, with some patients reaching a pensionable age. It is suggested that the child born today with cystic fibrosis will live into the sixth decade of life.367 There are three reasons for this hugely improved survival. Continuous expert pediatric and adult care delivered by a multidisciplinary team from a recognized CF center results in better nutrition and pulmonary function – the two main prognostic indicators for survival.368 Secondly, many more therapeutic options have become available over the last 25 years, all of which can be tailored to meet the clinical needs of the individual patient as their disease progresses (Table 9). Finally, as the disease progresses to the pre-terminal stages, various interventions, such as non-invasive ventilation (NIV), can bridge the survival gap for several years until limited donor organs can be procured for double lung transplantation. Nevertheless, pulmonary disease progression is inexorable and lethal.

Local management Few treatment options Small teams No supporting teams

Complex treatment Large teams Supporting team – Interventional radiology – Diabetes – Obstetrics – Renal – Gastroenterology – Transplantation – Non-invasive ventilation – Accountants

National organization Few CF centers No funding No lung transplantation in UK No standards/guidelines No CF training

Many CF centers Specialist funding Six transplant centers Multiple standards Training obligatory

Clinical features Cystic fibrosis can present at birth with acute small bowel obstruction due to a meconium ileus. Small bowel contents solidify and surgery is usually required. Otherwise, clinical diagnosis is made during early childhood due to failure to thrive as a consequence of pancreatic insufficiency, or due to recurrent chest infections. Diagnosis is confirmed with a sweat test and DNA typing to define the specific gene mutation. Neonatal screening is now available; mild cases are diagnosed early and patients receive expert care. Atypical mild cases may present later in life, usually with a lone symptom such as infertility. In these instances DNA typing confirms the presence of a recognized mutation. The progression of lung disease is relatively slow due to maximized treatment. Yet as time passes, opportunistic pathogens colonize the lungs, leading to chronic infection. Cough becomes persistent and troubles sleep, while the sputum becomes more purulent, occasionally blood-stained and greater in volume (Figure 52). Hemoptyses are common and can occasionally be massive (500 ml over 24 h) and life-threatening. The bronchial arteries to the upper lobes are characteristically enlarged in CF and embolization of these arteries can be life-saving (Figure 53). Inflammation of the pleura is frequent and pleuritic pain is extremely common in moderate to severe disease. Sinusitis

Figure 52. Large jar containing 24 hours of purulent sputum from CF adult with severe lung disease.

and nasal polyps can cause facial discomfort, headaches and nasal obstruction. As the lung disease progresses, the airways progressively narrow due to a combination of chronic infection and aggressive inflammation. The patient becomes increasingly breathless on exercise and then at rest due to hypoxia and requires supplemental oxygen. Pulmonary hypertension ensues with muscularization of the pulmonary arteries and enlargement

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

(b)

Figure 53. Limited angiogram showing bronchial arteries. (a) Pre- and (b) post-embolization for life threatening hemoptysis.

of the right ventricle (Figure 54). When medical treatment has failed, it is important to introduce palliative care. The decision as to the correct timing for this can be extremely difficult, especially if the patient is on an active transplant list. The clinical signs of respiratory disease are remarkably few. Clubbing of the fingers occurs in nearly all patients with severe disease. On auscultation, coarse inspiratory crackles can be heard over the upper lobes, where CF lung disease initially develops. Transient pleural rubs can be heard during infective exacerbations. Acute onset of breathlessness with chest pain and unilateral decreased air entry is diagnostic of pneumothorax. Pneumothoraces are due to perforation of ruptured cysts and indicate progressive disease and a poor prognosis. Unless the patient receives a transplant, death from respiratory failure is inevitable. At post-mortem the lungs are contracted, bronchiectatic and pus-filled (Figure 55).

Genetics and the basic defect Cystic fibrosis is the commonest lethal autosomal recessive disorder amongst Caucasians. In the UK, 1 in 20–40 people carry the gene for CF and there is an annual incidence of 1 in 2500 live births. Currently there are approximately 8000 people with CF in the UK, with equal numbers of adults and children. The race to identify the gene was concluded in 1989 when it was cloned and sequenced by an American and Canadian

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consortium.365,366 The gene is located on the long arm of chromosome 7. It is a 1480 amino acid protein. A single amino acid deletion, phenylalanine at position 508 (Δ F508) is the most frequent mutation and the overall incidence in the Caucasion population of Europe and North America is approximately 70%. The protein encoded by the mutant gene has been named the cystic fibrosis transmembrane conductance regulator (CFTR). It is located in the apical membrane of epithelial cells. Epithelial cells lie on the surface of all ductal systems in the body; the bronchus, the biliary tree, the pancreatic ducts, the gastrointestinal tract, the renal tubules and the vasa deferentia. The expression of abnormal CFTR in all ductal systems of the body accounts for CF being a systemic disease. The main biochemical difference between normal and abnormal CF cells is a decreased permeability of chloride transport across the epithelial membranes. It is now accepted that CFTR functions as a chloride channel. Over 1500 mutations of the CF gene have been recorded so far. These mutations are allocated to six different classes, based upon how the CFTR is processed.369 Class mutations 1, 2 and 3 are associated with more severe disease, poor pulmonary function and pancreatic insufficiency. Class mutations 4, 5 and 6 are associated with pancreatic sufficiency, milder disease and a better survival. The enormous number of mutations and division of these mutations into separate classes, according to degree of CFTR function, accounts for the great phenotypic variation and differential pathological expression of the disease.

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 55. Cystic fibrosis. Bronchiectasis and airway pus are obvious.

Figure 54. Thickened right ventricle wall in a CF adult with severe pulmonary hypertension

Pathophysiology of the airways and the basic defect

Folklore records “the child will die soon whose forehead tastes salty, when kissed” recognizing the lethal consequences of CF.370 Prior to genetic screening, a mother who had already had one child with CF would diagnostically kiss her second child at birth to see whether she had been blighted again. As noted, epithelial cells are expressed in all ductal systems in the body but progressive bronchial sepsis accounts for the considerable morbidity and almost total mortality in cystic fibrosis. Although a systemic disease, the main thrust and discipline of care and treatment is directed towards the chest. In the lungs, epithelial cells have both a re-absorptive and secretory function. The correct composition of water and electrolytes in epithelial secretions is essential to fulfill the cellular function of protection and lubrication. Lung health is maintained by the movement and progression of airway surface liquid (ASL) from the distal to the proximal airways. This movement and removal of the ASL is maintained by the regular coordinated beating of the ciliary escalator. Airway surface liquid is composed of two layers, an upper mucous layer, termed the gel, and a periciliary layer, the sol. Recent work has shown the ASL in CF is disrupted, as a consequence of the abnormal functioning of CFTR.371 Chloride impermeability leads to excessive absorption of sodium ions into the epithelial cell with dehydration and

reduced volume of the ASL on the bronchial luminal surface. Reduction and increased viscosity of the ASL impairs the efficiency of the ciliary escalator and the clearance of secretions from the airways. In addition, the constituents of the ASL, which act as a usual defense mechanism, are compromised and less efficient at eliminating opportunistic pathogens, such as Pseudomonas aeruginosa (PA). Over the last few years there has been increasing awareness of the importance of the submucosal glands, which express significant amounts of CFTR, in the pathogenesis of CF airways disease.372,373 Defects have been identified in submucosal gland anion secretion, altering the rheology of the mucus from the glands. These recent findings are compatible with the longstanding recognition that hyperplasia of the submucosal glands and mucin occlusion of the gland ducts are one of the earliest histological hallmarks of CF.374 Gland ducts can become occluded during the third trimester of fetal life.375

Infection and inflammation Apart from distended mucus glands, the lungs of fetuses with CF have normal histology at birth. However, during early childhood, the airways become chronically infected with unusual pathogens which continue as patients grow older. Early in life there is a persistent and self-damaging exuberant host inflammatory response. The cellular response in CF is mediated primarily by neutrophils. The airways demonstrate a neutrophilic bronchitis/bronchiolitis (Figure 56). There remains an unresolved debate as to whether the milieu of the CFTR disrupted airways produce a primary inflammatory response or the aggressive response is to the opportunistic pathogens.376 The initial infecting pathogens are Haemophilus influenzae and Staphylococcus aureus. By adulthood the majority (90%) of CF patients are chronically infected with PA (Figure 57). The CF airways with reduced defenses and a volume-diminished ASL are especially hospitable to PA. Once established in the airways, PA alters its phenotype, producing an alginate, which enmeshes the bacteria. Neither neutrophils nor antibiotics can penetrate the mucoid barrier and eliminate the pathogen (Figure 58). Infection with the group of Gram-negative bacteria, known as the B. cepacia complex, carries an especially sinister

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Figure 57. Cystic fibrosis. Typical mucoid P. aeruginosa on a culture plate. Figure 56. Cystic fibrosis with purulent bronchiolitis.

the most virulent and most transmissible organism of the B. cepacia complex, are disqualified from transplantation. The injured airways of CF patients are receptive to a Pandora’s box of unusual pathogens, including but not limited to Aspergillus species, atypical mycobacterium, methicillinresistant Staphylococcus aureus, Achromobacter, Ralstonia, Stenotrophomonas maltophilia and Pandorea. Additionally, all the usual respiratory viruses are highly pathogenic to CF airways, resulting in prolonged infective exacerbations and increasing the likelihood of chronic infection with PA. P. aeruginosa is more likely to adhere to damaged epithelium.377

Histopathology and histopathogenesis of lung disease Figure 58. Cystic fibrosis. Neutrophils are unable to penetrate alginate enmeshing a microcolony of P. aeruginosa.

prognosis for patients with cystic fibrosis. The organism exists in the general environment and is a cause of soft onion rot (Figure 59) (see Chapter 4). The B. cepacia complex can reduce the life expectancy of a CF patient by two decades. It exhibits multiple antibiotic resistance, can be highly transmissible between CF patients, and produces its own unique lethal inflammatory response know as the cepacia syndrome. The chest radiograph has a characteristic appearance of rapidly progressive consolidation, associated with fevers and no response to treatment. Death is inevitable within weeks (Figure 60). Cystic fibrosis patients infected with B. cenocepacia,

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Although the lungs of neonates are structurally normal, the earliest histological findings are mucus plugging of the small ducts of the serous and submucosal glands. This is followed by mucus gland hypertrophy and hypersecretion with mucus stasis, which provides a favorable environment for chronic bacterial infection (Figure 61). The bronchial wall becomes chronically inflamed and infiltrated by neutrophils with mucosal erosion and then ulceration. Fibrosis follows (Figure 62). Airway smooth muscle hypertrophies and is infiltrated by inflammatory cells (Figure 63). Inflammation leads to loss of cartilage and elastic tissue. Airways therefore become unstable and floppy (Figure 64). These changes start in the upper lobes but eventually involve all lobes leading to bronchiectasis (Figure 65). The hilar lymph nodes are enlarged as a consequence of chronic bronchial sepsis (Figure 66). Local areas of emphysema and bullae occur at the sites of lung destruction.

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 59. A brown rotten onion (right) infected with B. cepacia. (Image courtesy of Professor J. Govan, Edinburgh, UK.)

(a)

(b)

Figure 60. Two chest radiographs taken 1 week apart in a cystic fibrosis patient with necrotizing cepacia. (a) Moderate consolidation progressed to (b) total lung consolidation. The patient died 2 days after second chest radiograph.

As a consequence of airways narrowing, lung destruction and shunting of oxygen between the pulmonary and bronchial arteries, there is gross mismatching of ventilation and perfusion. Progressive hypoxia and the bronchopulmonary anastomoses lead to pulmonary hypertension (Figure 67) and right ventricular wall thickening (Figure 54).

a1-Antitrypsin deficiency

a1-Antitrypsin deficiency is the third major genetically inherited condition associated with significant lung pathology. a1-Antitrypsin protects tissues against various proteases, most notably neutrophil elastase. The protein is encoded on the protease inhibitor (Pi) locus on chromosome 14q and the gene is pleomorphic (see Chapter 17).378

Although a genetic disease, the presentation of A1AT deficiency in the pediatric age group is essentially confined to hepatic disease, associated with chronic cholestasis. Lung disease, with symptoms related to chronic obstructive airways disease, does not usually present until the third or fourth decade, by which time there may be significant pulmonary impairment. Earlier diagnosis of A1AT deficiency may sometimes occur following a screening diagnosis in another family member.379 There is a little evidence to suggest an increased prevalence of asthma in younger individuals with A1AT deficiency. Follow-up of a cohort screened and diagnosed with this deficiency in the neonatal period demonstrated little evidence of significant lung function impairment at the end of the second decade.380

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(a)

(b)

Figure 61. Cystic fibrosis. (a) Serous and mucosal gland hyperplasia, moderate smooth muscle hypertrophy and luminal mucus are common findings. (b) Dilated mucus glands contain viscid mucus. Lymphoplasmacytic infiltrates are plentiful.

Figure 62. Severe chronic inflammation in the bronchial wall with early ulceration. The lumen is filled by acute inflammation together with mucus.

Perinatal pathology At birth, the lungs must rapidly change from being largely redundant into a fully functioning respiratory organ. Perinatal pathology is dominated by the problems related to this transition. This is especially the case in the very preterm infant, where the lung is both structurally and biochemically immature. The advent of surfactant replacement therapy along with improving intensive care regimes have radically altered the

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Figure 63. Bronchial wall with hypertrophied smooth muscle. Overlying respiratory mucosa is simplified.

management and outcomes for these small infants. In parallel with these improvements, macro- and microscopic pathology have been altered. Additional acquired perinatal pathology is more likely due to adverse events occurring while the baby is in

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 65. Gross bronchiectasis in sectioned lung.

Figure 64. Destruction of cartilage by inflammatory cells which contributes to making the larger airways unstable and floppy.

Figure 67. Medial hypertrophy associated with pulmonary hypertension in a resected cystic fibrosis lung.

Figure 66. Enlarged hilar lymph node.

utero. Acquired infection and meconium aspiration are examples. The latter in particular is more a feature of term gestation than prematurity.

Hyaline membrane disease Hyaline membrane disease (HMD) and respiratory distress syndrome (RDS) are terms that are frequently used interchangeably. While this does not always matter, the two terms are not equivalent. Hyaline membrane disease is a pathological term describing the presence of hyaline membranes in the lung. Respiratory distress syndrome is a clinical term describing an acute illness usually of preterm infants with tachypnea, expiratory grunting and respiratory distress.381 Respiratory distress denotes intercostal, subcostal and sternal retraction. While there is near overlap between the presence

of RDS and HMD, it is not complete and there are occasions when one is present but the other is not. Respiratory distress syndrome is a clinical manifestation of surfactant deficiency. Surfactant is a mixture of lipid and protein that reduces surface tension at the air-liquid interface on the alveolar surface during expiration. Both the lipid and protein components are synthesized by type II pneumocytes. The lipid, mostly phosphatidylcholine, is stored together with the proteins in pneumocyte lamellar bodies. When secreted by exocytosis onto the alveolar surface, the lamellar bodies unravel and the lipid, together with surfactant protein A (SP-A) and surfactant protein B (SP-B), forms cross-hatched structures known as tubular myelin. There are four surfactant proteins, SP-A, SP-B, SP-C and SP-D. The four proteins have distinct functions. SP-B and SP-C are hydrophobic, are of relatively low molecular weight and interact closely with the surface layer of lipid. They have important surface tension regulatory functions.382 Surfactant proteins SP-A and SP-D do not have surface active functions. These hydrophilic proteins are of larger molecular weight and are structurally related to the collectin family of lectins. Collectins also have important immune functions and are capable of recognizing,

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Figure 68. Severe hyaline membrane disease. The lungs are deep red with liver-like texture. With surfactant replacement therapy, this is now an unusual finding.

inhibiting and inactivating a broad spectrum of pathogens.382,383 SP-A is involved in the production of tubular myelin and SP-D is involved with regulation of surfactant re-uptake. Recycling occurs mainly by resorption of surfactant, back across the type II pneumocyte membrane, via endocytosis. A small proportion (approximately 10%) is taken up and degraded by alveolar macrophages. Respiratory distress occurs in approximately 2–3% of infants. Of these, just over 1% have RDS, while the remainder have transient tachypnea of the newborn.380 The risk of RDS increases the earlier the gestation, so at 28 weeks gestation up to 50% of infants will suffer RDS at birth. Respiratory distress syndrome is largely associated with prematurity but there are other well-known risk factors, such as maternal diabetes and birth asphyxia. Hereditary factors play a role in disease development and severity in many different areas of lung function.384 Males are more likely than females to develop RDS, perhaps due to an androgen-induced delay in some biochemical pathways related to surfactant production. Caucasians are more at risk than non-white babies, perhaps due to allelic variation in SP-A production. The classic criteria of RDS are those of a respiratory rate above 60/min; grunting expiration; inspiratory indrawing of the sternum, intercostal spaces and lower ribs; and cyanosis without oxygen supplementation.381 Generally these signs develop before the infant is 4 hours old and last for more than 1 day. Improvement, often associated with a diuresis, commonly starts after 2–3 days, and is associated with recovery of the type II cells and resumption of surfactant production. The natural history of surfactant-deficient RDS is considerably modified by modern therapy, notably with the use of surfactant replacement and assisted ventilation. The diagnosis is now based on the radiographic appearance of premature infants.385 This may show diffuse fine granular opacification in both lungs with air bronchograms. These changes result from air-filled airways standing out against the background atelectatic alveoli.

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Figure 69. Very early phase of hyaline membrane disease in a 25 week gestation baby dying at 2 hours of age. Necrotic bronchiolar epithelium may be prominent.

Similar to the clinical presentation and perspective, there is a difference in the spectrum of pathology seen in the pre- and current surfactant era. Macroscopically, the lungs of babies dying with the clinical picture of severe RDS are collapsed, deep red-purple with a liver-like texture (Figure 68). Histologically, the earliest change is necrosis of distal bronchial and bronchiolar respiratory epithelium.386 This is often a particularly striking feature in the very preterm infants (approximately 24/25 weeks) surviving only a few hours (Figure 69). Desquamated epithelium may plug more distal airways. Early hyaline membrane formation will be found in parts of the lung, occasionally within half an hour of birth in the very preterm. In most cases hyaline membranes usually take more than 2 hours to form but are well established by 24 hours (Figure 70). The membranes line the distal airways, especially the respiratory bronchioles and alveolar ducts. The most peripheral parts of the lung, the terminal sacs or alveoli, are often collapsed and rarely lined by membranes. The eosinophilic membranes are predominantly composed of plasma proteins admixed with necrotic epithelial cell debris. Fibrin is scant, if present.387 Fibrin thrombi may be seen in pulmonary arterioles. The membranes may be stained yellow, particularly if the newborn’s bilirubin level is elevated. The presence of this “yellow hyaline membrane disease” has no further significance. Some membranes will show yellow staining on the luminal surface but the more typical eosinophilic appearance in the deeper layers. If the HMD is uncomplicated, then the host reaction proceeds rapidly from about 24 hours after birth. Epithelial regeneration is rapid and these cells may grow beneath or over the membrane. By 48 hours macrophages (membranophages) are a prominent feature in the airways and in the surrounding interstitium, ingesting the membranes (Figure 71). Neutrophils can be relatively inconspicuous histologically, although they are usually a prominent component of bronchoalveolar

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 70. Early hyaline membranes typically do not involve the most distal airspaces, which remain collapsed.

lavage specimens taken during the acute phase of HMD.388 Five days after onset of the disease only residual peripheral balls of membranes are likely to be found. Following the advent of artificial surfactant therapy, the initial pathology study suggested that the pattern of hyaline membrane disease was the same regardless of whether surfactant had been administered.389 However, Pinar et al. pointed out that the findings differed.390 While the presence of hyaline membrane disease was similar in treated and untreated infants, treated infants had less severe epithelial necrosis and fewer hyaline membranes. It is now very unusual to see the deep plum-colored lungs, while hyaline membranes tend to be scattered and thin. These findings coincide with the reduction in clinical severity of RDS, such that it is uncommon to consider HMD the cause of death in the absence of other complicating conditions or events. The pathogenesis of prematurity-associated HMD is similar to that noted in other processes with hyaline membranes. Hyaline membrane disease is a manifestation of acute lung injury, where the barriers between the airspaces and vascular lumina are damaged. Epithelial-endothelial integrity breakdown allows efflux of plasma components followed by partial resorption, resulting in the deposition of eosinophilic material (hyaline membranes) on the airway and alveolar walls. In the preterm infant, the attempt to breathe, either naturally or via mechanical ventilation, in the face of elevated intra-alveolar pressure and alveolar collapse creates sheer forces and uneven transpulmonary pressures. Alveolar duct and respiratory bronchiole dilatation ensue.390 Ischemia may play a role in the HMD of prematurity. Neonatal pulmonary vessels are very reactive, muscularized, and capable of vasoconstriction. Uneven lung expansion causes shunting of blood from atelectatic airway segments.386 Local ischemic injury to type II pneumocytes may enhance surfactant deficiency. After the period of vasoconstriction, hyaline membranes may form following the resumption of

Figure 71. Hyaline membrane disease at 5 days. Hyaline membranes are still visible but airspaces are re-epithelialized. Epithelium may grow under or sometimes over the membrane. Fibroplasia and macrophage reaction beneath membrane within the wall on the left are seen.

Figure 72. Hyaline membrane disease associated with overwhelming group B streptococcus infection.

blood flow. Increased pulmonary intravascular coagulative activity has also been documented in this scenario.392 Importantly, hyaline membranes in the neonate are not exclusively associated with surfactant deficiency and prematurity. They may be caused by a variety of insults to epithelialendothelial integrity. Group B streptococcal infection may clinically mimic the RDS of prematurity.393 Pathologically the lungs appear similar but the hyaline membranes may show a blue tinge, due to the overwhelming numbers of bacteria (Figure 72). Some neonates present with HMD at term. These cases are usually in association with birth asphyxia. In this subgroup, the clinical and pathological profile bears a greater resemblance to adult disease (acute respiratory distress syndrome

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 10 Surfactant dysfunction mutations and related disorders: dominant histological patterns

1.

SpB genetic mutations: pulmonary alveolar proteinosis and variant histology

2.

SpC genetic mutations: chronic pneumonitis of infancy, DIP and NSIP

3.

ABCA3 genetic mutations: pulmonary alveolar proteinosis, CPI, DIP and NSIP

4.

Cases with histology consistent with a surfactant dysfunction disorder without a recognized genetic disorder

Modified from512 DIP, desquamative interstitial pneumonia; NSIP, nonspecific interstitial pneumonia; CPI, chronic pneumonitis of infancy.

(ARDS) and diffuse alveolar damage (DAD)), than to the typical preterm disease.394–396 In this instance, epithelialendothelial injury may be primarily ischemic in origin, with toxic radical production, microthrombi and leukocyte accumulation in the microvasculature amplifying the initial damage. Injury to type II pneumocytes producing a secondary surfactant deficiency may further contribute to hyaline membrane formation (see Chapter 9). Complications from HMD may be acute and related to air leaks (see below). The overall mortality of RDS is improving and deaths in babies weighing more than 1500 g are rare. The overall mortality is 5–10%.385 There is significant morbidity in survivors. Chronic lung disease often extends into childhood and beyond (see below).

Pulmonary surfactant metabolic dysfunction disorders Surfactant-related protein variation is a particular area of study in an attempt to understand the influence of inherited factors on the development of acute infant respiratory distress. Inherited susceptibility has been associated with variations in proteins SP-A, SP-B, and SP-C.397–399 Similar susceptibility has been associated with SP-B and SP-C related gene mutations.384,398 The pathology is very different from the typical surfactant deficiency associated with prematurity and may include a number of interstitial lung diseases (Table 10) (see Chapter 10). There are over 150 mutations in the ATP-binding cassette A3 gene (ABCA3) located on chromosome 16. Most will cause severe neonatal lung disease and death within a few months of life. The pathology is usually that of congenital alveolar proteinosis. However, a few mutations are milder and are associated with chronic lung disease in childhood (Table 10). The lungs show interstitial disease sometimes with desquamative interstitial pneumonitis.382 As the gene is important in normal lipid transport, ultrastructural studies show an absence of normal lamellar bodies and a lack of normal lipid constituents. Immunohistologically, all

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Figure 73. Radiograph of fibrotic cystic lung typical of severe “old” bronchopulmonary dysplasia.

surfactant proteins may be detected. While the diagnosis is suggested by the above histological and ultrastructural features, definitive diagnosis is obtained by identifying the ABCA3 gene locus. A complete absence of SP-B may occur as an autosomal recessive condition and is usually fatal in the neonatal period. The gene for SP-B is located on chromosome 2. There are a number of recognized abnormal alleles and the homozygous state is associated with lethal lung disease for which lung transplantation may be required.400,401 In the presence of SP-B deficiency, the lungs will accumulate SP-A and SP-C in the alveoli, i.e., alveolar proteinosis, rather than an HMD picture.401,402 SP-B deficiency may be suspected in the clinical context of treatment-refractory severe lung disease, sometimes in a term infant and may progress to chronic lung disease (see Chapter 10).382 Inherited disorders of SP-C tend to be less severe than those of SP-B, with presentation in infancy or even in adulthood. The lungs may show interstitial fibrosis and/or pneumonitis. Autosomal dominant inheritance has been reported.403

Bronchopulmonary dysplasia Bronchopulmonary dysplasia (BPD) is a term that has been used to describe a constellation of clinical, radiological and pathological features following ventilatory therapy for HMD.404 In the early days of intensive care for neonates, the defining clinical features were an acute lung injury (usually HMD in premature infants), some form of continuing ventilatory support, and pulmonary radiographic abnormalities, such as persistent increased radiological opacities (Figure 73).405 While there was a spectrum of pathological changes, it usually entailed significant fibrosis. Such pathology often resulted in respiratory failure and/or cor pulmonale. Improved management of premature infants over the years has caused a gradual transformation of BPD from a

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 74. Major airway pathology such as (a) obliterative bronchiolitis and/or (b) squamous metaplasia usually accompanies only severe BPD.

clinically severe fibrosing condition of the lung into a milder condition, where continued respiratory support is still required but at a much diminished level. Along with this change, the diagnostic label has changed to “chronic lung disease of prematurity”. Others suggest designating this condition “new bronchopulmonary dysplasia”.406–409 Both “old” and “new” BPD are strongly associated with prematurity and infants have a continuing requirement for ventilatory support. Despite the changes in the nature of the condition over the years, the overall prevalence of BPD has remained relatively constant owing to the improved survival of infants born at an earlier gestational age.410 New BPD almost always occurs in infants less than 30 weeks gestation with birth weight less than 1500 g. Partly because of variations in definition, it is estimated that some 20–40% of very low birth weight infants (< 1500 g) may develop chronic lung disease of prematurity (new BPD).410–412 Chronic lung disease of prematurity or “new” BPD is essentially a clinical diagnosis. The criteria for diagnosis depend on the infant’s need for continued ventilatory support, such as supplemental oxygen, and the need for that support at a defined time. This is usually 28 days of age. Thirty-six weeks post-menstrual age (i.e. equivalent to 36 weeks gestation) is used as an alternative by those who believe it may be a better predictor of long-term outcome.413 There is no longer a requirement either for associated radiographic changes or even for an initial acute lung disease.414 The severity of the disease can be graded according to the extent of the ventilatory support required.

Pathology As indicated above, the clinical and radiological features of BPD have changed substantially over the years, such that almost two different conditions can be recognized. The pathology has also changed. The histological features evolved from a largely diffuse fibrosing condition to one in which the dominant theme is one of interference with normal lung development. The two ends of this spectrum will be discussed.

Old BPD Traditionally, the pathology of BPD has been described in terms of three stages:415 an exudative and early reparative stage, a subacute fibroproliferative stage and a chronic fibroproliferative stage. Exudative and early reparative phase This early phase occurs between 3 and 9 days of age. It represents a combination of primary damage, usually HMD, exaggerated repair, and lung reaction to aggressive ventilatory support. Large and small airway damage is often prominent with squamous metaplasia, submucosal muscular hyperplasia and, if particularly severe, an obliterative bronchiolitis (Figure 74). Many airways will show residual hyaline membranes, some of which incorporate into the fibroproliferative process. Unlike uncomplicated HMD described above, hyaline membranes may continue to form beyond the first day, and membranes may contain fibrin. Peripheral alveoli can also be involved. The interstitium is edematous with florid interstitial fibrosis, smooth muscle proliferation and type II pneumocyte metaplasia. The immediate cause of death may well be respiratory failure.

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Figure 75. Nodular pleural surface in a lung with bronchopulmonary dysplasia.

Subacute fibroproliferative stage This stage represents a transition from the early acute lung disease to the chronic phase. It is seen from the end of the second week up to the end of the first month. Obliterative bronchiolitis is still obvious and interstitial fibrosis becomes more pronounced. Re-epithelialization progresses with type II pneumocytes. Chronic fibroproliferative stage Macroscopically, the lung is firm with a nodular pleura (Figure 75). Examination of the pleural or cut surfaces may reveal emphysematous airspaces or a fairly uniform firm parenchyma. Histological appearances are variable. Large emphysematous airspaces may be adjacent to more normal-sized ones (Figure 76). This geometric airspace distortion probably contributes significantly to the degree of respiratory failure and may reduce gas exchange capacity to 25% of normal.51 Such variation may not be present, but the lung will show more uniformly enlarged alveolar spaces with a reduced alveolar surface area.416,417 This feature links clearly with the underlying pathology of “new” BPD (see below). Simplified lung structure may be responsible for the death of infants. Elastic stains reveal thickened elastin plaques at alveolar crests. Airspaces are frequently lined by hyperplastic type II pneumocytes. More proximal airways are relatively normal, although there may be persistent squamous metaplasia and smooth muscle hypertrophy. Twenty-five to 50% of cases feature glandular hyperplasia and patchy chronic inflammation.417,418 The vascular changes are a significant factor in the development of cor pulmonale. Smaller muscular arteries feature medial smooth muscle hypertrophy and an increase in adventitial fibrous tissue.241,419 Endothelial cells may remain plump and contribute to the vascular obstruction. A reduction in normal peripheral arterial recruitment in the immediate neonatal period may lead to a reduction in peripheral arterial numbers.

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Figure 76. Architectural distortion with emphysematous airspaces, interstitial fibrosis and metaplastic cuboidal alveolar epithelium was always the most consistent component of old BPD even if variable in degree. This may still be seen, though rarely as severe as illustrated here.

New BPD New BPD is much more a condition in which interference with normal lung development is the dominant feature. Compared with old BPD, there is little or no airway damage. Interstitial fibrosis is not obviously present, although elastic stains may demonstrate an increase in elastic tissue plaques. The acinus is simplified with large alveolar structures showing reduced complexity and diminished secondary crest formation. Severe pulmonary hypertensive changes typically seen in old BPD lungs are not uncommon in new BPD. Pulmonary vascular changes are more subtle and variable. There may be a “dysmorphic pattern” in which there are prominent “corner vessels”, adjacent dilated vessels, and sometimes reduced capillarization of alveolar walls. An abnormal distribution of alveolar capillaries with vessels more distant from the air surface has also been noted.420–422 There may be a significant reduction in the alveolar surface area and pulmonary microvasculature.414

Pathogenesis and outcomes The causes of BPD are multifactorial. Old BPD was linked very closely to barotrauma and high oxygen concentrations. It was suggested the very high ventilatory pressures needed in the early days of therapy were closely associated with the major obliterative airway lesions seen in the acute phase of BPD.423 The effects of mechanical forces on the development of new BPD are less clear, in part because of the complexity of current interventions. Surfactant has clearly reduced the requirement for the more tissue disruptive pressures. Mechanical ventilation is a life-saving intervention but the full effects of iatrogenic barotrauma are unclear.424,425 Preterm infants appear susceptible to oxidant stress. These infants have low levels of antioxidants. High inspired oxygen levels increase the risk of BPD. In animal models hyperoxia appears to worsen the severity of lung injury in the more

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

immature.424 Further oxidative stress may derive from activated phagocytes responding to infections, such as chorioamnionitis. It is possible that some of this oxidative stress acts antenatally.381,409,426 Initiation of inflammatory cascades has the net effect of impairing vasculogenesis and alveolarization.427 Tracheal aspirate analysis shows inflammatory factors are increased in ventilated preterm infants who subsequently develop BPD.428,429 Interleukin-8 (IL-8), which precedes neutrophil infiltration, has been the most extensively investigated chemokine in preterm infants. Raised levels are found in preterm infants who develop BPD compared with those infants who do not. Whether it is actively involved in BPD pathogenesis or simply a marker of illness severity is not clear.430 Normal lung development is dependent on epithelialendothelial interactions, the former producing a number of angiogenic factors. One such, vascular endothelial growth factor-A (VEGF), is critical for endothelial cell survival. In animal models, hyperoxia leads to reduced VEGF levels. Decreased VEGF expression has been associated with BPD in human infants.424 Genetic influences are a significant factor in susceptibility to BPD. Twin studies show that once other factors such as birth weight, gestational age and various complications of RDS are taken into account, the occurrence of BPD in one twin is a highly significant predictor of BPD in the other.429,431,432 Genetic predisposition could be partly expressed through variations in the level of antioxidant defenses.433 The prognosis of BPD is generally good. Death may occur from intercurrent infection but in severe BPD the cause of death may be respiratory failure or cor pulmonale. Morbidity is significant. Pulmonary morbidity may involve prolonged supplementary oxygen, sometimes given at home, for months or even years. Cough and wheeze may occur in up to 20% of very preterm (< 29 weeks) infant BPD survivors at 1 year of age.434 Respiratory impairment with wheeze may be found in up to 7% of pre-school age survivors.435 There is significant risk of re-hospitilization in the first 2 years of life. This is due to respiratory disorders including reactive airway disease, infectious bronchitis, especially respiratory syncytial virus (RSV), and pneumonia. Admissions for respiratory problems decrease after 4 to 5 years.436 Nevertheless there is detectable impairment of lung function for many years, with some of the worse affected suffering with airway obstruction through adulthood.437

Wilson-Mikity syndrome In 1960, Wilson and Mikity described a new form of lung disease.438 They reported five preterm infants, all weighing between 1200 and 1300 g, dying from a chronic lung disorder after a few months. One infant died after only 27 days. Further cases were reported in the subsequent decade.439,440 Clinical and pathological similarities to BPD were noted. An important aspect of the syndrome that persuaded authors to retain it as a separate disorder was the context in which the condition developed. All infants had either no or only very mild initial

lung disease. Infants lived for up to a month with no outward sign of pulmonary disease before the development of chronic lung disease. With time, the distinction between BPD, especially “new” BPD, and Wilson-Mikity syndrome has become less clear since one of the defining characteristics of Wilson-Mikity, the absence of early acute lung disease, has become a feature of atypical BPD. While it is reasonable to consider Wilson-Mikity part of the spectrum of atypical BPD,441 a recent report contends that it remains a distinct disorder.442 Wilson-Mikity syndrome overlaps with BPD in that it is predominantly a disease of the very preterm infant (< 30 weeks) with a low birth weight (< 1500 g). In these patients progressive lung disease starts at a few days of age or later in the absence of an acute lung injury. It may be distinctive, since chest radiographs at 2–4 of weeks of age demonstrate microcystic changes. This “bubbly” appearance is thought to be due to a unique sensitivity to mechanical disruption leading to interstitial air leaks. In some studies, WilsonMikity babies show elevated immunoglobulin M (IgM) levels, suggesting infection may play a role.443–444 If there is a significant inflammatory response, there may be progression to severe lung fibrosis including macrocysts and pulmonary hypertension. These features once again merge with features seen in BPD. Mortality may be greater than 10%.

Air leaks: pulmonary interstitial emphysema and pneumothorax Leakage of air from the alveoli into the delicate tissues of the preterm infant lung most commonly causes either pulmonary interstitial emphysema (PIE) or pneumothorax. Pulmonary interstitial emphysema can track along tissue planes and lead to pneumomediastinum, pneumopericardium and more rarely massive subcutaneous emphysema. Air leaks are not uncommon and, before the advent of surfactant therapy, occurred in approximately 20% of infants ventilated for respiratory distress syndrome.445 The incidence has reduced significantly with surfactant treatment and other strategies to reduce mechanical ventilation pressures. Mild degrees of PIE may be asymptomatic but if severe, interstitial air compresses surrounding alveoli and causes respiratory embarrassment. This may increase the degree of respiratory distress without distinct clinical symptoms or signs. Pneumothoraces may also be asymptomatic but large pneumothoraces can be under tension, leading to respiratory distress. A sudden deterioration with shock may occur. Pneumopericardium is rarely asymptomatic, usually causing tamponade. Pulmonary interstitial emphysema is most often a radiological diagnosis. The chest X-ray is considered diagnostic with hyperinflation and multiple diffuse small non-confluent, cystic radiolucencies. Transillumination may show increased light on the affected side, a feature also seen with pneumothorax. The chest X-ray of a pneumothorax shows absent lung markings, lung collapse and in the case of a large tension pneumothorax, mediastinal shift (Figure 77).385

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Figure 78. Subpleural pulmonary interstitial emphysema.

Figure 77. Chest radiograph of tension pneumothorax showing severe right mediastinal shift.

Figure 80. Encysted chronic pulmonary interstitial emphysema. Giant cells (inset) may be found lining the airspaces.

Figure 79. Acute pulmonary interstitial emphysema. This may be within interlobular spaces or lymphatics.

The diagnosis of air leaks at autopsy is usually straightforward. A pre-autopsy radiograph will usually be adequate to demonstrate a moderate or large pneumothorax. Otherwise, the escape of air bubbles from the hemithoraces, when opened underwater, is a straightforward alternative. Caution may be needed in diagnosis if a drain has been left in situ and remains open. Macroscopically, the lungs might be collapsed although this may only be more readily assessed when the pneumothorax is unilateral and there is a distinct difference between the two lungs. Pneumothorax is commonly associated with PIE, although the latter may be unimpressive if there has been time for the small cysts to decompress between death and autopsy. Small air or air and fluid-filled cysts are present on the pleural surface (Figure 78).

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On section, cysts may be present in the parenchyma, sometimes localized to the interlobular septa. Pulmonary interstitial emphysema may be bilateral, unilateral or even more localized. Microscopically, acute interstitial emphysema is not infrequently diagnosed by the pathologist in very immature lung, sometimes without having been recognized macroscopically (Figure 79). It simply appears as large circular or oval empty spaces surrounded by compressed lung. Distended lymphatics in interlobular septa are commonly seen. In some of the more immature infants, pulmonary hemorrhage into the emphysema may be a terminal event. Persistent PIE can be localized or diffuse. Localized disease may result in local surgical resection whereas diffuse disease is usually but not inevitably fatal.446 In resections, the clinical diagnosis often includes other cystic disorders, such as adenomatoid malformation. Persistent PIE stimulates fibrosis and there is often a foreign-body giant cell reaction (Figure 80).447

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Some cysts may become large, probably due to a ball-valve effect and pressure differences between the alveolus and interstitium.448,449 Air leaks occur most commonly as a complication of RDS and associated mechanical therapy, including high-frequency oscillatory ventilation.450 In more mature infants, air leaks are more likely to be associated with meconium aspiration. The occlusion of distal airways (ball-valve effect) causes emphysema distal to the incomplete obstruction. Spontaneous pneumothoraces can occur at birth because of the high pressures generated during the infant’s first breaths. Active resuscitation can also produce the same effect. Air leaks are rarely associated with spontaneous breathing.451,452 There is evidence suggesting increased mortality associated with PIE in very low birth weight infants453 and there is an increased risk of chronic lung disease.

Complications of other ventilatory techniques The main therapeutic complications of conventional ventilatory therapy in the preterm infant have been described above. Newer therapies, introduced after conventional ventilation has proved unsuccessful, attempt to avoid pulmonary hypoxia and barotrauma-related complications. There is little pulmonary pathology specific to these modalities and it is often impossible to untangle the effects of the therapy from the complex underlying pathology and additional therapies.

High-frequency jet ventilation High-frequency jet ventilation (HFJV) delivers gas into the trachea in short high-pressure bursts. The lower peak inflation pressures are intended to reduce BPD and air leaks.454 The main reported specific complication is necrotizing tracheobronchitis.455,456 This is probably an ischemic injury secondary to the high tracheal ventilator pressures. Some reports claim it may be present in up to 90% of infants dying after HFJV. Humidification appears to reduce the injury.457 The major histological characteristics of HFJV tracheobronchitis are ulceration with mucosal hemorrhage, inflammation and accumulation of luminal mucus. Some of the changes may be seen where the endotracheal tube is in contact with the trachea, but the presence of lesions distal to the tip of the tube, and indeed extending into the main bronchi, indicates that the ulcerations are not simply contact trauma. The pathogenesis is not entirely clear but may be related to excessive drying of tissues or the “jet-hammer” effect on tissues subject to HFJV.458

Extra-corporeal membrane oxygenation Extra-corporeal membrane oxygenation (ECMO) is a ventilatory modality used in respiratory failure when conventional therapy fails to maintain adequate ventilation. This process maintains oxygenation while allowing time for intrinsic recovery of the lungs or heart usually over a 3–10 day period. It is indicated in a number of neonatal conditions where the underlying lung

condition is considered reversible such as primary pulmonary hypertension of the newborn (PPHN), including idiopathic PPHN, meconium aspiration syndrome, RDS, group B streptococcus sepsis and diaphragmatic hernia.459 During ECMO, the right atrium is cannulated and the venous blood is drained and passed through an oxygenator. Gas exchange occurs via a countercurrent flow of blood and gas. This benefits the lung partly by protecting it from the effects of the underlying disease process. Tissue oxygenation and capillary perfusion are improved, thus reducing hypoxia-induced vasoconstrictive pulmonary hypertension. Although there are some data suggesting there may be an increase in pleural effusions and hemothoraces, there are few direct pathological data on the effect of ECMO on the lung. Chou et al. reported on lungs examined at autopsy following ECMO therapy.460 Although much of the pathology is either disease-related or a nonspecific response to lung injury, the authors argue that ECMO is important in the generation of these histological features. They believe much of the autopsy pathology arises during the ECMO treatment period, rather than as a consquence of either the underlying primary condition or prior conventional ventilation. Interstitial and intra-alveolar hemorrhages with hyaline membranes and type II pneumocyte hyperplasia are apparently the major morphological findings in the early stages of ECMO therapy. By day 7 interstitial fibroplasia is prominent and often accompanied by bronchial changes, including epithelial hyperplasia, squamous metaplasia and smooth muscle hyperplasia. Intraalveolar calcification appears after 7–15 days. In three cases where ECMO was used for between 2 and 3 weeks, mucinous metaplasia affecting terminal bronchioles was noted. These latter two features appear characteristic of ECMO lungs and are not seen secondary to meconium aspiration or conventional ventilation. Sebire et al.461 in a series of lung biopsies taken to assess the underlying pulmonary pathology, recognized only fresh or old hemorrhage as a likely specific consequence of ECMO.

Pulmonary hemorrhage Small histological foci of alveolar and interstitial hemorrhage are common in preterm infant lung. Clinically significant pulmonary hemorrhage may occur in up to 12% of very preterm infants with RDS or those treated with surfactant.462,463 Massive pulmonary hemorrhage may be heralded by an acute deterioration in the infant’s clinical condition. This is followed by the production of abundant bloody secretions from either the mouth or the endotracheal tube. Macroscopically, lungs from infants dying of massive pulmonary hemorrhage are firm, and red-purple with obvious areas of fresh parenchymal hemorrhage on cut section. Histologically, massive hemorrhage may be found with fresh blood involving large confluent areas of lung. Hyaline membranes may be found.

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Although hemorrhage may occur with trauma, in most instances the fluid is heavily blood-stained pulmonary edema fluid. This fluid has a lower hematocrit than blood.464,465 It is largely associated with cardiac dysfunction or fluid overload. While the cause is not always clear, in the term infant it is most likely to follow a hypoxic insult.466 In the preterm infant, a persistent patent ductus arteriosus or the reduction in lung compliance associated with surfactant administration may be significant factors.465,467 Other associated risk factors include the severity of the associated illness, growth restriction, and coagulopathy.468

Thrombosis and embolism The development of small arterial and venous thrombi is not an uncommon event in preterm infants dying from hyaline membrane disease or chronic lung disease. In most instances, these thrombi are probably not functionally significant. Most thromboembolic events are related to the presence of arterial or venous catheters.465,469 Infarction is associated with peripheral subpleural cyst formation and secondary pneumothorax.470 Aspirated meconium is associated with thrombosis and infarction, probably due to its vasoconstrictive effect.471 Lipid can sometimes be found in the pulmonary capillaries of infants. Although this probably results from parenteral nutrition lipid administration,472,473 some argue that lipid infusion is not a prerequisite.474 Histologically, it is most easily identified by lipid stains on fresh tissue, but is also usually apparent even after normal processing. Capillaries are round and are devoid of red cells. At vessel margins, slightly refractile material can usually be seen. The effect of lipid on lung function is difficult to assess.475 Very rare sources of emboli include fractured tips of venous catheters.476 Pulmonary cerebellar emboli are a rare complication of traumatic delivery.300,301,477 The pulmonary emboli may be sufficient to cause death, although if the embolism finds its way into the coronary arteries, this may be more critical in some cases.

Meconium aspiration syndrome Meconium aspiration syndrome (MAS) is largely a clinical diagnosis. It is defined as respiratory distress in a neonate born through meconium-stained amniotic fluid, whose symptoms cannot otherwise be explained.478 Meconium discharge is rare before 32 weeks of gestation and most infants are more than 37 weeks gestation. In one study,479 meconium-stained amniotic fluid was only found in 4.3% of pregnancies before 33 weeks of gestation. There was an association with prolonged membrane rupture but no indication of increased mortality. No listerial infection, a condition traditionally associated with preterm meconium release, was found. Up to 20% of term gestations and possibly 40% of post-terms are associated with meconium discharge. Ninety-eight percent of episodes occur after 37 weeks of gestation.480

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Figure 81. Meconium plug with squames and mucus in term infant airway.

Clinically, some infants may appear surprisingly normal at birth with good Apgar scores before subsequent deterioration.481 Otherwise, a mature or post-mature baby presents with respiratory distress. Meconium staining of the placental membranes is likely. The infant may show tachypnea with intercostal or subcostal retraction and sometimes with an expiratory grunt.385 Chest radiographs exhibit patchy infiltrates and occasional small pleural effusions. Complete opacification (“white-out”) may occur in severe cases.385 Pneumothorax, pulmonary hypertension and hypoxic-ischemic encephalopathy may also be apparent. At autopsy, the lung surfaces may not show any gross abnormalities in the acute phase, aside from nonspecific asphyxia-associated petechial hemorrhages. Major airways often contain residual sticky, dark-green meconium, although the suction and washing out associated with resuscitation can remove this material. With compression, the cut surfaces may exude meconium from smaller airways. Histologically, meconium is present throughout the lungs from the bronchi to the alveoli (Figure 81). It appears as a mixture of mucus with granular eosinophilic material, often containing small yellow “meconium bodies”. Squames are the most obvious feature of the aspirated material. Obstruction of smaller airways with tenacious meconium causes distal collapse. In some parts of the lung, emphysema may develop due to a “ball-valve” effect. This overdistension can precipitate pneumothorax (see above). An acute inflammatory response to meconium develops within hours. The ventilation/perfusion mismatch associated with meconium aspiration produces an acidosis and hypoxia, causing vasoconstrictive pulmonary hypertension. Some authors argue that the excessive muscularization of pre-acinar pulmonary arterioles and the extension of muscle into the normally muscle-free intra-acinar arterioles found in these infants is an in utero development. As such, the pulmonary hypertension is less a complication of the meconium aspiration but

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

both conditions share a common etiology of chronic intrauterine stress and hypoxia.482,483 Others suggest methodological defects have led to an underestimation of the normal muscularization of pre-acinar arterioles in normal infants and the vascular changes are of postnatal origin.484 Fetal distress, due to hypoxia or infection, could lead to meconium discharge but the evidence to support this is mixed.480,485–487 It is unlikely that significant aspiration of meconium occurs at birth during the first breath but it takes place mainly in utero. It has long been recognized that acute hypoxia can lead to a deep form of gasping type of fetal breathing. This could be the point at which meconium is aspirated. The relationship between fetal distress, meconium discharge and subsequent MAS is less than clear.488 Once inhaled, the pathology induced by meconium occurs from two main mechanisms. As noted above, obstruction of airways with distal collapse, hypoxia and a “ball-valve effect” causing emphysema and pneumothorax can occur from the simple physical presence of meconium. Secondly, chemical irritation may also be important. Meconium is largely composed of water, lanugo, desquamated cells, minerals, small quantities of various enzymes, and bile pigments, including bile acids.489,490 Bile salts have a direct toxic effect on pneumocytes491 and there is good evidence from the umbilical cord that meconium damages the cord.492 Focal myocyte necrosis within the cord vasculature is reported and there is experimental evidence that meconium may stimulate muscle contraction. Umbilical vessel “spasm” might cause fetal hypoxia.493,495 The inflammatory response itself further impairs lung function, as leakage of plasma proteins can inhibit surfactant and reduce lung compliance. Meconium may also directly inhibit surfactant function.496,497 This inflammatory process may be amplified by meconium-predisposing infections. There is experimental evidence to suggest pneumonias develop from bacteria inhaled with the meconium.498 Overall, the mortality associated with MAS is between 4 and 12%.385 In a recent study of infants with MAS treated by ECMO, only 3% died.459

Persistent pulmonary hypertension of the newborn Persistent pulmonary hypertension of the newborn (PPHN) is a clinical disorder in which pulmonary vascular resistance fails to drop or stay relatively low at birth.499 It occurs in approximately 1–2 newborn infants per thousand.500 There is a left to right intrapulmonary shunt, and shunting also occurs across the foramen ovale and ductus arteriosus, if these structures remain patent. For these reasons, PPHN was initially called persistent fetal circulation.484 Clinically, PPHN is characterized by severe hypoxemia in the presence of relatively mild lung disease with either right to left ductal shunting or substantial shunting across the foramen ovale. The former is suggested by an umbilical arterial (postductal blood) PaO2 that is significantly lower than the PaO2 in the

right radial artery (pre-ductal blood).385 Shunting across the foramen ovale may be demonstrated by echocardiography. Most PPHN is secondary and is associated with an increased vascular resistance. Severe hypoxia due to intrapartum asphyxia and meconium aspiration may cause intense pulmonary vasoconstriction. Vasoconstriction, probably due to vasoactive amines, also occurs with sepsis. The pulmonary vascular bed is reduced in pulmonary hypoplasia, so prolonged membrane rupture, possibly associated with both the oligohydramnios–pulmonary hypoplasia sequence and sepsis, is a risk factor in preterm infants.501 Chorioamnionitis in term infants may also be a marker for severe PPHN.502 Also, PPHN can occur in association with congenital heart disease or congenital alveolar dysplasia (congenital misalignment of the pulmonary veins; see above). Very rarely, there is no recognized underlying condition. In these cases it is appropriate to consider the pulmonary hypertension idiopathic or primary. Whether idiopathic PPHN or that associated with meconium aspiration syndrome (see above) is due to an intrauterine structural abnormality of the pulmonary vessels is a matter of debate.482–484,503 Excessive prenatal arterial muscularization of pulmonary arterioles may be a significant factor in these cases; however, etiological clues are lacking. Pathologically, pre-acinar and intra-acinar pulmonary arteries and arterioles demonstrate grossly thickened walls due to an increase in vascular smooth muscle. This process extends into the precapillary vessels. In some instances, muscularization of these vessels is so complete that lumina are almost obliterated.504 Normally, the postnatal fall in pulmonary vascular resistance associated with dilatation of the smaller pulmonary arteries is stimulated by a combination of increased oxygen tensions, ventilation, and sheer stress within the blood vessel. This leads to an eight-fold increase in blood flow. These factors stimulate the production of nitric oxide (NO) by activation of endothelial cell nitric oxide synthase. Nitric oxide acts on arterial smooth muscle cells and activates the production of cyclic guanosine monophosphate (cGMP), a powerful vasodilator. Nitric oxide may also have a role in angiogenesis. Suffice it to say, disruption of the NO-cGMP cascade is a major factor in the development of PPHN.499 Stimulating this NO-cGMP cascade with exogenous NO forms the basis of therapy for PPHN. Inhaled NO diffuses through to smooth muscle cells and directly stimulates cGMP production. NO binds with hemoglobin quickly, so it has no systemic effects. At or near term, this therapy improves infant oxygenation and reduces the need for ECMO.505 Some infants, especially those with pulmonary hypoplasia, do not respond, often predicting a poor outcome. Mortality related to PPHN is 10–20% overall506 but these figures are heavily influenced by the underlying condition.

Diffuse interstitial lung disease in children This category comprises a varied set of diseases manifesting with impaired gas exchange and diffuse radiographic infiltrates.507

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While the interstitium is usually affected, the disease process may also involve airways and airspaces. Hence, these processes are more diffuse than simply interstitial. These rare conditions have a combined incidence of 1.32 new cases per 1 million children per year,508 and most are diagnosed within the first year of life. Interstitial lung disease (ILD) in children can be broadly divided into two groups; those conditions more characteristic of infancy and those in which the histological pattern of the disease and underlying condition is more typical of older children or adults. In the former group, genetic factors, especially those associated with surfactant production, are prominent. The infant group of diseases also includes those conditions that interfere with normal growth. The groups of conditions, which usually occur after infancy, are poorly understood. The histological patterns of ILD, as described in adults, may occur in children but inconsistencies in past definitions have led to a less than clear picture (Table 11) (see Chapter 10). Further, these conditions are rare, thus clinical characteristics are not as well defined in the pediatric population. For instance, it is probable that many cases of usual interstitial pneumonia (UIP) have included examples of what now would be considered nonspecific interstitial pneumonia.509 This may account for the reported better prognosis of childhood UIP.510 Desquamative interstitial pneumonitis (DIP) is one of the commoner histological disease patterns in children. It tends to be more aggressive than in adults but while some cases are idiopathic many are associated with surfactant-related disorders (see above).511 Review of biopsies from young children and infants507,512 under the age of 2 years has led to the proposal of a classification system that may be more relevant to infants and children (Table 11). Most of the infant-associated conditions are related to processes and conditions interfering with normal postnatal growth or development512 and have already been discussed. However, there are two relatively recently described conditions that present post-natally, whose cause is less clear.

Pulmonary interstitial glycogenosis Pulmonary interstitial glycogenosis (PIG) was originally described in seven infants, four of whom were preterm.513 All presented with tachypnea, hypoxemia and diffuse interstitial infiltrates with overinflation on chest radiographs. Five of the infants presented on the first day and all within the first month of life. Subsequent reports have largely confirmed that this early presentation is typical and presentation after 6 months rare. It is more common in males and has been described in monozygotic twins.514 The lungs show a diffuse widening of the alveoli by bland, round to spindle-shaped cells with pale cytoplasm. Periodic acid-Schiff stains may show prominent glycogen, while electron microscopy readily reveals the glycogen. The nature of

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Table 11 Classification scheme of diffuse lung disease in children

Conditions prevalent in infancy

Disorders not specific to infancy

Diffuse developmental disorders Acinar dysplasia Congenital alveolar dysplasia Alveolar capillary dysplasia

Disorders related to systemic disease Immune-mediated/collagen vascular disorder Storage disease Sarcoid Langerhans cell histiocytosis Malignancy

Growth abnormalities with deficient alveolarization Pulmonary hypoplasia Chronic neonatal lung disease Related to chromosomal disorders Related to congenital heart disease

Disorders of the presumed immunocompetent host Infectious/post-infectious process Environmental agents Hypersensitivity pneumonitis Toxic inhalation Aspiration Eosinophilic pneumonia

Specific conditions Neuroendocrine cell hyperplasia of infancy Pulmonary interstitial glycogenosis

Disorders of immunocompromised host Opportunistic infections Related to therapy Transplantation and rejection Diffuse alveolar damage, unknown etiology

Surfactant dysfunction disorders SpB genetic mutations SpC genetic mutations ABCA3 genetic mutations Others

Other disorders masquerading as ILD, e.g. vasculopathies; lymphatic disorders or pulmonary edema

the cells is uncertain512 but they are probably mesenchymal. Vimentin is characteristically strongly positive. Pulmonary interstitial glycogenosis may be associated with other conditions, especially where there is a lung growth disorder, and it has been seen in association with meconium aspiration and cardiac abnormalities.512,515 The prognosis is usually good and corticosteroid therapy may be helpful. Histological resolution has been described.515

Neuroendocrine cell hyperplasia of infancy Pulmonary neuroendocrine cells (PNECs) have been recognized for many years but their function has been the subject of some speculation (see Chapter 1). Recently it is considered that these cells may modulate lung growth and differentiation in the fetus, and act as postnatal stem cells.516 They are

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

present in peripheral airways, usually singly (see Chapters 1, 17 and 31). Originally described by Deterding et al.,517 neuroendocrine cell hyperplasia of infancy was reported in a series of 15 infants presenting with chest retraction, tachypnea and daytime or nighttime hypoxia. The mean age at presentation was 4 months (range 0–11 months) and 80% of cases were term gestations. Those born prematurely had no associated chronic lung disease. Chest X-rays show hyperexpansion while HRCT features a distinctive geographic ground-glass opacity centrally, especially the right middle lobe and lingua, associated with diffuse air trapping.518 Light microscopy shows twice as many cell clusters as compared with age-matched controls. Increased numbers of bombesin-positive cells in greater than 70% of bronchioles are also noted. Although long-term oxygen may be required to support infants with this condition, possibly for many years, no deaths have yet been reported.512

Figure 82. Ascending infection in mid-trimester miscarriage (19 wks). Interstitial mononuclear cells are an indication of chronicity.

Infection Pulmonary infections are largely dealt with in Chapters 4 to 8. This section briefly discusses infections in the perinatal period.

Ascending infection In utero fetal infection is due mainly either to ascending infection, through the maternal os, or transplacentally. Ascending infection is more common. Amniotic fluid infection is common before 22 weeks of gestation and evidence of ascending infection with placental chorioamnionitis and/or cord funisitis may be found in some 20–30% of very preterm infants.519 Features at presentation may include maternal infection, such as pyrexia, sometimes with a history of prolonged membrane rupture. Infection may be associated with spontaneous abortion, stillbirth or early-onset neonatal pneumonia (see below). Pathologically, inflammation of the placental membranes is usual but the fetal lungs may also show neutrophils in the airways and developing terminal sacs (Figure 82). While some of these may be aspirated maternal cells, studies on male fetuses demonstrate inflammatory cells with a Y-chromosome and therefore represent a fetal reaction to the infective process.520 In response to infection, lymphoid aggregates may develop adjacent to bronchial epithelium; these are part of the mucosal-associated lymphoid system (see Chapter 1).521 Bacterial vaginal commensals, such as group B Streptococcus (GBS), are often isolated, but fungal infection with Candida may also occur (Figure 83). In the abortuses, despite a florid histological pneumonia, cultures may be negative. Mycoplasma or Ureaplasma infections have been suggested as the etiology for a proportion of these cases.522,523

Neonatal bacterial pneumonia Neonatal pneumonia is classified depending on its time of onset. The implication is that early infection is acquired either

Figure 83. Specific features are uncommon in ascending infections but occasionally a specific organism such as Candida is identified. Inset – periodic acid-Schiff stain demonstrates classic Candida morphology.

ante or intra-partum from aspirated infected or colonized material, such as amniotic fluid, blood or vaginal material. Late infection is more likely to be acquired postnatally. Early onset is often considered to be up to 48 h after birth but there are other definitions.524 During the first week of life, the timing and source of infection may depend on individual case review. One study recorded an incidence of early neonatal pneumonia of 1.79 per 1000 live births.525 While pneumonia is an important aspect of the process,524 recent studies tend to report sepsis rates rather than lung-confined infections. Early-onset sepsis in very low birthweight infants may be as high as 15–19 infants per 1000 livebirths.526 Early-onset pneumonia, especially in the preterm infant, may present with the clinical and radiological features of RDS.

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Figure 84. Pseudomonas sepsis. Hemorrhagic lung (some old) is commonly seen. One vessel shows a profusion of organisms in the wall (inset).

In the term infant, infection may present with respiratory distress accompanied by other features of sepsis. If infection is overwhelming, especially with GBS, it may mimic birth asphyxia. The commoner organisms causing early-onset pneumonia include GBS, Streptococcus pneumoniae, Haemophilus influenzae, enteric bacteria and Staphylococcus. Group B Streptococcus causes up to 70% of infections.525 Enteric bacteria are becoming more significant.526 Group B Streptococcus can present with overwhelming infection and death may occur rapidly. In the preterm infant, the characteristics of the infection can be clinically and pathologically very similar to HMD. At autopsy, the lungs are heavy and red/purple. Occasionally a blood-stained effusion is present. Histologically, the lung features a nonspecific pneumonia, but with severe GBS infection hyaline membranes may demonstrate with a blue-tinge, due to the overwhelming numbers of bacteria (Figure 72).527 Late-onset neonatal pneumonia results from infection by environmentally acquired organisms, rather than those acquired from the mother. These pneumonias usually afflict preterm infants on neonatal units towards the end of their first week of life. Pseudomonas and occasionally Proteus are implicated. Since Pseudomonas is a frequent colonizer of the infant airway, it may only be recognized as an etiological agent at autopsy. Characteristic involvement of vessel walls (Figure 84), thrombosis and hemorrhagic exudates suggest its pathological role.528,529 Pseudomonas may account for up to 11.7% of nosocomial acquired pneumonias.530 In septicemia, the lung may be a site of infection, possibly secondary to an in-dwelling catheter. More uncommon pneumonias may be due to atypical organisms such as Listeria monocytogenes.531,532 Often part of a disseminated infection, it leads to small abscess formation (Figure 85) with a gradually increasing proportion of macrophages and granuloma formation. Syphilis, acquired transplacentally, leads to disseminated infection with white patches

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Figure 85. Listeria monocytogenes abscess in preterm infant lung. Many neutrophils show karyorrhexis. Tissue Gram, silver and Dieterle stains demonstrate the Gram-positive pleomorphic rods.

identifiable on the cut surface of the lung. Histologically, there are areas of fibrosis and a mononuclear interstitial infiltrate. Bordetella pertussis is a rarely recognized cause of pneumonia in the very young (< 4 months of age) and may be associated with intractable pulmonary hypertension. The pathology is dominated by a necrotizing bronchiolitis with intra-alveolar hemorrhage and edema.533 Chlamydia trachomatis may cause a mild pneumonic illness with a mixed alveolar infiltrate of macrophages, lymphocytes, plasma cells, eosinophils and neutrophils. Rarely, bronchiolitis-associated lung collapse occurs.534,535 Candida is the commonest fungal infection and carries a high mortality rate (Figure 83).536–538 Aspergillosis is also implicated. Both these fungi may be associated with hemorrhagic pneumonias, due to vessel involvement.539,540

Viral infection Viral infections are a potential source of significant neonatal morbidity and mortality.541 Up to 1% of infants admitted to an intensive care unit suffered from viral respiratory tract infections.542

Cytomegalovirus The most serious cytomegalovirus (CMV) infections occur following early maternal primary infection with transplacental transmission to the fetus. In abortuses, the infection is often unsuspected, although it may present with hydrops. It is occasionally associated with pulmonary hypoplasia. At birth, up to 90% of neonates with congenital disease may be asymptomatic. Extra-pulmonary problems are more likely to predominate, with jaundice, hepatosplenomegaly and a purpuric rash.543 Neonatal disease is usually acquired from the mother, either during birth or via breast milk.544 Pulmonary disease in this instance is primarily confined to a self-limiting pneumonitis. A more severe pneumonitis can occur if the neonate is

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

Figure 86. CMV in a lung from a baby dying with chronic lung disease. The acquired CMV may have been contributory. In addition to the cell containing the typical inclusion, another cell is loaded with smaller inclusion bodies (arrow).

Figure 87. Herpes pneumonia. The lung features relatively bland irregular areas of necrosis to which there is often relatively little reaction. Inclusions may found at the margin but immunohistology for HSV can be helpful (inset).

transfused with infected blood. Nosocomially acquired CMV pneumonia also occurs in infants spending excessive time in neonatal units, such as those with BPD. Macroscopically, the lung pathology is nonspecific and may be relatively normal or show focal consolidation. Necrotizing bronchiolitis with parenchymal infarction is apparent. Typical CMV inclusions are detectable in epithelial cells, endothelial cells and macrophages (Figure 86) although they may be scant. Interstitial fibrosis may accompany the lung infection.

pneumonia, marked by, in some cases, respiratory distress and wheezing requiring hospitalization. Most RSV infection occurs in older infants but may be seen in the perinatal period, especially in association with chronic lung disease or congenital heart defects.547 Prematurity may predispose to later RSV infection, with up to 10% of infants of 29–32 weeks gestation requiring rehospitalization.548 After recovery from the acute illness, there is a longer-term morbidity with an increased risk of wheezing and asthma.546 Two broad patterns of histology can be found but these probably represent opposite ends of a spectrum.549–551 The bronchiolar pattern shows plugging of bronchioles by mucus and mixed inflammatory cells associated with epithelial desquamation. Air trapping can occur distal to the affected airway. Alveolar spaces are lined by giant cells, while proximal airways feature squamous metaplasia. The distal pattern of RSV infection shows more alveolar plugging by eosinophilic debris, more giant cells (Figure 88) and paranuclear globular inclusions that vary in size.551 Giant cells are not specific and may be found in other viral diseases, such as measles and parainfluenza. Respiratory syncytial virus culture from nasal washes, aspirates or throat swabs may produce a cytopathic effect in 3 to 7 days but PCR studies on secretions now provide a rapid and sensitive method of detection. The prognosis for most babies who acquire RSV is generally good but there is a higher mortality rate in preterm infants with chronic lung disease.552

Herpes simplex virus Herpes simplex virus (HSV), predominantly type 2, involves the lung as part of a disseminated disease. The virus is acquired primarily during the birthing process from maternal secretions, although up to 10% of infections may be acquired postnatally. Mothers with primary, rather than secondary, disease generate the greatest risk.545 Lung involvement, in addition to disseminated viral infection, usually presents after 1 week of life and up to 50% of infants die. Disseminated disease has been reduced significantly by the use of antiviral drugs. The lung may appear normal or consolidated with small white necrotic foci on the cut surface. Histologically, the necrotic foci are bland and inflammation may be remarkably absent (Figure 87). Hyaline membranes or a pneumonitis may be present and inclusions can usually be detected around the margins of the necrotic foci. Where there is any doubt, anti-herpes antibodies are now readily available for immunohistological confirmation.

Respiratory syncytial virus Respiratory synycytial virus (RSV) is essentially an infection of the younger child and most will have been infected by the age of 2 years.546 In most cases symptoms are mild, nonspecific and coryzal in nature. Others may develop a bronchiolitis or

Metapneumovirus Metapneumovirus is a relatively recently described virus. Serological evidence of infection is found in most children by the age of 5 years.553 Presentation is very similar to RSV, although the age at presentation tends to be slightly later. The symptomatology is also very similar with cough, wheezing and

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Figure 88. A giant cell pneumonia associated with respiratory syncytial virus infection.

exacerbation of asthma, with some reports suggesting that the infection may be slightly less severe when compared with RSV. There may be co-infection with other viruses and fatal cases have been reported.554,555 Histopathology of a lung biopsy556 suggests the virus primarily affects the airway epithelium, causing degeneration and necrosis. An acute inflammatory response and hemorrhage are also seen. An expansion of bronchus-associated lymphoid tissue, squamous metaplasia and an accumulation of foamy macrophages develop. Inclusions may be seen in bronchoalveolar lavage specimens.

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Figure 89. Adenovirus pneumonia may be very aggressive and superficially may even resemble a bacterial bronchopneumonia. Here there is necrosis with abundant karyorrhexis. There may be macrophages and some neutrophilic reaction. Very occasional very large inclusions may be present (inset).

pneumonitis. Focal hyaline membranes may be seen and some changes may be attributable to disseminated intravascular coagulation.562 In adenovirus, a necrotizing bronchiolitis may be prominent566 and desquamated bronchiolar epithelial cells may contain inclusions.567 A bronchopneumonic pattern of inflammation has also been described (Figure 89).568

Other viruses

Pediatric lung tumors Introduction

A variety of other viruses, including enterovirus, rhinovirus, adenovirus, and parainfluenza cause neonatal lung disease.557 Neonatal enterovirus, including coxsackie A, B and echovirus,557–561 causes neonatal pneumonia and can be acquired through aspiration or ingestion of infected secretions during birth. Tranplacental infection may occur but is less common. In general, vertically acquired congenital pneumonias are more serious and can be fatal. Most infants with enterovirus infection have a relatively mild course but about 10% will have a more serious systemic illness, which usually includes a pneumonitis. The severely ill infants tend to present on the first day of life, rather than at the end of the first week.558 Echovirus infections, most commonly type 11, may resemble acute bacterial infections.561–563 These have been associated with severe pulmonary hypertension.564 Adenovirus may cause a very severe acute pneumonia when congenitally acquired.565 The pathology of neonatal viral pneumonia is relatively nonspecific and most of these viruses give rise to similar pathology. Macroscopically, lungs are firm with patchy consolidation and hemorrhage. Like RSV, the microscopic appearance is a spectrum from a primarily airway disease with bronchiolar necrosis, epithelial plugging and air trapping to interstitial

Primary lung tumors are rare in the pediatric age group and metastatic tumors far exceed the number of primary lesions.569–571 Lung lesions in children are far more likely to be benign developmental or reactive lesions than neoplasms. These include the common malformations, such as pulmonary sequestrations and CPAM. The solid parenchymal lesions most often represent inflammatory, infectious or reactive processes.572,573 Metastases are by far the commonest malignancy of the lung in children and account for around 80% of all lung tumors.569 They comprise a different spectrum of tumors to those seen in the adult population. Metastatic tumors most commonly seen in children and adolescents are Wilm’s tumor, osteosarcoma, primitive neuroectodermal tumor, rhabdomyosarcoma and neuroblastoma. Primary pulmonary malignancy accounts for approximately 0.2% of all malignancies in children.569,571 While virtually any adult-type neoplasm, including small cell carcinoma, can arise in the pediatric population, the most commonly reported tumors include inflammatory myofibroblastic tumor (IMT), while the commonest primary malignancies of the lung in children are pleuropulmonary blastomas and carcinoid tumor.569

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Table 12 National Cancer Registration for England: primary lung cancer 0–16 years old 1995–2006 (51 primary cases in lung or pleura)

Table 13 National Cancer Registration for England: primary lung sarcomas 0–16 years old 1995–2006 (total 11 cases)

Malignant tumors NOS

11 cases (21.5%)

Rhabdomyosarcoma NOS

2 cases (3.9%)

Carcinoid tumors

12 cases (23.6%)

Rhabdomyosarcoma alveolar

2 cases (3.9%)

Sarcomas

11 cases (21.5%)

Rhabdoid tumor

1 case (2.0%)

Pleuropulmonary blastomas

11 cases (21.5%)

Desmoplastic small round cell tumor

1 case (2.0%)

Primitive neuroectodermal tumor

4 cases (7.8%)

Spindle cell sarcoma

1 case (2.0%)

Carcinomas

6 cases (11.8%)

Table 14 National Cancer Registration for England: primary lung carcinomas 0–16 years old 1995–2006 (total 6 cases)

Mucoepidermoid carcinoma

3 cases (5.9%)

Squamous cell carcinoma

3 cases (5.9%)

Data from the National Cancer Registration for England (Table 12) show there were 51 primary lung tumors in 0–16-year-olds between 1995 and 2006. Eleven cases of pleuropulmonary blastoma were reported. In addition there were 11 sarcomas (Table 13), including primitive neuroectodermal tumor, rhabdomyosarcoma and desmoplastic small round cell tumor, among others. Primary carcinomas in children are rare and only six were registered between 1995 and 2006 (Table 14). These are coded diagnoses and no specific pathological review was undertaken (see Chapter 24). Suffice it to say, virtually any benign or malignant neoplasm can afflict a child.

Pleuropulmonary blastoma In 1988, Manivel et al. were the first to describe pleuropulmonary blastoma (PPB) as a distinct intrathoracic/pulmonary neoplasm of childhood.574 This neoplasm has characteristic blastematous and sarcomatous features that differentiate it from the biphasic epithelial-stromal morphology of the classic adult type pulmonary blastoma and well-differentiated fetal adenocarcinoma. Pulmonary blastomas (PB) are rare malignant primary lung tumors, now classified in the latest version of the WHO classification as a subtype of sarcomatoid carcinoma.575,576 It is difficult to ascertain the true incidence of pulmonary blastomas in children. This is mainly due to the fact that the terms PB and PPB have been used interchangeably in many previously reported cases and reviews. No more than 5% of PB are diagnosed in children younger than 10 years of age. A neonatal PB was recently described by Reichman et al. and accepted by the International Pleuropulmonary Blastoma Registry (www.ppbregistry.org).577

Clinical and radiographic features Pleuropulmonary blastoma usually presents in the first 4 years of life but is rare after 10 years of age. As it is thought that the three pathological subtypes reflect tumor progression over time, it is not surprising that the median age of presentation

for type I tumors is approximately 9 months of age, while the type II and type III tumors are diagnosed at 31 and 42 months, respectively. The common clinical features of PPB at presentation are respiratory distress, fever, chest or abdominal pain, cough, anorexia and/or malaise. The main indication for surgery includes presence of pulmonary cysts (type I), spontaneous pneumothorax (type I) or an intrathoracic mass (types II or III). Radiographic abnormalities include peripheral cysts with or without accompanying densities or a homogeneous mass with partial or complete obliteration of the hemithorax. In these cases, the tumor can surround or infiltrate contiguous structures, including the great vessels and pericardium. Although most cases are reported to arise in a lobe of the lung, in up to 25% of cases the mass is extrapulmonary with attachment to the parietal pleura, and diaphragmatic or mediastinal involvement.578

Macroscopic pathology Type I tumors are multicystic with thin septations, while type III tumors are solid, friable masses often filling a hemithorax with a yellowish-white mucoid appearance (Figures 90 and 91). These tumors may measure up to 20 cm and weigh up to 1200 g. Type II lesions have a mixture of cystic structures with solid areas or thickened septa.574,578–581

Histopathology Type I PPB is the least common subtype (16%). It consists of multiloculated cysts lined by ciliated columnar respiratory epithelium separated by thin fibrous septa. A zone of condensed small, round to spindle-shaped immature cells with a cambium-like appearance of botryoid rhabdomyosarcoma are present beneath the epithelium (Figure 92). Variable numbers of large polygonal or elongated strap cells are admixed with the immature mesenchymal cells. Rhabdomyoblastic differentiation can be demonstrated by immunohistochemical staining for desmin, myo-D1 or myogenin (Figure 93). No solid areas are present in this subtype, but foci of immature cartilage can be present in the cyst wall. This type corresponds to those cases previously reported in the literature as “embryonal rhabdomyosarcoma in congenital lung cysts”, pulmonary blastoma with cysts or primary rhabdomyosarcoma of the lung.569,574,579,580

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 90. Type I PPB. Multiloculated cyst with thin septa and no solid areas. (Image courtesty of Dr Michael McDermott, Dublin, Ireland.)

Figure 91. Type III PPB. Solid tumor. (Image courtesy of Dr Michael McDermott, Dublin, Ireland.)

Type II PPB represents up to 44% of all PPB. These have areas of type I PPB morphology, but also solid areas consisting of irregular islands of blastematous cells (Figure 94). In addition, clusters of anaplastic cells, some with rhabdomyoblastic features, as well as scattered nodules of malignant/immature cartilage are seen. These anaplastic cells can also have intraand/or extracellular hyaline globules. Sheets of undifferentiated spindle cells with a vague fascicular fibrosarcomatous growth pattern are usually noted in solid areas. Entrapped respiratory epithelium should not be mistaken for a malignant

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epithelial component. The malignant stroma resembles that of Wilm’s tumor. The chondroid component is usually minor, but in occasional cases it can be prominent enough to raise the possibility of chondrosarcoma. S100 protein staining is confined to the areas of conspicuous cartilaginous differentiation, and cytokeratin is limited to the epithelium of the cystic spaces or entrapped respiratory epithelium or mesothelial cells.574,581 Type III PPB accounts for up to 40% of all cases in children. These entirely solid tumors have a spectrum of histological

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms Figure 92. Type I PPB. Cyst wall lined by ciliated columnar epithelium with condensed immature cells. (Case courtesy of Dr Lilian Boccon-Gibod, Paris, France.)

Figure 93. Type I PPB. Desmin staining demonstrates rhabdomyoblastic differentiation. (Case courtesy of Dr Lilian Boccon-Gibod, Paris, France.)

Figure 94. Type II PPB. Solid areas of this solid and cystic neoplasm contain undifferentiated spindle cells.

appearances (Figure 95). Some are predominantly blastematous, while others can resemble well-differentiated mesenchymal chondrosarcoma.571,581 This type of tumor can also show pericytomatous or liposarcomatous patterns. Myxoid degeneration may be prominent, while areas of necrosis leading to cyst formation should not be confused with type II PPB.574,579,581 These cystic areas do not have an epithelial lining. Rhabdomyosarcomatous elements usually have an embryonal appearance, although alveolar features in an otherwise typical PPB have also been described.582

Pathogenesis There are approximately 330 identified cases of PPB worldwide.583 It remains uncertain as to whether PPB arises in lungs with an underlying congenital pulmonary airway malformation

or if the tumor induces the formation of epithelial lined cysts (see below). Vargas et al. examined the cytogenetic and Tp53 profiles in CPAM and PPB. These authors concluded that although some may view CPAM as a PPB precursor, it is biologically distinct in terms of karyotypic and Tp53 status.584 However, there are numerous reports of sarcomatous and blastomatous transformation in CPAM,22 as well as cases of type 4 CPAM transforming to type III PPB.189,585–587,596 It is now considered that previously reported cases of rhabdomyosarcoma arising in congenital cysts actually represent type I PPB.579,589–584 Cases of type 4 CPAM with limited resection recurring as type III PPB most likely represent an incompletely excised Type I PPB.199,602 Thus type I PPB is a potentially deceptive lesion and all multicystic lesions in the lung should be extensively sampled and investigated with myogenic immunohistochemical markers.199,602 There is no evidence that PPB is a variant of pulmonary blastoma, as has been previously suggested.603 In fact no histological features of well-differentiated adenocarcinoma of fetal type or classic biphasic epithelial mesenchymal pulmonary blastoma have been described to date in cases of PPB. Malignant epithelial features are not described in PPB.580

Treatment and prognosis The therapeutic approach for PPB includes surgery, chemotherapy and radiotherapy.604 There is a correlation between the gross and microscopic features of PPB at presentation and prognosis. Patients with type I PPB have a better prognosis than those with either types II or III.600,601 However, if type I PPB recurs, it is usually as a type II or III PPB and it then has the same prognosis as these tumors.597 The 5-year survival rate of type I PPB is 80 to 85%, while types II and III PPB have a 5-year survival rate of only 45 to 50%.578 Based on clinical, gross and histological findings as well as observational survival data, it is reasonable to suggest that PPB progresses over time from type I to type III lesions.

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(a)

(b)

(c) Figure 95. Type III PPB. (a) This low magnification view demonstrates blastematous and less cellular areas of the neoplasm. (b) The different cell populations are apparent. (c) Blastematous areas with well differentiated mesenchymal chondrosarcoma-like areas. (Case courtesy of Dr Michael McDermott, Dublin, Ireland.)

Local recurrences and metastases describe the clinical course. Distant metastases are associated with types II and III tumors, the brain being the most common site. PPB may also spread to bones, lymph nodes, liver, pancreas, kidneys and adrenals.588,607 Metastases usually lack the morphological heterogeneity of the primary tumor. Extrapulmonary involvement at diagnosis confers a worst prognosis.578,605 Total resection of the tumor results in an overall better prognosis.605

Genetics Twenty-five percent of PPB occurs in a constitutional familial setting, in which PPB patients or young family members have other dysplastic or neoplastic conditions.608,609 Renal abnormalities,

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usually cystic nephroma, are common in families with PPB and are also associated with an increased incidence of bilateral pulmonary cysts and/or other tumors.608–612 The other commonly occurring tumors in family members include medulloblastoma, embryonal rhabdomyosarcoma, synovial sarcoma, germ cell tumors and hematological as well as thyroid malignancies.609,611–614 Cytogenetic abnormalities are common. Trisomies, particularly gains in chromosome 8, are reported. Other abnormalities include loss of 17p, loss of chromosome 10 or 10q, rearrangement of 11p, loss of chromosome X or Xp, and gain of chromosome/arms of 1q, 2 and 7q.608,615–623 Pleuropulmonary blastoma is often part of an inherited cancer syndrome. The specific genetic abnormality

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

(b)

Figure 96. (a) Embryonal rhabdomyosarcoma. Primitive spindle cells in a myxoid stroma allow diagnosis. (b) Alveolar rhabdomyosarcoma. The characteristic growth pattern is apparent.

predisposing to familial PPB is as yet unknown. However, germline mutations in DICER1 in familial PPB have been described. This gene is located on the long arm of chromosome 14 and encodes an endoribonuclease, critical to the generation of small noncoding regulatory RNAs. It is still not clear whether DICER1 haploinsufficiency contributes to pathogenesis.624

Differential diagnosis The variable morphological appearances of PPB can lead to misdiagnosis. As mentioned earlier, types I or 4 CPAM need to be distinguished from the multicystic type I PPB. Some suggest that type 4 CPAM should be considered as type 1 PPB. The identification of focal areas of increased cellularity, a cambium layer, rhabdomyoblasts and myogenin staining in the primitive mesenchymal cells all aid in differentiating these two lesions. However, absence of staining does not preclude the pathological diagnosis of type I PPB. In addition, the newly described fetal lung interstitial tumor should be distinguished from type I PPB. This solid to spongy lobar-based mass is circumscribed and composed of small airspace-like structures lined by a single layer of TTF-1 positive cuboidal epithelium and septal interstitium expanded by uniform polygonal cells with clear cytoplasm and capillaries.579 It is important to distinguish type I from type II PPB, because of the therapeutic implications for type II and the worse prognosis it carries. For types II and III PPB, the differential diagnosis includes primary and metastatic rhabdomyosarcoma, synovial sarcoma, other undifferentiated or spindle cell sarcomas and malignant teratoma. For tumors with a predominantly blastematous component, metastatic Wilm’s tumor needs to be considered. However, the clinical history and imaging studies should be helpful in arriving at the appropriate diagnosis.

Rhabdomyosarcoma Rhabdomyosarcoma (RMS) is the commonest soft tissue sarcoma in children under 15 years of age, and also one of the more common soft tissue sarcomas of adolescents and young adults. It accounts for 6–8% of all malignancies in children with an annual incidence in neonates and infants of 6.4 cases per million each year and a further 4.5 cases per million per year in children and adolescents.625 Rhabdomyosarcoma in the pediatric population usually presents in the head and neck or urogenital organs. Primary RMS of the lung is rare, and usually raises the differential diagnosis of pulmonary blastoma. As discussed above, examples of RMS arising in pulmonary cysts are likely to represent examples of pleuropulmonary blastoma.189,586,587,589,590 In fact, most cases initially reported as RMS have been retracted and reinterpreted as a pleuropulmonary blastoma.598,601 In 1998 the Intergroup Rhabdomyosarcoma Study Group (IRSG) reported only three primary lung cases out of more than 2000 total RMS cases.626,627 Several other welldocumented cases of primary pulmonary RMS have been reported.629 Primary chest wall RMS may invade into lung. The lung is the most common site for metastases, as almost half the patients have lung lesions. In 18% of metastatic RMS, the lung is the only metastatic site.629 It also arises in the chest wall with extensions into the thoracic cavity.630 Rhabdomyosarcoma in children is currently classified according to the International Classification of Rhabdomyosarcomas (IRS).631 The botryoid and spindle cell subtypes of embryonal RMS fall into the favorable prognostic group, the “classical” embryonal rhabdomyosarcoma in the intermediate prognosis group (Figure 96a), and the alveolar in the unfavorable group (Figure 96b). The alveolar RMS are characterized by balanced reciprocal translocation t(2;13)(q35;q14) and

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

(b)

Figure 97. Ewing sarcoma. (a) This small round blue cell tumor is mitotically active. (b) CD99 membranous staining confirms the diagnosis.

t(1;13)(p36;q14). Diagnoses are aided with immunohistochemical stains including desmin, myogenin and MyoD1. The grouping refers to the behavior of the different types of RMS in the current treatment specification protocol.

Ewing sarcoma Ewing sarcoma, primitive neuroectodermal tumors, Askin tumors (Ewing sarcoma affecting the chest wall) and extraosseous Ewing sarcoma are a family of tumors which share common pathological, immunohistochemical and genetic features (Figure 97). Thoracic tumors often invade into underlying lung. Rearrangement of the Ewing sarcoma gene with one of a variety of fusion partner genes, most commonly Fli-1, is produced by the characteristic t(11;22)(q24;q12) translocation. Primary lung Ewing sarcoma has been described,628–639 but is rare in children. The lung is, however, a common metastatic site, with up to 63% of cases involving the lung upon relapse.640

Synovial sarcoma Synovial sarcoma (SS) is the third commonest type of soft tissue sarcoma to occur in childhood. Nevertheless it is rare in children, with an annual incidence of 0.5 per million. Not more than one dozen cases are diagnosed in the United Kingdom each year.641–644 Across all ages, only about 20% of cases occur in patients less than 19 years old, mainly in the second decade.640 The incidence has changed over time, in part due to more accurate histological diagnosis and, more recently, because of the recognition of a characteristic translocation involving chromosomes 18 and X. This translocation results in a fusion gene (SYT-SSX1, SYT-SSX2, SYT, SSX4) in at least 90% of cases.641,642 While synovial sarcoma occurs predominantly in the extremities, it has been described at virtually every anatomic site, including the pleuropulmonary region (see Chapters 33

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and 36).648–653 Lung tumor morphology in children is more commonly monophasic than biphasic (Figure 98). The overall prognosis of SS appears to be better in children compared to adults, with an overall 5-year survival of 76%.654 Children and adolescents with SS originating at non-extremity locations, including lung, have a worse prognosis compared to limb SS.

Congenital peribronchial myofibroblastic tumor Congenital peribronchial myofibroblastic tumor (CPMT) had previously been described under several other names, including leiomyosarcoma and fibrosarcoma, implying aggressive biological behavior. However, complete surgical resection affords an excellent prognosis.656–658 This tumor is usually recognized shortly after birth or can be detected by imaging studies carried out in the prenatal period. Pregnancy can be complicated by polyhydramnios and non-immune hydrops fetalis.659–661 The tumors are circumscribed but non-encapsulated, measure up to 14.5 cm in diameter and weigh up to 100 g.575 Gray to yellow cut surfaces are noted. Histologically well-circumscribed lesions consist of uniform bland spindle cells arranged in a fascicular pattern. Mitotic activity is variable but can be brisk.575,657 Atypical mitoses have been described.657 A hemangiopericytomatous vascular pattern may be prominent while necrosis, cysts, extramedullary hematopoiesis and small irregular cartilage islands can be seen (Figure 99).662,663 Immunohistochemistry shows consistent staining with vimentin and desmin but smooth muscle actin is generally negative. There are some reports of positivity with various antibodies, including muscle-specific actin, smooth muscle actin, neuron-specific enolase, CD34, CD68, S100 protein, CAM 5.2 and Factor XIIIa.575 The CPMT and classic congenital mesoblastic nephroma (CMN) have many similar features, histologically as well as

Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

(b)

Figure 98. (a) Biphasic synovial sarcoma. This variant features clusters of hyperchromatic epithelioid cells with primitive gland formation along with a spindle cell component. (b) Monophasic synovial sarcoma. Despite the absence of glands, this spindle cell neoplasm has plump tumor cells.

The lung is one of the most common visceral organs involved, and in some cases pulmonary lesions are multifocal. Solitary and multiple lesions in soft tissue and bone carry a good prognosis with spontaneous regression. In newborns and infants with visceral lesions, the prognosis is less favorable as up to 75% of the patients die of respiratory distress soon after birth.664,665

Inflammatory myofibroblastic tumor

Figure 99. Congenital peribronchial myofibroblastic tumor. Fascicles of spindle cells and small irregular islands of cartilage are obvious. (Case courtesy of Dr. Cheryl M. Coffin, Nashville, TN, USA.)

prognostically. A consistent genetic abnormality has not been identified in CMPT.

Myofibromatosis These lesions, when presenting as solitary nodules, are found most commonly in the dermis and subcutis, in the region of the head and neck area, and in the upper and lower extremities. Multifocal lesions (myofibromatosis) are histologically similar to the solitary lesions but affect internal organs, as well as the skeleton.

Inflammatory myofibroblastic tumors (IMT) are rare neoplastic lesions that occur in children and young adults. They are the most common primary pediatric lung tumors and cases in childen account for up to 40% of all IMTs. Pulmonary IMT can involve the bronchus with protrusion into the lumen and also extension into lung parenchyma, but others simply involve lung parenchyma. This neoplasm is discussed in detail in Chapter 33. Inflammatory myofibroblastic tumors are cellular lesions composed of myofibroblasts arranged in short fascicles with a minor inflammatory component (Figure 100a). Nuclear atypia may be present. Immunostaining shows strong positivity for vimentin and focal staining for smooth-muscle actin. Focal desmin and cytokeratin positivity has been described in 69% and 36% of cases, respectively. ALK staining is characterized by diffuse cytoplasmic staining in myoblastic cells (Figure 100b). A chromosomal rearrangement involving the anaplastic lymphoma kinase gene locus on chromosome 2p23 with other partner genes (TPM3 in 17q 23 or TPM4 in 19p13) is noted in up to 75% of pulmonary tumors.666–668 The clinical behavior of IMT is unpredictable. Most are benign but up to 20% recur locally.668 Metastases occur.667 It

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Chapter 3: Congenital abnormalities and pediatric lung diseases, including neoplasms

(a)

(b)

Figure 100. Inflammatory myofibroblastic tumor. (a) Fasicles of cytoplasm-rich spindle cells feature scattered lymphocytes and plasma cells. (b) Cytoplasmic ALK1 staining is noted in up to 75% of cases.

has also been described following treatment for malignant disease in children.669

Disseminated juvenile xanthogranuloma Solitary dermal juvenile xanthogranuloma (JXG) is more common and does not progress to the disseminated form. The latter occurs by the age of 10, and represents half of all cases within the first year of life. There is a known association with neurofibromatosis type 1 and patients with both at higher risk of developing juvenile myelomonocytic leukemia. Disseminated JXG can involve many sites, commonly skin and soft tissue, but the central nervous system, lung, liver, lymph node and bone marrow are all well documented.671–675

Other tumors Bronchial neoplasms including carcinoid tumor, mucoepidermoid carcinoma and adenoid cystic carcinoma are among the

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Chapter

4

Pulmonary bacterial infections Mark Woodhead*, Mary Klassen-Fischer*, Ronald C. Neafie*, Ann-Marie Nelson, Jeffrey R. Galvin and Teri J. Franks

Background “Pneumonia”, wrote Laennec, “is one of the diseases most anciently known; and before pathological anatomy . . . had investigated the true nature of diseases, it was generally regarded as one of the internal afflictions most readily recognized.”1 Pneumonia occurs when the host mounts an inflammatory response, centered on the lung parenchyma, usually against a microorganism, but sometimes against another toxic agent, which has reached this normally sterile site. Bacteria are the most common causative microorganisms. The effect on the host is variable, ranging from complete absence of clinical manifestations to sudden death or a brief illness followed by sudden death. More typically the effects of the inflammatory response and the replacement of the normal gas-exchanging lung tissue cause a constellation of symptoms and signs. These are associated with, and may be diagnosed as, lung infection. Untreated, the condition progresses until the host dies or the inflammatory response overcomes the microbial threat and lysis of the toxic state is followed by gradual recovery. Recognition of the condition will usually lead to appropriate medical intervention, resulting in improved outcome in most cases. Although bacterial pneumonia is diagnosed most frequently without the aid of a microscope, the information in this chapter is organized in a way that we hope will be helpful to practicing pathologists charged with diagnosing the disease in tissue specimens. One objective of this chapter is to provide a foundation of knowledge, including the epidemiology, clinical manifestations, management, prognosis and natural history of bacterial pneumonia. This foundation along with the discussion of the routes of injury, pathogenesis and normal host defense will allow correlation with the histopathological findings. Whereas conventional texts on bacterial pneumonia have emphasized the gross anatomic features of the stages seen at autopsy, this chapter provides more practical information about the radiological manifestations in living patients. Note also that the purpose here does not include reiteration of the

* These authors contributed equally to this work.

details of clinical laboratory identification of bacteria, which are found in microbiology textbooks. This chapter is unique in its presentation of many useful photomicrographs depicting common and uncommon causes of bacterial pneumonia, as they appear in histological sections.

Epidemiology Lobar pneumonia and bronchopneumonia were terms used historically to classify the condition. These terms, although pathologically defined, are not clinically helpful, since they lack specific associations with individual causative organisms and infectious sources. Pneumonia is now usually classified according to the likely source of the causative organism and host immune status. These are the main factors which determine different spectra of causative microorganisms and may be useful as a broad basis for therapy. The main groups are community-acquired pneumonia (CAP), nosocomial (hospital or healthcare-acquired) pneumonia (NP) and pneumonia in the immunocompromised. Although microaspiration of oropharyngeal flora is probably the usual mechanism of onset of pneumonia, there is a separate category of aspiration pneumonia, which occurs after macroaspiration of larger volumes of oropharyngeal material. Pneumonia is common worldwide and can occur in any individual, even without obvious risk. In high-income countries, about one per thousand adults develop the condition each year, with higher frequencies in the first years of life and in the elderly.2 The frequency progressively rises from about the age of 50 years.3 Pneumonia kills more children than any other infectious disease, with 99% of those deaths occurring in developing countries. In high-economy countries, most cases of CAP will be managed outside hospital, with from 8%4 to 58%5 admitted to hospital and from 3%6 to 15%7 managed in the intensive care unit. A variety of microorganisms can be responsible for pneumonia in each category with geographic differences noted (Table 1). Pneumonia is usually mono-microbial, but sequential and mixed infections with more than one organism can occur.

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 4: Pulmonary bacterial infections Table 1 Causes (%) of Community-acquired pneumonia in recently published series in adults

Where performed:

UK16

USA53

Spain54

Chile55

S. Africa22

Thailand56

Number of cases studied

267

205

474

176

259

147

S. pneumoniae

48

15

13

24

29

22

H. influenzae

8

7

0.5

3

1

0

Legionella spp

3

3

2

2

0.5

5

Staph. aureus

2

3

0

1

3

3

M. catarrhalis

2

0

0

0.5

0

0

GNEB

1

7

0.5

1

26

18

M. pneumoniae

3

1

5

1

1

7

C. pneumoniae

13

3

8

1

NA

16

C. psittaci

2

0.5

0.25

0

NA

NA

C. burnetii

1

0

2

0

NA

NA

All viruses

25

3

10

13

1

NA

Influenza A and B

20

3

4

5

1

NA

Mixed

27

NA

NA

7

3

6

Other

2

9

4

0

0

5

None

25

37

59

44

24

29

GNEB, Gram-negative Enterobacteriaceae; NA, not available.

In studies of CAP, the largest patient group is usually those with no pathogen identified. There are reasons to believe that most of these patients have undiagnosed pneumococcal infection.8 This means in adults, 75–90% of cases are bacterial in origin, with the remainder caused by viruses. Fungal pneumonia occurs more frequently in the immunosuppressed. Streptococcus pneumoniae (pneumococcus) is the most common organism in adults worldwide. The organism is delicate and easily missed, so the exact frequency varies from study to study. The typical frequency is 25–50% of cases, but up to 76% of CAP pneumonias have been attributed to this cause.9 Streptococcus pneumoniae remains the most frequent cause of severe pneumonia and the commonest reason for pneumonic death. The organism is a normal resident in the upper respiratory tract of 12% of the population. This frequency rises to as much as 28% in young children.10 These children often act as the source for infection in older persons. This has recently been confirmed by the reduction in elderly pneumococcal bacteremias, resulting from vaccination of young children with conjugate pneumococcal vaccine.11 Nearly all pneumococcal pneumonias are sporadic but occasional outbreaks occur in vulnerable individuals e.g., residents of men’s shelters.12 Haemophilus influenzae is the other common bacterial cause of CAP, although much less frequent than Streptococcus pneumoniae. It has no specific characteristics and although once thought to be more common in those with underlying chronic lung disease, the evidence to support this is weak.13 Moraxella catarrhalis is often implicated as a cause of chronic obstructive pulmonary disease (COPD) exacerbation but is a very rare cause of CAP.

Mycoplasma pneumoniae, Chlamydophila, Coxiella and sometimes Legionella may be grouped as the “atypical organisms”. These organisms share the necessity or propensity (Legionella) for an intracellular existence within host cells. This property is partly responsible for a common lack of susceptibility to b-lactam antibiotics. For these reasons the organisms, but not the clinical illness they cause, continue to be grouped as “atypical”. Mycoplasma pneumoniae is probably the next most frequent organism, being particularly a cause of a non-severe infection in teenagers and young adults and it is therefore common in patients managed outside hospitals. In the United Kingdom, it has a curious periodicity, occurring in epidemics spanning two or three winters every 4 years. It can also be a cause of outbreaks in closed communities of young people, e.g., schools and colleges. The role of Chlamydophila pneumoniae (previously Chlamydia pneumoniae) as a cause of CAP continues to be debated. Studies appear to identify the organism with a high frequency.14 Its apparent frequency, however, depends very much on the methods used to detect it. Most methods have been serological and not well validated, and newer PCR-based methodologies appear to be no more reliable.15 When detected, other pathogens (e.g., Streptococcus pneumoniae) are often also present, and patients often recover with antibiotics to which the organism is not sensitive.16 Occasional cases of severe CAP, where Chlamydophila pneumoniae is the only organism found, have been described. Undoubtedly a cause of CAP, its overall importance is now generally thought to be limited.

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Chlamydophila psittaci is a rare cause of CAP. It is classically acquired from birds (ornithosis or psittacosis), especially of the parrot family, but may also be transmitted from ungulate animals, especially at parturition, and occasionally cats. If acquired by pregnant females it may give rise to abortion, in addition to pneumonia. Coxiella burnetii is another atypical pathogen that is generally a rare cause of CAP. It is another zoonosis, usually from ungulate animals, but occasionally cats or rabbits. It can be a common cause of CAP especially in sheep-rearing communities, e.g., north-west Spain and rural Nova Scotia, Canada.17,18 Many outbreaks have been described, apparently remote from such animal hosts. In most cases, materials (e.g., straw) contaminated with animal products prove to be the source. The organisms are particularly released at the time of parturition, which means the humans most likely to be affected are farmers or veterinarians. The Legionella genus is a curious group of bacteria, which are probably only accidental causes of pneumonia (Legionnaires’ disease). A large number of species have been described and many have been linked with human disease. Most cases are due to a single species Legionella pneumophila and serogroup 1 of this species accounts for 95% of cases. The organisms are often found in environmental water, where they live in close association, including intracellularly, with environmental amebae. They thrive only at warm temperatures. Man becomes infected usually only in situations where warm water first of all becomes stagnant, allowing the organism to multiply and then secondly aerosolized, so the organism can be inhaled. Many such sources have been described, including potable tap water, air conditioning systems, decorative fountains and even birthing pools. With the exception of the last situation, CAP due to this organism is almost uniquely an adult phenomenon. Because of these common sources, outbreaks are common, the largest to date being of 449 cases in Murcia, Spain.19 Many cases are travel-associated, because water-containing systems in hotels can be a source. For the same reasons, the organism can be a cause of nosocomial pneumonia and pneumonia in the immunosuppressed. Although most clinical cases manifest pneumonia, the organisms can also cause a milder, self-limiting flu-like illness, known as Pontiac fever. Sporadic cases of Legionella pneumonia tend to be severe. The second most common cause of Legionnaires’ disease is Legionella micdadei. Other important causes of CAP include Gram-negative bacteria and Staphylococcus aureus. These organisms, together with Legionella species, are often associated with severe illness and are more commonly encountered in patients managed in the intensive care unit. A variety of Gram-negative bacteria can cause CAP, including Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae and Proteus mirabilis. Colonization of the oropharynx is usually a precursor to pneumonia. In some parts of the world, Gram-negative bacteria appear to be common causes of CAP, especially severe disease.

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Staphylococcus aureus has a propensity to occur following influenza virus infection. It has long been known that a small proportion of Staphylococcus aureus organisms can produce toxins, such as the Panton-Valentine leucocidin (PVL), which can be responsible for a particularly severe and destructive pneumonia.20 Recent evidence suggests that, although rare, such organisms are increasing in frequency. In other parts of the world, variations on this basic causative pattern are found. Burkholderia pseudomallei is second only to pneumococcus as a cause of CAP in Southeaste Asia and Northern Australia,21 Klebsiella pneumoniae is particularly common in South Africa.22 Mycobacterium tuberculosis should never be forgotten as a mimic of CAP (see Chapter 6). In addition, there are many other bacteria which can rarely give rise to CAP, many of which are zoonoses, such as Brucella species (acquired from ungulate animals), Francisella tularensis (known as tularemia, acquired from rodents), Yersinia pestis (known as plague and acquired from rodents and cats), Bacillus anthracis (anthrax, usually from contaminated animal products) and Leptospira species (usually acquired from rats or rat-contaminated water). The pattern of causes of nosocomial pneumonia (NP) is quite different from that of CAP. Gram-negative bacteria, Staphylococcus aureus and Acinetobacter are the most frequent causes. In these organisms, antimicrobial resistance is a common phenomenon. Atypical organisms, with the exception of Legionella, and viruses do not usually cause NP.23 In the immunocompromised patient, organisms usually considered to be non-pathogenic can also cause pneumonia, with the pathogens dependent on the type of immune suppression. In aspiration pneumonia, anaerobic and Gram-negative bacteria are the most frequent causes. Anaerobic bacteria are also associated with severe illness and are more common in patients with chronic alcoholism.

Clinical manifestations Pneumonia usually presents as an acute illness of abrupt onset with fever, sometimes rigors (chills), cough, sputum production, which is often purulent (or “rusty”), breathlessness, anorexia, and sometimes pleuritic chest pain and hemoptysis. In the elderly the presentation may be much less specific, with alteration in mental function, falls and incontinence as dominant features with or without some of the above symptoms. None of the symptoms of pneumonia are unique to this condition, and it may be difficult to separate from other respiratory conditions such as acute bronchitis or a COPD exacerbation. The unique feature of pneumonia is consolidation within the lung parenchyma. Every medical student knows that dullness to percussion, bronchial breathing, sometimes with egophony (a peculiar broken quality of the voice sounds, like the bleating of a goat, heard at the upper level of a parapneumonic effusion) and whispering pectoriloquy, with or without a pleural rub, indicate underlying consolidation. Unfortunately, this classic association is present in only a minority of those with underlying consolidation, while many

Chapter 4: Pulmonary bacterial infections

have only localized lung crackles or no respiratory signs. Clinical features may also include fever, tachycardia and tachypnea, and sometimes, in severe cases, mental confusion. In contrast to Laennec’s statement at the beginning of this chapter, the accurate clinical diagnosis of pneumonia is often difficult. In some the presentation may be more indolent. Symptoms such as dry cough, muscle aches and arthralgia may develop over many days with minimal respiratory clinical examination findings and diffuse rather than focal radiographic shadowing. The identification of such features in association with infection by Mycoplasma pneumoniae first led to use of the term “atypical pneumonia syndrome”.24 Subsequently similar features were found in infections with Chlamydia and Coxiella and more recently Legionella. All four were then grouped under the “atypical” category. Indeed a clinical constellation, supposedly diagnostic of Legionella infection, was described, including mental confusion, hyponatremia, abnormal liver function and sometimes hematuria. Subsequent studies have shown that, although statistically true, these associations are part of a spectrum of presentations that occur in pneumonia.25 Extreme presentations can still be used to predict the likely pathogen but this approach will not work in the vast majority of cases. The features linked with Legionella infection are now recognized to be features of severe pneumonia, regardless of causative pathogen. For these reasons the term “atypical pneumonia” is no longer considered to be clinically useful.

Routes of injury Microorganisms typically enter the lungs and cause infection by one of three routes: most commonly the airways, but also the pulmonary vasculature, and by direct extension from the neck, mediastinum, chest wall, or across the diaphragm. Infections thus acquired give rise to characteristic patterns, which, particularly early in their course, can be recognized on radiological and pathological studies. Although the radiological and pathological patterns are most often nonspecific with regard to the causative organism, they are useful in delineating the route of infection. This focuses the clinical and physical interrogation of the patient and limits the list of possible organisms. The radiological-pathological correlation that establishes the route of infection begins with the anatomy of the secondary lobule, defined by Miller as the smallest lung unit surrounded by a connective tissue septum.26 Modern high-resolution computed tomographic (HRCT) scans of the lung accurately reflect the normal anatomy and pathological process at the level of the secondary lobule (Figure 1) as defined by Miller.27–29 Infection acquired through the airways most commonly occurs by aspiration or inhalation of microorganisms. Less commonly, airway involvement is the result of seeding from an infected source, e.g., peribronchial lymph nodes, bronchoscope,30 or tracheostomy site.31 Aspiration is the introduction of solid or liquid material into the lungs, which can lead to parenchymal damage by two mechanisms, based on volume. In large amounts (macroaspiration), aspirated material tends to

Figure 1. Secondary lobule. Schematic of the secondary lobule (left illustration) as described by Miller bounded by a fibrous septum through which runs pulmonary veins and one set of lymphatics. The centrilobular or core structures are situated in the center of the lobule and comprise the respiratory bronchiole, arteriole and a second set of lymphatics. Airway spread (center illustration) of infection commonly results in centrilobular (bronchiolocentric) nodules with sparing of the pleural surface. Hematogenous spread (right image) of bacteria during episodes of septicemia creates a “miliary” pattern of small nodules that is random in distribution and involves the pleural surface. (Courtesy of Aletta Ann Frazier, MD, Baltimore, MD, USA.)

cause injury to the lung by direct chemical or physical means, “so-called” aspiration pneumonitis. Once injured, the lung may become secondarily infected by bacteria, making identification of the initial pathogenic mechanism impossible. In small amounts (microaspiration), aspirated oral and nasal secretions containing microorganisms may be the cause of the pneumonia. It is generally diagnosed according to the specific causative organism, e.g., Gram-positive pneumonia, Streptococcus pneumoniae.32 Inhalation refers to breathing air contaminated with microorganisms, e.g., inhalation of contaminated aerosols produced by patients infected with bacteria, such as Mycobacterium tuberculosis, via talking, coughing, sneezing or singing.33 Events following deposition of bacteria on airway or alveolar epithelial surfaces vary depending on the virulence of the organism, the size of the microbial inoculum, the immune status of the host, and the presence or absence of underlying lung disease. These events can be organized into four groups: (1) transient infection, where the bacterial load remains low, the epithelial surfaces remain intact, and the primary lung defenses (mechanical barriers, mucociliary escalator, and cell junctional complexes) destroy and clear the organisms; (2) acute infection, where the bacterial load is higher, the primary pulmonary defenses fail and bacteria multiply. This results in an acute inflammatory response leading to recruitment of secondary lung defenses, particularly neutrophils (see Pathogenesis and normal host defense). The patient becomes acutely ill, and the inflammatory response sterilizes the airways and parenchyma leading to resolution of the infection; (3) colonization, where a low-grade, mild, inflammatory process typically occurs in a background of abnormal host defense. A balance is achieved between pulmonary lung defenses and bacterial replication, and patients often have acute self-limited exacerbations; (4) chronic infection, where the bacterial load is sufficient to cause a marked inflammatory response, but the response

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Chapter 4: Pulmonary bacterial infections

Figure 3. Necrotizing granuloma caused by Gram-positive cocci (H&E).

Figure 2. Escherichia coli. This gross specimen, from a patient with E. coli infection, demonstrates diffuse consolidation of the lung parenchyma and shows the secondary findings of hemorrhage and abscess formation.

fails to completely clear the organisms. This results in a self-perpetuating process that can lead to chronic airway injury, such as bronchiectasis.34 Bacterial infection of the lower respiratory tract, acquired through the airways, may be unilateral or bilateral and initially involves the conducting airways (trachea, bronchi, or bronchioles). Once established in bronchioles, spread of infection to surrounding alveolar spaces ensues and manifests as parenchymal consolidation. Grossly, consolidation is visible as well as palpable and is evidenced by pale, firm areas that on cut section protrude above the surrounding lung. Under pressure, airways within consolidative areas often yield purulent material. Initially consolidation is bronchiolocentric (centrilobular), but may become segmental, involve an entire lobe, multiple lobes, or the entire lung (Figure 2). As most bacterial infections of the lung are the result of aspiration or inhalation, consolidation is often most prominent in the lower lobes. Both gravity and physiological airflow to the lower lobes are responsible for the lower lobe predominance. Termed acute bronchopneumonia, bacterial infection of the lungs in immunocompetent patients is most commonly characterized histologically by the presence of pus, containing fluid, neutrophils, debris of dead cells, and fibrin in

150

conducting airways and alveolar spaces. The acute inflammation is variably accompanied by the secondary features of hemorrhage, necrosis, and abscess formation. In immunocompromised patients, the pattern may be altered and include granulomas (Figure 3). Clearing the acute bronchopneumonic exudate generally follows one of two paths. Most often, enzymatic liquefaction allows the exudate to be expectorated or absorbed into the lymphatics with return to normal pulmonary function. Less commonly, organization of the exudate occurs, evidenced by plugs of loose fibroblastic tissue within bronchioles and alveoli (so-called “organizing pneumonia” or “Masson bodies”). This is followed by fibrosis associated with variable loss of normal lung architecture. The remaining two routes of infection, through the pulmonary vasculature and by direct extension from the neck, mediastinum, chest wall, or across the diaphragm, are less common than via the airways. Infection acquired through the pulmonary vasculature most often arises from an extrapulmonary source secondary to sepsis (organisms circulating free in the blood) or septic emboli (organisms associated with a thrombus). Hematogenous spread of organisms by sepsis and septic emboli both cause parenchymal nodules, but with different distribution patterns, based on size of the circulating particles. In sepsis, circulating single organisms or small clusters of organisms are sieved out in similarly sized pulmonary capillaries, resulting in numerous small 1 to 5 mm parenchymal nodules. These are randomly distributed from the center of the secondary lobule to the pleura and are referred to as the “miliary pattern” of infection. By contrast, septic emboli, which are larger and more variable in size, are sieved out in the dichotomous pulmonary arteries anywhere from the main pulmonary artery to the small pulmonary arteries at the level of the terminal bronchioles. These result in parenchymal nodules that are fewer in number and are associated with vessels, the so-called “feeding vessel sign”. Hematogenous spread of organisms is characterized histologically early in

Chapter 4: Pulmonary bacterial infections

(a)

(b)

Figure 4. Infectious bronchiolitis “tree-in-bud”. (a) Coronal reconstruction of axial computed tomographic date in a 63-year-old male with bacterial bronchiolitis demonstrates a typical (left image) tree-in-bud pattern with branching structures (arrowhead), and (right image) indistinct small (1–3 mm) nodules (arrowhead) that are predominantly centrilobular. (b) This gross specimen shows bronchiolocentric (centrilobular) pale areas of acute bronchopneumonia that correlate with the similarly distributed areas of consolidation on imaging.

the disease by intravascular collections of organisms with or without acute inflammatory cells and thrombus. Once organisms and the acute inflammatory response breach the vascular wall, spread to the surrounding parenchyma ensues. When parenchymal involvement is extensive, it may be impossible to ascertain the initial pathogenic mechanism. Pulmonary infection can occur via direct extension from extrapulmonary sources in the neck, mediastinum, chest wall, or across the diaphragm. Lung infection acquired by this route of injury is contiguous with the source and most commonly manifests as an abscess.

Radiological manifestations The key to the definite diagnosis of pneumonia is the presence of lung shadowing on the chest radiograph that is likely to be new and unexplained by prior disease or an alternative cause. Use of radiographic change to define pneumonia is fundamental to hospital practice. It is not always appropriate outside this setting, where access to radiology is not always easy, and a clinical diagnosis must often be accepted. Such lung shadowing may be patchy or homogeneous and may occupy part of a lobe, a whole lobe (“lobar pneumonia”), a single lung or both lungs. The lower lobes are most commonly affected but pneumonia can occur in any part of the lung. Separation of consolidation from other forms of lung shadowing is aided, when present, by the presence of an air bronchogram, where the patent air-filled bronchi contrast with the surrounding consolidated lung. It is important to recognize that a persistent area of consolidation should raise the possibility of malignancy. Air bronchograms are commonly identified in pulmonary adenocarcinoma and do not reliably distinguish infection from a primary lung malignancy. Associated radiographic features of infection include pleural effusion, lymphadenopathy and cavitation. Volume loss or

collapse is uncommon in uncomplicated bacterial infections of the lung. Such loss should prompt a close examination of the airways leading to the collapsed segment to exclude inspissated secretions, foreign body or endobronchial neoplasm. As with clinical features, there is no unique relationship between radiographic pattern and causative organism,35 although some features occur more frequently with certain organisms. Thus cavitation is more common in pneumonia caused by Staphylococcus aureus, anaerobic bacteria and tuberculosis, a swollen lobe may occur with Klebsiella and true lobar shadowing with Streptococcus pneumoniae. Lung shadowing that extends across tissue boundaries is very unusual and may be a feature of infection with Actinomyces israelii. In general the more extensive the shadowing, the more severely ill is the patient. Patchy extensive shadowing may be a feature of Mycoplasma infection, where the patient is often not severely ill. As described above, radiological imaging has its greatest diagnostic utility in highlighting the likely source of bacteria: airway, bloodstream or direct extension. Bacterial infections that reach the lung via the airways create centrilobular inflammatory nodules that involve the terminal and respiratory bronchioles and the surrounding alveoli. Collections of these nodules, as demonstrated on HRCT, resemble the branches of a tree in spring before it blooms and are referred to in the radiology literature as a “tree-in-bud” pattern.36 The periphery of the lobule is spared along with the pleural surface (Figures 1 and 4). If the infection progresses, there is coalescence of nodules, creating broader areas of consolidation (Figure 5). Hematogenous spread of infection to the lung during episodes of septicemia results in a random distribution of 1–5 mm nodules within the secondary lobule (Figures 1 and 6). The periphery of the lobule and pleura are commonly involved. Larger quantities of infected material embolize from infected

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Chapter 4: Pulmonary bacterial infections

Figure 5. Staphylococcal pneumonia. A 35-year-old male with Staphylococcus aureus pneumonia and bilateral areas of consolidation demonstrated on a PA radiograph of the chest (left image). Coronal reconstruction of axial CT data (right image) demonstrates centrilobular nodules in the right lower lobe consistent with small airway spread of infection (arrowhead). An area of coalescing nodules is noted in the right upper lobe (curved arrow) near an area of cavitation. An area of dense consolidation is noted in the left upper lobe (asterisk).

Figure 6. Hematogenous spread of bacteria. High-resolution axial (left image) and coronal CT (right image) of the right lung acquired at the level of the bronchus intermedius in a 26-year-old with bacterial septicemia demonstrating widespread distribution of small (1–2 mm) nodules with a random or “miliary” pattern and pleural involvement.

heart valves, infected venous catheters and peripheral sites of septic phlebitis. The material lodges in distal vessels, creating a typical pattern of peripheral lung nodules that are often cavitary and demonstrate an attached “feeding vessel” (Figure 7).37 Direct infection of the lung either from trauma or from primary infection of the neck, mediastinum, chest wall or subdiaphragmatic region is clearly delineated by CT.

Pathogenesis and normal host defense Host-pathogen interactions have evolved over a long period of time and are extremely complex. The host has a variety of

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means that act both in sequence and in parallel to repel the pathogen, while the pathogen has evolved multiple methods to evade the host defenses. It is this complex interaction which leads to the clinical illness we know as pneumonia. In health, microbial pathogens, while present in the aerodigestive tract above the larynx, are absent from the airways and lung parenchyma. For pneumonia to develop, microorganisms must first bypass a number of mechanical barriers and establish themselves as a cause of pulmonary inflammation. Microbial features (large infecting dose or specific virulence factors) and host features (temporary, e.g., post-viral, or permanent, e.g., squamous metaplasia of surface epithelium)

Chapter 4: Pulmonary bacterial infections

Figure 7. Septic emboli. Coronal reconstruction of axial CT data in a 45-yearold male with septic emboli from intravenous drug abuse. There are numerous nodules of varying size, the majority of which are associated with blood vessels (arrowheads).

may contribute to their capacity to do this. Colonization of the oropharynx may serve as a bridgehead for this invasion of the lower respiratory tract with either a new bacterial species or serotype. The causative serotype of Streptococcus pneumoniae usually first appears in the nasopharynx in the month before pneumonia. Prolonged nasopharyngeal carriage leads to the production of type-specific antibody, which protects against subsequent pneumonia. Mechanical barriers include the nasal vibrissae (hairs), which filter out large (greater than 5 µm) particles, the nasal turbinate bones and the carinae between bronchial divisions in the lungs, where particles impact due to turbulent airflow and the cough and sneeze reflexes, which expel particles deposited in both upper and lower airways. Particles smaller than 0.5 µm are exhaled, as a result of which only particles between 0.2 and 2 µm usually reach the alveoli. In addition, the glottis prevents macroaspiration, but small amounts of microaspiration are normal. The next barrier is the respiratory epithelium with the associated mucociliary escalator. The airways are lined by pseudostratified, columnar epithelium, topped by cilia, which propel the airway lining mucus. The apical junctional complex between cells completes the barrier to the sub-epithelial layers. The airways are lined by a 5–25 µm thick layer of surface liquid, which traps particles, including microorganisms. This liquid provides the stream through which they can then be moved by the surface cilia towards the upper airways. In addition the particles are engaged by antimicrobial molecules within the liquid. A number of different proteins, including lactoferrin, lysozyme, fibronectin, immunoglobulins and complement, and peptides, including defensins, cathelicidins and collectins, which include the surfactant proteins, are found within the airway surface liquid. Some are constitutive, others inducible. Their actions include bacterial membrane damage, opsonization, iron-binding, cytokine release, stimulation of

mucus production, interference with protein synthesis, direct DNA damage, and chemotaxis for neutrophils, monocytes, mast cells and T cells. Together with the low pH of the surface fluid, these proteins form an important antimicrobial defense. To counteract this defense, bacteria may replicate within the liquid and produce molecules (e.g., Streptococcus pneumoniae produces pneumolysin) which interfere with ciliary function and disrupt intracellular junctions, on which normal mucus production depends. Some viruses destroy surface epithelial cells and facilitate bacterial adherence to these cells. Any hostmediated interruption to this system, such as endobronchial obstruction (e.g., by foreign body or tumor), anatomic variant (e.g., inherited ciliary dysfunction, bronchiectasis) or exogenous toxins (such as tobacco smoke), that promotes reduction in ciliary function and loss of ciliated cells facilitates infection. The next step in the host response is the recognition of foreign material, including microorganisms.38–40 Without this identification, the inflammatory response cannot follow. Microbes are detected by surveillance cells, equipped with pattern-recognition receptors. The key surveillance cells are the alveolar macrophages and dendritic cells. The alveoli are patrolled by mobile macrophages, and dendritic cells are found throughout the respiratory tract. The latter have a limited ability to kill microorganisms and are more important as conduits for information to lymphocytes and epithelial cells. These then recruit the most important effector cells, the neutrophils. A variety of different pattern-recognition receptors, including Toll-like receptors, serving this purpose have been described. Some are specific to single microbial types, others pluripotent. There is much overlap and redundancy in this system because of its importance. This means that an individual bacterium may be recognized by a variety of different receptors. The signals generated from these pattern-recognition receptors converge on common intracellular pathways, such as those mediated by nuclear factor-kB, which mediates the transcription of chemokines, adhesion molecules, colonystimulating factors and other cytokines. Interleukin-1 and tumor necrosis factor-a are important cytokines released from macrophages. These activate epithelial cells and lead to NF-kB activation. Macrophages may also activate T-lymphocytes, which then activate epithelial cells, via interleukin-17. Neutrophils are recruited into the lungs and migrate from capillaries into the alveoli. There they phagocytose microorganisms, which are killed by antimicrobial proteins, degradative enzymes and reactive oxygen species. Neutrophils also trap extracellular bacteria in a chromatin and protein mesh – the neutrophil extracellular trap. Neutrophils also generate proinflammatory messengers, including interleukin-1, TNFa and chemokines. These messengers recruit and activate dendritic cells and attract B- and T-lymphocytes. Unchecked, the inflammatory response may self-perpetuate through positive feedback pathways mediated via a triggering receptor expressed on myeloid cells-1 (TREM-1). The inflammatory response may lead to tissue damage, as well as microbial death. For this reason molecules with negative

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Chapter 4: Pulmonary bacterial infections

feedback effects, such as p50, and molecules that interfere with pattern-recognition cells are produced. Many of these pathways and mechanisms constitute the innate immune response. Numerous microorganisms have adapted to evade this response. Microorganisms are able to detect markers of the inflammatory response and may counteract its effectiveness. Alternatively they may change into forms against which the inflammatory response is less effective, such as biofilms, e.g., in Pseudomonas infection. It is the adaptive immune response that protects the host from such organisms. The B- and T-lymphocytes, which mediate the humoral and cell-mediated immune responses respectively, are the key cellular players. B cells arise in the bone marrow and migrate to peripheral lymphoid organs. Interaction of antigens with immunoglobulin molecules on their surface leads to their activation and antibody production. This Tcell-independent activation is important for antigens, such as bacterial cell wall components, e.g., pneumococcal polysaccharide and lipopolysaccharide. B cells can also be activated by a T-cell-dependent mechanism, involving macrophages and dendritic cells. When first stimulated, B cells may take weeks before antibody is produced. The T-cell-dependent activation route also leads to production of B cells with memory that can produce antibody within hours of subsequent antigen exposure. Antibodies serve to opsonize, neutralize proteins, render microorganisms inactive, prevent colonization and activate the complement cascade. Immunoglobulins G, A, M and to a lesser extent E are the most important in defense against lung infection.

Management Once the condition has been diagnosed, treatment is directed at the causative microorganism and the physiological and symptomatic derangements caused by the host response to the organism. Patient management guidelines exist in most developed countries.41,42 It is generally agreed that care should be guided by illness severity,23,43 which directs the location, investigation, antibiotics and additional treatment steps. Briefly, non-severely ill patients can be managed in the community, more severely ill in hospital, and the very ill in the ICU. Investigations should cover possible microbial etiologies, markers of illness severity, including measures of renal function and gas exchange, and markers of underlying disease, such as a chest radiograph to exclude underlying lung cancer. Symptomatic relief for pleuritic pain may be required. Antibiotics are the mainstay of therapy. Since the causative organism is unknown at presentation and is often never identified, antibiotic therapy is usually empirical and based on the knowledge of likely causes and resistance patterns, in addition to illness severity. Fluid replacement and oxygen to improve gas exchange may be required. In more severely ill patients intubation and assisted ventilation may be necessary. Despite the central role of the host response in the causation of the clinical syndrome, much research has failed as yet to identify any anti-inflammatory interventions such as

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steroids, activated protein C, or granulocyte colony-stimulating factor that definitely modify outcome.

Prognosis and natural history As indicated earlier, although pneumonia may resolve by “crisis” in a minority, the risk of death is high, if untreated. Despite knowledge of likely causative pathogens, sophisticated microbiological tests, specific antibiotics and advanced lifesupport systems, patients still die from pneumonia. Of those admitted to hospital with CAP, the mortality is between 5% and 10%, rising to up to 50% in the subgroup who reach the intensive care unit. In those who recover, the acute illness lasts some 7 to 10 days. Clinical recovery takes much longer and may only occur over 4 to 6 months, with lethargy being the slowest symptom to recover. In general radiographic resolution follows clinical recovery. Failure to recover should lead to consideration of incomplete therapy for the infection, complications, including empyema or underlying pathology, such as lung malignancy. Recovery is slower in those with underlying comorbidity and the elderly. There is some evidence of specific persistent morbidity associated with Legionella and Coxiella infection.

Microbiological work-up Microbiological work-up of suspected bacterial pneumonia is a standard part of pneumonia management.44 Although tissue diagnosis is the subject of this chapter, correlation of tissue findings with culture diagnosis is optimal for appropriate therapy. If an unusual pathogen is suspected, the laboratory should be notified to include specific media or cell culture, direct fluorescent antibody (DFA) or other special handling. Overall the yield of routine microbiological investigation, as opposed to that as part of research studies, is poor, with pathogens, principally bacteria, found in only 23 to 26% of cases of communityacquired pneumonia.45,46 Treatment is influenced by such results in only 6 to 8% of cases.45,46 Bacteria may be identified by non-culture methods, but culture is preferred when possible as antibiotic sensitivity can be determined. Sputum is the standard specimen used, as it is available in most patients, but often gives a poor return. Adequacy of the sample may be assessed with a Gram stain to determine cell count (> 25 neutrophils and < 10 epithelial cells per lowpower field). Bronchial secretions, lung aspirates and tissue provide higher yields and lower rates of contamination by oral flora. If anaerobic bacteria are suspected, sputum cannot be used. Although often foul-smelling, tracheal aspirates, empyema or pleural fluid is recommended instead. Interpretation of sputum results is complicated when the bacterium isolated could be either a commensal or a pathogen. Blood cultures provide a sample from a normally sterile site and thus have greater specificity than respiratory tract secretions, but a low sensitivity of about 5 to 15%.46,47 Antigen detection methods are preferred for bacteria that are difficult to culture, e.g., Legionella, but may also be used for

Chapter 4: Pulmonary bacterial infections

other bacteria such as Streptococcus pneumoniae. Serological methods may also be helpful for such organisms and are preferred for Mycoplasma pneumoniae, Coxiella burnetii and rarer bacteria. Nucleic acid amplification tests (NAATS), including PCR, for bacterial detection have entered clinical practice. Their extreme sensitivity is both an advantage and a disadvantage. Very small amounts of bacterial nucleic acid can be detected, which means that scrupulous methodology and quality control is required to prevent contamination. The problem of distinguishing commensal from pathogenic bacteria remains a problem, but NAATS are beginning to be used for non-commensal bacteria, such as Mycoplasma and Coxiella.

Histological features of bacteria that cause pneumonia Under certain circumstances histology is a valuable tool in the diagnosis of pneumonia. Often biopsy specimens from patients suspected of malignancy are fixed entirely in formalin, precluding the possibility of bacterial cultures. Some of these patients have an infection, and it becomes the task of the surgical pathologist to help determine the likely cause. Although it may not always be possible to determine the bacterial species by histology, the findings may direct further definitive testing. Another circumstance in which histology is helpful is in the verification that an unusual or normally nonpathogenic organism is causing an infection. Observation of certain bacteria in inflamed tissue can corroborate ambiguous culture results. There is tremendous variation in the staining of bacteria in tissue sections. For example, Nocardia asteroides and Treponema pallidum are never identified in H&E-stained sections. Staphylococcus aureus may stain well with H&E or may not stain at all. Many of the smallest bacteria are not visible and will be missed by the pathologist, unless they are viewed under the 100
oil immersion objective. Although many bacteria can be seen in H&E-stained sections, special stains are needed to adequately determine their precise morphology and Gram-staining characteristics. Surgical pathologists need to know the special stains that demonstrate bacteria in tissue. The Brown-Hopps (BH) tissue Gram stain stains Gram-positive organisms dark blue, dark purple or almost black and Gram-negative organisms red. It is not as sensitive for the detection of Gram-positive organisms as the Brown-Brenn (BB) tissue Gram stain, which also stains Gram-positive organisms dark blue, dark purple or almost black but does not usually stain Gram-negative organisms at all. Distinguishing Gram-positive from Gram-negative bacteria is not always straightforward. Dead Gram-positive bacteria may appear Gram-negative; Gram-negative bacteria may appear Gram-positive if not adequately decolorized. Grocott methenamine silver (GMS) is useful for detecting Gram-positive bacteria, including the carcasses of dead organisms that may not be seen in Gram-stained sections. Silver impregnation stains, like the Warthin-Starry (WS) stain, demonstrate Gram-

negative bacteria well, including many that are too small to be seen with BH. Many bacteria will appear larger on sections stained with WS than with Gram stains. Although it is technically more difficult, WS is superior to other techniques, such as Dieterle or Steiner. Periodic acid Schiff (PAS) may stain Gram-positive organisms, but its greatest usefulness is in the detection of Whipple disease. Some non-mycobacterial organisms are acid-fast or partially acid-fast and can be stained with Ziehl-Neelsen (ZN), Fite-Faraco (FF) or Coates-Fite (CF). Histological features of acute or granulomatous inflammation should prompt a search for microorganisms. Extensive tissue necrosis due to pneumonia has sometimes been misinterpreted as Wegener granulomatosis but vasculitis away from the area of inflammation is absent (see Chapter 19). The major features and images of the common and uncommon bacteria that cause pneumonia are presented in Table 2, while additional characteristics are presented in the following section.48–50 The size measurements are based on our observations of bacteria in tissue sections or are those reported in Bergey’s manuals.51,52 All images of bacteria are from tissue sections of lung, unless otherwise noted (Figures 8–108).

Common bacterial causes of pneumonia Anaerobic bacteria The anaerobic bacteria include Gram-positive and Gramnegative cocci and bacilli, including the anaerobic Actinomyces. If anaerobic infection is suspected it is essential to alert the clinical laboratory and obtain appropriate specimens and rapid processing for anaerobic cultures. Many infections are polymicrobial. Bacteroides fragilis is the most common anaerobe isolated from respiratory samples. Others include Porphyromonas spp., Prevotella spp. and Fusobacterium spp.

Clostridium spp. Clostridial pneumonia develops as a complication of penetrating chest injuries or as part of a mixed bacterial infection due to aspiration. The presence of Clostridium spp. in cultures from autopsy specimens must be interpreted with caution, because these organisms can escape the intestinal tract and invade tissues shortly after death. Clostridium perfringens Clostridium perfringens (formerly Clostridium welchii) are large rods that often appear boxcar-shaped in tissue. The most common infections due to C. perfringens are gas gangrene, bacteremia and emphysematous cholecystitis. It may be a component of the normal intestinal flora. Clostridium septicum Clostridium septicum are also large rods that can become very long. Spores are more common than in Clostridium perfringens but do not usually occur in the long forms. Clostridium

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Chapter 4: Pulmonary bacterial infections Table 2 Common and uncommon causes of bacterial pneumonia

Name

Gram stain

Diameter or length (mm)

Morphological features

Useful special stains

Figure no.

Bacteroides fragilis



1 to 4

Single, CB, B

BH, GMS

8

Clostridium perfringens

þ

B 3 to 9, F up to 19

Single, pairs, B with blunt ends and rare subterminal spores, long B, F

BB, BH, GMS

9, 10

Clostridium septicum

þ

B 3 to 14, F up to 35

Single, pairs, B with common subterminal spores, very long B, F

BB, BH, GMS

11, 12, 13

Peptostreptococcus spp.

þ

0.3 to 1.2

Chains, pairs, clumps, C

BB, BH, GMS

14, 15

Chlamydia trachomatis (uncommon in adults)



Less than 1

Intracellular vacuole, granules

BH, WS

16, 17, 18

Chlamydophila pneumonia



Less than 1

Intracellular vacuole, granules

BH, WS

Chlamydophila psittaci



Less than 1

Intracellular vacuole, granules

BH, WS

Escherichia coli



1 to 6

Single, pairs, pleomorphic B, some bipolar

BH, WS

19, 20

Klebsiella pneumoniae



1 to 6

Single, pairs, chains, B, some bipolar

BH, WS

21, 22

Proteus mirabilis



1 to 3

Single, pairs, B

BH, WS

Serratia marcescens (uncommon)



1 to 3

Single, B

BH, WS

23

Haemophilus influenzae



0.5 to 2

Single, short chains, pleomorphic CB, B, C

BH, WS

24, 25, 26, 27

Legionella micdadei (uncommon)



1.0

Intracellular, B

ZN, BH, WS

28, 29, 30

Legionella pneumophila



1.5 to 3

Intracellular, single, pairs, B

BH, WS

31, 32, 33

Mycoplasma pneumonia

NA

Up to 0.8

NA

NA

Pseudomonas aeruginosa



B 1.5 to 5, F up to 40

Single, pairs, straight to slightly curved B, F

BH, GMS

34, 35, 36, 37

Staphylococcus aureus

þ

0.5 to 1.5

Clusters, single, pairs, C

BB, BH, GMS

38, 39, 40, 41, 42

Non-coagulase-producing Staphylococcus spp.

þ

0.5 to 1.5

Clusters, single, pairs, C

BB, BH, GMS

Common ANAEROBIC BACTERIA

ENTEROBACTERIACEAE

Staphylococcus spp.

Botryomycosis

43, 44, 45

STREPTOCOCCACEAE Enterococcus spp.

þ

0.6 to 1.0

Single, pairs, short chains, C

BB, BH, GMS

Streptococcus agalactiae

þ

0.6 to 1.0

Pairs, short chains, C

BB, BH, GMS

Streptococcus pneumoniae

þ

1.0

Pairs, short chains of pairs, lanceolate C (diplococcus)

BB, BH, GMS

46, 47

Streptococcus pyogenes

þ

Up to 1.0

Pairs, short chains, C

BB, BH, GMS

48, 49, 50

Streptococcus viridans groups

þ

0.6 to 1.0

Chains, C

BB, BH, GMS

Botryomycosis

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51, 52, 53

Chapter 4: Pulmonary bacterial infections Table 2 (cont.)

Name

Gram stain

Diameter or length (mm)

Morphological features

Useful special stains

Figure no.

Acinetobacter calcoaceticusbaumannii complex



1 to 5

Pairs, single, pleomorphic plump B, CB, C (diplococcus)

BH, WS

54, 55, 56

Actinomyces israelii

þ

Up to 40

Branching, beaded F, not acid-fast

BB, BH, GMS

57, 58

Nocardia asteroides

þ

Up to 40

Branching, beaded F, partially acid-fast

CF, BB, BH, GMS

59, 60, 61, 62, 63, 64

Bacillus anthracis

þ

Up to 3.5

Single, pairs, long chains, B

BB, BH, GMS

65, 66

Bacillus cereus

þ

3 to 5

Single, short chains, B

BB, BH, GMS

67, 68

Bacillus sphaericus

þ

1.5 to 5

Single, B

BB, BH, GMS

69, 70

Bartonella spp.



1 to 2 (on WS)

Single, clumps, chains, CB, B may appear branching

WS

Brucella spp.



Up to 2 (on WS)

Single, CB, C, B

WS

71

Burkholderia pseudomallei



1.5

Single, short chains, B

BH, WS

72, 73

Burkholderia cepacia



3.2

Single, pairs, chains, B

BH, WS

74, 75, 76

Chromobacterium violaceum



1.5 to 3.5

Single, pairs, short chains, B with rounded ends, frequent bipolar

BH, WS

77, 78, 79

Coxiella burnetii



0.3 to 0.7

Intracellular, pleomorphic CB

NA

Francisella tularensis



0.2 to 0.7

Mostly intracellular, CB

BH, WS

80, 81, 82

Leptospira interrogans

NA

6 to 20

Single, S, hook at one or both ends

WS

83, 84

Micrococcus spp.

þ

Up to 2

Tetrads, pairs, C

BB, BH, GMS

85, 86

Moraxella catarrhalis



0.6 to 1.0

Pairs, C (diplococcus)

BH, WS

87, 88, 89

Neisseria meningitidis



0.6 to 1.0

Pairs, C (diplococcus)

BH, WS

90, 91

Neisseria mucosa



0.6 to 1.0

Pairs, C (diplococcus)

BH, WS

92, 93, 94

Pasteurella multocida



1 to 2

Single, CB, B

BH, WS

95, 96

Rhodococcus equi

þ

1 to 2

intracellular, pleomorphic CB, C, B, partially acid-fast

CF, BB, BH, GMS

97, 98, 99

Anaplasma phagocytophilum



0.5

Intracellular in neutrophils, pleomorphic C, CB

BH

Ehrlichia chaffeensis



0.5

Intracellular in macrophages, pleomorphic C, CB

BH

Rickettsia conorii



1

Intracellular, B

BH

Rickettsia rickettsii



1 to 2

Intracellular in endothelial cells, B

BH

Orientia tsutsugamushi



1

Intracellular, B

BH

Salmonella spp. (ENTEROBACTERIACEAE)



2 to 5

Single, straight B, some bipolar

BH, WS

103

Treponema pallidum

NA

5 to 20

Single, S

WS

104

Tropheryma whipplei

þ

1 to 2

Intracellular, NA

PAS

105, 106

Yersinia pestis



1 to 3

Single, chains, CB, C, oval C, B

BH

107, 108

Uncommon

RICKETTSIAE

100, 101, 102

NA, not applicable; B, bacillus; C, coccus; CB, coccobacillus; F, filament; S, spirochete; BB, Brown-Brenn; BH, Brown-Hopps; CF, Coates-Fite; GMS, Gomori methenamine silver; PAS; Periodic acid-Schiff; WS, Warthin Starry; ZN, Ziehl-Neelsen.

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Chapter 4: Pulmonary bacterial infections

158

Figure 8. Bacteroides fragilis. Gram-negative rods in liver (Brown-Hopps).

Figure 9. Clostridium perfringens. Rods (H&E).

Figure 10. Clostridium perfringens. Gram-positive rods (Brown-Hopps).

Figure 11. Clostridium septicum. Abscess (H&E).

Figure 12. Clostridium septicum. Rods with subterminal spores (H&E).

Figure 13. Clostridium septicum. Gram-positive rods with subterminal spores (Brown-Hopps).

Chapter 4: Pulmonary bacterial infections

Figure 14. Peptostreptococcus. Abscess (H&E).

Figure 15. Peptostreptococcus. Gram-positive cocci in short chains (Brown-Hopps).

Figure 16. Chlamydia trachomatis. Clusters of organisms in tubular epithelium of epididymis (H&E). Figure 17. Chlamydia trachomatis. Cluster of organisms in tubular epithelium of epididymis (Brown-Hopps).

Figure 18. Chlamydia trachomatis. Clusters of organisms in tubular epithelium of epididymis (Warthin-Starry).

Figure 19. Escherichia coli. Acute inflammation and necrosis (H&E).

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Chapter 4: Pulmonary bacterial infections

160

Figure 20. Escherichia coli. Pleomorphic gram-negative rods (Brown-Hopps).

Figure 21. Klebsiella pneumoniae. Rods (H&E).

Figure 22. Klebsiella pneumoniae. Gram-negative rods (Brown-Hopps).

Figure 23. Serratia marcescens. Gram-negative rods (Brown-Hopps).

Figure 24. Haemophilus influenzae. Pleural exudate (H&E).

Figure 25. Haemophilus influenzae. Intracellular coccoid forms (Brown-Hopps).

Chapter 4: Pulmonary bacterial infections

Figure 26. Haemophilus influenzae. Rod forms (Brown-Hopps).

Figure 27. Haemophilus influenzae. Pleomorphism (Warthin-Starry).

Figure 28. Legionella micdadei. Abscess (H&E).

Figure 29. Legionella micdadei. Acid-fast rods (Ziehl-Neelsen).

Figure 30. Legionella micdadei. Single weakly acid-fast rod (arrowhead) (Ziehl-Neelsen).

Figure 31. Legionella pneumophila. Acute bronchopneumonia (H&E).

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Chapter 4: Pulmonary bacterial infections

Figure 32. Legionella pneumophila. Intracellular Gram-negative rods (Brown-Hopps).

Figure 34. Pseudomonas aeruginosa. Filamentous forms (H&E).

Figure 36. Pseudomonas aeruginosa. Rod forms (H&E).

162

Figure 33. Legionella pneumophila. Extracellular Gram-negative rods (Brown-Hopps).

Figure 35. Pseudomonas aeruginosa. Filamentous forms (Brown-Hopps).

Figure 37. Pseudomonas aeruginosa. Gram-negative rods (Brown-Hopps).

Chapter 4: Pulmonary bacterial infections

Figure 38. Staphylococcus aureus. Abscess (H&E).

Figure 39. Staphylococcus aureus. Abscess with cocci (H&E).

Figure 40. Staphylococcus aureus. Abscess with dead organisms staining red and live organisms staining blue (arrowhead) (H&E).

Figure 41. Staphylococcus aureus. Abscess with cocci (GMS).

Figure 42. Staphylococcus aureus. Abscess with cocci (Brown-Brenn).

Figure 43. Botryomycosis. Grain (H&E).

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Chapter 4: Pulmonary bacterial infections

164

Figure 44. Botryomycosis. Cocci in grain (H&E).

Figure 45. Botryomycosis caused by Staphylococcus aureus (Brown-Hopps).

Figure 46. Streptococcus pneumoniae. Acute bronchopneumonia characterized by neutrophils filling alveolar spaces (H&E).

Figure 47. Streptococcus pneumoniae. Diplococci with pointed ends (arrowhead) (Brown-Hopps).

Figure 48. Hemolytic Streptococcus. Abscess (H&E).

Figure 49. Hemolytic Streptococcus. Short chains (Brown-Hopps).

Chapter 4: Pulmonary bacterial infections

Figure 50. Hemolytic Streptococcus. Staphylococcus aureus is also present (Brown-Hopps).

Figure 51. Botryomycosis. Grain (H&E).

Figure 52. Botryomycosis. Bacteria in grain (H&E).

Figure 53. Botryomycosis. Grain with Streptococcus Gram-positive cocci in chains (Brown-Hopps).

Figure 54. Acinetobacter calcoaceticus. Acute bronchopneumonia (H&E).

Figure 55. Acinetobacter calcoaceticus. Intracellular bacteria (H&E).

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Chapter 4: Pulmonary bacterial infections

166

Figure 56. Acinetobacter calcoaceticus. Intracellular and extracellular pleomorphic Gram-negative rods (Brown-Hopps).

Figure 57. Actinomycosis. Grains (H&E).

Figure 58. Actinomycosis. Gram-positive filaments (Brown-Hopps).

Figure 59. Nocardia asteroides. Abscess (H&E).

Figure 60. Nocardia asteroides. Acid-fast branching filaments (arrowheads) (Coates-Fite).

Figure 61. Nocardia asteroides. Gram-positive filaments (Brown-Brenn).

Chapter 4: Pulmonary bacterial infections

Figure 62. Nocardia asteroides. Filaments (GMS).

Figure 63. Nocardia asteroides. Granulomatous reaction with multinucleated giant cells (H&E).

Figure 64. Nocardia asteroides. Filaments in multinucleated giant cell. (Brown-Hopps).

Figure 65. Bacillus anthracis. Filamentous rods (H&E).

(a)

(b)

Figure 66. Bacillus anthracis. (a) Gram-positive rods (Brown-Brenn). The predominant lung findings in anthrax infection include (b) pulmonary edema, which typically has little in the way of inflammatory infiltrates (H&E) and (c) bacilli completely filling lymphatic spaces (H&E).

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Chapter 4: Pulmonary bacterial infections

(c) Figure 66. (cont.)

168

Figure 67. Bacillus cereus. Rods (H&E).

Figure 68. Bacillus cereus. Gram-positive rods (Brown-Hopps).

Figure 69. Bacillus sphaericus. Inflammatory mass lesion. (H&E).

Figure 70. Bacillus sphaericus. Gram-positive rod (arrowhead) (Brown-Brenn).

Chapter 4: Pulmonary bacterial infections

Figure 71. Brucella melitensis. Coccoid forms in testis (Warthin-Starry).

Figure 72. Burkholderia pseudomallei. Acute bronchopneumonia (H&E).

Figure 73. Burkholderia pseudomallei. Gram-negative rods (Brown-Hopps).

Figure 74. Burkholderia cepacia. Abscess and necrosis (H&E).

Figure 75. Burkholderia cepacia. Rods (H&E).

Figure 76. Burkholderia cepacia. Gram-negative rods (Brown-Hopps).

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Chapter 4: Pulmonary bacterial infections

170

Figure 77. Chromobactericum violaceum. Non-caseating granuloma (H&E).

Figure 78. Chromobactericum violaceum. Two Gram-negative rods (arrowhead) (Brown-Hopps).

Figure 79. Chromobactericum violaceum. Gram-negative rods in bronchiolar lumen (Brown-Hopps).

Figure 80. Francisella tularensis. Abscess (H&E).

Figure 81. Francisella tularensis. Hemorrhage (H&E).

Figure 82. Francisella tularensis. Acute bronchopneumonia (H&E).

Chapter 4: Pulmonary bacterial infections

Figure 83. Leptospirosis. Hemorrhage (H&E).

Figure 84. Leptospirosis. Two spirochetes in kidney (Warthin-Starry).

Figure 85. Micrococcus. Tetrads (H&E).

Figure 86. Micrococcus. Tetrads, note black septations (GMS).

Figure 87. Moraxella catarrhalis. Abscess (H&E).

Figure 88. Moraxella catarrhalis. Abscess with intracellular bacteria (H&E).

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Chapter 4: Pulmonary bacterial infections

Figure 89. Moraxella catarrhalis. Gram-negative diplococci (Brown-Hopps).

Figure 90. Neisseria meningitidis. Intracellular Gram-negative diplococci (arrowhead) (Brown-Hopps).

Figure 91. Neisseria meningitidis. Small Gram-negative diplococcus (arrowhead) and larger Gram-positive Staphylococcus aureus (curved arrow) (Brown-Hopps).

Figure 92. Neisseria mucosa. Grain (arrowhead) (H&E).

Figure 93. Neisseria mucosa. Grains (H&E).

172

Figure 94. Botryomycosis caused by Gram-negative diplococci of Neisseria mucosa (arrowhead) (Brown-Hopps).

Chapter 4: Pulmonary bacterial infections

Figure 95. Pasteurella multocida. Acute bronchopneumonia in a background of BALT hyperplasia and interstitial fibrosis (H&E).

Figure 96. Pasteurella multocida. Intracellular Gram-negative rods (arrowhead) (Brown-Hopps).

Figure 97. Rhodococcus equi. Acute inflammation and foamy macrophages (H&E).

Figure 98. Rhodococcus equi. Gram-positive coccoid forms (Brown-Hopps).

Figure 99. Rhodococcus equi. Coccoid forms (Brown-Brenn).

Figure 100. Rickettsia rickettsii. Vascular congestion and edema (H&E).

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Chapter 4: Pulmonary bacterial infections

Figure 101. Rickettsia rickettsii. Endothelial cells lifted off the basement membrane (H&E).

Figure 102. Rickettsia rickettsii. Four Gram-negative bacilli in an endothelial cell (arrowhead) (Brown-Hopps).

Figure 103. Salmonella. Gram-negative rods in necrotic tissue from amputation specimen (Brown-Hopps).

Figure 104. Treponema pallidum. One spirochete in testis (Warthin-Starry).

Figure 105. Whipple disease. Histiocytic reaction in lymph node (H&E).

174

Figure 106. Tropheryma whipplei. Organisms in lymph node (periodic acid-Schiff).

Chapter 4: Pulmonary bacterial infections

Figure 107. Yersinia pestis. Abscess (H&E).

Figure 108. Yersinia pestis. Gram-negative rods in spleen (Brown-Hopps).

septicum also causes gas gangrene and bacteremia and is part of the intestinal flora.

Enterobacteriaceae

Peptostreptococcus Peptostreptococcus spp. are cocci that can sometimes be small (0.3 to 0.5 mm in diameter). They and other anaerobes of the oral cavity are isolated as mixed cultures.

Chlamydiaceae The Chlamydiaceae group includes very small obligate intracellular pathogens that have a unique life cycle including two stages: reticulate and elementary bodies. The reticulate body (0.8 to 1.0 mm in diameter) is the intracellular replicative form that infects columnar epithelial cells. Reticulate bodies have morphological and functional characteristics of Gram-negative organisms, but lack a rigid cell wall. They are visible with Giemsa or Papanicolaou in cytological specimens and with H&E, BH or WS in histological sections. Electron microscopy (EM) can be useful. The elementary body (0.35 mm in diameter) is the metabolically inactive, infectious form and is capable of surviving outside the host. Chlamydia trachomatis The histology of Chlamydia trachomatis is similar in various infected tissues (genital, ocular or pulmonary). Infected columnar epithelial cells may be misinterpreted as goblet cells because the reticulate bodies appear as granules within a clear vacuole in sections stained with H&E, BH or WS. Chlamydophila pneumoniae A single serovar of Chlamydophila pneumoniae is associated with human infection. Chlamydophila psittaci (ornithosis or psittacosis) Psittacosis is a nationally notifiable disease in the United States.

Gram-negative rods are the most common isolates from clinical cultures overall and the second most common from patients with CAP (Table 1). The organisms are short and broad. Ubiquitous in the environment, they are often implicated in nosocomial infections. Escherichia coli Escherichia coli rods may show bipolar staining, that is darker staining at both ends of the rod. Both fecal strains of Escherichia coli and extraintestinal pathogenic strains can cause infection in normal and immunocompromised hosts. Klebsiella pneumoniae Most Klebsiella pneumoniae organisms are encapsulated rods, some of which may show bipolar staining. It is capable of causing pneumonia in otherwise healthy individuals; however, most patients have underlying conditions or nosocomial infection. Pneumonia due to Klebsiella pneumoniae is often complicated by necrosis and abscess formation. Sputum from patients with this infection is often mucoid and brick-red in color. Proteus mirabilis Proteus spp. may colonize the upper respiratory tract in hospitalized patients and can cause rapidly progressive pneumonia. Serratia marcescens Pneumonia due to Serratia marcescens is similar to pneumonia due to Klebsiella pneumoniae, although less likely to be complicated by abscess formation. It has been transmitted by contaminated respiratory care equipment. Some strains of Serratia marcescens produce red pigment, causing the sputum to be tinged with red, mimicking hemoptysis.

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Chapter 4: Pulmonary bacterial infections

Other Gram-negative bacteria

Gram-positive bacteria

Haemophilus influenzae Haemophilus influenzae organisms may appear larger (2 mm) on sections stained with WS. Encapsulated forms are implicated in infections of the lower respiratory tract. Non-encapsulated, non-typeable strains may cause CAP in persons with underlying conditions. Culture from sterile sites may be required to determine clinical significance of isolates due to colonization of upper respiratory tract by Haemophilus spp. Due to the poor viability, specimens should be cultured as soon as possible onto appropriate media.

Staphylococcus spp. are cocci that usually form grapelike clusters. Organisms may be Gram-variable in tissue, especially in patients on antibiotic therapy. GMS is helpful in staining non-viable organisms in tissue sections. There are multiple species of Staphylococcus, but Staphylococcus aureus (high virulence) and Staphylococcus lugdunensis (low virulence) are pathogenic for humans; Staphylococcus epidermidis and Staphylococcus saprophyticus cause device-associated and urinary tract infections.

Legionella spp Legionella spp. are slender bacilli that do not grow on routine blood agar, so the laboratory should be notified of a suspected case. Legionellosis is a nationally notifiable disease in the United States. Legionella micdadei (Pittsburgh pneumonia agent) Histological sections of lung infected with Legionella micdadei show predominantly intracellular acid-fast bacilli in acute suppurative pneumonia, without granuloma formation or caseating necrosis. It is clinically similar to pneumonia due to Legionella pneumophila, although it tends to occur in immunocompromised patients.

Staphylococcus aureus Staphylococcus aureus produces various tissue-destructive products and surface adhesion molecules that are associated with invasive disease. Staphylococcus spp. (coagulase-negative) The non-coagulase-producing Staphylococcus spp. colonize and persist on catheters and other biomaterials by forming a biofilm. However, reported cases of pulmonary disease do not meet strict culture requirements of a true pathogen.

Botryomycosis

Legionella pneumophila (Legionnaires’ disease) The most common species of Legionella isolated from the lung, Legionella pneumophila, is a facultative intracellular pathogen. Although usually found as single bacilli in tissue, it can form chains or long filaments up to 20 mm long in cultures. It is rarely weakly acid-fast with partial acid-fast stains in tissue.

Botryomycosis is a grain-forming bacterial infection, which is most often caused by Staphylococcus aureus, but which can also be due to chronic localized infection by other non-filamentous bacteria, such as Streptococcus spp., Neisseria mucosa or Pseudomonas aeruginosa. Because of the similarity to mycetoma, it is referred to as a pseudomycosis. The role of bacterial virulence and host defense in the development of this condition is ill-understood. Diagnosis of the bacterial cause is by tissue Gram stain or cultures of the grains. The bacteria are almost always confined to the grains and not present in the surrounding exudate or tissue.

Pseudomonas aeruginosa

Streptococcaceae

Pseudomonas aeruginosa can cause acute bronchopneumonia or botrymycosis. Virulence factors are multiple and have complex interactions with host factors, site of infection and comorbid conditions.

Members of the family Streptococcaceae are cocci, most of which form chains. The Streptococcus spp. have been classified over the years, based on hemolysis and Lancefield antigens. The current classification includes Streptococcus pyogenes, Streptococcus agalactica and Streptococcus pneumoniae species, as well as the Anginous, Bovis, Mutans, Salivarious and Mitis “Viridans” groups. The enteric streptococci have been renamed Enterococcus species.

Mycoplasmas Mycoplasmas are the smallest known free-living forms. They lack a cell wall. Mycoplasma pneumoniae Mycoplasma pneumoniae is a short rod not visible in tissue by Gram stain. Diagnosis is usually made by PCR or antigen capture on throat swabs or sputum. Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium and Ureaplasma spp Mycoplasma hominis, Mycoplasma fermentans, Mycoplasma genitalium and Ureaplasma spp. are similar to Mycoplasma pneumoniae. These organisms are common contaminants.

176

Staphylococcus spp.

Enterococcus spp. The Enterococcus spp. are members of Lancefield Group D. Enterococcus faecalis and Enterococcus faecium account for most infections. They are often part of polymicrobial bacteremia, but pneumonia is rare. They do not have known intrinsic virulence factors, but produce various substances that promote adherence and inflammation. Streptococcus agalactiae Streptococcus agalactiae is a member of Lancefield Group B. It is an important cause of perinatal pneumonia.

Chapter 4: Pulmonary bacterial infections

Streptococcus pneumoniae (pneumococcal pneumonia) Streptococcus pneumoniae is an encapsulated diplococcus. Gram stains often show Gram-positive or Gram-variable lanceolate cocci in pairs and sometimes short chains of pairs. The capsule inhibits phagocytosis, and requires opsonins (antibody, complement). Other reported virulence factors include pneumolysin, which inhibits phagocytosis, and cell wall polysaccharides that assist in adherence and activate inflammation. Streptococcus pyogenes Streptococcus pyogenes is an encapsulated coccus that is a member of Lancefield Group A. The capsule and M protein help to retard phagocytosis. It is an uncommon cause of pneumonia, occurring as a complication in other conditions, such as influenza or drug abuse, or as a nosocomial infection. Streptococcus viridans group Most organisms in this diverse group constitute a large part of the normal human microbiota and have low virulence; and hence clinical significance is based on culture from a sterile site. Infection is often part of polymicrobial aspiration pneumonia.

Uncommon bacterial causes Acinetobacter spp. Acinetobacter spp. are encapsulated pleomorphic bacteria related to Moraxella spp. Its diplococci may have adjacent sides flattened, giving them a coffee bean-like appearance like Moraxella spp. and Neisseria spp. It grows well on routine media as strict anaerobes. They are opportunists that are able to grow in devitalized tissue. The capsule may inhibit phagocytosis. Acinetobacter calcoaceticus-baumannii complex are the most frequent clinical isolates.

Actinomyces and Nocardia spp. Both Actinomyces spp. and Nocardia spp. are branching filaments with alternating segments of Gram-positivity and Gram-negativity. They may morphologically resemble fungi, although they are much narrower. The presence of these organisms in clinical specimens should alert the laboratory to use specialized media. These bacteria are found in soil and aquatic environments. Actinomyces israelii Actinomyces israelii is present within grains and not in the surrounding polymorphonuclear exudate or tissue. It may be vaguely seen in H&E-stained sections and is not acid-fast. These grains are visible grossly as yellow so-called “sulfur granules”. It is an endogenous organism in the mouth, but it can cause a suppurative infection in traumatized tissue, such as following a tooth extraction. The abscesses can spread to the thorax and abdomen. This infection is often not suspected in the lung and not cultured. Diagnosis is usually

based on tissue examination. If culture is requested, the specimen must be transported appropriately for anaerobic culture. Nocardia asteroides Nocardia asteroides is morphologically identical in tissue sections to Actinomyces israelii, except that Nocardia asteroides is not visible in H&E-stained sections, is usually partially acid fast and not found in grains. Single filaments of Nocardia asteroides and Actinomyces israelii would be morphologically indistinguishable. Nocardia asteroides may potentially be confused with mycobacteria, because of its partial acid-fast properties. They are well-stained by GMS. Although infection with Nocardia asteroides is not characterized by grains, other Nocardia spp. may form grains. The laboratory should be advised when this infection is suspected.

Bacillus spp. Bacillus anthracis (anthrax) Bacillus anthracis is a wide bacillus that may form central or subterminal oval endospores, although the spores are not usually seen in tissue sections. Virulence factors are responsible for death, especially in inhalational and gastrointestinal disease. These toxins include capsule polyglutamates that inhibit phagocytosis, edema factor and lethal factor. Anthrax is a nationally notifiable disease in the United States. Lung cultures are rarely positive. The species must be confirmed by PCR in a public health reference lab following strict criteria (www.bt.cdc.gov/anthrax). Reference laboratories may have immunoperoxidase antibodies to detect bacterial antigens in tissue of suspected cases. Bacillus cereus Bacillus cereus is a rare cause of pneumonia in patients with bacteremia. Its toxins are related to vomiting and diarrhea, not pulmonary disease. Bacillus sphaericus Bacillus sphaericus can form spherical endospores. It is a rare cause of pulmonary infections. Bartonella spp. (bacillary angiomatosis and bacillary peliosis) Bartonella spp. are small organisms that usually stain poorly with tissue Gram stains. Silver impregnation (e.g., WS) is preferred for tissue. In immunocompetent patients, Bartonella spp., especially Bartonella henselae, usually cause cat scratch disease with stellate microabscesses in lymph nodes with variable numbers of organisms. In immunocompromised patients, Bartonella spp. usually cause bacillary angiomatosis with vascular proliferation and inflammation in various tissues with large numbers of organisms. In such peliotic tissues, masses of organisms appear as “granular clouds” that are blue (not acidfast) on ZN. Immunohistochemical stains are not

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Chapter 4: Pulmonary bacterial infections

commercially available. The laboratory should be alerted if Bartonella infection is suspected.

subtropics, but may be more common among patients with chronic granulomatous disease.

Brucella spp. Brucella spp. are minute, slender, organisms that stain with WS but are difficult to detect in tissue. They are facultative intracellular pathogens of the reticuloendothelial system. Manipulation of these organisms requires a BSL3 (biosafety level 3) laboratory, so any suspected infection should be specified for appropriate handling of clinical specimens and cultures. Diagnosis is rarely made from respiratory specimens; blood and bone marrow culture have the highest yield. Species are related to the predominant zoonotic reservoir: Brucella abortus (cattle), Brucella suis (swine) and Brucella melitensis (goats).

Francisella tularensis (tularemia) Francisella tularensis is a faintly staining coccobacillus that is rarely detected in tissue sections. The organism is highly contagious (infective dose is 10 to 25 bacteria). For this reason BSL3 laboratory conditions are required to handle specimens and cultures.

Burkholderia spp. Burkholderia pseudomallei (Melioidosis) Burkholderia pseudomallei is a tiny or short and slender organism that is often scarce. They are facultative intracellular pathogens that can invade neutrophils, macrophages and some epithelial cell types where they are able to survive in a latent state due to capsular polysaccharides. The organisms display a tropism for the lung. Acute melioidosis is characterized by the formation of microabscesses, which become confluent and form a necrotizing bronchopneumonia. Varying numbers of macrophages and scattered giant cells are also present. Defective neutrophil function in conditions such as diabetes, renal disease and alcohol abuse predispose to progressive disease. The term “pseudomallei” or false glanders is derived from the fact that the pulmonary form of the disease resembles glanders in horses (a disease caused by Burkholderia mallei). The organisms are present in the environment and usually affect the lung as a disseminated infection, but inhalation does occur. Culture is required for definitive diagnosis and is easy to perform, but low frequency of the pathogen in non-endemic areas may lead to misidentification as a related Pseudomonas spp. Selective media should be used for sputa to inhibit growth of contaminants. Burkholderia cepacia Burkholderia cepacia is a slender rod. Patients with cystic fibrosis may be colonized with Burkholderia (formerly Pseudomonas) cepacia. Pneumonia due to Burkholderia cepacia in patients without cystic fibrosis is rare. It has been associated with disorders of neutrophil function, such as chronic granulomatous disease. The histological appearance may be similar to that of melioidosis, ranging from acute suppurative pneumonia to chronic granulomatous inflammation with extensive necrosis. Chromobacterium violaceum Chromobacterium violaceum is a common environmental bacillus. It is a rare cause of infection in the tropics and

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Leptospira spp. Leptospira spp. are helical rods. Although they can be up to 20 mm in length, they are usually less than 10 mm in tissue sections. Silver impregnation and immunohistochemistry are used for tissue diagnosis. The organism has fewer coils than Treponema spp. Seven species cause disease in humans; Leptospira interrogans is the most common. Micrococcus spp Micrococcus spp. divide in more than one plane. The distinctive cross-shaped septations are easily seen in GMS-stained sections. Although Micrococcus rarely causes infections in immunocompetent hosts, it can cause cavitating pneumonia and fatal pulmonary hemorrhage in immunocompromised patients. Moraxella (Branhamella) catarrhalis Moraxella (formerly Branhamella) catarrhalis is a diplococcus with adjacent sides flattened, giving them a coffee bean-like appearance. Molecular testing may be required to differentiate it from Neisseria spp. The organisms may colonize the upper or lower respiratory tract prior to infection. Neisseria spp Neisseria spp. are diplococci with adjacent sides flattened, giving them a coffee bean-like appearance like Moraxella spp. Neisseria meningitidis causes meningitis, pneumonia, endocarditis and arthritis. Neisseria mucosa is a species of so-called “non-pathogenic” Neisseria that is part of the normal upper respiratory flora but occasionally causes infections, including pneumonia. Pasteurella spp. Pasteurella spp. are very small, slender organisms. Pathogenic strains are usually encapsulated. Pasteurella multocida is the most common pathogen of the respiratory tract and often colonizes the upper respiratory tract. Rhodococcus equi (malakoplakia) Rhodococcus equi is a small bacterium that is intracellular in tissue sections. For isolation, selective media are required to differentiate from normal microbiota. Molecular studies may be required to confirm identification. Rhodococcus equi is one of the causes of malakoplakia, a condition resulting from

Chapter 4: Pulmonary bacterial infections

accumulation of phagolysosomes that contain partially degraded bacteria. The phagolysosomes eventually calcify to form Michaelis-Gutmann bodies, which are stained by PAS and Von Kossa stains. Salmonella spp. Salmonella spp. belong to the Enterobacteriaceae. They are usually an enteric pathogen and pulmonary infection occurs as part of a septicemic process. Non-typhoidal Salmonella spp. are isolated from respiratory or blood cultures in patients with underlying conditions, such as pulmonary malignancies, sickle cell anemia or HIV infection. Treponema pallidum (syphilis) Treponema pallidum spp. pallidum is a spirochete with tight or loose, regular or irregular coils observed in WS stained sections of tissue. Syphilis is a sexually transmitted or congenitally acquired infection that can affect almost any organ in the body. Syphilis is a nationally notifiable disease in the United States. Tropheryma whipplei (Whipple disease) Tropheryma whipplei organisms are pleomorphic bacteria of low virulence that usually infect the intestinal tract but may occur in many other organs, including the lung. Whipple disease is characterized by the accumulation of foamy macrophages that contain PAS-positive sickle-shaped inclusions. It is important to distinguish Whipple disease from Mycobacterium avium-intracellulare infection in patients with AIDS and from muciphages. Yersinia pestis (plague) Yersinia pestis organisms reproduce rapidly in tissue, producing enormous bacterial burdens. These are seen as amorphous masses on H&E and Gram stain in smears or in tissue. Virulence factors include cell wall lipopolysaccharides that induce inflammatory cytokines, antigens and outer membrane proteins with antiphagocytic and anti-inflammatory activity and are cytotoxic. Suspected isolates should be referred to a public health laboratory.

Rickettsiales Rickettsiales are tiny bacteria that are host-associated pathogens and often arthropod-borne. The subgroups include the Rickettsiae, Erhlichiae and Orientia. As culture is extremely difficult, immunofluorescent serology and immunohistochemistry are often used for diagnosis. Cross reactivity is observed. Anaplasma phagocytophilum (anaplasmosis) Anaplasma phagocytophilum is similar in form and size to Ehrlichia chaffeensis (below), except that the morulas are seen in neutrophils. Anaplasmosis was previously called human

granulocytic ehrlichiosis and is a nationally notifiable disease in the United States. Coxiella burnetii (Q fever) The organism is usually not seen in tissue sections. It survives in the phagolysosomes of infected macrophages. Histological findings in pulmonary infections include interstitial, peribronchial and perivascular inflammation, numerous plasma cells, intra-bronchiolar neutrophils and intra-alveolar mononuclear cells. Q fever is a tick-borne zoonosis of domestic animals acquired by humans by inhalation of contaminated aerosols. The organism can survive in formalin-fixed tissue and has been recovered from paraffin blocks in the spore stage. Diagnosis is usually serological, because most laboratories are unable to isolate the organism due to its highly infectious nature and the need for BSL3 facilities. Ehrlichia chaffeensis (monocytotropic ehrlichiosis) Ehrlichia chaffeensis is a small bacterium that forms clustered inclusions (morulas) in cytoplasmic vacuoles of macrophages and monocytes. The organisms can be seen in interstitial macrophages when pneumonitis is present. The organism is transmitted by ticks. PCR is the most sensitive detection method. Immunohistochemisty may provide early diagnosis if an adequate specimen is available. Ehrlichiosis is a nationally notifiable disease in the United States. Rickettsia conorii Disseminated Rickettsia conorii infection causes endothelial damage and can cause coagulopathy and pulmonary hemorrhage, especially in immunocompromised hosts. Immunostaining of endothelial cells in skin biopsy or circulating in the bloodstream can be used for early diagnosis. This tickborne infection of the spotted fever group has several geographic names (e.g. Boutonneuse fever, Mediterranean spotted fever and Kenya tick typhus). Rickettsia rickettsii (Rocky Mountain spotted fever) Rickettsia rickettsii organisms are extremely small obligate intracellular pathogens that target endothelial cells, where they are visible in BH-stained sections as slender rods arranged parallel to the long axis of the cell. Immunohistochemical staining can identify the organisms in endothelial cells. The organism enters the host via the skin and spreads through the lymphatics. Pneumonitis can occur in up to 15% of patients and can manifest as pulmonary edema or ARDS. Rocky Mountain spotted fever is a tickborne zoonosis and a nationally notifiable disease in the United States. Orientia tsutsugamushi (scrub typhus) Orientia (formerly Rickettsia) tsutsugamushi is transmitted by mites and is the cause of scrub typhus.

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References 1. Laennec R. A Treatise on Disease of the Chest. New York: Samuel Wood & Sons, 1830. 2. Woodhead MA, Macfarlane JT, McCracken JS, et al. Prospective study of the aetiology and outcome of pneumonia in the community. Lancet 1987;1:671–4. 3. Jokinen C, Heiskanen L, Juvonen H, et al. Incidence of community-acquired pneumonia in the population of four municipalities in eastern Finland. Am J Epidemiol 1993;137:977–88. 4. Bochud PY, Moser F, Erard P, et al. Community-acquired pneumonia. A prospective outpatient study. Medicine (Baltimore) 2001;80:75–87. 5. Marrie TJ, Peeling RW, Fine MJ, et al. Ambulatory patients with communityacquired pneumonia: the frequency of atypical agents and clinical course. Am J Med 1996;101:508–15. 6. Holmberg H. Aetiology of communityacquired pneumonia in hospital treated patients. Scand J Infect Dis 1987;19:491–501. 7. Marrie TJ, Carriere KC, Jin Y, et al. Mortality during hospitalisation for pneumonia in Alberta, Canada, is associated with physician volume. Eur Respir J 2003;22:148–55. 8. Farr BM, Kaiser DL, Harrison BD, et al. Prediction of microbial aetiology at admission to hospital for pneumonia from the presenting clinical features. British Thoracic Society Pneumonia Research Subcommittee. Thorax 1989;44:1031–5. 9. Macfarlane JT, Finch RG, Ward MJ, et al. Hospital study of adult community-acquired pneumonia. Lancet 1982;2:255–8. 10. Foy HM, Wentworth B, Kenny GE, et al. Pneumococcal isolations from patients with pneumonia and control subjects in a prepaid medical care group. Am Rev Respir Dis 1975;111:595–603. 11. Kyaw MH, Lynfield R, Schaffner W, et al. Effect of introduction of the pneumococcal conjugate vaccine on drug-resistant Streptococcus pneumoniae. N Engl J Med 2006;354:1455–63. 12. Mercat A, Nguyen J, Dautzenberg B. An outbreak of pneumococcal pneumonia

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associated pneumonia. Am J Respir Crit Care Med 2005;171:388–416. 24. Reimann HA. An acute infection of the respiratory tract with atypical pneumonia. JAMA 1938;111:2377–84. 25. Woodhead MA, Macfarlane JT. Comparative clinical and laboratory features of legionella with pneumococcal and mycoplasma pneumonias. Br J Dis Chest 1987;81:133–9. 26. Miller W. The Lung. Springfield IL: Charles C. Thomas, 1937.

15. Wellinghausen N, Straube E, Freidank H, et al. Low prevalence of Chlamydia pneumoniae in adults with community-acquired pneumonia. Int J Med Microbiol 2006;296:485–91.

27. Itoh H, Tokunaga S, Asamoto H, et al. Radiologic-pathologic correlations of small lung nodules with special reference to peribronchiolar nodules. AJR Am J Roentgenol 1978;130:223–31.

16. Lim WS, Macfarlane JT, Boswell TC, et al. Study of community acquired pneumonia aetiology (SCAPA) in adults admitted to hospital: implications for management guidelines. Thorax 2001;56:296–301.

28. Itoh H, Murata K, Konishi J, et al. Diffuse lung disease: pathologic basis for the high-resolution computed tomography findings. J Thorac Imaging 1993;8:176–88.

17. Marrie TJ, Haldane EV, Faulkner RS, et al. The importance of Coxiella burnetii as a cause of pneumonia in Nova Scotia. Can J Public Health 1985;76:233–6. 18. Sobradillo V, Ansola P, Baranda F, et al. Q fever pneumonia: a review of 164 community-acquired cases in the Basque country. Eur Respir J 1989;2:263–6. 19. Garcia-Fulgueiras A, Navarro C, Fenoll D, et al. Legionnaires’ disease outbreak in Murcia, Spain. Emerg Infect Dis 2003;9:915–21. 20. Fridkin SK, Hageman JC, Morrison M, et al. Methicillin-resistant Staphylococcus aureus disease in three communities. N Engl J Med 2005;352:1436–44.

29. Müller NL, Miller RR. Diseases of the bronchioles: CT and histopathologic findings. Radiology 1995;196:3–12. 30. Ramsey AH, Oemig TV, Davis JP, et al. An outbreak of bronchoscopy-related Mycobacterium tuberculosis infections due to lack of bronchoscope leak testing. Chest 2002;121:976–81. 31. Harlid R, Andersson G, Frostell CG, et al. Respiratory tract colonization and infection in patients with chronic tracheostomy. A one-year study in patients living at home. Am J Respir Crit Care Med 1996;154:124–9. 32. Fraser RS, Müller NL, Colman N, et al. Fraser and Pare’s Diagnosis of Diseases of the Chest. Philadelphia: WB Saunders, 1999.

21. Boonsawat W, Boonma P, Tangdajahiran T, et al. Communityacquired pneumonia in adults at Srinagarind Hospital. J Med Assoc Thai 1990;73:345–52.

33. Tang JW, Li Y, Eames I, et al. Factors involved in the aerosol transmission of infection and control of ventilation in healthcare premises. J Hosp Infect 2006;64:100–14.

22. Feldman C, Ross S, Mahomed AG, et al. The aetiology of severe communityacquired pneumonia and its impact on initial, empiric, antimicrobial chemotherapy. Respir Med 1995;89:187–92.

34. Stockley RA. Lung infections. 1. Role of bacteria in the pathogenesis and progression of acute and chronic lung infection. Thorax 1998;53:58–62.

23. Guidelines for the management of adults with hospital-acquired, ventilator-associated, and healthcare-

35. Macfarlane JT, Miller AC, Roderick Smith WH, et al. Comparative radiographic features of community acquired Legionnaires’ disease, pneumococcal pneumonia,

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mycoplasma pneumonia, and psittacosis. Thorax 1984;39:28–33.

evaluation. Infect Dis Clin North Am 2004;18:791–807.

36. Rossi SE, Franquet T, Volpacchio M, et al. Tree-in-bud pattern at thinsection CT of the lungs: radiologicpathologic overview. Radiographics 2005;25:789–801.

44. Stralin K. Usefulness of aetiological tests for guiding antibiotic therapy in community-acquired pneumonia. Int J Antimicrob Agents 2008;31:3–11.

37. Kuhlman JE, Fishman EK, Teigen C. Pulmonary septic emboli: diagnosis with CT. Radiology 1990;174:211–3.

45. Ewig S, Bauer T, Hasper E, et al. Value of routine microbial investigation in community-acquired pneumonia treated in a tertiary care center. Respiration 1996; 63:164–9.

38. Mizgerd JP. Acute lower respiratory tract infection. N Engl J Med 2008;358:716–27. 39. Strieter RM, Belperio JA, Keane MP. Cytokines in innate host defense in the lung. J Clin Invest 2002;109:699–705. 40. Wunderink RG, Waterer GW. Community-acquired pneumonia: pathophysiology and host factors with focus on possible new approaches to management of lower respiratory tract infections. Infect Dis Clin North Am 2004;18:743–59, vii. 41. Mandell LA, Wunderink RG, Anzueto A, et al. Infectious Diseases Society of America/American Thoracic Society consensus guidelines on the management of communityacquired pneumonia in adults. Clin Infect Dis 2007;44 Suppl 2:S27–S72. 42. Woodhead M, Blasi F, Ewig S, et al. Guidelines for the management of adult lower respiratory tract infections. Eur Respir J 2005;26:1138–80. 43. Woodhead M. Community-acquired pneumonia: severity of illness

46. Woodhead MA, Arrowsmith J, Chamberlain-Webber R, et al. The value of routine microbial investigation in community-acquired pneumonia. Respir Med 1991;85:313–7. 47. Chalasani NP, Valdecanas MA, Gopal AK, et al. Clinical utility of blood cultures in adult patients with community-acquired pneumonia without defined underlying risks. Chest 1995;108:932–6. 48. Koneman EW, Allen SD, Janda WM, Schreckenberger PC, Winn WC. Color Atlas and Textbook of Diagnostic Microbiology; 5th edn. Philadelphia: Lippincott, Williams & Wilkins, 1997. 49. Isenberg HD. Essential Procedures for Clinical Microbiology. Washington DC: American Society of Microbiology, 1998. 50. Mandell GL, Bennett JE, Dolin R. Mandell, Douglas and Bennett’s

Principles of Infectious Diseases, 5th edn. Philadelphia: Elsevier, Churchill Livingstone, 2006. 51. Bergey DH, Buchanan RE, Gibbons NE. Bergey’s Manual of Determinative Bacteriology, 8th edn. Baltimore: Williams & Wilkins, 1974. 52. Holt JG. Bergey’s Manual of Determinative Bacteriology, 9th edn. Baltimore: Williams & Wilkins, 1994. 53. Mundy LM, Auwaerter PG, Oldach D, et al. Community-acquired pneumonia: impact of immune status. Am J Respir Crit Care Med 1995;152:1309–15. 54. Almirall J, Boixeda R, Bolibar I, et al. Differences in the etiology of community-acquired pneumonia according to site of care: a population-based study. Respir Med 2007;101:2168–75. 55. Diaz A, Barria P, Niederman M, et al. Etiology of community-acquired pneumonia in hospitalized patients in Chile: the increasing prevalence of respiratory viruses among classic pathogens. Chest 2007;131:779–87. 56. Wattanathum A, Chaoprasong C, Nunthapisud P, et al. Community-acquired pneumonia in southeast Asia: the microbial differences between ambulatory and hospitalized patients. Chest 2003;123:1512–9.

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5

Pulmonary viral infections Richard L. Kradin and Jay Fishman

Introduction As the portal between the ambient atmosphere and the internal milieu, the respiratory tract is a frequent site of infection. Factors predisposing to respiratory viral infections include distortions in airway anatomy, decreased mucociliary clearance, disorders of ciliary function, factors that bypass the normal defenses of the lung, e.g. mechanical ventilation, and innate abnormalities of cellular and humoral immunity.1–4 In recent years, iatrogenic immunosuppression and human immunodeficiency virus (HIV)-1 infection have substantially increased the incidence of opportunistic viral infections (Table 1).5–7 Viral respiratory infections are caused by hundreds of antigenically distinct viruses of multiple genera. Recently, novel pulmonary viral pathogens have emerged, including a coronavirus that causes the severe acute respiratory syndrome (SARS), a hantavirus that specifically targets the lung, the metapneumovirus, and a novel variant H1N1 swine influenza. It is anticipated that epidemics of respiratory viral illness will emerge as a result of changes in the global migratory patterns of people, animals and insect vectors, the disruption of previously isolated ecological habitats, and changes due to global warming.8

Patterns of respiratory viral disease Respiratory viral infections may be divided into those that affect the upper respiratory tract (acute tracheobronchitis) or the lower airways (pneumonia) (Table 2). The viruses commonly associated with these syndromes can be subclassified based on epidemiology, i.e. as community-acquired or nosocomial, age distribution, i.e. childhood versus adult infections, level of host immunocompetence, or specific viral etiology.9,10 In the normal host, acute tracheobronchitis is a self-limited illness, characterized by cough and increased sputum production. Patients with acute tracheobronchitis may develop spasmodic bronchospasm or croup, or persistent wheezing. Upper respiratory viral illnesses can produce constitutional symptoms, including myalgias, headache and diarrhea, but high

Table 1 Factors predisposing to respiratory viral infections

Anatomical distortion Bronchitis/bronchiectasis Interstitial fibrosis Decreased mucociliary clearance Smoking Particulate inhalation Ciliary dyskinesias Mechanical ventilation Immunosuppression Iatrogenic HIV-1infection Cytomegalovirus infection Humoral dysfunction T-cell-mediated disorders

fevers are uncommon, except in influenzavirus infection, or as an indication the upper airway infection has extended into the lung to cause pneumonia. As discolored sputum may result from the sloughing of respiratory epithelial cells and local inflammation, a finding of purulent sputum does not help to distinguish upper from lower respiratory tract infections. In most cases, however, the presence of pulmonary consolidation on physical examination or infiltrates on chest radiographs accurately separates tracheobronchitis from pneumonia.

Epidemiology Viruses are the preeminent cause of respiratory infection in all age groups. They account for the majority of childhood pneumonias, but for only ~10% of lower respiratory tract infections in adults. On average, young children will suffer five to nine distinct viral respiratory illnesses per year; adults three to five.9 Respiratory syncytial virus (RSV) accounts for most viral respiratory infections in children; influenza A is the most common respiratory infection in adults.9 Adenovirus is a

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 5: Pulmonary viral infections Table 2 Human respiratory viral infections

a

Virus

Family

Seasonality

Diseases

Commentsa

Influenza virus (A, B)

Orthomyxoviridae

Yes (winter)

Bronchitis, influenza, croup, pneumonia

Superinfection common, epidemic infection

Parainfluenza virus (PIV)

Paramyxoviridae

Yes (winter)

Croup (PIV1, PIV2), pneumonia (PIV3)

Respiratory syncytial virus (RSV)

Respiroviridae

Yes (winter)

Bronchiolitis, pneumonia

Adenovirus

Adenoviridae

No

Croup, pneumonia, viremia

Metapneumovirus (hMPV)

Respiroviridae

Yes (winter)

Bronchiolitis, pneumonia

Children, immunocompromised hosts

Measles

Paramyxoviridae

No

Croup, pneumonia (giant-cell pneumonia)

Children

Rhinovirus

Picornaviridae

Yes (spring, fall)

Common cold

Generally mild

Coronavirus (CoV)

Coronaviridae

No

Common cold (OC43), pneumonia (HuCoV-SARS)

Epidemic severe acute respiratory syndrome (SARS)

Enterovirus

Picornaviridae

Yes (summer, fall)

Common cold, bronchitis

Echovirus, coxsackie, poliovirus

Hantavirus

Bunyaviridae

No (may have common source)

Pneumonia; hantavirus cardiopulmonary syndrome (HCPS)

Rodent vectors; deer mouse for sin nombre virus; single cases, some clusters

Herpes simplex (HSV)

Herpesviridae

No

Pneumonia, cold sores

With oral involvement

Varicella zoster virus (VZV)

Herpesviridae

No

Pneumonia, chickenpox pneumonia, shingles

Dissemination life-threatening, facial dermatomes

Cytomegalovirus (CMV)

Herpesviridae

No

Pharyngitis, pneumonia, viremia

Solid organ and hematopoietic transplant recipients; infants

Epstein-Barr virus (EBV)

Herpesviridae

No

Pharyngitis, “infectious mono”, viremia, lymphocytic interstitial pneumonitis

Post-transplant lymphoproliferative disorder (PTLD)

Human herpesvirus 6

Herpesviridae

No

Pneumonitis

HHV-6A and HHV-6B; immunocompromised hosts

Bocavirus

Parvoviridae

No

Bronchiolitis

Uncommon

Parechovirus (HPeV)

Picornaviridae

Unknown

Bronchitis, pneumonia

Children, rash, paralysis

Children < 5 years; immunocompromised

All infections amplified in immunocompromised hosts; common manifestations noted.

well-recognized cause of viral pneumonia in military trainees but it also causes sporadic cases of severe pneumonia.11,12 Host factors are important determinants of disease severity. Chronic cardiopulmonary disease, smoking, immunosuppression, pregnancy, vaccination status and immune deficiency have all been implicated in an increase in both morbidity and mortality due to viral pneumonias.2,13–21 Vaccination status plays an important role in the incidence of viral respiratory infections. Decreased childhood vaccination for measles in industrialized countries has recently led to a resurgence of this disease and its pulmonary complications. Atypical measles pneumonia has been seen in those who

were previously vaccinated with inactivated rather than live attenuated virus.22 Conversely, as varicella vaccination of adults has been more broadly implemented, the incidence of varicella pneumonia has diminished.23 Most viral respiratory illnesses are mild, and a definite viral etiology, while suspected, is confirmed in less than half of fatal cases of putative viral pneumonia.10,24,25 Published data with respect to viral respiratory illnesses tend to be biased towards the reporting of severe infections and to outbreaks with large numbers of cases. Respiratory viral infections are readily transmitted in schools, military bases, hospitals and day care centers. Nosocomial transmission of viral respiratory

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infections is also common, and hospital outbreaks tend to result from aerosolized droplet spread or contact with fomites. While death due to acute viral respiratory infection is uncommon, it is a significant cause of mortality amongst socioeconomically disadvantaged children, and in the immunocompromised host. In epidemic influenza, mortality depends on several factors, including vaccination status, immune competence and bacterial superinfection.

Seasonality The incidence of respiratory viral infections is most common in mid-winter and early spring, possibly as a result of the dissemination of aerosolized virus in enclosed spaces.10 However, pandemic influenza A (H1N1 “swine flu” of 1918 and 2009) can persist despite warming temperatures. Rhinoviruses and parainfluenza infections are largely non-seasonal, but they show an increased incidence in spring and fall. Parainfluenzavirus type 3 (PIV-3) infections occur most commonly in the spring, whereas PIV-1 and -2 tend to produce outbreaks in fall to early winter. Some viruses, e.g., herpesviruses, show no seasonal variation.

Radiographic appearances The radiographic appearances of viral pneumonia are variegate and nonspecific.26 Complex radiographic changes can be due to concomitant bacterial or fungal superinfection or to the presence of an underlying chronic pulmonary disease, e.g., emphysema.27 Bronchial wall thickening, “ground-glass” reticulonodular interstitial opacities and asymmetric patchy airspace consolidation with migratory infiltrates are all commonly observed. In severe viral pneumonia, the diffuse bilateral infiltrates that characterize the acute respiratory distress syndrome (ARDS) are often seen (Figure 1). Large pleural effusions, while unusual, may occur with adenovirus and parainfluenza pneumonias. Pulmonary nodular disease is seen in measles and varicella pneumonia, and lobar consolidations may develop with coronavirus infections of SARS, and in adenovirus and hantavirus infections.

Pathophysiology Viral infections exhibit distinct clinical presentations based on the route of infection, host factors and virulence of the organism. Respiratory viruses acquired via the airways tend to involve the proximal tracheobronchial tree but may invade into the distal bronchioles and gas-exchanging alveoli. Rhinoviruses characteristically produce an upper respiratory tract infection with little airway epithelial injury. Influenza and RSV cause an acute tracheobronchitis but they may also “blossom” into diffuse alveolar damage (DAD). Viral infections that target the conducting airways tend to denude the lining respiratory epithelium, leading to luminal plugging by mucus

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Figure 1. Diffuse pulmonary infiltrates in patient with novel H1N1 influenza pneumonia. (Courtesy S. Digumarthy, Boston, MA, USA).

and cellular debris, and alternating regions of air trapping and atelectasis. Hematogenous viral dissemination occurs in cytomegalovirus (CMV) and herpesvirus pneumonia, particularly in solid organ allograft recipients. In hematopoietic allograft recipients, CMV pneumonitis results from the cell surface membrane display of CMV antigens together with MHC Class I antigens on the infected pulmonary cells of immunologically naive patients. This produces an extensive pattern of disease, as compared to CMV infections that result from the direct spread of virus from an infected solid allograft. In the arboviral hemorrhagic fevers, e.g., dengue and Ebola, hemorrhagic pulmonary edema may be a prominent feature.28

Pulmonary defenses Although the majority of respiratory viruses initially attack the upper airways, their minute size allows penetration into the distal lung. Viruses differ in their ability to elicit host humoral and cellular immune responses. Humoral factors, including secretory immunoglobulin A (sIgA), and defensins released by airway cells limit the penetration of viruses into the gasexchanging pulmonary acinus. Airway mucosal dendritic cells (DC) entrap viral antigens and transport them to regional lymph nodes to be presented to T- and B-lymphocytes (Figure 2).29 Natural killer (NK) and CD3þ T-cell-mediated immunity are well recognized to play key roles in the elimination of viral infected pulmonary cells.29 The gas-exchanging alveolar spaces are normally maintained sterile by resident macrophages that scavenge inhaled viruses and secrete monokines, including interleukin-10 (IL-10) and transforming growth factor-beta (TGFb), which locally suppress inflammation and promote immunotolerance. However, when alveolar lining epithelial cells are injured, or when the viral load exceeds the phagocytic capacities of

Chapter 5: Pulmonary viral infections

AIRWAY WALL NL Mast cell DC

DC + Ag

Ag

DC LYMPH NODE

or

NL

Afferent Lymphatic Channel Δ Ag

Surface Macrophage

Efferent Lymphatic Channels

Ag DC

ALVEOLUS (Bronchial mucosa) INFLAMMATION

DC

ML

AM ML

ML

ML

Venous Blood

Alveolar or Bronchial Capillary

Figure 2. Pulmonary immune anatomy. Ag, antigen; DC, dendritic cell; NL, naive lymphocytes; ML, memory lymphocytes; AM, alveolar macrophages.

resident alveolar macrophages, neutrophils and activated exudate macrophages are recruited from the circulating blood and accumulate at sites of pulmonary infection.30 Even small amounts of viral antigen can evoke substantial host inflammation, via the amplification of immune activities due to chemokines, cytokines, and complement. These host defenses promote the clearance of infection but they can also produce bystander injury to the lung. Respiratory viral infections can also be complicated by bacterial or fungal superinfection. Primary viral infection promotes bacterial and fungal colonization by increasing the adhesion of these pathogens to respiratory epithelial cells, and by reducing mucociliary clearance and cell-mediated phagocytosis. All of these effects enhance the invasion of pathogens into normally sterile sites, including the paranasal sinuses, middle ear and lower respiratory tract.31

Patterns of lung injury due to viral infection Clinicians and radiologists may apply classification schemas to pulmonary viral infection that are different from those used by pathologists. For example, the findings of viral pneumonias may be referred to as consistent with atypical pneumonia,

differentiating them from those of a typical bacterial infection.32 However, the clinical and radiographic features of an atypical pneumonia may primarily involve the pulmonary interstitium, small airways or the alveolar spaces. The actual microanatomy of disease can only be established reliably by microscopic examination.

Tracheobronchitis/bronchiolitis Viruses evoke a variety of patterns of pulmonary inflammation. These depend on host factors and the virulence mechanisms of the offending virus (Table 3). The pathological changes seen in tracheobronchitis include airway mucosal congestion, edema, acute and chronic inflammation and the denudation of the lining pseudostratified columnar ciliated respiratory epithelium. Whereas viral infections evoke predominantly lymphohistiocytic cell-mediated inflammatory responses, herpesviruses can elicit primarily neutrophilic exudates (Figure 3). The reparative process in response to acute airway injury includes squamous metaplasia of the denuded respiratory surfaces. In the absence of persistent inflammation, the metaplastic squamous cells tend to revert to normal-appearing ciliated

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Chapter 5: Pulmonary viral infections Table 3 Injury patterns seen in lung viral infection

Tracheobronchitis Bronchiolitis (acute, chronic, necrotizing) Bronchopneumonia (acute, chronic, necrotizing) Pulmonary hemorrhage Pulmonary edema Diffuse alveolar damage Pulmonary nodules and micronodules

Figure 3. Acute herpetic bronchiolitis showing neutrophilic exudate in the lumen of a small airway.

respiratory epithelium. However, when ulceration of the airway mucosa has ensued, a fibroproliferative plugging of the airway lumina, i.e., obliterative bronchiolitis, develops. This can result in persistent scarring with airway distortion and expiratory air-trapping.

Figure 4. Consolidated lung with the beefy red appearance of diffuse alveolar damage in a patient who died of influenza pneumonia.

Diffuse alveolar damage Diffuse alveolar damage represents global injury to the gasexchange surfaces with disruption of the blood-air barrier (see Chapter 9).33 The acute findings in the exudative phase of DAD include proteinaceous edema, hemorrhage and inflammation. Over the course of several days these may resolve if the insult has been limited or progress to fibrosis with severely compromised pulmonary mechanics. Grossly, the lungs in DAD are congested, edematous and airless, with total combined lung weights that usually exceed 2000 g. This is attributable to extravascular exudation of fibrin that acts as a gel entrapping free lung water (Figure 4). The sine qua non of DAD is the hyaline membrane – necrotic alveolar lining cells embedded in an extravascular fibrin coagulum apposed to an ulcerated alveolar septa (Figure 5). Pulmonary angiography detects large in situ pulmonary vascular thrombi

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Figure 5. Hyaline membrane lining an alveolar duct in DAD.

Chapter 5: Pulmonary viral infections Table 4 Changes seen in viral infected lung cells

Organism

Cytopathic change

Influenza

No cytopathic change

SARS (coronavirus)

No cytopathic change

Respiratory syncytial virus

Polykaryons, inconspicuous cytoplasmic inclusions

Parainfluenza

Polykaryons, intracytoplasmic inclusions

Measles

Polykaryons, intranuclear inclusions

Adenovirus

Intranuclear inclusions (smudge cells)

Herpesvirus

Intranuclear inclusions, polykaryons

Cytomegalovirus

Intranuclear and cytoplasmic inclusions

Varicella zoster

Intranuclear inclusions

EBV

No cytopathic change

Figure 6. Lung in reparative phase of acute lung injury showing highly atypical alveolar lining cells with changes that mimic viral infection.

in nearly half of cases, microvascular fibrin thrombi are present in 90%,33 and systemic disseminated intravascular coagulation (DIC) occurs in 20%. Diffuse alveolar damage is the most frequent underlying pathological pattern seen in ARDS, but extensive bacterial or fungal bronchopneumonia and acute pulmonary hemorrhage can also produce ARDS. In addition, the pathology of the exudative phase of non-infective DAD can focally mimic acute bacterial superinfection, so caution is counseled in suggesting the diagnosis of acute infection in this setting.34 Viruses are the most common infectious cause of DAD, and surgical pathologists should carefully examine the lung in all cases of DAD for evidence of viral-induced cytopathic changes. However, as most RNA viruses do not produce nuclear cytopathic changes, their absence does not exclude a viral etiology, and ancillary studies may be indicated (Table 4). A common diagnostic pitfall in surgical pathology is to

mistake the hyperplastic reparative alveolar type II cells of DAD for viral-infected cells, as epithelial repair produces prominent reactive nucleolar changes and these are particularly evident in rapidly frozen sections (Figure 6). The lung in acute injury also shows megakaryocytes entrapped in the pulmonary microvasculature. This is due to their translocation from a stressed bone marrow, and their appearance can result in an erroneous interpretation of viral infected nuclei (see Chapter 2).35

Individual viruses Influenzavirus Influenzaviruses are members of the Orthomyxoviridae family, and include influenza A, influenza B and influenza C viruses.36 Influenzavirus is composed of an RNA core surrounded by a lipid envelope and a virion that appears segmented by ultrastructural analysis (Figure 7). Hemagglutinin (HA) and neuraminidase (NA) epitopes project as spikes from the surface of its lipid envelope. Hemagglutinin promotes entry into host epithelial cells, whereas NA cleaves sialic acid residues, which fosters the egress of viral progeny from infected cells. The antiviral amantadine disrupts the internal pH balance of the virus, whereas oseltamivir (Tamiflu) and zanamivir target neuraminidase activity. Influenza A and B viruses cause typical influenza, whereas influenza C causes a milder respiratory disease, predominantly in children. The nomenclature for influenza viruses includes the HA and NA specificity, serotype (A, B or C), geographic origin, strain number, year of isolation and subtype of virus based on its major antigenic epitopes, e.g., H3N2 (A/Sydney/5/97-like). Influenzavirus is primarily a zoonotic infection of birds. It generally crosses over into mammals when birds are in proximity to swine. Swine serve as mixing vessel hosts in which the influenzavirus genome is modified by genetic re-assortment, combining with previously acquired human genetic material to produce a novel viral genome potentially showing enhanced infectivity for man. The highest risk of eventual transmission to man occurs in agrarian settings in which birds, swine and man live together at close quarters. An antigenic change in both the HA and NA of influenza A is referred to as antigenic shift, minor seasonal changes in the HA or NA as antigenic drift. The former has occurred several times in modern history; in 1918, when H1N1 viruses replaced H3N2 viruses; in 1957, when H2N2 viruses replaced H1N1 viruses; and in 1968, when H3N2 viruses replaced H2N2 viruses. Most recently, human infections with avian (H5N1, 2005) and swine influenza viruses (H1N1, 2009) have been replacing endemic H3N2 subtypes (Figure 8). Ascertaining a definite source of a pandemic outbreak of a new influenzavirus is a difficult task. Whereas the antigenic drift that gives rise to seasonal influenza appears to arise each year in China and Southeast Asia, evidence suggests that novel

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strains capable of causing pandemics may result from complex genetic recombinations taking place at locations separated by distance and over prolonged periods.37,38 The continued public health concerns with H5N1 avian influenza are based on its increasing prevalence in bird populations and on small numbers of lethal cases in Asia that have resulted from direct human exposure to infected birds.39

Although its precise provenance is uncertain, the current epidemic of novel H1N1 influenza/2009 appears to have started as an outbreak in rural Mexico, where a number of fatal cases were first reported.40 Rapid spread of infection was documented due to air travel and on June 11, 2009, the World Health Organization (WHO) signaled that a global pandemic of a novel influenza A (H1N1) was under way, by raising the worldwide pandemic alert level to its highest level, Phase 6. The first wave of disease has in general been mild, with most people recovering without hospitalization; however, excess deaths occurred, particularly in children, otherwise healthy young adults who are immunologically naive with respect to the virus, diabetics, pregnant women and immunosuppressed hosts (Table 5).

Table 5 Risk factors for developing novel H1N1 influenza

Adults and children over 6 months of age with long-term health conditions: Chronic lung disease Chronic cardiac disease Chronic renal disease Chronic hepatic disease Chronic neurological disease Immunosuppression from any cause Living in the same household as an immunocompromised patient Pregnancy in any stage Obesity Limited herd immunity due to low vaccination rates Figure 7. Influenza virus A (original magnification 45 000). (Courtesy M. Selig, Boston, MA, USA.)

Based on criteria of the UK National Health Service, medical literature, and personal communication from Professor P. Hasleton.

Figure 8. Neuraminidase and hemagglutinin expression of influenza correlates with epidemic outbreaks.

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The severity of disease caused by a novel influenzavirus is difficult to determine with accuracy. Once the disease becomes widespread, the reporting of new cases often diminishes, so establishing the prevalence of infection is impossible.41 Nevertheless, in some countries, the early death rate from novel influenza A (H1N1) was estimated to be as high as ~10%. Severe disease was observed in developing regions with low vaccination rates. In patients who required hospitalization, ~25% required support in an intensive care unit.42 However, the risk and types of bacterial superinfection appear to have been comparable to those of seasonal influenza. Influenzavirus is spread by large droplets, small aerosol particles, and contact with fomites.9,10,17,27 The incubation period from exposure to viral shedding is 1–3 days, depending on the size of the inoculum. Shedding persists for 5–10 days, or longer in immunocompromised hosts and in young children. The clinical features of influenza A and B virus infection are similar. The onset of “flu” is abrupt, with prominent systemic symptoms including fever, myalgias and fatigue. But symptoms may also be limited to rhinorrhea, sore throat, fever, conjunctival injection and non-productive cough. The early onset of dyspnea is a poor prognostic sign, as it may progress rapidly to respiratory failure. Acute influenza causes diffuse inflammation of the larynx, trachea and bronchi, with mucosal injection and edema. Chest pain is a reflection of necrotizing tracheobronchitis. Rates of serious morbidity and death are highest among persons older than 65 years, children aged less than 2 years and persons of any age who have medical conditions that place them at increased risk for complications from influenza.16,17,21 Children are prone to gastrointestinal symptoms, including nausea and vomiting, and febrile seizures are common in young hospitalized children.43 Uncommon complications include myocarditis, pericarditis, myositis, myoglobinuria, encephalopathy, transverse myelitis and Reye syndrome.10 Due to high infectivity and aerosolized/droplet mode of transmission, influenza attacks large numbers of individuals in the community at once. Immunocompromised hosts tend to develop disease earlier in the fall, but the bulk of disease occurs in the winter and early spring. Influenza pneumonia tends to develop in elderly, non-vaccinated or immunocompromised individuals, and bacterial superinfection is common in these settings.15,31,44 However, in the 1918 influenza epidemic and in the recent H1N1 outbreak, it was the young – not the old – who suffered most. Diagnosis rests predominantly on the immunological detection of viral antigens in respiratory secretions. Most tests are designed to detect both influenza A and B. Viral culture is also both sensitive and specific. Antiviral medications are an adjunct to vaccination and may be effective when administered as treatment or used as chemoprophylaxis following an exposure to influenzavirus. Oseltamivir and zanamivir are the only antiviral medications that are currently recommended for use in the United States. Amantadine or rimantidine should not be used

Figure 9. Lung from patient who died in the 1918 influenza epidemic showing DAD with no cytopathic changes or evidence of bacterial superinfection.

Figure 10. Lung in patient with DAD due to influenza showing prominent squamous metaplasia of terminal airways.

for either the treatment or prevention of influenza, until evidence of susceptibility to these antiviral medications can be established. The pathology of seasonal influenza includes an acute diffuse tracheobronchitis/bronchiolitis in which the normal ciliated respiratory epithelial lining is sloughed.45,46 The development of DAD carries a high mortality even in the absence of acute bacterial superinfection (Figure 9).47 In DAD caused by influenza, the small airways may show prominent squamous metaplasia, in addition to alveolar hyaline membranes, hemorrhage, edema and microvascular thrombi (Figure 10). Although these findings are characteristic, they are also nonspecific, and immunohistochemical stains, in situ hybridization, electron microscopy or viral antigen detection are required to confirm the diagnosis in situ (Figure 11). Superinfection by pyogenic bacteria, including H. influenza, group A streptococcus and staphylococcus, is a well-recognized

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complication, and occurs in up to 50% of cases. Bacterial infection can mask evidence of a previous influenza infection, and there may be multiple bacterial pathogens in some cases. In patients dying with novel influenza A (H1N1) the lungs at autopsy show DAD, and, like other forms of influenza, the virus produces no diagnostic cytopathic changes (Figure 12).48

Severe acute respiratory syndrome Worldwide epidemic severe respiratory disease spread from southern China in the spring of 2003 as the result of a novel zoonotic coronavirus, referred to as the human SARS coronavirus (HuCoV-SARS).49,50 HuCoV-SARS is a positivestranded RNA virus of the Coronaviridae family. Virions form along the endoplasmic reticulum or Golgi apparatus and next bud into cisternae, where they acquire a lipid envelope.49

According to the WHO, a total of 8098 people worldwide became sick with SARS during the 2003 outbreak. Of these, 774 died, so the overall case fatality rate approached 10%. HuCoV-SARS likely developed in the palm civet or a related animal. Subsequent transmission took place largely in the hospital setting, where transmission was by droplet spread and required close contact. Severe acute respiratory syndrome begins with a high fever (> 38.0 C), headache, malaise, and myalgias with mild cough or diarrhea. After 2–7 days, patients develop a dry cough and progressive dyspnea over 4–7 days. Upper respiratory viral symptoms of rhinorrhea or sore throat are uncommon and most patients develop pneumonia. Chest radiographs show unilateral or bilateral infiltrates, either ground-glass opacities or consolidation, progressing to bilateral airspace consolidation. In some cases, chest radiographs are normal or show ground-glass infiltrates with septal and interstitial thickening seen best by computed tomography (CT). Laboratory findings include elevations in lactate dehydrogenase, transaminases and creatine kinase, with lymphopenia and thrombocytopenia. Pulmonary interstitial fibrosis is a common sequela.50 In patients dying of SARS, the lungs show DAD with scattered multinucleated giant cells that lack inclusion bodies and are of uncertain diagnostic significance. The virus appears to target airway epithelium and alveolar type II cells. However, in one study, viral antigens could not be detected in situ, probably as a result of the prolonged interval (> 2 weeks) between the primary infection and death. Otherwise, the virus produces no cytopathic changes and infection is histologically indistinguishable from DAD due to influenza.51–53

Figure 11. Immunostain confirms the presence of influenza A.

Figure 12. Lung in patient dying of influenzavirus A (H1N1). (A) Trachea showing denudation of respiratory epithelium and squamous metaplasia (arrow). (B) Lung with DAD and extensive hemorrhagic infarction secondary to pulmonary arterial in situ thrombosis. (C) Lung with DAD and squamous metaplasia of airways (arrow).

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Respiratory syncytial virus Respiratory syncytial virus (RSV) is a 120 nm enveloped negative-sense RNA strand and a member of the Paramyxoviridae family. Respiratory syncytial virus causes most cases of viral bronchiolitis and pneumonia in children.27,54 Severe disease may affect immunocompromised children and adults.17,55 Two serological subgroups, A and B, are recognized and type A is responsible for most severe disease. Surface fusion proteins, F and G, mediate attachment and multikaryon formation by infected cells.27 The virus spreads by aerosolized droplets and infection develops with an average incubation period of 4 to 5 days (range 2 to 8 days). Immune assays, such as immunofluorescence (EIA) or enzyme-linked immunosorbent assay (ELISA), and nucleic acid-based techniques, including hybridization and polymerase chain reaction (PCR), can rapidly establish the diagnosis. Ribavirin, administered via either intravenous or aerosol routes, has been used to treat immunocompromised hosts with lower tract disease. The efficacy of antiviral therapies remains uncertain, and hyperimmune globulin or monoclonal antibodies may alternatively provide treatment for severe disease. Other respiratory viruses can mimic the bronchiolitis of RSV, including influenza, parainfluenza, mumps, and rhinoviruses. Adenovirus is an uncommon cause of acute bronchiolitis, but must be considered in the setting of severe disease. Human metapneumovirus (hMPV) causes mild to severe bronchiolitis as well as pneumonitis in all age groups, comparable to RSV.54,56–60 The recently described human parvovirus and human bocavirus have also been implicated as pathogens in young children with upper airway disease.61 Respiratory syncytial virus typically causes necrosis of bronchiolar epithelium, with loss of ciliated epithelial cells, marked peribronchiolar mononuclear inflammation and submucosal edema (Figure 13). The virus produces syncytial

Figure 13. Acute bronchiolitis in RSV.

epithelial giant cells with non-prominent and poorly defined eosinophilic cytoplasmic inclusions (Figure 14).62 Fatal cases generally show necrotizing bronchiolitis and interstitial pneumonitis. Diffuse alveolar damage with giant cells (giant cell pneumonia) can develop in immunosuppressed hosts (Figure 15).63 The histopathology of hMPV has not yet been adequately described but it appears to produce changes comparable to those seen in RSV and it must be considered in the histological

Figure 14. Multinucleated epithelial cells in RSV contain inconspicuous eosinophilic cytoplasmic inclusions (arrow).

Figure 15. Multinucleated cells in DAD due to RSV. (Courtesy of Dr S. Yousem, Pittsburgh, PA, USA.)

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differential diagnosis. Confirmation of hMPV infection requires RT-PCR or evidence of serological conversion.58

Parainfluenza viruses Parainfluenza viruses are the most common cause of childhood croup, although a similar syndrome may be seen with RSV, influenza, measles, rhinoviruses, and adenoviruses, and in Chlamydia pneumoniae and Mycoplasma pneumoniae infections.64 Immunosuppressed patients are at an increased risk of parainfluenza infection.16,65,66 The virus is a single-stranded, negative-sense RNA virion and a member of the Paramyxovirus family. It is 150–300 nm, polymorphic,67 and its lipid envelope shows HA, NA and fusion protein “spikes” (Figure 16). Viral laryngotracheobronchitis, i.e., croup, generally develops in young children with underlying upper respiratory tract symptoms of rhinorrhea and sore throat. A mild cough progresses to a persistent “barking cough” with fever. Children may develop inspiratory stridor and wheezing when lower respiratory tract involvement ensues. Both the cough and stridor are signs of inflammation, while edema of the larynx and trachea is primarily localized at the subglottic level. Hypoxia develops with involvement of the lower respiratory tract. Specific viral diagnosis can be established by viral culture. However, it is not routinely performed, as the clinical syndrome is generally characteristic, and management does not depend on identification of the specific agent. Croup must be distinguished from other serious causes of airway obstruction, such as bacterial epiglottitis and tracheitis. Epiglottitis, most often due to vaccine-preventable Hemophilus influenzae type B or to alpha hemolytic streptococci, causes acute respiratory

distress and drooling without cough. Stridor can result from peritonsillar or retropharyngeal abscess, diphtheria or the aspiration of a foreign body. Parainfluenza produces acute bronchiolitis (Figure 17) and DAD with syncytial giant cells and epithelial cell intracytoplasmic inclusions.6862 The latter are both larger and more frequent than those due to RSV (Figure 18). Similar histological findings can at times be seen following viremia in other organs, including pancreas, bladder and kidney.

Measles The exanthem of measles can be complicated by a clinically severe pneumonia in a small percentage of healthy adults.69 Measles is a member of the Paramyxoviridae family; it is 120–150 nm, spherical and pleomorphic. Its lipid envelope includes an HA and a fusion protein.70

Figure 17. Acute bronchiolitis in parainfluenza infection.

Figure 16. Parainfluenza virus (original magnification 19 000). (Courtesy M. Selig, Boston, MA, USA.)

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Figure 18. Parainfluenza-infected epithelial cell showing eosinophilic inclusions that are both larger and more frequent than in RSV (arrow).

Chapter 5: Pulmonary viral infections

Figure 19. Chronic inflammation involving a small airway in patient with measles.

Measles is transmitted by droplet infection and is highly contagious. Passive maternal humoral immunity tends to protect children for the first year of life. In the pre-vaccine era, most children were infected by the age of six.71 Following a 1– 2 week prodrome, the infection blossoms as coryza, cough and conjunctivitis, followed by the appearance of Koplik spots – raised erythematous papules that appear characteristically on the buccal mucosa.72 Subsequently, a morbilliform rash begins on the face and spreads rapidly to the trunk and extremities. The disease generally resolves uneventfully in about a week but complications of progressive disease or bacterial superinfection may arise. Mortality is approximately 1 per 1000 cases, but this figure is higher among malnourished or immunocompromised patients.73 In resource-limited settings, measles can be a major determinant of subsequent lifethreatening acute protein malnutrition, in the forms of both marasmus and kwashiorkor. Fatal measles pneumonia can develop in the absence of rash (Hecht’s giant cell pneumonia), notably in the immunocompromised host and in pregnancy.74 Pneumonia is also common in atypical measles, which may develop in individuals previously vaccinated with inactivated measles vaccine.75 It is characterized by high fever and by a centripetal, petechial, pustular or vesicular skin rash that involves the palms and soles. Koplik’s spots are absent but mucous membrane involvement (strawberry tongue) is common. Patients may develop either diffuse or nodular pulmonary infiltrates with a nonproductive cough. Bacterial superinfection is common. In response to the high morbidity of this unusual infection, the inactivated measles vaccine was withdrawn from commercial use in 1968. The pathogenesis of measles has been extensively investigated. The virus can bind to respiratory epithelial cells, via the CD46 and the signaling lymphocyte-activation molecule (SLAM) cell surface membrane receptors.76 A primary viremia infects the reticuloendothelial tissues and the virus next disseminates, while attached to circulating lymphocytes and monocytes.

Figure 20. Multinucleated measles-infected cell showing glassy nuclear Cowdry type A inclusions.

The pathology of pulmonary infection ranges from acute or chronic bronchiolitis (Figure 19) to DAD.78,79 The virus produces multikaryons with prominent glassy eosinophilic nuclear Cowdry type A inclusions (Figure 20).78 The differential diagnosis of giant cell pneumonia includes RSV, varicella zoster and parainfluenza infection, as well as hard-metal pneumoconiosis (see Chapter 14).80 The Warthin-Finkeldey cell is a multinucleated histiocyte characteristic of measles infection, located primarily in lymphoid tissues; however, it is not specific to this infection and may be seen with other systemic viral infections.77

Adenovirus Adenovirus is a cause of viral pneumonia outbreaks in military recruits, as well as an uncommon but significant cause of serious respiratory infection in normal adults.11,27 The virus is a nonenveloped, double-stranded DNA in a 70–90 nm icosahedral capsid (Figure 21). There are more than 50 known serotypes and six subgroups (A–F) of adenoviruses – approximately half of these have been linked to human disease, including pneumonia.81 In the normal host, progression of infection is generally gradual (2–3 weeks). However, in immunocompromised individuals disease progression may be rapid and lead to death, in part as a result of superinfection by bacteria or fungi. Adenovirus pneumonia mortality rates approach 60% in the immunosuppressed host, and 15% in immunocompetent patients.82–85 Severe cases can develop multisystem involvement and DIC.86 Adenovirus produces a variety of pathological changes in the airways and lung.87 The lungs are generally congested and edematous. Characteristic changes include (1) a necrotizing ulcerative bronchiolitis (Figure 22), (2) mixed neutrophilic and monocytic pneumonitis (Figure 23), (3) acute intrapulmonary necrosis with hemorrhage (Figure 24) and (4) DAD. Infected airway cells show amphophilic intranuclear inclusions with perinuclear clearing and marginated chromatin that

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Figure 23. Neutrophilic pneumonia due to adenovirus.

Figure 21. Adenovirus (original magnification 11 000). (Courtesy M. Selig, Baston, MA, USA.)

Figure 24. Necrotizing hemorrhagic adenovirus pneumonitis.

Figure 22. Ulcerative bronchiolitis in adenovirus infection.

mimics herpesvirus infection. These nuclear inclusions tend to enlarge to cause the nuclear membrane to bulge, yielding diagnostic “smudge cells” (Figure 25).88 The appearance of “smudge cells” can be mimicked by other viruses, pulmonary cytotoxic drug injury and the epithelial repair seen in the early proliferative phase of DAD. For this reason and because of the morphological overlap with herpesvirus, the diagnosis of adenovirus infection should be confirmed by either immunohistochemical staining, ultrastructural examination or viral isolation. Polymerase chain reaction detects adenoviral nucleic acid in respiratory secretions and in formalin-fixed or paraffin-embedded tissues.

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Figure 25. “Smudge cell” showing extrusion of nuclear contents beyond the confines of the nuclear membrane in adenovirus infection.

Cytomegalovirus Cytomegalovirus causes a severe pneumonitis in individuals infected with AIDS and in immunosuppressed patients, particularly transplant recipients.20,27,89,90 With a genome of 230 kb pairs, CMV is the largest of the herpesviruses. It shows

Chapter 5: Pulmonary viral infections

Figure 26. Focus of miliary infection in CMV pneumonitis.

Figure 27. Alveolar type II lining cells showing cytomegaly, Cowdry type B inclusions and cytoplasmic inclusions (arrow)

the characteristic icosahedral nucleocapsid structure of other herpesviruses and has a lipid envelope. Cytomegalovirus infection is common; between 50 and 90% of the North American population show serological evidence of previous infection.91 It has been estimated that as many as half of all Epstein-Barr virus (EBV)-negative cases of the mononucleosis syndrome are the result of CMV. The virus remains dormant in macrophages and endothelial and mucosal epithelium but can be reactivated.92 The highest risk group for developing infection appears to be the transplant population. Infection tends to develop at 1–3 months, post-solid organ transplantation, in patients who have not received antiviral prophylaxis, following the completion of antiviral prophylaxis, or with intensification of immune suppression.20,93 Clinical signs include fever, myalgias, arthralgias, dry cough, tachypnea and systemic toxicity. Leukopenia and

thrombocytopenia are common, as are elevations of liver transaminases, so-called transaminitis, with viremia. Diffuse interstitial pneumonitis is the most common radiographic pattern, but nodular infiltrates may occur. At times, superinfection due to bacteria, Aspergillus, Nocardia or Pneumocystis, can complicate the radiographic appearance. The risk of CMV infection is greatest in seronegative and immunologically naive organ transplant recipients receiving seropositive organs. Conversely, in hematopoietic stem cell recipients, pneumonia is most common in seropositive recipients of seronegative hematopoietic grafts.20 Diagnosis is established by lung biopsy or by the presence of a consistent clinical picture with documented viremia. Cytomegalovirus pneumonia occurs at the extremes of age, and is common in HIV/AIDS.94 The number of pulmonary cells showing cytopathic changes can vary considerably and tends to parallel the severity of infection. Cytomegalovirus primarily targets pulmonary macrophages and endothelial cells, although virtually any cell can be infected. The most common distribution of CMV pneumonitis is miliary disease with hemorrhage and patchy necrosis due to viremia (Figure 26); however, bronchiolitis and DAD also occur. At times, CMV-infected cells can be seen without a prominent host inflammatory response.95 The diagnostic features of infection are all distinguishable in routine H&E-stained sections and include (1) cytomegaly, (2) intranuclear basophilic inclusions with characteristic Cowdry type B inclusions (Figure 27) and (3) ill-defined 1–3 mm amphophilic and granular intracytoplasmic inclusions that can be seen on H&E, PAS or GMS stained sections (Figure 28). In patients receiving prophylactic treatment with anti-viral medications, CMV infected cells may not exhibit characteristic cytopathic changes. In this setting, immunohistochemical stains or in situ hybridization may be necessary to identify intracellular CMV antigens (Figure 29).96 Cytomegalovirus is both a frequent co-pathogen in the immunosuppressed patient and a cause of immunosuppression in its own right.91 Cytomegalovirus infection predisposes to other viral infections, Pneumocystis jiroveci and opportunistic fungal infections (Figure 30).

Herpesvirus Herpesvirus (HSV) infection of the lower airways may be caused by the spread of virus from infected oral or esophageal lesions, by hematogenous dissemination, or following transplantation of infected organs.97–99 Herpesvirus is a large (100– 110 nm) enveloped double-stranded DNA with an icosahedral nucleocapsid (Figure 31). The viral particle is invested with a lipid envelope, as a result of viral budding at the level of the nuclear envelope.100 Both herpesvirus types 1 and 2 can infect the lung.101 HSV-1 is the most frequent cause of primary disease, whereas HSV-2 results from viremic viral spread. Infection is long-lived as virions persist in dormant states within sensory nerves and ganglia. Reactivation begins in the nerve cells and virus is

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Figure 30. Patient with CMV infection and cryptococcal pneumonia (arrow) complicating HIV/AIDS.

Figure 28. Cytoplasmic CMV inclusions stain positive with GMS.

Figure 29. Immunostain demonstrates CMV antigen in transplant patient treated prophylactically with ganciclovir.

transported via sensory nerve axons to mucosal surfaces, where they can produce vesicular eruptions. Infants, airway trauma, airway burns, prolonged mechanical ventilation and immunosuppression are risk factors for

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developing herpetic pneumonia.102,103 Newborn infants develop disseminated disease with pulmonary infiltrates, and mortality approaches 30% even following acyclovir therapy.104 In adults, HSV can be isolated from secretions in ~30% of mechanically ventilated patients, and herpesviruses may be an important factor in severe exacerbations of chronic obstructive pulmonary disease.52 Dyspnea, cough and hypoxemia are common in this setting. Acute respiratory distress syndrome can develop with virulent herpetic infection and bacterial and fungal superinfections are both common and fatal when they supervene. Herpesvirus can also complicate ARDS.105 The chest radiographic appearances of herpesvirus infection range from mucosal thickening of the pulmonary airways, multifocal areas of consolidation and diffuse bilateral pulmonary infiltrates. Herpesvirus tracheobronchial infections tend to complicate labial and esophageal disease, with virus spreading via the aspiration of oropharyngeal secretions.106,107 Intubated patients receiving chronic ventilatory support are at risk as a consequence of local mucosal barotrauma from inflated endotracheal tubes. The respiratory mucosa is the primary target, where herpesvirus characteristically produces ulceration and extensive necrosis.108 The ballooning of infected cells, cell karyorrhexis and the piling up of the infected cells suggest the diagnosis. There may also be a prominent neutrophilic response that mimics a pyogenic bacterial infection (Figure 32). Diagnostic cytopathic changes include the presence of either type A or type B Cowdry nuclear inclusions, molding of adjacent cells and multikaryon formation (Figure 33). When there is extensive necrosis, immunohistochemistry for herpes viral antigen often demonstrates high background staining. While this suggests the diagnosis, it can also potentially obscure it (Figure 34).96 If necessary, the examination of paraffin-embedded tissues by electron microscopy can demonstrate the presence of diagnostic virions. Immunosuppressed patients with herpes viremia may develop miliary foci of pulmonary hemorrhagic necrosis with

Chapter 5: Pulmonary viral infections Figure 31. Herpesvirus-1 (original magnification 7500). (Courtesy M. Selig, Boston, MA, USA.)

Figure 32. Neutrophilic exudates shows herpesvirus-1 infected cell (arrow).

prominent alveolar fibrin exudates (Figure 35).109 Foci of infection may be paucicellular but scattered neutrophilic exudates are generally present. Diagnostic cytopathic changes may be difficult to identify but generally they will be seen upon detailed examination of the involved foci. The diagnosis of airway disease can be established by viral isolation from respiratory secretions, bronchoalveolar lavage fluids, or mucosal biopsies of ulcerated sites (Figure 36). As immunohistochemistry shows antigenic overlap between HSV-1 and HSV-2 infections, PCR methods may be required for accurate speciation.

Varicella zoster virus Varicella zoster (HSV-3 or VZV) is genomically closely related to HSV1/2 and is both morphologically and ultrastructurally indistinguishable from HSV. Viral pneumonia with VZV usually develops within a week of the onset of rash during primary varicella and disseminated zoster infections.107,110,111 The primary infection is the result of aerosol droplet spread or direct contact with fomites.112 The cutaneous exanthem of chickenpox is generalized and pruritic. Following resolution of the rash, the virus becomes dormant in sensory nerves, but it may be reactivated with age, stress or decreased cellular immunity, to

Figure 33. Polykaryons with nuclear inclusions in herpetic bronchitis. Squamous metaplasia is also apparent.

produce the painful vesicular rashes of zoster or “shingles” along sensory nerve dermatomes. Pneumonia without rash rarely occurs.113 Severe infection may afflict immunocompromised hosts, the elderly, pregnant women and heavy smokers. Symptoms include cough, dyspnea, pleuritic chest pain and hemoptysis. Physical findings other than fever and tachypnea are generally modest.111,114 Bronchial and bronchiolar involvement is common and bacterial superinfection may occur. The chest radiograph shows characteristic nodular (1 to 10 mm) infiltrates with hilar adenopathy, pleural effusions and peribronchial infiltrates, while areas of pulmonary infarction can develop. The pulmonary lesions heal via scarring and dystrophic calcification, producing miliary calcific densities that resemble “buckshot” (Figure 37).115 Cases that come to biopsy or autopsy show either miliary nodules of hemorrhagic necrosis in the lung and pleura, or

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(a)

(b)

Figure 34. (a) Ulcerated bronchial lesion showing (b) intense immunostaining for herpesvirus-1 The diagnosis was confirmed by ultrastructural examination demonstrating diagnostic virions.

(a)

(b)

Figure 35. (a) Hemorrhagic necrotizing pneumonia in immunosuppressed patient with herpesvirus-1 viremia. (b) Multiple infected cells immunostaining for herpesvirus-1.

DAD (Figure 38).115,116 Hemorrhagic inflamed foci can involve the pulmonary interstitium, airways or vessels. Infected cells with primarily eosinophilic Cowdry type A inclusions are generally seen at the edges of these lesions, but they are often more difficult to identify than in herpesvirus pneumonia. In disseminated disease, comparable foci can be seen at virtually any site.117 As the clinical diagnosis of varicella zoster is generally straightforward, lung biopsies are rarely required. The exception is in the immunosuppressed host, in whom pulmonary

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symptoms can precede the diagnostic rash. Viral isolation is the method of choice in order to confirm the diagnosis.118–121

Epstein-Barr virus Epstein-Barr virus (EBV) is a herpesvirus that has been implicated in disorders ranging from the mononucleosis syndrome to malignant lymphoid neoplasia.122 The mononucleosis syndrome includes pharyngitis, lymphadenitis and hepatosplenomegaly. Epstein-Barr virus is an uncommon cause of pneumonia, even

Chapter 5: Pulmonary viral infections

in immunocompromised individuals.123 While pneumonitis may develop in individuals with infectious mononucleosis, it is generally self- limited unless bacterial superinfection ensues.124 Chest radiographs may reveal diffuse bilateral ground-glass infiltrates, often with pleural effusions. Although pulmonary infection is rarely biopsied, EBV pneumonia produces patchy peribronchiolar and interstitial polyclonal lymphoid infiltrates with scant interstitial and intra-alveolar fibrinous exudates (Figure 39). The concept of persistent EBV pulmonary infection is controversial. The diagnosis is generally established serologically by EBV antigen titers but can also be confirmed in tissues by in situ hybridization. Other pulmonary diseases associated with EBV infection

include lymphomatoid granulomatosis and post-transplant lymphoproliferative disorder (see Chapters 20 and 34).

Human herpesvirus-6 Studies of lung tissues and bronchoalveolar lavage specimens from patients with pneumonia have led some investigators to propose that Human herpesvirus 6 (HHV-6) is a clinical cause of pneumonia.125 Most cases are most accurately described as “HHV-6-associated” pneumonia. Both mild and severe cases of pneumonia and bronchiolitis obliterans organizing pneumonia have been reported in immunosuppressed individuals with HIV and following bone marrow transplantation. Some studies have demonstrated co-infection with other viruses. No systematic evaluation of treatment regimens is currently available and controlled prospective studies are required in order to confirm HHV-6 as a primary pulmonary pathogen.

Hantavirus pulmonary syndrome

Figure 36. Herpetic inclusions in squamous respiratory epithelium of a chronically intubated patient.

A number of viral hemorrhagic fevers can attack the lung. Hantavirus, a member of the Bunyaviridae family, is 70–130 nm and spherical.126 Unlike other Bunyaviridae, hantavirus produces characteristic granular filamentous inclusions in macrophages and endothelial cells that can be detected by ultrastructural examination.127 Hantavirus causes hemorrhagic fever renal syndrome (HFRS), which primarily targets the kidneys, and the pulmonary syndrome (HPS), which affects the lungs.128,129 Figure 37. CT scan showing multiple calcifications in a patient with healed varicella pneumonia. (Courtesy R. Greene, Boston, MA, USA.)

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Figure 38. Hemorrhagic pneumonia due to varicella zoster.

Figure 39. Dense lymphoid infiltrate in patient with EBV pneumonia.

Figure 40. Pulmonary edema in patient with hantavirus pulmonary syndrome.

Figure 41. Pulmonary vessel with intraluminal atypical lymphocytes (arrow) in hantavirus pulmonary syndrome.

The virus was responsible for a limited outbreak in 1993 of fatal respiratory disease in the four corners region of the southwestern USA.130 The infection is a zoonosis transmitted primarily by infected Perimyscus maniculatus or by the feces of other rodents. The most common radiographic presentation is diffuse pulmonary edema with pleural effusions that can mimic congestive heart failure Following a brief prodrome of influenza-like symptoms, including fever, dizziness, myalgias, cough, nausea, abdominal pain and headache,129 patients develop a mild, non-productive cough with progressive dyspnea and tachycardia. Respiratory symptoms can progress to ARDS and systemic hypotension may lead to circulatory collapse and death. Laboratory abnormalities included hemoconcentration, thrombocytopenia with coagulopathy, and leukocytosis with circulating atypical immunoblasts. In the absence of trauma or surgery, clinical bleeding is unusual. Liver enzyme abnormalities are common, with elevated serum lactate dehydrogenase and aspartate aminotransferase. Histologically, the lung shows pulmonary edema with scant, poorly formed hyaline membranes (Figure 40).127,131 Atypical lymphocytes may be identified in both the pulmonary

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interstitium and the lumina of the pulmonary microvasculature (Figure 41).132 Histological involvement of splenic red pulp, lymph nodes, peripheral blood and hepatic portal triads is common, as evidenced by the presence of atypical immunoblasts. Confirmation of the diagnosis requires specific immunohistochemistry, serological evidence of hantavirus-specific IgM, or PCR. Ultrastructural identification of the characteristic viral inclusions is diagnostic but the inclusions are infrequently found.

Human immunodeficiency virus-1 A number of pulmonary syndromes have been associated with infection by HIV-1 (Figure 42) in the absence of an identifiable co-pathogen. As HIV-1-infected patients often receive antiviral and other medications, distinguishing possible HIV-1-induced pulmonary changes from those due to drugs or other infections can be exceedingly difficult. The reported pulmonary changes seen with HIV-1 are listed in Table 6. Lymphoid interstitial pneumonia (LIP) is the commonest and it is the most characteristic form of

Chapter 5: Pulmonary viral infections Table 6 Pulmonary abnormalities associated with HIV-1 infection

Lymphoid interstitial pneumonia Nonspecific interstitial pneumonia Hypersensitivity pneumonitis Organizing pneumonia Sarcoidosis Emphysema Pulmonary arterial hypertension Malignancies

Figure 42. Human immunodeficiency virus-1 (original magnification 12 000). Note the inner formed “core” which contains a nucleoid, a tapering conical shell and a capsid. (Courtesy M. Selig, Boston, MA, USA.)

Figure 44. Mixed cellular/fibrotic nonspecific interstitial pneumonitis (NSIP) in a patient with HIV-1 infection.

Figure 43. Patient with HIV-1 infection and lymphoid interstitial pneumonitis (LIP).

interstitial lung disease seen in HIV infection (see Chapter 34).133 Lymphoid inter-stitial pneumonia occurs in 25–40% of children with perinatally acquired HIV, usually in the second or third year of life. It is an AIDS-defining condition in children, but not in adults. The pathology of LIP shows polymorphic, non-clonally restricted, interstitial, lymphoplasmacytic infiltrates (Figure 43). Pulmonary cyst formation is common, and poorly formed sarcoidal granulomas are seen in approximately 20% of cases.

Nonspecific interstitial pneumonia (NSIP) was reported in 38% of patients with AIDS in one study, and nearly half of these patients had normal chest radiographs.134 Another study showed that since the introduction of highly active antiretroviral therapy, 7% of patients with HIV and interstitial lung disease have NSIP.135 The reported pathologies of NSIP in patients with HIV is variable and includes cellular, fibrotic and mixed subtypes (Figure 44). Other forms of interstitial lung disease, including sarcoidosis, hypersensitivity pneumonitis, organizing pneumonia and emphysema, are uncommon. HIV-related pulmonary arterial hypertension has been described in patients in whom an alternative cause could not be identified. More the 200 cases have been reported in the medical literature since its initial description in 1987. It is a

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rare complication of HIV infection, occurring in approximately 1 out of every 200 HIV-infected patients. Yet this prevalence is 6–12-times greater than that seen in patients

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varicella zoster virus in a neonate. Arch Pathol Lab Med 1989;113(2):201–3. 117. Johnson HN. Visceral lesions associated with varicella. Arch Pathol 1940;30:292–307. 118. Ziegler T. Detection of varicella-zoster viral antigens in clinical specimens by solid-phase enzyme immunoassay. J Infect Dis 1984;150:149. 119. Drew WL, Mintz L. Rapid diagnosis of varicella-zoster virus infection by direct immunofluorescence. Am J Clin Pathol 1980;73:699. 120. Hyman RW, Ecker JR, Tenser RB. Varicella-zoster virus RNA in human trigeminal ganglia. Lancet 1983;2:814. 121. Schmidt NJ, Gallo D, Devlin V, et al. Direct immunofluorescence staining for detection of herpes simplex and varicella-zoster virus antigens in vesicular lesions and certain tissue specimens. J Clin Microbiol 1980; 12:651. 122. Marzouk K, Corate L, Saleh S, Sharma OP. Epstein-Barrvirus-induced interstitial lung disease. Curr Opin Pulm Med 2005; 11:456–460. 123. Gutermann KSA, Hair LS, Morgello S. Epstein-Barr virus and AIDS-related primary central nervous system

lymphoma. Clin Neuropathol 1996;15:79. 124. Fermaglich DR. Pulmonary involvement in infectious mononucleosis. J Pediatr 1975; 86(1):93–5. 125. Cone R, Huang M, Hackman R. Human herpesvirus 6 and pneumonia. Leuk Lymphoma 1994;15:235–41. 126. Zaki SR, Greer PW, Coffield LM, et al. Hantavirus pulmonary syndrome. Pathogenesis of an emerging infectious disease. Am J Pathol 1995;146:552. 127. Goldsmith CS, Elliott LH, Peters CJ, Zaki SR. Ultrastructural characteristics of Sin Nombre virus, causative agent of hantavirus pulmonary syndrome. Arch Virol 1995;140(12):2107–22. 128. Butler JD, Peters CJ. Hantavirus and hantavirus pulmonary syndrome. Clin Infect Dis 1994;19:387. 129. Duchin JS, Koster FT, Peters CJ, et al. Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. The Hantavirus Study Group.[see comment]. N Engl J Med 1994; 330(14):949–55. 130. Duchin JD, Koster FT, Peters CJ, et al. Hantavirus pulmonary syndrome: a clinical description of 17 patients with a newly recognized disease. N Engl J Med 1994;330:949.

131. Zaki SR, Khan AS, Goodman RA, et al. Retrospective diagnosis of hantavirus pulmonary syndrome, 1978–1993: implications for emerging infectious diseases. Arch Pathol Lab Med 1996;120(2):134–9. 132. Nolte KB, Feddersen RM, Foucar K, et al. Hantavirus pulmonary syndrome in the United States: a pathological description of a disease caused by a new agent. Hum Pathol 1995;26:110. 133. Joshi V, Oleske J, Minnefor A, Brown R. Pathologic pulmonary findings in children with AIDS. Hum Pathol 1985;16:241–8. 134. Suffredini A, Ognibene F, Lack E, et al. Nonspecific interstitial pneumonitis: a common cause of pulmonary disease in the acquired immunodeficiency syndrome. Ann Intern Med 1987;107:7–13. 135. Narayanswami G, Narasimhan M, Rosen M. Pulmonary complications of HIV disease in the era of highly active antiretroviral therapy (HAART). Chest 2002;122:34S. 136. Sitbon O, Lascoux-Combe C, Delfraissy J-F, et al. Prevalence of HIVrelated pulmonary arterial hypertension in the current antiretroviral therapy era. Am J Respir Crit Care Med 2008; 177:108–13.

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6

Pulmonary mycobacterial infections Luciane Dreher Irion and Mark Woodhead

Introduction Tuberculosis (TB) is the name given to a spectrum of clinical syndromes caused by a small number of mycobacterial species of the Mycobacterium tuberculosis complex. Most of the other
130 described mycobacterial species are environmental organisms of which a few can cause disease only in very specific circumstances. These are principally associated with impaired host defense. M. tuberculosis, and less commonly the other organisms of the M. tuberculosis complex, M. bovis, M. africanum and M. microti, by contrast, can affect immunocompetent individuals, and are highly effective pathogens. Evidence to support this statement comes from history, including archeological evidence of TB in bony human remains from 500 000 years ago. That one-third of the current world’s population is estimated to be infected with TB is further evidence. In addition, the global human immunodeficiency virus (HIV) epidemic amplifies the importance of tuberculous infection, as co-infection is quite common. Infection with M. tuberculosis may lead immediately to progressive clinical illness, but it may also be asymptomatic or cause a minimal self-limiting illness. The organism then lies dormant in macrophages (latent infection). In 10% of such latent cases the disease reactivates. Clinical tuberculosis can affect any organ system, but the lungs and lymph nodes are most commonly affected in the UK.1 Tuberculosis is curable, yet 1.6 million people died from the disease in 2005. The key steps in TB management are the detection and appropriate treatment of infectious cases, as well as detection and management of latent infection. Cure requires compliance with a prolonged course of multitablet treatment with a high frequency of side effects. Mycobacterial resistance to one or more drugs makes treatment failure more likely and successful treatment more complex and expensive. This is especially true for multi-drug resistant (MDR) TB (resistance to at least isoniazid and rifampicin) and the recently described extensively drug resistant (XDR) TB (resistance to any fluoroquinolone, and at least one of three injectable second-line drugs, capreomycin, kanamycin and amikacin, in addition to MDR-TB).

Of the environmental mycobacteria, otherwise known as atypical mycobacteria, non-tuberculous mycobacteria or “mycobacteria other than tuberculosis” (MOTT), the M. avium complex (MAC) (principally M. avium and M. scrofulaceum), M. kansasii, M. xenopi and M. malmoense are most likely to cause pulmonary disease. A key difference from M. tuberculosis infection is that these organisms are not usually transmissible from one person to another.2 They typically cause clinical illness only in the presence of damaged host defenses, either locally after colonization of previously damaged lungs or systemically, as in the immunosuppressed including those with HIV infection.

Epidemiology TB occurs worldwide with an estimated 9.2 million new cases reported in 2006 and an incidence of 139/100 000.3 It is estimated that only 56%, ranging from 41% in Africa to 78% in Europe, of the estimated total are reported.3 It is most prevalent in low- and middle-income countries. Its frequency is increased by poverty and poor social conditions and is often associated with war and migration. HIV is largely responsible for the high TB prevalence, especially in sub-Saharan Africa. The largest number of new cases occurs in India, China and Indonesia. The incidence is highest in sub-Saharan Africa. The 2006 incidence in South Africa was 940/100 000 (Figure 1).3 The lowest incidence is in the Americas. Worldwide the incidence is slowly falling, but this is more than offset by population growth, leading to an increased number of total cases. Drug resistance is a worldwide problem, with the highest number of cases in the Russian Federation and parts of China and India.4 The frequency ranges from 0% in several Western European countries to 56% in Baku, Azerbaijan. The rate can be as high as 80% in previously treated patients. Multi-drug resistant TB cases range from 0% in eight Western European countries to 22% in Azerbaijan and 19% in Moldova. Data on XDR-TB are incomplete, but it appears to follow a similar distribution to MDR TB. Infection with environmental mycobacteria is not reportable and distinguishing infection from contamination is

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Figure 1. Worldwide estimated incidence of tuberculosis in 2007. Modified from reference3.

difficult, so incidence figures are not generally available. However, it appears that such infections are becoming more common, especially in countries with lower M. tuberculosis rates.5,6 Exact species distribution varies between countries.5

Microbiological diagnosis Microbiological investigation (Table 1) is desirable to confirm the diagnosis, exclude environmental mycobacterial infection and determine antibiotic sensitivity. Largely due to cost, but to a lesser extent organizational issues, such investigation is seldom available in most low-income countries of the world, where tuberculosis is most prevalent. At best, smear confirmation of acid-fast bacilli may support the clinical or radiographic diagnosis. More sophisticated investigation is usually only available in high-income countries. Sputum is the sample most frequently assessed microbiologically. The same investigations can be applied to lower respiratory tract secretions obtained bronchoscopically, via pleural fluid, and tissue samples from lung, mediastinal node or pleura. As identified by Koch over a century ago, the mycolic acid content of the mycobacterial wall endows the organism with unusual staining properties. Stains used in conventional microbiology are seldom taken up by mycobacteria. Stains resistant to acid or alcohol removal (acid-fast) taken up by the organism led to the use of the term acid/alcohol-fast bacilli or AAFB. The red bacilli stained with an arylmethane stain stand out better with a green or blue counterstain. This

counterstain colors the background material but not the mycobacteria. The whole process follows the modified approach, devised by Ziehl and Neelsen. Although bacteria other than mycobacteria may be AAFB-positive, this finding in the correct clinical context provides very strong support for the diagnosis (see Chapter 4). AAFB-positivity is dependent on bacterial numbers, and although likely to be positive in cavitary pulmonary disease, the paucibaciliary nature of tuberculosis at other lung sites (e.g., pleural fluid, pleural tissue, lymph node) means that AAFB positivity is less common in such situations. In addition, AAFB-negative results do not exclude the diagnosis.7 Although staining has the advantage of speed, it is still relatively labor-intensive and many laboratories employ an initial fluorescent screening stain and apply the Ziehl-Neelsen (ZN) stain only to those cases with positive fluorescence. Mycobacteria grow slowly and culture has depended, until relatively recently, on the appearance of mycobacterial colonies on Lowenstein-Jensen slopes from 2 to 6 weeks. Once an organism has cultured, antibiotic sensitivity can be determined. The advent of liquid culture is now replacing solidphase cultures in laboratories in high-prevalence countries. Commercial rapid culture systems combine liquid culture with either radiometric or fluorimetric/colorimetric detection of changes in oxygen consumption/carbon dioxide production.8 Such systems can often now produce results within 2 weeks of sample inoculation. Environmental mycobacteria can be demonstrated by either ZN or auramine-rhodamine stains and usually show

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Chapter 6: Pulmonary mycobacterial infections Table 1 Comparison of benefits and drawbacks of diagnostic tests in tuberculosis

Test

Benefits

Drawbacks

AAFB smear

Cheap result within hours

Positive depends on large numbers of bacilli – negative result does not exclude TB Unable to distinguish live from dead mycobacteria

Mycobacterial culture

Allows definitive bacterial identification Allows antibiotic sensitivity determination

Slow – up to 6 weeks with conventional culture Expensive liquid culture gives results within 2 weeks

Serology

Insensitive and not specific

Nucleic acid amplification tests

Highly sensitive Useful for rapid detection of resistance Useful for species determination

False positives without scrupulous quality control Cannot distinguish intact bacteria from nucleic acid fragments

Tuberculin skin test

Detects immune response to mycobacteria Detects mycobacterial infection regardless of anatomical site

False positive due to prior BCG or environmental mycobacterial exposure False negative in active TB sometimes and in the immunosuppressed Experience required to perform and read test Requires two visits

Interferon-g releasing assays

Detects immune response to mycobacteria Not influenced by BCG or most environmental bacteria

Requires a blood test Relatively expensive False negatives in very young children and the immunosuppressed

Tissue pathology

Relatively quick result

Granulomas of sarcoid and other diseases indistinguishable unless AAFB positive

Figure 2. Microscopy of M. Kansasii. Organisms are notably curved and beaded (ZN stain).

similar characteristics to M. tuberculosis. M. kansasii can be slightly curved and longer than M. tuberculosis (Figure 2).9 Polymerase chain reaction (see below) or culture is still recommended for more precise speciation. As an increasing number of species have been identified and with more sensitive and specific diagnostic techniques available, cases of simultaneous involvement by M. tuberculosis and an environmental mycobacterium10 or with more than one environmental mycobacteria in the same sample have been detected. It is important to perform additional special stains, as mycobacteria can be found concomitantly with other microorganisms, especially but not

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exclusively in samples from immunocompromised patients. A wide range of bacteria, fungi or viruses may be identified in such co-infections. For these organisms the distinction between sample contamination and true infection is important. Identification from multiple samples and close correlation with the clinical and radiological picture is required.11 Serological tests for antibodies against mycobacterial components have been and continue to be assessed in TB diagnosis, but none has so far been found to be useful. The measurement of adenosine deaminase in body fluids has been shown to be useful in diagnosis in some reports.12,13 Nucleic acid amplification tests (NAATS) are now widely available to assist in tuberculosis diagnosis. These depend on the amplification and detection of genes or gene fragments unique to the mycobacterial and specifically the M. tuberculosis genome. Because of the extreme sensitivity of the tests, scrupulous care in sample handling is required to prevent contamination. Rigorous and continued control and monitoring are essential. Due to the persistence of mycobacteria in latent infection and the continuous shedding of mycobacterial genetic fragments in such cases, NAATS have not fulfilled the initial promise in diagnosis of active tuberculosis, especially in individuals from high-prevalence areas. However, these tests have an important and growing role in the identification of specific mycobacterial species in smear-positive samples, detection of resistance and genotyping in the assessment of case clustering, and the identification of potential sources of infection.8 Identification of mycobacterial species depends on the detection of DNA sequences unique to that species. Antibiotic

Chapter 6: Pulmonary mycobacterial infections

resistance can be determined by a large number of different gene mutations. Fortuitously, rifampicin resistance is encoded by one particular mutation in up to 90% of cases.14 This means that a NAAT specific for this mutation can be used to rapidly detect rifampicin resistance. In the UK, 90% of rifampicinresistant isolates are also isoniazid-resistant, i.e., MDR TB, and this probe can be used as a rapid surrogate for MDR tuberculosis diagnosis. Yet such results do not replace conventional sensitivity testing and should always be supported by the findings of conventional results. Detection of repetitive gene sequences (e.g. IS6110 in M. tuberculosis) is used to specifically genotype M. tuberculosis isolates.8 This allows separation of bacteria otherwise believed to be identical according to conventional methods. Bacteria with the same genotypes are likely to originate from the same source and this leads to assessment of clustering and source detection in outbreaks. Until recently the standard test for the detection of latent tuberculosis has been the tuberculin skin test (TST). This involves measurement of the size of the area of skin induration on the forearm after the intradermal injection of a standard dose of purified protein derivative (PPD) prepared from a reference strain of M. tuberculosis. The test is imperfect, since it is neither sensitive nor specific. This leads to a significant number of false-positive reactions in those previously BCG vaccinated or previously exposed to environmental mycobacteria. False-negative results are seen in young children and the immunosuppressed.15 Overall sensitivity and specificity values vary according to the age, immune status and BCG use within any particular population, so it is not possible to give average figures. One spin-off from the elucidation of the M. tuberculosis genome16 has been the identification of M. tuberculosisspecific gene products. Such antigens, based in the RD1 region of the M. tuberculosis genome, have been used to develop M. tuberculosis-specific tests. These are based on the in vitro production of interferon-g from host T-lymphocytes challenged with these particular antigens. Interferon-g releasing assays (IGRAs) have similar sensitivity and improved specificity, compared to the TST in TB contacts.13 They are being introduced into clinical practice.17 Their role in other situations, such as diagnosis of active TB and detection of latent infection in specific subgroups, such as children and the immunosuppressed, continues to be evaluated.

Clinical and laboratory manifestations The distinction between primary and postprimary tuberculosis (see below) is of no relevance to clinical management and the two entities may be clinically indistinguishable. Primary disease is usually asymptomatic or associated with an inconsequential self-limiting febrile illness. This is the normal pattern in children.18 Occasionally it can develop into progressive pulmonary disease or disease at another site. The clinical features in this setting are as for postprimary disease.

Four clinicoradiographic syndromes of pulmonary TB, namely pulmonary, pleural, miliary and multifocal, are described. In all, systemic symptoms may be present, usually for weeks or even months by the time of presentation. Weight loss, fever and night sweats are the usual systemic symptoms. The hallmark of typical pulmonary tuberculosis is a persistent, usually productive, cough. Sputum is usually mucoid, but may be bloodstained, and frank hemoptysis can occur. Breathlessness is a late feature, and only encountered with extensive disease. Pain is not common, unless disease is complicated by pneumothorax. When symptom duration is short it can resemble community-acquired pneumonia. Clinical examination may reveal signs of weight loss, but with little in the way of chest signs apart from raised respiratory rate, despite often very extensive radiographic change. In pleural disease, the local symptoms are usually breathlessness, sometimes with pleuritic pain and dry cough. The pleural effusion causes reduced chest expansion, dullness to percussion, reduced breath sounds and vocal fremitus on the affected side. In miliary disease the systemic symptoms may occur alone or with dry cough. Occasionally the systemic symptoms occur alone without radiographic chest findings, or indeed obvious disease elsewhere; so-called cryptic tuberculosis. Cryptic tuberculosis may be confirmed by liver or bone marrow examination or sometimes only by response to empirical anti-tuberculous therapy. Occasionally tuberculosis is multifocal, with disease outside the chest, such as lymph nodes or bone, coexisting with pulmonary disease. The features of pulmonary tuberculosis complicating HIV infection are the same as in the immunocompetent host, with the proviso that systemic features may be more prominent and widespread dissemination is more common. Therefore, the clinical symptoms may be many and varied. Pulmonary infection by environmental mycobacteria manifests with very similar symptoms and signs to those of M. tuberculosis infection. Cough, sputum production and failure to thrive are common.19 Such symptoms may be obscured when such infection occurs as a complication of underlying lung disease, such as bronchiectasis or cystic fibrosis. Careful integration of clinical, radiographic and microbiological information is required to make the diagnosis. The American Thoracic Society sets out specific criteria for the diagnosis of pulmonary environmental mycobacterial infection, based on data for M. avium, M. kansasii and M. abscessus infection.11 There are insufficient data to know whether they apply to other organisms in this group. Laboratory investigations, apart from microbiological tests (see below), are not diagnostic of TB, but aid in excluding other processes in the clinical differential diagnosis. Inflammatory markers (e.g. peripheral blood leukocyte count, C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR)) are usually, but not always, elevated in TB. However, leukocyte counts and CRP are not as high as in,

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(a)

(b)

Figure 3. Primary pulmonary tuberculosis. (a) Frontal chest radiograph of a young asymptomatic adult demonstrating a left upper lobe calcified nodule (line). Non-calcified nodules smaller than 1cm are not commonly visible in conventional chest radiographs. (b) Axial CT image at lung window settings from the same individual reveals a calcified granuloma in the superior segment of the left upper lobe, which is a common sequel of primary TB complex. A calcified hilar lymph node was noted in another slice of the same examination.

for example, community-acquired pneumonia. The ESR, in contrast, is sometimes greatly elevated. Abnormal liver function may be a pointer to hepatic involvement. Gas exchange measures, such as oxygen saturation, will only be impaired in very extensive disease. Pleural fluid will usually have the characteristics of an exudate with raised protein levels compared to the blood, a low glucose and raised adenosine deaminase and g-interferon levels in addition to a lymphocytosis.20,21

Figure 4. Typical radiographic features of pulmonary tuberculosis with right upper lobe cavitating consolidation (arrows) and additional minor consolidation in the right lower and left mid zones. Sputum smears and cultures were positive.

Radiological manifestations The chest radiograph is the key clinical investigation. Radiographic films may suggest the diagnosis or confirm a clinical suspicion. Primary tuberculosis may not be visible at the time of infection or appear as a small localized area of consolidation. It is more likely to be visible after the process has healed and a small, high-density, calcified opacity, usually in the lung periphery, becomes apparent (the so-called Ghon focus) (Figure 3). Typical parenchymal pulmonary tuberculosis can affect any area of the lung but is more common in the better ventilated regions, i.e., the upper lobes or apical segments of the lower lobes (Figure 4). In the early stages of disease, changes are not visible on chest radiographs, but only on computed tomograms (CT). One typically sees small foci of consolidation along an airway wall resembling a “tree in bud” (Figure 5). By the time of clinical presentation, consolidation, typically with cavitation (Figure 4),

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is usual, but in the early stages the cavitation may be difficult to see. With time, volume loss appears. Pleural effusion and less often pneumothorax can occur. An acute respiratory distress syndrome-like picture is infrequent (see Chapters 9 and 36). A tuberculous pleural effusion is indistinguishable from other etiologies (Figure 6). Effusions are usually unilateral

Chapter 6: Pulmonary mycobacterial infections

Figure 5. Ten millimeter collimation axial CT revealing multiple acinar shadows distributed along the bronchovascular bundles in a sub-segment of the anterior segment of the right lobe. Note the normal aspect of the vessels (arrow) and the areas of abnormal lung parenchyma (circle). The presence of multiple small ill-defined nodular opacities accompanying the arteries is called “tree-in-bud” pattern, and is almost pathognomonic of pulmonary infection, not only TB, but a very common finding in active phases of the disease.

Figure 6. Tuberculous right-sided pleural effusion.

Figure 7. Millet seeds for reference and comparison with usual size of nodules in miliary TB.

and small or moderate in size. A massive effusion is less common. Miliary disease refers to the widespread distribution of the typically millet seed-sized nodules, seen throughout the lung parenchyma of all lobes (Figure 7). These changes may be subtle and are better visualized on CT scans (Figure 8). This investigation may point to the diagnosis, especially in a patient with fever of unknown origin. Nodal disease is usually asymmetric, with nodal involvement limited to one hilum but seldom both (Figure 9). The latter is commoner in sarcoidosis. Nodes in the right paratracheal region are the commonest radiological appearance of mediastinal disease. Such disease is better visualized at CT scan, when nodal disease is usually more extensive than suspected from the plain chest radiograph (Figure 10). In coincident HIV infection, the radiographic features are more often atypical, with more infiltrates, nodal involvement and effusions but less prominent cavitation.

Figure 8. Miliary nodulation on CT scan.

Environmental mycobacterial infection typically produces radiographic appearances similar to cavitary pulmonary tuberculosis. There are additional changes related to the background structural lung pathology, e.g., emphysema, previous tuberculosis. M. avium, in particular, can also cause mid and lower zone nodular shadowing, often associated with bronchiectasis. A syndrome of nodular diffuse infiltrates on plain chest radiography and ground-glass or mosaic patterns on CT scanning has been associated with a hypersensitivity-like reaction to inhalation of aerosolized M. avium – so called “hot tub lung” (see Chapter 12).22

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Figure 9. Tuberculous right hilar lymphadenopathy (arrow). This asymptomatic patient from a high-prevalence country had complete radiographic resolution after anti-tuberculous treatment.

Pathogenesis In nearly all cases M. tuberculosis bacilli enter the lung in inhaled infected droplets. The exception is hematogenous spread of bacilli, leading to miliary tuberculosis. Larger droplets impact in the airways and are removed by the mucociliary escalator, usually to be swallowed and destroyed in the gastrointestinal tract. Smaller droplets reach the alveoli. From this point onwards mycobacteria have mechanisms that subvert the normal host response, which leads to bacterial multiplication.23 This together with the host response causes the clinical disease. In the alveoli mycobacteria encounter macrophages, dendritic cells and type II pneumocytes.24 These cells recognize and ingest the mycobacteria through pattern recognition, and receptors, including Toll-like receptors. Once ingested, the bacteria-containing phagosome is immunologically activated by interferon-g, leading to fusion with a lysosome. Such exposes the organism to a low pH and antibacterial substances (principally reactive oxygen and nitrogen molecules). However M. tuberculosis has the ability to stall phagosome maturation and to prevent phagosome-lysosome fusion, allowing the mycobacteria to persist and multiply within the phagosome. The mycobacterial cell wall limits the penetration of toxic molecules. It is also able to detoxify these host-produced reactive oxygen and nitrogen molecules and to repair the damage caused by these molecules.25 The extent to which these processes occur depends on mycobacterial virulence factors, as well as genetic factors, which determine the host response. Many complex molecular pathways are likely to be involved in mycobacterial virulence and it is beyond the scope of this work to go into these in

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Figure 10. Frontal chest radiograph of a young adult revealing a massive enlargement of the upper mediastinum and right hilum due to lymphadenopathy. The diagnosis of nodal TB was confirmed. No obvious abnormality other than the hilar lymphadenopathy was noted in the lung parenchyma.

detail. Unlike many other bacteria, M. tuberculosis does not produce soluble virulence factors. Instead it is the interaction between the mycobacterium and the macrophage. Perhaps most importantly, the ability of the mycobacterium to evade the host immune response and to persist within the macrophage (i.e., latent infection) is the key to the virulence of the organism.24,26 Since much of the mycobacterial genome codes for complex glycolipids, it is likely that these are important in mycobacterial virulence. Ingestion of bacteria into macrophages should be the key step to the death and elimination of these bacteria. Subversion of this process is essential to the virulence of mycobacteria. A very large number of different mechanisms have been identified whereby the mycobacterium might do this. The ability of M. tuberculosis to undertake non-opsonic binding to the macrophage surface via surface molecules, such as the mannose receptor and complement receptor type 3 (CR3), is an important initial step. Important factors in mycobacterial persistence within the macrophage, many of which are interrelated, are the arrest of phagosome development, inhibition of mycobacterial activation, mycobacterial resistance to the effects of reactive oxygen and nitrogen intermediates, inhibition of MHC Class II-dependent antigen-presenting processes and other cell signaling pathways, phagosome persistence through inhibition of phagosome-lysosome fusion and inhibition of host cell apoptosis (programmed cell death). Modulation of antigen processing is one factor in the arrest of phagosome development. Mycobacterial products such as lipoarabinomannan (LAM), a 25 kDa glycoprotein and a 19 kDa lipoprotein attenuate MHC Class II-dependent antigen

Chapter 6: Pulmonary mycobacterial infections Figure 11. Interaction among inflammatory cells in tuberculosis. The inflammatory lesion within the lung is a dynamic environment containing a variety of protective and regulatory cells. Effector T lymphocytes (purple) mediate control of bacterial growth and the mononuclear composition of the granuloma. Regulatory T lymphocytes (orange) also accumulate in the lesion and limit the ability of the acquired response to stop bacterial growth. Infected phagocytes elaborate cytokines and effector molecules that limit the activity of the lymphocyte response. B cells (blue) accumulate within the lesion in the form of nascent lymphoid follicles; these cells can affect bacterial control and the immunopathologic consequences of infection. Mtb (red) can modulate the inflammatory response via the modification of trehalose dimycolate (TDM) expressed at the bacterial surface. (Reproduced with permission from Cooper AM. Cell-mediated immune responses in tuberculosis. Annu Rev Immunol 2009;27:393–422 © Annual Reviews 2009.)

presentation. LAM may be important in the suppression of macrophage activation. SapM, PknG and PtpA are other M. tuberculosis proteins that have been linked to the arrest of phagosome development. Resistance to reactive nitrogen intermediates may be mediated via KatG, a catalase-peroxidase, which can inactivate reactive oxygen, but other genes such as ahpC, glbN and msrA have also been implicated in mycobacterial resistance to such reactive molecules. In addition the mycobacterial proteosome may be able to repair proteins damaged by reactive oxygen and nitrogen molecules. M. tuberculosis may produce its own anti-apoptosis genes (e.g. nuoG) as well as upregulate host anti-apoptosis genes to facilitate persistence of the infected macrophage. Glycolipids such as phosphatidylinositol 3-phosphate (PI3P) may be important in the inhibition of phagosome-lysosome fusion. The lipid trehalose 6,60 -dimycolate (TDM or cord factor) may inhibit angiogenesis, modulate macrophage function and play a role in lung granuloma formation and cavitation. Also, mycobacterial heat shock proteins modify nuclear transcription factors within the macrophage. While much is known about possible virulence factors of M. tuberculosis, since most research is based on in vitro or laboratory animal work, the exact mechanisms of virulence relevant to man are not yet well understood. Important host genes include natural-resistance-associated macrophage protein 1 (NRAMP1),27 the vitamin D receptor gene and MHC genes. Ultimately reactive nitrogen intermediates, as well as reactive oxygen intermediates, lysosomal enzymes and toxic peptides are responsible for mycobacterial killing. If the mycobacteria survive, they will multiply until the macrophage bursts and the released organisms are ingested by other macrophages. Macrophages present M. tuberculosis antigens to effector T cells via major histocompatibility complex (MHC) I and II molecules. This antigen presentation process is interfered with at many levels by M. tuberculosis.28

The release of chemokines from macrophages attracts further macrophages and dendritic cells to the site of infection. Dendritic cells migrate to regional lymph nodes, and release chemokines, including IL12, that attract and activate T lymphocytes. Activated T cells then return to the site of infection in the lung via the bloodstream. While CD8 lymphocytes play a role through production of cytolytic porforin and granzymes, CD4 lymphocytes are vital in the response to mycobacteria. This is shown by the importance of tuberculosis in CD4-deficient individuals, such as those with HIV infection. Of a number of CD4 subclasses, the TH1 cells are most important as the producers of interferon-g and tumor necrosis factor-α (TNFα). The central role of the latter cytokine has been highlighted by the increased occurrence of clinical tuberculosis in patients with latent tuberculous infection treated with drugs that block TNFα. These cytokines activate M. tuberculosis-infected macrophages to produce large amounts of nitric oxide synthase 2. Reactive nitrogen intermediates then kill the infectious organisms. These delayed-type hypersensitivity and cell-mediated immune responses usually contain the mycobacteria within granulomas. Within both the macrophages and the solid caseous centers, organisms are either inactive or replicate very slowly. The granuloma is a well-organized structure, which effectively seals off the mycobacteria from the rest of the host. Within this there is a complex interaction among the involved cells (Figure 11).29 This homeostatic interaction between the organism and the host can continue indefinitely and characterizes latent tuberculous infection. However, any reduction in the host’s delayed-type hypersensitivity response will tip the balance in favor of the mycobacterium. Liquefaction of the central caseous necrosis creates an inviting extracellular environment for M. tuberculosis replication and both local and distant spread. This reactivation tuberculosis causes clinical illness. The pathogenesis of environmental mycobacterial infection has been little studied. However, since the pathology is similar,

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(b)

Figure 12. (a) Advanced TB changes with combined cavitation, consolidation and focal miliary component. (b) Close view of cavitated areas with extensive adjacent consolidation. (Courtesy of Professor E. Marchiori, Rio de Janeiro, Brazil.)

the mechanisms are likely to be the same, with the exception of the virulence factors found in M. tuberculosis.

Pathology Macroscopy The macroscopy of pulmonary TB varies greatly according to the location (lung, lymph nodes or pleura), the presence of caseous necrosis or cavitation, fibrosis, inflammatory response and carbon pigment. An isolated tuberculoma can be seen in surgical specimens as part of investigation of a single solid lesion. The surgical biopsy can be performed if AAFB have not been demonstrated by other diagnostic methods and the differential diagnosis includes neoplasia. Often surgical specimens of a known TB case contain more advanced cavitated lesions combined with adjacent consolidation or associated with a miliary component (Figure 12). Frequently cavitated lesions are seen in upper zones and vary in size from 1–2 cm to as large as 10 cm.

Histopathology Microscopically, pulmonary tuberculous lesions are similar to those elsewhere in the body (Figure 13). The initial nonspecific histological finding is a neutrophilic infiltrate. In late-stage disease, most of the inflammation has resolved and has been replaced by fibrosis. The likelihood of demonstrating AAFB is decreased at this time. Classic histology is airway-centered necrotizing granulomatous inflammation. Infected epithelioid histiocytes may remain uninucleate or fuse into giant cells, otherwise known

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as Langhan’s-type giant cells. These organism-harboring histiocytes are surrounded by varying numbers of lymphocytes, ranging from scattered to a dense lymphocytic “belt”. The centers of these granulomas feature striking parenchymal necrosis but granulomas may remain non-necrotizing. The areas of necrosis usually exhibit a round, oval or geographic shape. The necrotic foci are commonly seen partly or totally replacing bronchioles. Tuberculous necrotizing granulomatous inflammation can affect pulmonary vessels and manifest as vasculitis (Figure 14). The granulomas usually involve the media and intima. This reaction should not be confused with Wegener granulomatosis, where neutrophils are present in the necrotic foci (see below). The granulomatous reaction can also affect lymph nodes and may mimic sarcoidosis (Figure 15). The cellular response in immunosuppressed/immunocompromised individuals may be decreased and a full granulomatous response may not occur. Extensive areas of necrosis with little or no cellular response can be seen. On histological preparations, mycobacteria can be demonstrated in sections stained with either ZN or auraminerhodamine, the latter requiring a fluorescent microscope. They are highlighted as bright red-purple thin 3–4 mm long bacilli. Organisms tend to be identified in central necrotic zones (Figure 16). Organisms are rare in lymph nodes.

Clinicopathologic classification The pathological presentation of pulmonary TB and severity of the host inflammatory reaction vary according to the host’s immune status and according to the virulence of the M. tuberculosis strain. Animal models have been of great importance

Chapter 6: Pulmonary mycobacterial infections

(a)

(b)

(c)

Figure 13. (a) This classic necrotizing granuloma obliterates a large portion of a lobule. Note the geographic necrosis and focal perilesional inflammatory infiltrate. (b) The wall of the lesion contains histiocytes, multinucleated giant cells scattered lymphocytes and plasma cells. (c) Although the temptation to search for organisms in the giant cells is great, most are found in the central necrosis.

Figure 14. Vasculitic process in a pulmonary artery branch in a patient with tuberculous consolidation.

Figure 15. Well-demarcated non-necrotizing granulomas in a mediastinal lymph node resemble sarcoidosis.

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Chapter 6: Pulmonary mycobacterial infections Figure 17. Calcified hilar lymph node (arrow) from a healed primary complex.

Figure 16. Thin, bright red bacilli in an area of caseous necrosis demonstrated with Ziehl-Neelsen stain.

for identifying variations in the clinical course and histopathological features associated with various M. tuberculosis strains at different phases of the disease.30,31

Primary pulmonary tuberculosis

The hallmark of primary pulmonary TB is the Ghon focus – a localized granulomatous lesion with caseous tissue necrosis, formed as a result of inhalation of M. tuberculosis by a person not previously exposed and sensitized to the organism. After the primary inhalation, a brief acute inflammatory response occurs, followed by a delayed cell-mediated hypersensitivity reaction which leads to the granuloma formation. The Ghon focus is usually subpleural or around fissures and can occur in either lung. Mycobacteria also drain to hilar lymph nodes, which may become enlarged by rapidly established caseous and granulomatous lymphadenitis. By 2 to 8 weeks, the Ghon focus is more defined and radiologically detectable. The primary pulmonary lesion associated with the regional hilar lymph node granuloma is known as the primary or Ghon complex. The primary complex usually develops and resolves silently. It is clinically uneventful in individuals with a competent immune system. The primary lung lesion, as well as the involved lymph nodes, eventually fibrose and calcify (Figure 17). In infants, who may not mount a mature immune response, a rapid spread of a primary complex can lead to tuberculous bronchopneumonia, due to erosion of the granulomatous lesion into a bronchus, atelectasis and air trapping. If erosion occurs into a blood vessel, miliary disease, including meningitis, may develop. In extreme cases, tuberculous lymphadenopathy may lead to erosion or perforation of the esophagus by a tracheoesophageal fistula.32 Primary lesions may also be associated with pleural effusions in adults. The average age for tuberculous pleurisy varies among case series but young adults and the elderly appear to be the most affected age groups.33 If necrotizing granulomas

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Figure 18. Subpleural dormant tuberculoma in a patient with a history of breast carcinoma. This lesion, while not calcified, is longstanding given the focally prominent fibrous capsule.

involve the pleura, empyema can occur. On rare occasions, granulomatous nodules on the pleural surface without parenchymal or nodal association have been described. Granulomatous pleuritis should be considered TB until proven otherwise.

Secondary pulmonary tuberculosis The usual pathway to secondary pulmonary tuberculosis follows further inhalation of M. tuberculosis by a sensitized person. In some cases, secondary pulmonary TB may result from reactivation of a dormant primary lesion (Figure 18). These secondary lesions should be located at the same site as the primary (i.e., subpleural location in an upper lobe).

Chapter 6: Pulmonary mycobacterial infections Figure 20. Cavitated pulmonary TB (top right). Also note the presence of bronchiectasis in the lower segments (bottom left and right). (Courtesy of Professor E. Marchiori, Rio de Janeiro, Brazil.)

Figure 19. Reactivated TB lesion. The infectious process has eroded into an airway and involves an adjacent intraparenchymal lymph node. Figure 21. Tuberculous bronchopneumonia. Multiple geographic areas are noted. (Courtesy of Professor E. Marchiori, Rio de Janeiro, Brazil.)

The second exposure to M. tuberculosis triggers a type IV hypersensitivity reaction through a complex interaction of cytokines, as previously discussed. Caseating tissue necrosis follows. The reaction is rapidly followed by recruitment of lymphocytes, plasma cells, epithelioid histiocytes and Langhan’s giant cells around the focus of necrosis. This is an attempt to neutralize and isolate the necrosis from the surrounding lung. A fibrous coat is formed around the area, acting as a physical barrier between the necrosis and adjacent lung tissue. Secondary lesions, also known as tuberculomas (Figure 19), may erode into adjacent bronchial walls. Cavitation and spillage of infectious material into the airway follows (Figure 20). Persistent discharge of caseous debris containing tubercle bacilli can be expectorated in the sputum. As the necrotic material from a tuberculous cavity accesses the patient’s airway, organisms spread into other bronchial subdivisions. Aspiration is a common mechanism

leading to tuberculous bronchopneumonia (Figure 21). The foci of tuberculous pneumonia can vary from small focal lesions to widespread consolidation occupying an entire lobe. The area involved depends on the amount of caseous material inhaled and the patient’s immunological response. Aspirated infectious material can reach terminal bronchioles, resulting in granulomas followed by fibrosis. This pattern of granulomatous pneumonia in bronchiolar terminal units potentially reflects the so-called “tree-in-bud” pattern described radiologically (Figure 5). As in primary disease, miliary TB can develop (Figure 22). Secondary lesions can erode into blood vessels. Thrombosis and infarction are complications of extensive granulomatous vasculitis.34,35 In secondary pulmonary TB, unlike primary TB, intrapulmonary lymphatic disease and hilar lymph node involvement are rare. If focal lymphatic permeation occurs, a few small tuberculous granulomas may form close to the main lesion. In most cases, there is a slow healing process, and the whole lesion is replaced by fibrosis and eventually calcifies. Nevertheless, if secondary TB presents as hilar or mediastinal lymphadenopathy only, underlying sarcoidosis should be excluded. Endobronchial TB can occur and should be included in the differential diagnosis of a necrotic endobronchial biopsy. The endobronchial lesion may be seen either as a tumor or as inflammation (Figure 23). Occasionally, it may present with bronchial stenosis associated with anthracosis. This process has a predilection for the right middle lobe.36 This condition, also known as bronchial anthracofibrosis, tends to occur in non-smoking elderly women with longstanding exposure to wood smoke. The association between TB presenting as anthracofibrosis and wood smoke exposure is not completely understood. Some hypothesize that longstanding exposure to such smoke damages mucociliary function or decreases macrophage clearance activity, consequently weakening natural defenses against infectious agents, including TB. Pleural TB may develop if a tuberculoma or a necrotic tuberculous lymph node ruptures into the pleura. Most cases

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(a)

(b)

(c)

Figure 22. (a) Miliary tuberculosis. Small lesions truly resemble millet seeds (see Figure 7). (b) Scattered small interstitial granulomas litter the lung interstitium. (c) Individual necrotizing granulomas maintain the same architectural form as large lesions.

are associated with an effusion. Miliary TB or a caseous focus from the thoracic spine or the chest wall37 may also be complicated by pleural disease. Especially in endemic areas, this can be complicated further by a chest wall abscess, which in half the cases can involve the ribs. The pleura is thickened, fibrotic and contains characteristic granulomas, often associated with a dense lymphocytic infiltrate. A needle biopsy is a good source of diagnostic material if pleural involvement is suspected and if other areas or methods fail to provide diagnostic AAFB. The diagnostic yield of needle biopsy may increase with the guidance of imaging techniques. In some undiagnosed cases, thoracoscopic pleural biopsy may be necessary to obtain diagnostic tissue. Secondary TB presenting as a spindle cell pseudotumor is rare. The first case reported described post-mortem findings of a renal and pancreatic transplant patient with ill-defined pulmonary spindle cell nodules.38 No granulomas were present but AAFB were demonstrated on Ziehl-Neelsen stain (Figure 24). Young children may sometimes present with epituberculosis. This peculiar condition is characterized by tuberculous

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lymphadenopathy causing bronchial obstruction and atelectasis. Such should not be mistaken for a tuberculous bronchopneumonia.39

Environmental mycobacteriosis Environmental mycobacterial infections have a broad clinical spectrum, including tuberculous-like infiltrates, solitary nodules and chronic bronchitis (Table 2). The pathology of environmental mycobacteriosis may be very difficult to differentiate from TB.40,41 Although some clinicopathological patterns are known, it is extremely difficult and unwise to predict the mycobacterial species causing the infection based on clinical presentation alone. While a number of mycobacterial species can present as a typical tuberculous-type granuloma, M. avium complex (MAC) or intracellulare can also be associated with variable clinicopathological presentations in different individuals, who may often suffer from pre-existing lung disease.42 Typical tuberculous-type granulomatous inflammation with necrosis is the most frequent histological presentation for environmental mycobacteriosis. Non-necrotizing granulomas are also often present. In other cases, usually involving

Chapter 6: Pulmonary mycobacterial infections

(a)

(c)

Figure 23. (a) Bronchoscopy on a patient with endobronchial TB. Note the edematous narrowing of the bronchus emerging on the right. (b) Bronchial biopsy showing marked nonspecific chronic inflammation associated with endobronchial TB. (c) Another microscopic field with focal granuloma and marked chronic inflammation. Note the presence of inflammatory cells also within the epithelium. (d) Ziehl-Neelsen stain highlighting the AAFB.

(b)

(d)

Figure 24. Secondary TB presenting as a spindle cell pseudotumor. This predominantly spindle cell proliferation from a kidney transplant patient lacks discrete granulomas. A ZN stain highlighted many organisms in the eosinophilic cytoplasm.

immunocompromised patients, the only finding may be nonspecific chronic inflammation. This is often associated with disorganized histiocytes, organizing pneumonia or fibrosis.43

Necrotizing granulomas may be associated with granulomatous bronchitis and vasculitis. Granulomas can sometimes be found in the pleura overlying the dominant lesion. Basal pleurisy is not often evident and only a few cases of environmental mycobacterial pleuritis with effusion have been reported.44,45 Although these tend to be associated with MAC, empyema following bronchopleural fistula, caused by other species, can occasionally occur even in immunocompetent patients.46 Granulomas are rarely seen in mediastinal nodes. However, on rare occasions mycobacteria can be demonstrated within a spindle cell pseudotumor type of lesion, particularly in HIV-positive or other immunocompromised patients. Association of mycobacterial pseudotumor in mediastinal lymph nodes and simultaneous Kaposi sarcoma has been reported.47 A ZN stain and immunohistochemistry (e.g. CD31, CD34, CD68, S100 protein) are helpful in establishing the diagnosis. In histopathological descriptions of pulmonary environmental mycobacteriosis, MAC appears to be the most frequently cited species in the recent English literature. This is presumably because MAC is the most prevalent type of environmental mycobacteria in the United States. The predominant species of environmental mycobacteriosis causing the infection, however, varies widely from country to country. Therefore, particular findings may be common in some places and unusual in others, and pathological features described with common species, such as MAC, M. kansasii and M. xenopi, may not always be present. M. avium complex can be associated with some specific disorders, such as Lady Windermere syndrome and

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hypersensitivity-like disease or so-called “hot tub lung”. The former tends to occur in the middle lobe or the lingula of elderly women as a result of poor clearance of these regions by avoidance of deep coughing (as per “good manners code”) in this gender and age group (Figure 25).48 Hypersensitivity-like disease refers to a pulmonary pathological reaction following exposure to water contaminated with MAC (e.g. from hot tubs, saunas or rarely showers) (see Chapter 12). It is characterized by non-necrotizing centrilobular and bronchiolocentric granulomas and organizing pneumonia. Finding granulomas in airways and airspaces helps differentiate this condition from sarcoidosis or other hypersensitivity pneumonitis (Figure 26).49 Nevertheless, there is still some controversy in the literature about whether MAC-associated penumonitis is a direct manifestation of the mycobacterial infection or a classic hypersensitivity pneumonitis. A few cases of MAC-associated pneumonitis have been reported without evidence of hot tub

exposure or other known microorganism sources.50 In such cases, structural abnormalities of the background lung acting as reservoirs may have a role in triggering the hypersensitivity pneumonitis.

Clinicopathological correlation The clinical features of tuberculosis are usually due to a combination of systemic features together with local effects at the site of disease. Occasionally a hypersensitivity response may cause features distant from the local site of TB infection.

Table 2 Most common clinicopathological patterns in pulmonary environmental mycobacterial infections

(a)

Clinico-radiological findings

Histological findings

Mycobacterial type

Solitary pulmonary nodules

Granulomas with necrosis (TB-like) Mediastinal LN usually without granuloma

Most species

Bilateral diffuse interstitial infiltration (association with immunosuppression)

Interstitial fibrosis and organizing pneumonia

MAC, M. gordonae, M. simiae

Multiple discrete infiltrates

Necrotizing granulomatous vasculitis

MAC

(b)

Figure 25. Lady Windemere syndrome. This transbronchial biopsy demonstrates peribronchiolar and interstitial granulomas along with florid mononuclear cell infiltrates and scattered foci of organizing pneumonia.

(c)

Figure 26. (a) Low magnification of typical “hot tub lung” features prominent centrilobular inflammation. (b) Interstitial lymphoid infiltrates and loosely formed non-necrotizing granulomas distort lung parenchyma. Intra-alveolar disease contributes to the consolidative appearance. (c) Scattered intra-alveolar granulomas are commonly seen in “hot tub lung”.

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The main systemic features are fever, night sweats and weight loss. Fever is mainly generated by the macrophagederived pyrogens interleukin-1, TNFα and interferon-α. All three may play a part in the other systemic features, with TNF being important in weight loss. The exact pathogenesis of night sweats is not known. In pulmonary disease cough, sputum production and hemoptysis are caused by the inflammatory response to bacteria in the alveoli and the presence of foreign material within the airways. The destructive nature of alveolar inflammation and bronchial/bronchiolar ulceration in TB leads to hemoptysis. Breathlessness is not usually a feature unless a very large proportion of the total alveolar space is occupied by inflammatory material. This is the case only in very advanced pulmonary disease, or in the presence of pleural disease with a large pleural effusion causing lung compression. Breathlessness may occur with lesser degrees of alveolar inflammation in those where lung function is already compromised by unrelated chronic lung disease, such as chronic obstructive pulmonary disease. Parietal pleural involvement may lead to pleuritic pain. Occasionally breathlessness with wheezing may occur due to bronchial compression by tuberculous involvement of mediastinal lymph nodes and rarely such nodes may rupture into a bronchus, causing cough, expectoration and hemoptysis. Examples of distant hypersensitivity-mediated features are erythema nodosum and uveitis.

Differential diagnosis A definitive diagnosis is based on demonstration of the AAFB on culture, PCR or histology sections. In the absence of bacilli, a number of differential diagnoses should also be considered.

Sarcoidosis The differential diagnosis between TB and sarcoidosis can be challenging, as one may mimic the other or even both pathologies may coexist (see Chapter 13). Ziehl-Neelsen and fungal stains, such as methenamine silver, are advisable in every case of suspected sarcoid. Histologically, sarcoid granulomas are usually non-necrotizing but might feature necrosis and tend to track along the lymphatics in the pleura, interlobular septa and bronchovascular bundles. They can include multinucleated giant cells and these may contain nonspecific inclusion bodies, such as asteroid or Schaumann bodies (see Chapters 2 and 13). Such inclusion bodies can be seen in TB. In typical sarcoid granulomas, vasculitis may be present involving the adventitia and media but vascular necrosis is not a common feature. Necrotizing sarcoid granulomatosis, as the name suggests, may contain large areas of necrosis along with nonnecrotizing granulomas. The granulomatous process involves bronchioles, as well as blood vessels, and the vasculitic component may sometimes be prominent. This process is histologically indistinguishable from TB and differentiation relies on clinical and microbiological findings (see Chapter 13).

Fungal infection Several fungal organisms cause necrotizing or non-necrotizing granulomas. Fungi causing pulmonary granulomas include Pneumocystis jiroveci, histoplasma, cryptococcus, blastomyces, coccidioidomyces, South American blastomyces, adiospiromyces and sporothrix (see Chapter 7).51 A fungal stain, preferably methenamine silver or Grocott with an adequate control, should be performed in each and every sample with granulomatous inflammation.

Other infections/infestations Rarely bacteria such as Bartonella can cause necrotizing granulomatous inflammation. This infection usually manifests with suppurative morphology. Cases of extranodal involvement by cat scratch disease tend to occur in immunocompromised patients; however, several cases of pulmonary involvement have been reported in immunocompetent individuals.52,53 Special stains may be helpful in the differential diagnosis but positive culture is often required (see Chapter 5). Cases of congenital pulmonary syphilis usually show numerous spirochetes and the differential diagnosis may not be a problem. On the other hand, in adults gummas may cause confusion. Multiple lesions may fuse to form a large solitary lesion with many plasma cells and only a few multinucleated cells. Nematode infestation (e.g., Dirofilaria immitis) may occasionally result in granulomatous inflammation. This finding is secondary to thromboembolic dead worms (see Chapter 8).51 Eosinophils may or may not be seen. Worms can potentially be identified within the necrosis.

Wegener granulomatosis The hallmarks of Wegener granulomatosis are necrotizing pneumonitis, vasculitis and giant cells (see Chapter 19). Geographic necrosis with a basophilic hue and many neutrophils may suggest this diagnosis rather than TB. Elastic-van Gieson and Martius Scarlet Blue stains are often informative in these cases. Serological studies may also aid in the differential diagnosis, especially when renal disease is absent.

Churg-Strauss syndrome Churg-Strauss syndrome is a rare systemic condition seen in asthmatics and may affect the lung (see Chapter 19). Microscopic features can be variable but include necrotizing, granulomatous inflammation with palisading histiocytes and vasculitis. Eosinophils can be numerous. Differential diagnosis can sometimes be difficult due to overlapping features, which also include the eosinophilic infiltrate. A positive P-ANCA test and high blood and tissue eosinophilia are useful for diagnostic confirmation.

Bronchocentric granulomatosis This disease is usually associated with allergic bronchopulmonary fungal disease in asthmatics (see Chapter 15). Infectious

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processes of other etiologies, including tuberculosis, may present with bronchocentric granulomas. Elastic tissue stains are useful in demonstrating the bronchocentric nature. Serial sections for fungal stains are also important.

Rheumatoid nodule Pulmonary rheumatoid nodules are identical to those in the subcutaneous tissue and are composed of palisading histiocytes arranged around central areas of necrosis with or without vasculitis (see Chapters 14 and 21). This histology may be seen in TB. Pleural involvement is common. Special stains, such as ZN and fungal stains, and culture are also important to exclude an infectious etiology. Rarely the two may coexist.

Foreign-body-type cells from aspiration Organic or non-organic aspirated material triggers an inflammatory reaction, which may vary substantially in intensity but is usually associated with a foreign-body-type multinucleated giant cell reaction. These giant cells differ from the Langhan’stype giant cells seen in tuberculous granuloma. Identification of the foreign-body material is also helpful in the differential diagnosis (see Chapter 17).

Granulomatous inflammation in lymph nodes draining cancer Granulomas can sometimes occur in lymph nodes draining a primary tumor, usually a carcinoma (e.g., breast, lung, larynx, stomach, colon, etc.). The epithelioid granulomatous reaction may be found even in the absence of neoplastic cells. The actual cause for this granulomatous reaction pattern is unknown but possible factors include direct reaction to tumor cells, therapy-related (e.g. BCG, interferon), underlying systemic granulomatous disease, and foreign-body reaction to a previous procedure (see Chapter 13).

Clinical diagnosis In low-income countries where most tuberculosis occurs, the diagnosis of pulmonary tuberculosis is usually simple and the only clinicopathological correlation is the knowledge of the frequency of tuberculosis, the common presenting symptoms and the sputum AAFB smear result. This will be sufficient to lead to treatment in the majority of cases. Occasionally this work-up will be supplemented by a chest radiograph. In high-income countries a chest radiograph will always be part of the initial clinical assessment and with typical upper zone cavitary change and typical symptoms, a positive sputum smear will be enough to initiate anti-tuberculous therapy. This may later be modified when sputum cultures and bacterial antibiotic sensitivity become available. In high-risk cases this may be supplemented by early use of gene probes for rifampicin resistance. In sputum smear-negative cases treatment will still be started if radiology and symptoms are typical. It is only in less typical cases that further pathological information is required. Bronchoscopic wash and bronchoalveolar lavage

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specimens may be helpful in those with non-productive or absent cough. Only rarely is lung biopsy required and often this is in those where the diagnosis is not expected. Pleural tuberculosis is usually paucibacilliary and so pleural fluid smears are usually negative. Pleural fluid lymphocytosis or an elevated adenosine deaminase level raises one’s index of suspicion. Either finding may suffice to commence treatment. In the non-severely ill the results of pleural fluid culture can be awaited; however, a pleural biopsy, especially in the absence of typical symptoms, may be required to commence therapy. In mediastinal lymph node disease, the inaccessibility of these nodes often means treatment is commenced without pathological confirmation. This is especially the case in those from high-prevalence countries with typical symptoms. Histopathological confirmation may be required more often as the differential diagnoses of sarcoid, lymphoma or metastatic cancer are difficult to exclude. Increasingly, diagnostic evidence is provided by fine-needle aspirate material obtained from relatively non-invasive transbronchial or transesophageal sampling with or without ultrasound guidance. It is in the environmental mycobacterial infections where clinicopathological correlation must be applied most carefully, both to distinguish infection from contamination and to separate these infections from those with M. tuberculosis. Overlap between clinical and radiological presentations and AAFB sputum smear results means that these infections are often only diagnosed when the sputum culture result is obtained after 6 weeks of a year-long treatment for presumed M. tuberculosis infection. Correlation of the clinical, radiographic and microbiological findings is critical to correct patient management in such infections.

Clinical management Efficacious management of TB infection relies on accurate clinicopathological diagnosis supported by microbiological confirmation. Drug sensitivities should ideally guide therapy. The existence of mycobacteria in different physiological states and the risk of resistance necessitates combination antibiotic therapy. This is based on randomized controlled trials and captured in various international and national guidelines.54–57 All expert groups recommend an initial, usually 2 month, usually four-drug, intensive phase of treatment followed by a continuation phase, which is less intensive, usually with two drugs and for a minimum of 4 months. Six months is thus the minimum duration of therapy. Drugs used combine bactericidal activity with sterilizing ability and the ability to prevent resistance development. The initial combination usually includes rifampicin, isoniazid, pyrazinamide and either ethambutol or streptomycin. Daily treatment is the norm, but twice and three times weekly regimes are available. Compliance with therapy for the full period is essential and for this reason the World Health Organization (WHO) recommends directly observed therapy (DOTS) for all. In high-income countries this may be reserved for patients at risk of non-compliance. Adjunctive therapy with steroids is

Chapter 6: Pulmonary mycobacterial infections

usually recommended in certain settings, including CNS, renal tract and pericardial disease. Management of drug-resistant TB is more complex, lengthy, labor-intensive and costly than treating sensitive infection. Again guidelines are available.58 Ideally treatment should be targeted according to the known drug sensitivities of the organism in each individual case. This may be possible in high-income countries, but not in the poorer areas of the world. Key aspects of treatment include using drugs from five different classes of anti-tuberculous agents plus at least four drugs to which the organism is likely to be sensitive. Therapy should continue for 18 months after culture conversion. There are a number of case series of surgical resection of localized disease to reduce disease burden in both MDR and XDR tuberculosis.59 This has not been examined by randomized controlled trial and its exact place in treatment is not known. Antituberculosis drugs are associated with significant nonpulmonary side effects such as rashes and hepatitis and also include interactions with drugs that the patient may be taking for other purposes. Such effects may occur in one-third of patients and be sufficient to stop or modify therapy in 5–10% of patients. Careful monitoring is again required for this reason. Tuberculosis in the context of HIV infection responds to therapy as effectively as in immunocompetent patients. Thus, treatment is similar. Side effects, particularly neuropathy, are more common in HIV patients, and anti-tuberculosis drugs may interact with antiretroviral agents, especially proteinase inhibitors. If highly active antiretroviral therapy (HAART) is started in a patient treated for latent TB or active TB, the recovery of the host immune response may lead to worsening of the clinical illness through the immune response inflammatory syndrome (IRIS).60,61 This phenomenon must be distinguished from tuberculosis progression. These patients are managed with additional steroid therapy and temporary cessation of HAART.

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Prevention of active disease by bacillus Calmette-Guérin (BCG) immunization, and detection and prophylaxis of latent tuberculosis is part of the anti-TB program in many countries. Accurate diagnosis is particularly important for environmental mycobacterial infection. Active disease must first be distinguished from colonization and sample contamination. Ideally before treatment is commenced the patient should be confirmed to have clinical and radiographic features compatible with environmental mycobacterial infection plus the identification of the mycobacterium from multiple respiratory samples. There is a poor correlation between in vitro bacterial sensitivities and clinical response for environmental mycobacteria. Treatment should follow guidelines.56 The combination of a rifamycin plus ethambutol with or without a macrolide, continued for at least a year following negative cultures, is the usual recommendation. Outcomes are, however, poor largely due to the severity of underlying disease.62

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25. Ehrt S, Schnappinger D. Mycobacterial survival strategies in the phagosome: defence against host stresses. Cell Microbiol 2009;11(8):1170–8.

38. Sekosan M, Cleto M, Senseng C, Farolan M, Sekosan J. Spindle cell pseudotumors in the lungs due to Mycobacterium tuberculosis in a

transplant patient. Am J Surg Pathol 1994;18:1065–8. 39. Gans B. Bronchoscopic treatment of atelectasis in children. Arch Dis Child 1952;27:254–6. 40. Farhi DC, Mason UGI, Horsburgh CRJ. Pathologic findings in disseminated Mycobacterium avium-intracellulare infection. A report of 11 cases. Am J Clin Pathol 1986;85:67–72. 41. Teirstein AS, Damsker B, Kirschner PA, et al. Pulmonary infection with Mycobacterium avium-intracellulare: diagnosis, clinical patterns, treatment. Mt Sinai J Med 1990;57(4):209–15. 42. Waller E, Roy A, Brumble L, et al. The expanding spectrum of Mycobacterium avium complex-associated pulmonary disease. Chest 2006;130(4):1234–41. 43. Marchevsky A, Damsker B, Gribetz A, Tepper S, Geller SA. The spectrum of pathology of nontuberculous mycobacterial infections in open-lung biopsy specimens. Am J Clin Pathol 1982;78:695–700. 44. Yanagihara K, Tomono K, Sawai T, et al. Mycobacterium avium complex pleuritis. Respiration 2002;69:547–9. 45. Shu CC, Lee LN, Wang JT, et al. Non-tuberculous mycobacterial pleurisy: an 8-year single-centre experience in Taiwan. Int J Tuberc Lung Dis 2010;14(5):635–41. 46. Hsieh HC, Lu PL, Chen TC, Chang K, Chen YH. Mycobacterium chelonae empyema in an immunocompetent patient. J Med Microbiol 2008;57(5):664–7. 47. Logani S, Lucas DR, Cheng JD, Ioachim HL, Adsay NV. Spindle cell tumors associated with mycobacteria in lymph nodes of HIV-positive patients: ‘Kaposi sarcoma with mycobacteria’ and ‘mycobacterial pseudotumor’. Am J Surg Pathol 1999;23(6):656–61. 48. Reich JM, Johnson RE. Mycobacterium avium complex pulmonary disease presenting as an isolated lingular or middle lobe pattern. The Lady Windermere syndrome. Chest 1992;101:1605–9. 49. Khoor A, Leslie KO, Tazelaar HD, Helmers RA, Colby TV. Diffuse pulmonary disease caused by nontuberculous mycobacteria in immunocompetent people (hot tub lung). Am J Clin Pathol 2001; 115(5):755–62.

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50. Zota V, Angelis SM, Fraire AE, et al. Lessons from Mycobacterium avium complex-associated pneumonitis: a case report. J Med Case Reports 2008;2:152. 51. El-Zammar OA, Katzenstein A-L A. Pathological diagnosis of granulomatous lung disease: a review. Histopathology 2007;50(3):289–310. 52. Margileth AM, Baehren DF. Chest-wall abscess due to cat-scratch disease (CSD) in an adult with antibodies to Bartonella clarridgeiae: case report and review of the thoracopulmonary manifestations of CSD. Clin Infect Dis 1998;27(2):353–7. 53. Marseglia GL, Monafo V, Marone P, et al. Asymptomatic persistent pulmonary infiltrates in an immunocompetent boy with cat-scratch disease. Eur J Pediatr 2001;160(4):260–1. 54. World Health Organization. Treatment of Tuberculosis. Guidelines for National Programmes Geneva: WHO, 2003.

55. Treatment of tuberculosis. MMWR Recomm 2003;52(RR-11):1–77.

resistant tuberculosis. Thorac Cardiovasc Surg 2009;57(4):222–5.

56. Griffith DE, Aksamit T, BrownElliott BA, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175(4):367–416.

60. Lawn SD, Wilkinson RJ, Lipman MC, Wood R. Immune reconstitution and “unmasking” of tuberculosis during antiretroviral therapy. Am J Respir Crit Care Med 2008; 177(7):680–5.

57. The National Collaborating Centre for Chronic Conditions at the Royal College of Physicians. Tuberculosis. Clinical Diagnosis and Management of Tuberculosis and Measures for its Prevention and Control. London, 2006.

61. Meintjes G, Lawn SD, Scano F, et al. Tuberculosis-associated immune reconstitution inflammatory syndrome: case definitions for use in resource-limited settings. Lancet Infect Dis 2008;8(8):516–23.

58. World Health Organization. Guidelines for the Programmatic Management of Drug-resistant Tuberculosis. Emergency Update Geneva: WHO, 2008. 59. Orki A, Kosar A, Demirhan R, Saygi A, Arman B. The value of surgical resection in patients with multidrug

62. Jenkins PA, Campbell IA, Banks J, et al. Clarithromycin vs ciprofloxacin as adjuncts to rifampicin and ethambutol in treating opportunist mycobacterial lung diseases and an assessment of Mycobacterium vaccae immunotherapy. Thorax 2008;63(7):627–34.

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Pulmonary mycotic infections Said Khayyata, Caroline B. Moore, Malcolm D. Richardson, Philip Hasleton and Carol Farver

Introduction

Table 1 Common fungi causing human pulmonary disease

Although innumerable species of fungi are known and the spores of some are ubiquitous, very few affect humans. However, the pulmonary mycoses have assumed greater importance as the number of immunocompromised patients has risen over the past few decades. Emerging fungal organisms previously thought to be non-pathogenic and extremely rare pathogens are now recognized as playing significant roles in the increased incidence of invasive fungal disease.1 While Aspergillus species (particularly Aspergillus fumigatus) are the most common cause of invasive fungal pneumonia, in recent years there are increasing numbers of severe invasive mycoses caused by hyaline septate molds such as Fusarium, Scedosporium, Trichoderma and Paecilomyces, infections caused by dematiaceous molds including Dactylaria/ Ochroconis, Wangiella/Exophiala and Cladophialophora, and emerging agents of mucormycosis such as Apophysomyces elegans, Cunninghamella bertholletiae and Saksenaea vasiformis. The main fungi causing pulmonary disease in humans are listed in Table 1. Some are saprophytic while others cause primary invasive infections in immunocompetent individuals without other predisposing factors. Considerable progress has been made in our understanding of fungal pathobiology, largely due to the sequencing of fungal genomes and the utilization of animal models to study the various components of fungal virulence and host immune response. The spectrum of clinical, radiographic and pathological findings is broad and some organisms may manifest clinically and pathologically in many different ways (Table 2). Recognizing risk factors for disease may be quite helpful when dealing with either a patient or specimen that may feature fungal-induced pathology (Table 3). This chapter will present current information on the most common pulmonary fungal diseases. For general information regarding the clinical manifestations, diagnosis and management of fungal infections the reader is referred to standard works.2–6

Organism

Disease

Aspergillus spp.

Spectrum of pulmonary aspergillosis

Mucorales

Mucormycosis

Scedosporium spp.

Usually fungus ball

Fusarium spp.

Fusariosis

Histoplasma capsulatum

North American histoplasmosis

Cryptococcus neoformans and C. gattii

Cryptococcosis

Blastomyces dermatitidis

North American blastomycosis

Coccidioides spp.

Coccidioidomycosis

Paracoccidioides brasiliensis

Paracoccidioidomycosis

Candida spp.

Candidiasis

Pneumocystis jiroveci

Pneumocystis pneumonia

Sporothrix schenckii complex

Sporotrichosis

General epidemiology The epidemiology of invasive fungal infections is difficult to determine from the literature because of the different definitions used, the different risk groups studied, and variation from institution to institution.7 Our current knowledge of the global distribution of systemic mycoses does not depict their true prevalence. It largely reflects the geographic distribution of medical mycologists or other investigators engaged in the study of fungal diseases and their research interests.8 The use of antifungal chemotherapy alters the incidence of different fungal infections. Fluconazole, introduced about 20 years ago, prevented many Candida infections but invasive aspergillosis has become commoner.7

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 7: Pulmonary mycotic infections Table 2 Tissue findings in common pulmonary fungal infections

Table 3 Risk factors for development of fungal disease

Organism

Form

Size (μm)

Histological features

Prolonged or profound neutropenia

Aspergillus spp.

Hyphae

3–6

Septate hyphae, acute-angle branching

Solid organ transplant recipients

Usually nonseptate hyphae, rightangle branching

Chronic disease

Septate hyphae, acute-angle branching

Chemotherapy including immunosuppressive and immunomodulating agents

Mucorales

Scedosporium spp.

Hyphae

Hyphae

10–25

2–5

Fusarium spp.

Hyphae

3–8

Septate hyphae

Histoplasma capsulatum

Yeast

2–5

Spherical to oval, narrowbased single bud

Cryptococcus neoformans

Yeast

2–20

Pleomorphic, capsule, narrowbased single bud

Blastomyces dermatitidis

Yeast

8–15

Doublecontoured with broad-based single bud

Coccidioides spp.

Spherule Endospore

10–60 1–2

Mature and immature spherules, often empty

Paracoccidioides brasiliensis

Yeast

10–60

Rounded with multiple narrow-based buds

Candida spp.

Yeast Pseudohyphae

3–5 3–5

Budding oval yeast. Rare septate hyphae

Pneumocystis jirovecii

Cysts Trophozoites

3–5 1–5

Round, oval with collapsed crescent forms

Sporothrix schenckii complex

Yeast

2–6

Oval to elongated

The most comprehensive multicenter epidemiological surveillance study of invasive fungal infections comes from the Transplant-Associated Infection Surveillance Network (TRANSNET) database. It is a network of 23 United States centers performing hematopoietic cell and/or solid organ transplantations.9–12 The overall incidence during a 12-month period for invasive fungal infections among hematopoietic cell transplant recipients is 3.4% (range 0.9–13.2%).11 This relative low

Hematopoietic cell transplant recipients Acquired immunodeficiency syndrome Chronic alcoholism Hereditary immunodeficiency states

Intensive care unit

incidence is probably due to the standard use of antifungal prophylaxis. TRANSNET data showed an overall 3-month invasive fungal infection mortality rate of 51%. More recent TRANSNET data demonstrate small bowel transplants with the highest risk of invasive fungal infection at 11.6%, followed by lung and heart-lung transplants (8.6%), liver (4.7%), and pancreas and pancreas-kidney (4%).12 Heart transplant recipients have a lower risk at 3.4%, and kidney the least at 1.3%. Most invasive fungal infections occur more than 90 days post-transplant. Invasive fungal infections are also increasing in intensive care unit (ICU) patients because of the growing use of complex surgical procedures, invasive medical devices, long-term broadspectrum antibiotics and the fact that these patients are immunosuppressed. One study showed fungi accounted for 20.9% of microorganisms recovered from positive cultures from ICU patients in Western Europe.13 Autopsy data, from a single-center review at the University Hospital of Frankfurt am Main for the time period 1993–2005, noted rising invasive fungal disease rates from 6.6% in 1993– 1996 as compared to 10.4% during the 2001–2005 period.14 Patients with underlying diseases included hematological malignancy, solid tumors, transplants, acquired immune deficiency syndrome (AIDS), and “other diagnoses”. The highest prevalence was found in patients with hematological malignancy (33%), followed by transplants (22.9%), AIDS (19.7%), solid tumors (4.8%), followed by “other diagnoses” (3.5%). The commonest fungi were Aspergillus species, followed by Candida species. Others (e.g., Cryptococcus species and species of Mucorales) comprised 1% or less of the cases reviewed. A similar type of study undertaken in Japan reached slightly different conclusions. From 1989 until 1994 the frequency of mycotic infections fell from 4.5% to 3.2%. There was an increase in 1993 due to a lack of effective antifungal agents. By 2001, the incidence had risen and has remained stable at 4.4% to 6% since. The age group most affected were between 60 and 79 years, though Candida showed a decrease in this age group. There is also an increasing tendency to find fungi in infants less than 1 year of age. These authors found the commonest organisms were Candida and Aspergillus. Candida showed a gradual decrease but Aspergillus increased. Even in

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2007, 12.6% of their cases were due to an “unknown fungus”. Between 3.1 and 5.1% of cases showed infection with multiple fungi.15 It should be noted the number of post-mortems halved between the years 1989 and 2007. The commonest underlying diseases in this study were leukemia and myelodysplastic syndrome. The frequency of infection was highest in the lungs followed by the central nervous system (CNS). However, Candida was commonest in the kidney in this study. There were small numbers of cases of trichosporonosis and histoplasmosis. It is possible Trichosporon was diagnosed as Candida or an undiagnosed fungus.15 Sub-Saharan Africa, which has just over 10% of the world’s population, is home to more than 25 million people living with human immunodeficiency virus (HIV)/AIDS. Opportunistic pulmonary infections are major causes of morbidity and mortality among HIV-infected adults in the subcontinent. Tuberculosis (TB) is the commonest infection, followed by Streptococcus pneumoniae but the prevalence of Pneumocystis jirovecii pneumonia is increasing, due to improved recognition of its clinical and radiographic features and diagnostic interventions. Combined infections, usually including TB, are common. Pulmonary nocardiosis, cryptococcosis and (possibly) histoplasmosis appear to be infrequent, but are probably underdiagnosed.16 In India there is endemic occurrence of blastomycosis, histoplasmosis and Penicilliosis marneffei. The last organism has emerged as a major endemic mycosis of AIDS patients in Southeast Asia. It has manifestations simulating those of Histoplasmosis capsulati, and the organism may spread to other regions with enlarging AIDS populations. Other important systemic mycoses reported from India include aspergillosis, cryptococcosis, candidiasis and mucormycosis.8 In China the pathogenic fungi isolation rates and species obtained from 1986 (n ¼ 9096), 1996 (n ¼ 19 009), and 2006 (n ¼ 33 022) suggest Trichophyton rubrum was the commonest organism cultured in the 1980s (45.4%) and 1990s (34.5%), but Candida albicans increased significantly and reached its peak (26.9%) in the 2006 survey. This organism has become the most common isolate of fungal infections in China currently.17 In the Mediterranean Aspergillus spp. are the leading cause of mold infections but the Mucorales and Fusarium species are increasing in frequency, and are associated with high mortality rates. Many of these emerging infections occur as breakthrough infections in patients treated with new antifungal drugs. The causative pathogens, incidence rates and severity are dependent on the underlying condition, as well as on the geographic location of the patient population. France and Italy show the highest incidence rates of Fusarium infections in Europe, following the USA, where numbers are still increasing. Scedosporium prolificans, which is primarily found in soil in Spain and Australia, is most frequently isolated from blood cultures in a Spanish hospital. Geotrichum capitatum is another species predominantly found in Europe, with especially high rates in Mediterranean countries.18

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Pulmonary aspergillosis Introduction Aspergillus is an important cause of pulmonary disease in debilitated patients.19–22 The lung is the most important portal of entry and also the commonest site for infection.21–23

Organisms Although there are several hundred species of Aspergillus, fewer than 40 cause diseases in humans or animals, and some of these have been reported only once. The fungus receives its name from the shape of the fruiting head, which resembles the brush used for sprinkling holy water in Roman Catholic and Anglican ceremonies (aspergillum). Normally, the vegetative form is found in human lesions, but conidiophores (fruiting bodies) develop if there is sufficient oxygen tension in the tissue (Figure 1). A. fumigatus still accounts for most human cases of aspergillosis, with A. flavus the second most frequent pathogen. A. terreus is resistant to amphotericin B, and is increasingly being reported as a cause of invasive disease in immunocompromised patients.24 A. fumigatus is an important human allergen, while Aspergillus clavatus and A. fumigatus are among the occupational causes of extrinsic allergic alveolitis. The histological diagnosis of Aspergillus infection depends on the identification of characteristic Aspergillus hyphae. In sections, well-preserved hyphae are septate (unlike Mucorales, which are non-septate) and homogenous with uniform 3–6 μm width (Figure 2). They have parallel contours, without constrictions at points of septation. Branches are dichotomous (of a size similar to the hyphae of origin) and diverge at a 45º angle. The hyphae produce conidial heads when exposed to air (Figure 3). When numerous, as in some angioinvasive lesions and fungus balls, the hyphae can be easily identified in hematoxylin-eosin (H&E) stained sections. The morphology is demonstrated best with a Grocott-methenamine silver

Figure 1. Aspergillus. Conidiophore and conidial head (fruiting body) of Aspergillus niger from a culture plate (methylene blue stain).

Chapter 7: Pulmonary mycotic infections

Figure 3. Aspergillus conidial head forming in a pulmonary fungus ball (Grocott methenamine silver stain). Figure 2. Aspergillus fungal hyphae with septae and acute angle branching (Grocott methenamine silver stain).

(GMS) stain and more rapidly with a periodic acid-Schiff (PAS) stain. Specific identification can be achieved by immunohistochemistry using monoclonal antibodies.

Epidemiology Molds of the anamorphic genus Aspergillus are widespread in the environment, growing in soil, on plants, and on decomposing organic matter.25–27 Thus a cultured species of Aspergillus should not be accepted as a pathogen, unless it fits with the clinical, radiological and pathological findings.28 These molds are often found in outdoor and indoor air, in water, on food, and in dust. In immunocompromised individuals, inhalation of spores can cause life-threatening invasive pulmonary or sinus infection and dissemination to other organs. This condition is termed invasive aspergillosis.23,29–31 Infection has also been reported by exposure and inhalation of water aerosols contaminated with Aspergillus conidia.32,33 In non-immunocompromised persons, these molds can cause localized infection of the lungs, sinuses and other sites.34 Human disease can also result from non-infectious mechanisms: inhalation of spores of these ubiquitous organisms can exacerbate allergic symptoms in both atopic and non-atopic individuals. A great increase in the number of pulmonary Aspergillus infections has followed the widespread introduction of immunosuppressive, steroid and cytotoxic drugs, and after solid organ transplantation (SOT). These drugs artificially depress both humoral and cellular immunity and, often coupled with the natural immunosuppressive effects of the primary disease, predispose the host to severe forms of Aspergillus infection. Aspergillus infections pose the most serious infectious risk to patients with hematological malignancies and those undergoing bone marrow transplants.22,35 Invasive aspergillosis is now the commonest invasive fungal infection in hematopoietic cell transplantation patients and A. fumigatus is

the most common Aspergillus species diagnosed, representing almost half of all cases.7 Traditionally, patients at highest risk of infection and invasive disease were those with either profound or prolonged neutropenia. Changing practices in oncology and stem cell transplantation have decreased both the duration and the frequency of neutropenia, and this may be further reduced with protocols including granulocyte colony-stimulating factors (G-CSF). The temporal association of invasive disease has thus shifted to a bimodal distribution, with the highest risk early after transplantation (< 20 days), and a second peak occurring more than 100 days post-transplantation. This later peak in incidence is due primarily to the use of high-dose corticosteroid therapy in the treatment of acute or chronic graft versus host disease (GVH).36 Up to 44% of lung transplant patients suffer with fungal infections while no more than 23% of patients with other organ transplants are affected. The median time to onset of invasive aspergillosis is 184 days.7 The reasons the lung is a “preferred” site of infection includes previous colonization with Aspergillus in the native or transplanted lung, a reduction in ciliary clearance capability, and continued lung exposure to the environment.37 Patients with AIDS seem to be at low risk of aspergillosis, since their major functional deficit is of T cells, with lesser defects of neutrophils and/or macrophages. Defects in the latter may allow the development of invasive aspergillosis, which is a life-threatening condition in advanced disease. Hereditary immunodeficiency states such as chronic granulomatous disease, oral corticosteroids or therapy with tumor necrosis factor-alpha (TNFa) inhibitors, such as infliximab, also appear to predispose to invasive disease.38–40

Genetics Recent studies have shed light on the mechanisms by which Aspergillus spp. is sensed by the host innate immune system.41 Pattern-recognition receptors (PRRs) on host immune cells

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Chapter 7: Pulmonary mycotic infections

detect conserved moieties called pathogen-associated molecular patterns (PAMPs) on the fungal cell wall. Human Toll-like receptors (TLRs) 2 and 4, as well as C-type lectin receptor dectin-1, are involved in immune recognition of A. fumigatus. Polymorphisms of these PRRs exist and lead to variant immune responses to the pathogen. TLR 4 donor haplotype consisting of co-segregated D299G/T399I single nucleotide polymorphism (SNP) confers increased susceptibility to invasive aspergillosis in HSCT recipients. These findings are not incidental, as the TLR4 D299G/T399I SNP is linked to chronic cavitary pulmonary aspergillosis and fungal colonization. The underlying pathophysiology remains to be elicited. It is unknown whether the consequence of the D299G/T399I SNP is related directly to susceptibility or indirectly via co-pathogens, like cytomegalovirus, or through disposition of antifungal therapy. This emerging knowledge on the immunogenetics of invasive aspergillosis may be utilized in the foreseeable future to risk-stratify individual patients and to tailor patient-specific preventive strategies against the disease.41

Pathogenesis Inhalation of airborne conidia is the primary route of human infection. In healthy individuals, conidia not removed by mucociliary clearance encounter epithelial cells or alveolar macrophages. The latter cells are primarily responsible for the phagocytosis of Aspergillus conidia. Conidia that evade macrophage killing and germinate are targeted by infiltrating neutrophils. The risk of developing invasive aspergillosis results primarily from a dysfunction in these host defenses, in combination with fungal attributes that permit A. fumigatus survival and growth in this pulmonary environment.42 Corticosteroids affect phagocyte function, including impairment of phagocytosis, phagocyte oxidative burst, production of cytokines and chemokines, and cellular migration (reviewed in43). Steroids impair the functional ability of phagocytes to kill A. fumigatus conidia and hyphae.44 Despite the effects of steroids on innate immune cell function, neutrophils are recruited to the lung and prevent hyphal invasion but create an inflammatory environment that results in tissue injury. This exacerbated inflammatory response is often the cause of death, which contrasts with uncontrolled fungal growth observed in neutropenic hosts. Dramatic differences in both fungal development and host responses under different immunosuppressive regimens highlight the importance of studying Aspergillus pathogenesis within the context of host immune status and subsequent response to fungal infection.45 Interactions between Aspergillus and the soluble components of the mucociliary escalator and respiratory epithelia are of paramount importance. A. fumigatus may facilitate colonization in otherwise healthy lung tissue via secreted products, including proteases, that alter epithelial function and viability.45 In addition A. fumigatus has defenses of its own by masking (1,3)-glucan and delaying macrophage activation. Resting A. fumigatus conidia are resistant to

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Table 4 Clinical manifestations of pulmonary aspergillosis

Bronchial asthma Eosinophilic pneumonia Mucus impaction of bronchi Allergic bronchopulmonary aspergillosis (ABPA) Bronchocentric granulomatosis (BCG) Extrinsic allergic alveolitis Aspergilloma Chronic pulmonary aspergillosis Chronic cavitary pulmonary aspergillosis (CCPA) Chronic necrotizing pulmonary aspergillosis (CNPA) Chronic fibrosing pulmonary aspergillosis (CFPA) Angioinvasive-disseminated aspergillosis Granulomatous (tuberculoid) pulmonary aspergillosis Tracheobronchial aspergillosis Diffuse pneumonic and suppurative aspergillosis Pulmonary aspergillus overlap syndromes

macrophage killing. The protective role of the pigment melanin against host defenses, specifically via scavenging reactive oxygen species, has been described for many pathogenic fungi.46,47

Different forms of aspergillosis Pulmonary disease due to Aspergillus can take a number of different forms depending on the immune status of the individual, pre-existing changes in the underlying lung architecture, and the degree of tissue invasion. These forms may show some degree of overlap. Aspergillus infection may be divided into non-invasive and invasive forms (Table 4).

Allergic forms of pulmonary aspergillosis These include asthma, eosinophilic pneumonia, mucus impaction of the bronchi (Figure 4), allergic bronchopulmonary aspergillosis (ABPA), bronchocentric granulomatosis (Figure 5) (BCG), and extrinsic allergic alveolitis (see Chapters 12, 15 and 17). Often, these separate manifestations are combined to produce a clinicopathological spectrum of disease. Many patients have longstanding asthma or cystic fibrosis.21,22 In fact, they may present with asthma, cough, fever and patchy consolidation or an infiltrate on chest imaging. There is often pronounced peripheral blood eosinophilia. Patients are frequently atopic. Patients with cystic fibrosis are particularly prone to develop hypersensitivity to Aspergillus. The Aspergillus syndromes may co-exist or progress from one to another.48 Mucus impaction of bronchi almost always presents as part of ABPA and may show as a worsening of bronchial asthma or have the same symptoms as APBA. Proximal bronchi are occluded by plugs of thick mucus. These are visible on chest radiographs. Mucus plugs are frequently expectorated and

Chapter 7: Pulmonary mycotic infections

(a)

(b)

(c)

Figure 4. Allergic forms of pulmonary aspergillosis. (a) Mucus plug extracted from lung with allergic bronchopulmonary aspergillosis. Layers of inflammation and mucus give a laminated appearance to the plug. (b) Scattered fungal hyphal forms consistent with A. fumigatus are seen within the mucus (Grocott methenamine silver stain). (c) Allergic mucin contains many eosinophils and Charcot-Leyden crystals.

(a)

(b)

(c)

Figure 5. Bronchocentric granulomatosis. (a) An exquisitely bronchocentric inflammatory process is appreciated at low magnification. (b) Airway mucosa is replaced with granulomatous inflammation and the lumen is filled with fibrin and fungal hyphal forms. (c) Intralumenal fungal hyphal forms consistent with Aspergillus spp. are apparent on a Grocott methenamine silver-stained tissue section.

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Chapter 7: Pulmonary mycotic infections

appear as bronchial casts up to 2 cm in diameter. Airways distended by impacted mucus show a variable inflammatory infiltrate but often return to normal if the plug is removed. There is a patchy and exudative tissue reaction in chronic eosinophilic pneumonia, involving predominantly the alveoli and terminal bronchioles. The inflammatory infiltrate is mainly composed of plasma cells, eosinophils and histiocytes. Allergic bronchopulmonary aspergillosis is the commonest and clinically important form of allergic aspergillosis. Most patients are young and atopic, with a history of severe asthma. There is serological evidence of hypersensitivity to Aspergillus, i.e., elevated levels of immunoglobulin E or serum precipitins, and eosinophilia.49 It may represent an unsuspected diagnosis in the occasional patient who undergoes bronchoscopy for a lung mass, discovered to be mucoid impaction. Allergic bronchopulmonary aspergillosis is caused by a complex hypersensitivity reaction to Aspergillus organisms. The fungi proliferate in the airway lumen, resulting in the production of a constant supply of antigen (Figure 4b). A type I hypersensitivity reaction with IgE and IgG release occurs. Immune complexes and inflammatory cells are deposited in the bronchial mucosa, causing necrosis and eosinophilic infiltrates (type III reaction). There is bronchial wall damage and consequent bronchiectasis.49 Excessive mucus production and abnormal ciliary function cause mucoid impaction. Radiographically one sees homogeneous finger-in-glove areas with increased opacity in a bronchial distribution. Abnormalities usually involve the upper lobes while these shadows can migrate from one region to another. Occasionally, isolated lobar or segmental atelectasis may occur. Computed tomography findings in APBA consist primarily of mucoid impaction and bronchiectasis mostly involving segmental and subsegmental upper lobe bronchi. In approximately 30% of patients, the impacted mucus has high attenuation or demonstrates calcification.50 Histologically, major bronchi feature intraluminal thick tenacious mucus plugs, sloughed bronchial epithelial cells and cellular debris (Figure 4a). In fully developed lesions, damage to the bronchial wall becomes irreversible, with replacement of most respiratory epithelium by inflamed granulation tissue. Remaining mucosa features squamous metaplasia. Eosinophils are numerous and histiocytes conspicuous along with scattered histiocytic giant cells. Surrounding airspaces may feature eosinophilic pneumonia. Allergic bronchopulmonary aspergillosis is difficult to diagnose. Laboratory features of ABPA include peripheral blood eosinophilia (> 1000/mm3), immediate cutaneous reactivity to Aspergillus, elevated levels of total serum IgE (> 1000 ng/ml), serum precipitins to A. fumigatus, and elevated serum antibodies of IgE, IgG or both to A. fumigatus. Elevated levels of specific antibodies to A. fumigatus are considered the hallmark of disease and are measured by fluorescent enzyme immunoassay tests or enzyme-linked immunosorbent assay (ELISA). Lung biopsy is rarely performed for diagnosis, but

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it can be important for patients with atypical presentations. Since these entities are hypersensitivity responses to Aspergillus, the fungus is not abundant in the tissues. Recognition of mucin with many eosinophils, Charcot Leyden crystals and scanty fungal hyphae offer support for a diagnosis of ABPA (Figure 4c). Skin tests with Aspergillus antigens are only useful in the diagnosis of allergic aspergillosis. Patients with uncomplicated asthma due to Aspergillus give an immediate type I reaction. Those with ABPA give an immediate type I reaction and 70% also give a delayed type III reaction. Reagents from different manufacturers may give differing results. Tests for Aspergillus antibodies are often helpful in the diagnosis of the different forms of allergic aspergillosis in the non-immunocompromised patient. Available tests include immunodiffusion (ID), indirect hemagglutination and ELISA. The ID test is simple and precipitins can be detected in up to 70% of patients with ABPA, but beware that over 90% of those with pulmonary aspergilloma or chronic pulmonary aspergillosis also have detectable precipitins.

Aspergilloma Introduction An aspergilloma can develop in a pre-existing cavity formed during the course of diseases, such as sarcoidosis and tuberculosis. Rarer causes are bronchiectasis, lung abscesses, cavitated neoplasms, infarcts, cavities resulting from other fungal disease, notably histoplasmosis, bronchogenic cysts, pulmonary sequestrations or pneumatoceles secondary to Pneumocystis jirovecii.50 It is best to use the term “fungus ball”, until the organism has been identified, since macroscopically it is impossible to be certain whether one is dealing with Aspergillus or another organism. Additional fungi capable of producing fungus balls include Candida, Nocardia, Sporothrix and Scedosporium species. Clinical features Patients are often asymptomatic for years, but may present with chronic cough, malaise and weight loss. Hemoptysis is the most common symptom, occurring in 50 to 90% of cases. Most patients have infrequent episodes of small amounts of bleeding, but up to 25% suffer massive life-threatening hemoptysis. Lesions most commonly occur in the upper lobes and may be multiple. Chest radiographs and computed tomograms (CT) show a rounded opacity with a radiolucent crescent of air outlining the upper border (Monod sign) (Figure 6). If peripheral, localized pleural thickening overlying a cavity is seen (Figure 7). The mass often moves as the patient changes position (Figure 8). Pathology Aspergillus colonizes necrotic debris-lined cavities. Saprophytic surface growth leads to the accumulation of layers of fungus, cellular debris, fibrin and inflammatory cells to form a brownish-yellow mass (Figure 9). Expansion of the cavity can occur

Chapter 7: Pulmonary mycotic infections

Figure 6. Pulmonary aspergillomas. This anterior-posterior chest radiograph features bilateral upper lobe apical cavities.

Figure 8. Pulmonary aspergilloma. This computed tomogram of patient lying prone oblique demonstrates a right apical cavity with movable solid contents. (Image courtesy of Dr Richard Sawyer, Manchester, England.)

as the fungus ball grows. This is accompanied by increasing fibrosis with dense pleural adhesions and pleural fibrosis. Approximately 10% of aspergillomas resolve spontaneously. Reversibility of the pleural thickening corresponding to the resolution of intracavitary fungal material has been demonstrated at follow-up radiography.51 This reversibility suggests

Figure 7. Pulmonary aspergillomas in a patient with cystic fibrosis. This computed tomogram demonstrates bilateral thin-walled cavities with solid material and air. (Image courtesy of Dr Richard Sawyer, Manchester, England.)

that the cavity wall and pleural thickening represent a hypersensitivity reaction. The intra-cavitary mycotic membrane may calcify, producing a broncholith, part of which may be expectorated if bronchial obstruction is incomplete. Rarely, the fungus invades the cavity wall and underlying lung. The aspergilloma may also rupture into the pleural cavity, forming a mycotic empyema. Zonation is characteristic of aspergillomas (Figure 10a). Hyphae from the center of the mass appear degenerate and dilated. They stain poorly and may resemble Mucorales. At the periphery, hyphae are more typical, with regular septa and dichotomous branching at 45 angles (Figure 10b). Conidiospores are formed if the cavity communicates with a bronchus. Splendore-Hoeppli material is frequently deposited around the margins. This is brightly eosinophilic amorphous material, which contains immunoglobulin and complement. It outlines the periphery of colonies and can be seen around the tips of individual hyphae. Extensive local deposition of calcium oxalate, secondary to oxalic acid production by the fungus, can be seen. The crystals are birefringent under polarized light (Figure 11). This finding does not distinguish between different species. Diagnosis The clinical diagnosis of aspergilloma may be suspected by the triad of hemoptysis, positive serology and the radiological demonstration of an intra-cavitary mass. Virtually all patients with this syndrome have serum precipitating antibodies to Aspergillus antigens, which serves as a useful confirmatory test. However, culture of respiratory secretions yields an Aspergillus species in only 50% of cases.

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Figure 9. Pulmonary aspergilloma. (a) This upper lobe smooth-walled cavity is filled with tan grumous material. Overlying pleura is thickened. (b) An old fungal cavity with no fungal ball but well-formed fibrous walls and surrounding airway scars.

Figure 10. Pulmonary aspergilloma. (a) Fungal hyphal forms have alternating basophilic and eosinophilic rings. (b) Typical hyphal forms are seen around the edges of the fungal ball.

parenchymal invasion, and its absence of vascular invasion and parenchymal infarction. A new nomenclature, based on clinical, radiological and histopathological findings, separates patients with chronic pulmonary aspergillosis into three groups: chronic cavitary pulmonary aspergillosis (CCPA), chronic necrotizing pulmonary aspergillosis (CNPA) and chronic fibrosing pulmonary aspergillosis (CFPA). Further refinements to this classification have been suggested, but the boundaries between some forms of chronic pulmonary aspergillosis and subacute invasive aspergillosis remain indistinct.

Figure 11. Aspergillus. Polarizing microscopy reveals characteristic pattern of calcium oxalate crystals in A. niger lung infection.

Chronic pulmonary aspergillosis Introduction A number of chronic forms of Aspergillus lung infection have been described.52 In the past, distinctions between these different clinical forms were ill-defined. These progressive, locally destructive forms occupy an intermediate position in the spectrum between colonizing and invasive processes. Chronic pulmonary aspergillosis is distinguished from invasive pulmonary aspergillosis by its limited extent of

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Clinical features Chronic pulmonary aspergillosis usually affects mildly immunocompromised patients, including those with diabetes mellitus, connective tissue disorders, poor nutrition, chronic obstructive lung disease or those taking low-dose corticosteroids. It is an indolent, progressive condition that tends to occur in middle-aged or older persons with an underlying lung disease, such as inactive tuberculosis, bronchiectasis, sarcoidosis or pneumoconiosis. More men than women are affected. Chronic cavitary pulmonary aspergillosis and CFPA typically affect patients without obvious immunological defects, but CNPA has been described in individuals with some degree of immunocompromise, including persons with AIDS, diabetes mellitus and alcoholism, and those who have received low-dose corticosteroid treatment.

Chapter 7: Pulmonary mycotic infections Figure 12. Chronic cavitary pulmonary aspergillosis. Several fungal balls with adjacent parenchymal destruction are seen. Fibrosis is not apparent.

Diagnosis The diagnosis of chronic pulmonary aspergillosis is suggested by the clinical course and the isolation of the fungus from pulmonary secretions; negative cultures for other pathogens and failure to respond to antibacterial or anti-mycobacterial therapy are characteristic. The diagnosis is confirmed by pathological evidence of tissue invasion by the fungus or a response to specific antimycotic therapy. Since the mycelial tissue invasion is local and limited, transbronchial biopsy has low diagnostic yields in this form of invasive aspergillosis.

Angioinvasive-disseminated aspergillosis

The most frequent presenting symptoms include chronic productive cough and weight loss, with mild hemoptysis, dyspnea, malaise and fatigue. Fever is absent and chest pain uncommon. The earliest radiological changes are ill-defined areas of consolidation, typically within the upper lobes, that progress to multiple well-defined cavities. These may or may not contain fungal balls, debris or fluids. In some cases the cavities have thick walls and are adjacent to thickened pleura, but more commonly they are thin-walled without pleural thickening. Pathology Macroscopically, the major findings include a cavity that contains well-formed fungus ball with destruction of surrounding lung parenchyma (Figure 12). Microscopically, the diagnosis of chronic pulmonary aspergillosis requires demonstration of parenchymal invasion and destruction of non-cavitary lung tissue in the appropriate clinical and radiological setting. Chronic cavitary pulmonary aspergillosis is characterized by the formation and expansion of multiple cavities, some of which may contain fungal balls. Infection may progress to marked and extensive pulmonary fibrosis, and then is termed CFPA. In some cases, there is local extension into the pleura, either as a result of direct invasion or as fibrosis. In CNPA there is progressive enlargement of a single cavity, usually with a thin wall, in some cases to substantial dimensions, occurring slowly over months or rapidly in weeks. Growth is due to tissue necrosis. Patients with a “simple aspergilloma” of their lungs have a similar clinical profile and it is sometimes difficult to distinguish between the two disorders. The difference between aspergilloma and CCPA is that the former arises in a pre-existing cavity. In addition, there is usually less pleural thickening in CCPA than in aspergilloma, but more pulmonary fibrosis or an enlarging cavity is seen.

Introduction Angioinvasive-disseminated aspergillosis is a fulminating and highly lethal type of pulmonary infection with little tissue reaction to the invading fungus. It may be terminally associated with bloodstream dissemination. A primary invasive form has been described in individuals exposed repeatedly to an overwhelming number of spores. More frequently, infection occurs in severely immunosuppressed patients, i.e. those who are profoundly granulocytopenic, have hematological malignancies or are receiving corticosteroids, cytotoxic agents or broad-spectrum antibiotics.53 Aspergillus fumigatus and A. flavus are the most common causative species. Traditionally, invasive pulmonary aspergillosis is seen 10 to 14 days after hematopoietic stem cell transplantation (HSCT) and associated with profound granulocytopenia.54 Recent reports suggest the shifting epidemiology of this infection, with less than one-third of all patients diagnosed with invasive pulmonary aspergillosis being neutropenic at the time of diagnosis. This impacts on both the clinical presentation and the period of high risk for infection.55 Clinical features Patients are usually chronically ill and debilitated. They have a dry cough, pleuritic chest pain, hemoptysis, pyrexia and dyspnea. Chest radiographs show a variety of abnormalities including patchy, multifocal, diffuse, bilateral consolidation or nodules, peripheral wedge-shaped pleural-based infiltrates in an infarct pattern or, rarely, bilateral miliary nodules.56 Pleural effusions may be seen. Up to one-third of patients may have normal radiographs. Follow-up films may show cavitation of the nodules and infarcts. Invasive disease may also reveal a ‘‘halo’’ sign (an area of low attenuation surrounding a pulmonary nodule). These areas may later cavitate and give rise to the ‘‘air-crescent’’ sign.57 Although these radiographic findings are not specific for diagnosis and can be seen in a variety of other fungal and bacterial infections, they may be of assistance in the evaluation of high-risk patients.36 A careful history must be taken in those at risk of invasive pulmonary aspergillosis, given their frequent inability to mount an immunological response including a fever.

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Figure 13. Invasive pulmonary aspergillosis. Multiple tan-yellow vasocentric nodules with intervening hemorrhagic lung are seen.

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Pathology Macroscopically, affected lung is dark red and hemorrhagic. Punctate yellow-tan vasocentric nodules are often seen (Figure 13). In AIDS patients, upper lobe cavitation may be seen along with parenchymal consolidation. Histologically, the lesions are typically angiocentric, with intravascular spread producing hemorrhagic infarcts with a segmental or lobar distribution (Figure 14a,b). Areas of hemorrhagic coagulative necrosis contain nuclear debris. The original structure of the lung is no longer recognizable or there may be only a few stainable elastic and reticulin fibers. Inflammatory cells are inconspicuous, with scattered neutrophils and lymphocytes. An occluded necrotic artery is often identified within or at the edge of the lesion, with abundant hyphae extending through the vessel wall and invading surrounding lung (Figure 14c).

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Figure 14. Invasive pulmonary aspergillosis. (a) Arteritis with surrounding fibrinous pneumonia is a constant finding. (b) Hemorrhagic infarction accompanies the vascular disease. (c) Hyphal forms infiltrate arterial walls (Grocott methenamine silver stain). (d) Fungal hyphal forms do not elicit a host response and overrun necrotic lung tissue (Grocott methenamine silver stain).

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Chapter 7: Pulmonary mycotic infections Figure 15. Tracheobronchial aspergillosis. A yellowgreen membrane covers the entire trachea from a lung transplantation patient.

Figure 16. Tracheobronchial aspergillosis. The upper lobe bronchi are occluded with fungi (arrows). The artery contains a thrombus. Small parenchymal and pleural nodules are also noted.

Hyphae may be scattered through the necrotic tissue and are difficult to identify without a GMS stain (Figure 14d). If they are numerous, they may form small colonies attached to alveolar walls. These are best seen at the edges of the necrotic zone bounded by alveoli filled with fibrinous material. Bronchi at the edges and within the lesion contain friable-brown material, including tangled mycelia. This is considered the primary lesion and fungal portal of entry into the lung. Invasive pulmonary aspergillosis can lead to rapid hematogenous dissemination to other organs, particularly the brain and heart. Fulminating necrotizing pulmonary aspergillosis is also associated with bacterial septicemia due to Pseudomonas pyocyaneus. Diagnosis Establishing the diagnosis of invasive aspergillosis in patients with hematological malignancies and transplant recipients is often difficult. In most cases, the diagnosis is based on a combination of clinical, radiological, microbiological and histopathological findings.19,52 These might include a positive culture for a specimen obtained from a normally sterile and clinically or radiologically abnormal site, or histopathological or cytopathological examination showing hyphae consistent with Aspergillus. Unfortunately, patients at highest risk of invasive infection are also at highest risk of complications from the invasive procedures needed to make a definitive diagnosis. Therefore, in many clinical settings the diagnosis is presumptively based on clinical signs and chest CT findings in patients with recognized risk factors. Several non-culturebased methods of diagnosis have been evaluated in recent years. These include tests for detection of Aspergillus antigens in blood and other body fluids, as well as molecular methods to detect circulating Aspergillus DNA.

Granulomatous (tuberculoid) pulmonary aspergillosis In this unusual variant of invasive pulmonary aspergillosis, the cellular response is similar to that seen in tuberculosis. Tubercle-like granulomas consist of a central area of necrosis, surrounded by histiocytic giant cells with lymphocytes and neutrophils. Fungal hyphae are present both in the necrotic tissue and in giant cell cytoplasm. Organisms are best demonstrated by a GMS stain. Tracheobronchial aspergillosis The incidence of this form of pulmonary aspergillosis is increasing in immunosuppressed patients. There are nonspecific signs with cough, tracheitis, fever and hemoptysis. Affected individuals present with bronchial obstruction and the diagnosis is made at bronchoscopy, where a felt-like membrane covers large portions of the tracheobronchial tree and occludes airways (Figures 15 and 16). Airway mucosa bleeds on contact due to ulceration. In lung transplants, there may be dehiscence of the anastomotic site (see Chapter 20). Histologically a carpet of fungal hyphae replaces and invades into the airway wall. Other forms of pulmonary aspergillosis Diffuse pneumonic and suppurative aspergillosis are acute pneumonias with marked neutrophilic response. Macroscopically, there are multiple small abscesses filled with yellow pus and hemorrhagic borders. The abscess cavities are filled with neutrophils, cellular debris and fungal hyphae, encircled by a lymphoplasmacytic infiltrate with macrophages and foreign-body-type giant cells. In most cases the fungal infection is superimposed upon a bacterial pneumonia, often staphylococcal, with abscess formation. Immunosuppressed or debilitated patients are at risk since fungal spore inhalation may not be cleared.

Clinical laboratory diagnosis of Aspergillus While clinical history, radiographic findings and histological evidence often support a diagnosis of Aspergillus lung disease,

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laboratory confirmation is essential in virtually all cases. Many modalities are available.

Culture Isolation of Aspergillus from a normally sterile site is the best way to confirm the pathological diagnosis. The presence of Aspergillus in cultures must be interpreted with caution, as these organisms may be present as saprophytes in material from the respiratory tract. Consideration should be given to the clinical situation. A positive sputum culture from a patient with severe neutropenia is suggestive of invasive disease.

Antibody tests Tests for Aspergillus antibodies have been evaluated in invasive aspergillosis, but their role remains uncertain. The detection of precipitins in a neutropenic patient with an unresponsive fever or a pulmonary infiltrate is often sufficient to prompt the initiation of antifungal treatment. A positive ID test result is not proof of infection. Nor does a negative test result preclude aspergillosis in an immunosuppressed patient, because such individuals are often incapable of mounting a detectable antibody response. Antibodies may appear following successful treatment.

Biomarker detection Galactomannan

Tests for the detection of Aspergillus antigen in blood and other biological fluids offer a rapid means of diagnosing aspergillosis in the immunocompromised.58 Numerous clinical evaluations show low concentrations of galactomannan, a major cell wall component of Aspergillus, which can be detected in serum, urine and bronchoalveolar lavage (BAL) specimens from HSCT recipients and neutropenic patients with hematological malignancies and invasive aspergillosis. Less information is available regarding the usefulness of antigen testing in other populations, such as SOT recipients and ICU patients. A sandwich ELISA is available for detection of Aspergillus galactomannan. This test is included in the revised consensus definitions for diagnosing invasive fungal disease.1,52 The Platelia Aspergillus ELISA results are reported as a ratio between the optical density of the patient’s sample and that of a control with a low but detectable amount of galactomannan (1 ng/ml). The optimal cut-off value to maximize test sensitivity and specificity was debated but a value of 0.5 is now accepted worldwide. This greatly increases the sensitivity of the test, albeit at some loss of specificity. False-positive results have been reported with the Platelia Aspergillus ELISA, including in patients receiving piperacillin-tazobactam and other betalactam antibiotics, including ampicillin, amoxicillin and amoxicillin-clavulanic acid. Piperacillin and amoxicillin are semi-synthetic drug-derived natural compounds produced by molds of the genus Penicillium that contain galactomannan in the cell wall. Other causes of false-positive results include neonatal gastrointestinal colonization with Bifidobacterium,

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and enteral feeding with a liquid nutrient containing soybean protein. The FDA has recently licensed the Platelia Aspergillus ELISA for testing of BAL fluid samples.59,60 As with serum, a cut-off value of 0.5 is considered positive.

b-D-Glucan detection (1,3)-b-D-Glucan is a cell wall component of many fungi. The Fungitell assay (Associates of Cape Cod, Falmouth, MA, USA) is approved by the FDA for the presumptive diagnosis of invasive fungal disease, including aspergillosis, candidiasis and trichosporonosis. The negative predictive value of the test appears to be high. False-positive results have been reported in bacteremia, those who have recently received albumin or immunoglobulin products, or undergone hemodialysis with cellulose membranes, and those exposed to other sources of glucan, such as certain parenteral antibiotics.

Molecular diagnostics Numerous nucleic acid amplification methods have been developed for detection of Aspergillus DNA in blood, serum, BAL and other clinical specimens.61–63 Most effort has focused on the ribosomal DNA genes (18S and 28S) or the internal transcribed spacer regions. Other performance variables include amplification platforms and detection of amplicons. Molecular diagnostics appear promising for the rapid detection of Aspergillus infection directly from tissue or body fluid specimens. Standardized PCR assays are commercially available in some countries (MycAssay Aspergillus PCR, Myconostica Ltd., Manchester, UK).

Differential diagnosis The hyphae of other fungi, notably Fusarium species and Scedosporium apiospermum, are both branched and septate, and can be mistaken for Aspergillus hyphae. Isolation of the fungus in culture is essential to confirm the diagnosis. Fusarium hyphae are slightly wider than Aspergillus and branch at right angles, rather than acute angles. Scediosporium hyphae are slightly narrower than those of Aspergillus and their pattern of branching is haphazard rather than progressive.

Treatment and prognosis Amphotericin B, echinocandins, and azole antifungals, voriconazole, itraconazole and posaconazol, are typical antifungal agents for the treatment pulmonary aspergillosis. Conventional amphotericin B can cause bronchospasm, vomiting and real toxicity. The three lipid-based formulations of amphotericin B are approved as second-line agents for patients with invasive aspergillosis. The antifungal triazoles inhibit ergosterol synthesis through their inhibition of the fungal cytochrome P450 enzyme. This causes fungal cell membrane dysfunction with inhibition of cell growth and ultimately fungal cell death. The azoles have side effects on the liver,

Chapter 7: Pulmonary mycotic infections

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Figure 17. Mucormycosis. (a) and (b) Rhizopus species in necrotic lung. Hyphae are large, septate, and show haphazard right angle branching. (Image (a) courtesy of R Neafie, Washington DC, USA.)

blood, skin and gastrointestinal tract but not usually the lung. Voriconazole is recommended for invasive pulmonary aspergillosis. Side effects of itraconazole are elevation of the hepatic transaminases, hypertriglyceridemia, hypokalemia, nausea and vomiting.64 Resistance of Aspergillus to the triazoles has been reported. The echinocandins (caspofungin, micafungin, and anidulafungin) inhibit synthesis of 1,3-b-glucan, a polysaccharide in the cell wall of many pathogenic molds. Caspofungin is the only drug of this group currently approved by the US Food and Drug Administration, as second-line treatment for invasive aspergillosis. Mortality rates range from 40% to 90% in high-risk populations and are dependent on factors such as host immune status, the site of infection, and the treatment regimen.65

Pulmonary mucormycosis Introduction The term mucormycosis is now used to refer to infections caused by molds belonging to the order Mucorales. Traditionally, this order was assigned to the phylum Zygomycota together with the order Entomophthorales, and the different forms of disease caused by the two groups of organisms were often referred to as zygomycosis. However, following molecular analysis, the phylum Zygomycota is no longer accepted due to its polyphyletic nature. The sub-phylum Mucormycotina has been proposed to accommodate the Mucorales and the sub-phylum Entomophthoromycotina has been created for the Entomophthorales. Mucormycosis is the second most frequent mold infection in immunocompromised individuals. Fungi of the order Mucorales can cause rhino-cerebral, pulmonary, gastrointestinal, cutaneous or disseminated disease in predisposed

individuals. The different clinical forms are often associated with particular underlying disorders.72–74

Organisms The commonest causes of human Mucorales infection are Rhizopus oryzae (R. arrhizus) and R. microsporus. Other etiological agents include Apophysomyces elegans, Cokeromyces recurvatus, Cunninghamella bertholletiae, Lichtheimia (formerly Absidia) corymbifera, Rhizomucor pusillus, Saksenaea vasiformis and Syncephalastrum racemosum. In tissue and culture, these molds form characteristic broad, non-septate or sparsely septate hyphae with right-angled branching. All these molds cause similar diseases in humans, and the diagnostic and therapeutic approaches are similar. Tissue identification is a very important diagnostic tool and is indispensable in determining whether there is blood vessel invasion. The microscopic demonstration of Mucorales in clinical material from necrotic lesions, sputum or BAL is more significant than isolation in culture because Mucorales are environmental isolates, which contaminate laboratory surfaces or colonize mucosal surfaces without causing invasive infection. Identification of fungi in biopsy material is based on the presence of the characteristic broad 10 to 25 μm non-septate hyphae with random right-angled branching (Figure 17). Mucorales genera produce non-pigmented thin-walled, ribbonlike hyphae with few septations (pauciseptate) and right angled branching.4 The hyphae may vary in width, appear folded or crinkled, and be sparse or fragmented. In lesions exposed to air, thick-walled spherical structures can form at the ends of the hyphae. Unlike most filamentous fungus, the Mucorales are best seen in tissue sections stained with H&E. Iron hematoxylin demonstrates them particularly well. The usual fungal stains, GMS and PAS, are confirmatory but produce variable staining.

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Epidemiology Analysis of hospital records provided a population-based estimate of mucormycosis incidence and trends over a 10-year period at a national level in France.75 Data showed an increasing incidence from 0.7/million in 1997 to 1.2/million in 2006. The authors showed the incidence of mucormycosis increased, particularly in patients with hematological malignancies or bone marrow transplants. The role of previous exposure to antifungal drugs lacking activity, especially voriconazole, could explain this increase but does not appear exclusive. The incidence also increased in the population of patients with diabetes mellitus.75 The average incidence rate in France is lower than the 1.7/million incidence reported in the 1992–1993 populationbased study by Rees et al. in California.76 The French results are based on a passive routine system, whereas active laboratory-based surveillance was implemented in the San Francisco Bay area at a time of high HIV prevalence. In a 2005 Spanish survey, the incidence was 0.43/million on a representative sample of 50 participating hospitals covering one-third of the country’s population, i.e., approximately 14 million inhabitants.77 The species isolated from cases of human and animal infection are thermotolerant, and many are ubiquitous in the soil and on decomposing organic matter. These molds are found in indoor and outdoor air, on food items, and in dust. The disease is rare in healthy individuals.78–80 Those with diabetes mellitus, leukemia or lymphoma are at increased risk, along with patients undergoing immunosuppressive or antibiotic therapy, particularly transplant patients, and patients with burns or severe neutropenia. Hematopoietic stem cell transplantation patients are probably in one of the highest risk categories. These patients typically have severe, prolonged neutropenia and are frequently receiving broad-spectrum antibacterial agents for unremitting fever.81 Most diabetics present with rhino-orbital or rhino-orbitalcerebral mucormycosis.67 Diabetes is also associated with pulmonary mucormycosis but not to the same degree as upper respiratory tract disease.82–84 In fact, most pulmonary mucormycosis in diabetics is co-existent with sinus, orbital or brain disease and it may follow a more indolent, subacute course than is typically seen in patients with neutropenia. Prophylaxis with itraconazole or voriconazole is implicated as predisposing to mucormycosis, while use of statins for elevated cholesterol may be protective given this class of drugs’ in vitro inhibitory activity against many of these organisms.85–87

Clinical features The organism may reach the lungs by inhalation or via the bloodstream or lymphatics. Mucormycosis may present with pneumonia, abscess, empyema or endobronchial mycosis. Patients present with dyspnea, cough and fever, often with pleuritic pain. Productive cough may produce white, yellow, blood-stained or grossly bloody sputum. Patients are often

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Figure 18. Mucormycotic abscess. This gelatinous abscess cavity is surrounded by parenchymal consolidation.

profoundly ill with marked gas exchange abnormalities and respiratory failure. Spread of infection is hematogenous and then rapidly fatal. Radiographic findings include lobar consolidation, isolated masses, nodular disease, cavitation or wedge-shaped infarcts. Computed tomography is the best method of determining the extent of pulmonary mucormycosis. Invasive mucormycosis may be difficult to differentiate from aspergillosis but multiple pulmonary nodules (i.e., > 10), pleural effusion or concomitant invasive sinusitis favor a diagnosis of mucormycosis. A recent study reported that the reversed halo sign, a focus of ground-glass surrounded by a solid ring of consolidation on CT scan, may be useful in differentiating the two fungal infections.88,89

Pathology The lung pathology of mucormycosis bears a close resemblance to aspergillosis. Usually, the fungus invades the bronchial wall, producing a surface film of exudate and hyphae, with occasional sporing structures. Fungi then penetrate the bronchial wall to invade pulmonary veins and arteries, causing thrombosis and infarction, producing the typical appearance of patchy, hemorrhagic consolidation. Abscess formation and cavitation can follow (Figure 18), and erosion into a vessel may cause fatal hemorrhage (Figure 19). A pyogenic inflammatory response is usual. If tissue is infarcted, the response is reduced. Chronic lesions feature granulomatous inflammation. Rarely, fungal hyphae are surrounded by eosinophilic sheaths, i.e., the Splendore-Hoeppli phenomenon. Endobronchial mucormycosis is the rarest form of the disease and usually presents in diabetics. Friable yellow tissue lines a main bronchus and protrudes into the lumen as a gelatinous plug. Subacute obstructive pneumonia often develops. Mediastinal disease with superior vena caval obstruction is also recorded. The fungi may complicate bacterial inflammatory lesions to produce a mycetoma. Typically, this has a yellowish-white, concentrically layered appearance within a fibrous walled cavity.

Chapter 7: Pulmonary mycotic infections

rates as high as 87% may be due to underlying conditions and/ or the inability to remove involved tissue.90 Four factors are critical for eradicating mucormycosis: rapid diagnosis, reversal of underlying predisposing factors (if possible), appropriate surgical debridement of infected tissue, and appropriate antifungal therapy.72,90 Early diagnosis is important because small, focal lesions can be surgically excised before they progress to involve critical structures or disseminate. Thrombosis and resulting tissue necrosis during mucormycosis results in poor penetration of antifungal agents to the site of infection. Delayed initiation of appropriate antifungal therapy in patients with mucormycosis is associated with increased mortality.91 Amphotericin B is active but not voriconazole while posaconazole is inferior to amphotericin B.89 Figure 19. Mucormycosis eroding into a lobar pulmonary artery. The aggressive infection enlarged until it encroached upon and invaded the pulmonary artery.

Diagnosis Because mucormycosis is such an aggressive infection, early diagnosis is essential for successful management. Tissue identification is a very important diagnostic tool and is indispensable to define whether there is blood vessel invasion. Isolation in culture is required for specific identification. Attention to using gentle processing is important, since aggressive grinding of the tissue may render the fragile fungal elements non-viable. Mucorales genera are fast-growing fungi but unfortunately the yield of cultures is low. Despite their predilection for hematogenous dissemination, blood cultures in all forms of mucormycosis are always negative. Nasal, palatal and sputum cultures are seldom helpful. Because Mucorales are common contaminants, isolation of these organisms from material obtained from a necrotic lesion or from sputum or BAL fluid must be interpreted with caution. However, if the patient is diabetic or immunosuppressed, the isolation should not be ignored. Fungus in cultures from the respiratory tract of an immunocompromised patient with clinical evidence of pulmonary infection is highly suggestive but not diagnostic of invasive mucormycosis.

Differential diagnosis These organisms are distinguished from other molds, such as Aspergillus, Fusarium, Candida and Scedosporium species, by their characteristic wide, non-septate hyphae with right-angled branching. Other molds have thinner, septate hyphae that typically branch at acute angles. Abundant septation and acute-angle branching suggests the diagnosis of aspergillosis or a disease caused by another hyaline septate mold, while yeasts with pseudohyphae should suggest Candida species. Poor staining with GMS should suggest mucormycosis.

Treatment and prognosis Prognosis for patients with pulmonary mucormycosis is worse than for those with rhino-orbital-cerebral disease. Mortality

Pulmonary scedosporiosis Introduction Scedosporium species cause a broad spectrum of human disease ranging from transient colonization of the lungs to localized subcutaneous or deep tissue infection, and widespread disseminated infection.29,92–95

Organism Until recently, only two species of Scedosporium were recognized; S. apiospermum (the anamorph or asexual state of the ascomycete Pseudallescheria boydii) and S. prolificans. The latter species is an important cause of phaeohyphomycosis. Based on molecular analysis, S. apiospermum and P. boydii are now considered two distinct species: S. apiospermum (teleomorph or sexual state, P. apiosperma) and S. boydii (teleomorph, P. boydii).96,97 Several new phylogenetic species have recently been delineated among clinical isolates previously identified as S. apiospermum, including S. aurantiacum and S. dehoogii.96 In tissue samples P. boydii hyphae are septate, branched, and 2 to 5 μm wide. Hyphae can produce thin-walled vesicles while conidia can be found in the less cellular areas or near the periphery (Figure 20a,b). The conidia are often single, and brown on hematoxylin and eosin-stained tissue sections and measure 3 to 7 by 6 to 10 μm (Figure 20c).

Epidemiology Scedosporium species are ubiquitous saprophytic fungi found in soil, manure, sewage and polluted fresh water. S. apiospermum is found in nutrient-rich soil and brackish water. It has a worldwide distribution. S. prolificans has a less extensive distribution.98 These two infections are becoming commoner in immunosuppressed patients. Disseminated Scedosporium infection has been reported in immunocompetent individuals.99,100 Trauma is the major risk factor for localized Scedosporium infection. Acquisition can occur in both the community-acquired and nosocomial settings.101 Underlying cystic or cavitating lung disease is a major factor predisposing an individual to Scedosporium

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Figure 20. Pseudallescheria boydii. (a) Branching hyphae with alternating zones of mycelial cellularity in the periphery (Grocott methenamine silver stain). (b) Pseudallescheria boydii hyphae show considerable variation in this lung lesion. (Grocott methenamine silver stain). (Image courtesy of R Neafie, AFIP, Washington, USA.) (c) The conidia appear brown on H&E staining.

fungus ball formation. This condition, radiologically indistinguishable from the commoner aspergilloma, has been described in patients with residual tuberculous or bronchiectatic cavities. Scedosporium colonization of the respiratory tract occurs in 6 to 10% of patients with cystic fibrosis but invasive infection is rare in these patients, even when they are immunosuppressed after lung transplantation. These molds have emerged as a significant cause of invasive disease in immunocompromised patients, in individuals with impaired anatomical barriers (trauma, burns, etc.), and in massive inoculation (near drowning or inhalation of polluted water). Scedosporium species are ubiquitous in the environment and the likelihood that infection will occur following inhalation or implantation of spores largely depends on host factors. Those at greatest risk in the immunocompromised include neutropenic cancer patients, particularly those with acute leukemia, allogeneic HSCT recipients undergoing treatment for GVH disease, and those receiving corticosteroids. Less commonly, disseminated Scedosporium infection has been seen in other groups of immunocompromised individuals, including persons with AIDS and solid organ transplant recipients. There are also several

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reports of acquisition via contaminated intravascular devices. The incubation period is unknown. The incidence of serious Scedosporium infection has increased in recent years, particularly among neutropenic cancer patients and those undergoing allogeneic HSCT. According to one recent report from a North American cancer hospital, the incidence increased from 0.82 cases per 10 000 patient days between 1993 and 1998 to 1.33 cases per 10 000 patient days between 1999 and 2005. In a retrospective analysis of 80 cases among HSCT and SOT recipients, patients who received transplants after 1999 developed the infection later (median time to onset, 6 months) than those who were transplanted in 1999 or earlier (median time to onset, 1.2 months). No fewer than 39% of infections among SOT recipients occurred more than 6 months after transplantation, compared with 25% of those among HSCT recipients.

Clinical features The lung is the commonest site of Scedosporium infection. Clinical manifestations range from transient local colonization,

Chapter 7: Pulmonary mycotic infections

to fungus balls and invasive infection. Patients can be asymptomatic or present with various combinations of fever, cough, night sweats, weight loss, or chills.94,102,103 Pulmonary findings may be localized or may be part of a more disseminated disease.102 In the immunocompetent individual, isolation of these molds from sputum or BAL most commonly represents transient (or more prolonged) colonization of the bronchi or lungs or both. In some cases a clinical picture indistinguishable from ABPA occurs. Scedosporium pneumonia is rare in immunocompetent persons. It may follow aspiration of contaminated water by previously healthy individuals. Patients usually present within a few days to several weeks after the incident, often after a temporary improvement in their condition. In the immunosuppressed, Scedosporium infection has a high rate of dissemination and a poor outcome. The clinical manifestations of Scedosporium infection of the lungs are similar to those of invasive aspergillosis. The commonest presentation in the neutropenic patient is a persistent fever unresponsive to broad-spectrum antibacterial treatment. Hematogenous dissemination to other organs is common. Radiographic findings are not distinctive and include pulmonary infiltrates, lobar consolidation, mass lesions, nodules with or without cavitation, necrotizing pneumonias and pulmonary abscesses.89

isolates to the species level is difficult and may require molecular methods.

Treatment and prognosis P. boydii is susceptible to the triazoles voriconazole and posaconazole in vitro, and is somewhat less susceptible to amphotericin B deoxycholate (AmB).104 Voriconazole is used for P. boydii infections refractory to other antifungals, and is associated with a strong trend to superior survival compared with treatment with AmB.29 S. prolificans tends to be highly resistant to azoles, and most other antifungal options.105 The overall mortality rate from Scedosporium infection is high, ranging from 65 to 75% in most reports. In patients with hematological malignancies, mortality is associated with dissemination and persistent neutropenia.

Pulmonary fusariosis Introduction Fusarium species have become major human pathogens, especially in immunocompromised hosts.106–108 They can cause a broad spectrum of human infections, including superficial, locally invasive and disseminated disease.

Pathology

Organism

Macroscopically, upper lobe and sometimes bilateral fibrotic cavitating lesions contain yellow-black fungus balls, while the whole fungal colony appears to the naked eye as a yellow grain. Histologically, the chronically inflamed cavity walls may be partly epithelialized or consist of granulation tissue. There is a considerable neutrophilic response. As the infection spreads, honeycomb change develops. The cavity contains a tangled mass of branching, sparsely septate hyphae in alternating zones of mycelial hypercellularity and hypocellularity. Invasive disease manifests as a necrotizing pneumonia with abscesses, mycelial vascular invasion and pulmonary hemorrhagic infarction. Hyphal forms are scattered amidst the pneumonia or infarcted lung tissue. Invasive disease may give rise to cavitary lesions.

Morphological identification of Fusarium is difficult and often confusing, even for specialists. It has been estimated that less than one-third of clinical isolates can be identified to species level using morphological characteristics. A three-locus DNA sequence database has recently been established to facilitate molecular identification of the 69 Fusarium species associated with human or animal infection. Analysis of this database shows that all pathogenic members of the genus Fusarium fall within one of eight species complexes. The two most important of these species complexes are F. solani and F. oxysporum. F. solani species complex includes more than 45 distinct species in three major clades, but human isolates are restricted to one of these clades. F. oxysporum, F. solani and a third major pathogen, F. moniliforme, are the commonest infectious agents that involve the lung. Histology reveals 3 to 8 μm in diameter hyphae with hyaline and septate filaments that branch at acute and right angles, similar to Aspergillus and P. boydii. Fusarium species do not show the regular, dichotomous branching of Aspergillus species (Figure 2). Sporulation may be present in tissue infected by Fusarium species. Finding hyphae with small, single-celled chlamydoconidia, yeast-like or microconidial structures is highly suspicious for this organism (Figure 21).109 Chlamydoconidia can be seen in cavitating lung lesions.

Diagnosis The definitive diagnosis of Scedosporium infection rests on the isolation of the fungus from clinical specimens. This is because the branching septate hyphal tissue form of these molds cannot be distinguished from that of other etiological agents of hyalohyphomycosis or aspergillosis in wet preparations or histopathological sections. This distinction is important because Scedosporium species are intrinsically resistant to amphotericin B. Scedosporium can usually be isolated from infected organs but blood and CSF cultures are seldom positive. These molds can be easily recovered on routine mycological media without cycloheximide (actidione). Morphological identification of

Epidemiology These molds are found throughout nature as soil saprophytes and plant pathogens as well as in water, usually as part of a

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(a)

biofilm. They are also found in hospital water sources, such as showers, and faucets.110 Organisms can cause locally invasive infections in both immunocompetent and immunocompromised individuals, usually in the skin or eye, by trauma, or a foreign body. Invasive fusariosis is essentially a nosocomial disease of the immunocompromised. Fusarium species have emerged as the second most frequent cause of life-threatening disseminated fungal infection, after aspergillosis, in severely immunocompromised individuals, particularly among neutropenic patients with hematological malignancies and HSCT recipients. Infections are also found in patients receiving high-dose corticosteroids and/or those who have a long history of profound neutropenia or T-cell immunodeficiency, including lung transplants. These patients are especially susceptible to the invasive or disseminated forms of the disease and usually have concomitant bacterial, viral or other fungal, e.g., Aspergillus or Candida, infections. The principal portal of entry is through airways, followed by the skin or mucosal membranes at sites of tissue breakdown.

Clinical features Airborne conidia inhaled into the sinuses and the lungs cause acute sinusitis and pneumonia, respectively. Some patients develop skin lesions, including painful nodules and ulcers.111 Pulmonary involvement is common in patients with invasive Fusarium infection. The presenting signs are nonspecific and include pleuritic chest pain, non-productive cough, shortness of breath and hemoptysis. The radiological presentation ranges from nonspecific alveolar or interstitial infiltrates to nodules or cavitating lesions.112 Nonspecific interstitial or airspace infiltrates are also seen.113 Chest radiographs can be negative in 25% of cases with an abnormal HRCT.114 The clinical and radiological findings are difficult to distinguish from invasive pulmonary aspergillosis; however, the halo sign, characteristic of the early stage of invasive pulmonary aspergillosis, is seldom seen in neutropenic patients with Fusarium infection.

Pathology Lesions may contain neutrophilic abscesses, granulomas and necrosis due to vascular invasion. The fungal hyphae are usually intermixed with the necrotic debris (Figure 22).

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Figure 21. Fusariosis. (a) Fusarium spp. with irregular, haphazard branching hyphae and prominent varicosities and terminal chlamydoconidia (GMS stain). (b) Thick-walled chlamydoconidia produced by the mycelium. (Case courtesy of Joseph F. Tomashefski, Jr., MD, Cleveland, OH, USA.)

(b)

Figure 22. Fusarium species hyphae show much variation in this lung lesion (GMS stain). (Image courtesy of R. Neafie, Washington DC, USA.)

Diagnosis The definitive diagnosis of Fusarium infection depends on the isolation of the etiological agent in culture from clinical specimens. Fusarium species are usually recovered on routine microbiological and mycological media without cycloheximide (actidione). The choice of medium can have a profound influence on colonial appearance and sporulation. Identification of isolates to the species level is difficult and may require PCR-based techniques and sequencing.115 Unlike other invasive molds, Fusarium often grows on blood culture.

Treatment and prognosis The prognosis of infected patients relates to their immune status. Mortality rates ranging from 60 to 80% are seen in those with disseminated infection. The most effective therapy for fusariosis has not been defined. Successful outcomes have been documented with voriconazole or with AmB or its lipid formulations.116 Posaconazole salvage therapy may be useful for refractory disease.117 Various combination regimens, including echinocandin-polyene, azole-polyene, and polyenes or azole plus terbinafine, have been described in case reports.116

Chapter 7: Pulmonary mycotic infections

Pulmonary histoplasmosis Introduction Histoplasmosis is the systemic fungal infection caused by the dimorphic fungus Histoplasma capsulatum var. capsulatum.2,118

Organism Histoplasma capsulatum var. capsulatum is a thermally dimorphic fungus. It grows as a mold in soil and in culture at 25 C, and as budding yeast in culture at 37 C as well as in human tissues. The spore, or conidium, is the infectious form and germinates to form hyphae. These divide to create yeasts, which are ovoid and measure 2 to 5 μm in diameter. The tissue yeast form lacks a true capsule and multiplies by narrowbased, unequal budding (Figure 23). These stain strongly with GMS stains, though nonviable yeast forms in necrotic debris are also strongly argyrophilic. The fungi are not seen on H&E except in the more florid cases of disseminated disease. The yeast form, when senescent, can become quite large, averaging 7 to 15 μm in diameter.

Epidemiology Histoplasmosis is worldwide in distribution but most prevalent in regions of North, Central and South America, as well as parts of Africa and Asia.119 It is the commonest endemic mycosis in North America, but it is also found throughout Central and South America.120,121 In the USA, the disease is most prevalent in the region around the Mississippi and Ohio Rivers, but is also found in foci throughout the eastern half of the continent.122 Thus, there is some overlap with the endemic region for blastomycosis (see below). In Central and South America, the disease is found in many countries, including Mexico, Guatemala, Nicaragua, Panama, Venezuela, Colombia, Brazil and Argentina. Other endemic regions include the Caribbean islands, parts of Africa,

Figure 23. Histoplasma capsulatum. Yeast and scattered budding forms (GMS stain).

Australia and eastern Asia, in particular India and Malaysia. Sporadic cases have also been diagnosed in other countries among individuals who had previously resided in or visited an endemic region. Histoplasma capsulatum is inhaled from soil contaminated with guano and other debris by nesting blackbirds, bats and chickens. The organism thrives in this environment and is spread via the dermal appendages of birds and bats, the intestinal contents of bats and, most importantly, by wind. Of note, post-transplantation histoplasmosis is rare, with less than one case per 1000 transplant-person-years, even in endemic areas. Interestingly, even SOT recipients with latent infection do not develop disseminated disease when prophylaxis is given.123,124

Pathogenesis Infection occurs after microconidia are aerosolized and inhaled. In the alveoli conidia are phagocytized by macrophages. Inside these cells, conidia convert to yeasts. During the first couple of weeks, Histoplasma yeasts multiply inside alveolar macrophages and spread throughout the reticuloendothelial system.125 Dendritic cells kill the yeast and present Histoplasma antigen to and stimulate naive T-lymphocytes.126,127 Dendritic cells line the airways and are involved early in uptake and processing of Histoplasma.128 Within 2 to 3 weeks, a potent T-cell-mediated immune response is generated which is responsible for halting dissemination by assisting intracellular killing of the yeast by effector macrophages.125 This orchestrated response appears to depend on a number of cytokines such as TNF-a,129,130 interferon-g131 and interleukin (IL)-12.132 Regulatory cytokines IL-10, IL-17 and IL-23 also probably play a major role in coordinating an effective immune response to this infection.133 If cellular immunity is defective, the fungus proliferates and disseminates throughout the body, causing tissue destruction and multiorgan failure.134

Clinical features The clinical manifestations of histoplasmosis depend on the size of the inoculum and the immune status of the host.135,136 In fact, approximately 75% of people with skin test positive for histoplasmin lack clinical symptoms or radiological evidence of primary infection. Human histoplasmosis can take five main clinical forms: (1) acute primary, (2) latent, (3) chronic cavitary pulmonary, (4) disseminated and (5) cicatricial. This disease spectrum is similar to tuberculosis, from which it can sometimes be difficult to distinguish clinically. Other entities that may require treatment but not antifungal therapy include pulmonary nodule, mediastinal mass, mediastinal fibrosis, broncholithiasis and inflammatory syndromes (pericarditis, arthritis, erythema nodosum) (Table 5).134 In an immunocompetent host acute primary disease occurs 2 weeks after a relatively large exposure. Most symptomatic acute infections present as a 5 to 14 day course with nonspecific symptoms including fever, chills, cough, dyspnea and chest pain. Usually lobar or patchy bilateral pulmonary

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Chapter 7: Pulmonary mycotic infections Table 5 Clinical forms of pulmonary histoplasmosis

Figure 24. Chronic pulmonary histoplasmosis. An autopsy specimen radiograph showing central hilar calcified lymph nodes and scattered calcified nodules through lung parenchyma.

Acute primary Latent Chronic cavitary pulmonary Disseminated Cicatricial Pulmonary nodule Mediastinal mass Mediastinal fibrosis Broncholithiasis Inflammatory syndromes

infiltrates are seen on chest imaging and mediastinal adenopathy is often present. Some patients with heavy exposure have severe dyspnea and hypoxemia with diffuse pulmonary infiltrates, mimicking hypersensitivity pneumonitis. These radiological changes heal without residue or leave multiple focal calcific foci, visible as “buckshot” on chest X-ray. Exudative pericarditis with effusion may ensue but usually resolves spontaneously or after tapping. In contrast to tuberculosis, pleural effusions are rare.134 Clinical manifestations usually disappear without treatment. Latent histoplasmosis is less common than latent tuberculosis but disease may reactivate with corticosteroids or immunosuppression and may give rise to disseminated disease. Progressive disseminated histoplasmosis can follow massive fungal exposure in an immunocompetent individual but is more frequent in immunodeficient hosts. It is more often a disease of young children, patients receiving corticosteroids or chemotherapy, or with malignant lymphoma. The disease also involves the liver, spleen, central nervous system, gastrointestinal tract and adrenal glands, in addition to other organs. Immunocompetent patients develop immunity in the first 2 weeks of infection but immunodeficient patients lack the cellular immune response necessary to clear the infection. AIDS patients and those on anti-TNF agents are especially at risk.137,138 Chest radiographs show reticulonodular or diffuse interstitial infiltrates. The diagnosis is frequently established by bone marrow biopsy or culture. Antigen testing in blood and urine is positive in 90% of patients.139 Lung biopsy or culture is positive in a minority of patients. Chronic cavitary pulmonary histoplasmosis is localized in character and involves the lung apices.140 Risk factors include chronic obstructive lung disease, older age, male sex, white race and immunosuppression.141 The clinical course of chronic pulmonary histoplasmosis is generally slower than that of tuberculosis and the systemic illness is less pronounced. Spontaneous remission is possible and cavitary lung lesions may heal. Cicatricial histoplasmosis is detected on chest X-rays when solitary layered coin lesions are seen. These lesions tend to

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calcify and are usually accompanied by multiple calcifications in hilar lymph nodes, the spleen and adrenal glands (Figure 24). Calcifications in the hilar nodes can eventually erode into the lumina of the adjacent bronchi and result in broncholithiasis (see Chapter 17). Mediastinal node involvement can cause superior vena cava syndrome and a sclerosing mediastinitis-like presentation. A mass may be visible on chest radiographs, but CT scans are often required to localize the process.

Pathology The pathology of the five main clinical forms of the disease is a result of a common pathological pathway, which includes inflammatory lesions that evolve from an exudative response to both necrotizing and non-necrotizing granulomatous patterns. The lesions range in size from miliary to large necrotic nodules and cavitary lesions. In acute histoplasmosis, the yeast elicits an epithelioid and giant cell granulomatous response with or without necrosis (Figure 25). In these lesions yeasts may be sparse. Laminated fibrous nodules represent the cicatricial form of the disease with central calcification (Figure 26). Biopsy may reveal only dense fibrous tissue since organisms are rare in these lesions. Sometimes only degenerating forms are noted. Necrosis and multinucleate giant cells should raise one’s suspicion for H. capsulatum infection. In progressive disseminated histoplasmosis, yeast forms are abundant and multiply inside macrophages throughout the

Chapter 7: Pulmonary mycotic infections

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(a)

Figure 25. Acute histoplasmosis. (a) A solitary necrotizing granuloma may be an incidental finding. (b) Large confluent necrotizing granulomas are clinically relevant. Nevertheless, organisms may be sparse.

(b)

Figure 26. Cicatricial histoplasmosis. (a) This calcified nodule was an incidental finding. (b) Biopsy samples often demonstrate bronchocentric hyalinized nodules with focal calcifications.

(b)

body, without granuloma formation. Lung may feature fibrinous pneumonia or even vasculitis (Figure 27). Systemic disease features hepatosplenomegaly, interstitial pneumonitis and mediastinal lymphadenopathy. Chronic pulmonary histoplasmosis demonstrates a mixture of granulomatous, interstitial and obstructive lesions, including emphysematous bullae. The yeast forms are found only within bronchocentric necrotizing granulomas. Granulomatous lesions with many free-floating organisms cavitate before fibrous walls surround and entomb the infection. In patients with normal immune function, H. capsulatum may take advantage of abnormal lung structure to cause chronic disease. Inhalation of spores into emphysematous bullae causes chronic apical and subapical disease, which may be mistaken for tuberculosis. The walls of the bullae become fibrotic and the pleural space is obliterated. The lung is progressively destroyed, causing volume loss and hilar retraction. Many organisms are identified in the walls of these cavities. Smaller rounded fluidfilled lesions with sparse organisms develop when the fungus colonizes centrilobular emphysematous spaces. Infected spaces are also associated with pneumonic lesions, i.e., segmental parenchymal consolidation and necrosis. Only rarely does a primary lesion cause a granulomatous pneumonia with widespread consolidation. On involution these fibrotic airway lesions form calcified fibrotic nodules, which can cause irreversible obstructive airway disease.

Diagnosis Microscopic examination of wet preparations of clinical material, such as sputum, BAL or pus, is not a suitable method for the diagnosis of histoplasmosis. All material should be examined as stained smears.142 If microscopic examination of Wright-stained peripheral blood or bone marrow smears, or stained tissue sections of lung, liver, lymph node or other specimens from individuals who have resided in, or visited, endemic areas reveals small oval yeast cells with narrow-based buds often clustered in monocytes and macrophages, histoplasmosis should be suspected.

Culture The definitive diagnosis of histoplasmosis depends on isolation of the fungus in culture. Mold colonies are white to brown and can usually be obtained after incubation at 25–30 C for 4 to 6 weeks on Sabouraud’s dextrose agar supplemented with cycloheximide (actidione). It is often difficult to distinguish the mycelial colonies of H. capsulatum from those of Blastomyces dermatitidis and species of Chrysosporium and Sepedonium. Unequivocal identification of a mycelial isolate as H. capsulatum requires conversion to the yeast form. More rapid identification is obtained by molecular testing of the mold culture with the AccuProbe test (Gen-Probe Inc., San Diego, CA,

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(b)

(c)

Figure 27. Progressive disseminated histoplasmosis in an immunocompromised patient. (a) Fibrinous pneumonia may lack granulomas. (b) Granulomatous vasculitis is not much different than what one sees in pulmonary vasculitic syndromes. (c) Yeast forms within alveolar macrophages and alveolar wall capillaries do not always elicit a host response.

USA), which can be completed within a few hours. However, H. capsulatum var. duboisii also gives positive results in the AccuProbe test. H. capsulatum has been isolated from the sputum or BAL fluid specimens in 60–85% of cases of chronic pulmonary histoplasmosis if multiple specimens are tested. In disseminated disease, useful specimens for culture include blood, urine, lymph node and bone marrow samples. The Isolator lysiscentrifugation system (Wampole Laboratories, Princeton, NJ, USA) is the most sensitive method for recovering H. capsulatum from blood. Bone marrow samples are positive in over 75% of cases. Cerebral spinal fluid samples are positive in only 25–50% of cases of Histoplasma meningitis.

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Serological tests The histoplasmin skin test is not recommended for diagnosis of histoplasmosis, because a positive result does not distinguish present from past infection. A negative result does not exclude recent infection. Moreover, it can induce the formation of antibodies, making the results of subsequent serological tests difficult to interpret. Serological tests for antibodies are most useful in the diagnosis of subacute pulmonary histoplasmosis, chronic pulmonary histoplasmosis, granulomatous mediastinitis and pericarditis. False-negative reactions may occur during the first 2 months after acute exposure, as well as in immunosuppressed patients with disseminated disease.

Chapter 7: Pulmonary mycotic infections

The immunodiffusion test is a qualitative method performed with mycelial-phase culture filtrate antigen (termed histoplasmin). The ID test detects precipitins to the H and M glycoprotein antigens of H. capsulatum present in histoplasmin. Precipitins to M antigen appear first at 4 to 8 weeks after exposure and can be detected in up to 75% of persons with acute histoplasmosis. M precipitins may persist for many months after the initial infection and may also be found in nearly all patients with chronic pulmonary histoplasmosis, as well as in those who have had a recent histoplasmin skin test. Precipitins to H antigen are specific for acute pulmonary histoplasmosis, but only occur in 10 to 20% of cases and disappear within 6 months of infection. H precipitins are seldom, if ever, found in the absence of precipitins to M antigen. Precipitins to both H and M antigens are highly suggestive of active histoplasmosis, regardless of other serological results. The complement fixation (CF) test is a quantitative procedure in which two antigens are employed: histoplasmin and a suspension of intact killed H. capsulatum yeast cells. The CF test is more sensitive, but less specific than the ID test in histoplasmosis. It can be performed with serum and CSF and is useful in the diagnosis of acute, chronic and disseminated disease. The CF test becomes positive 2 to 6 weeks following infection; CF titers of 1:8 or greater with either antigen are considered presumptive evidence of histoplasmosis. Titers above 1:32 or a four-fold rise in titers between paired specimens offer stronger evidence of active infection. These test results can be difficult to interpret because cross-reactions can occur with serum from patients with blastomycosis or coccidioidomycosis, when titers usually range between 1:8 and 1:32. Similar titers may be obtained in tests with serum from patients with proven histoplasmosis. Titers of CF antibodies to H. capsulatum decrease following resolution of infection, but increase in individuals with chronic progressive disease. A semi-quantitative latex agglutination test using histoplasmin as antigen is available, which detects IgM antibodies. It is used primarily for the presumptive diagnosis of acute histoplasmosis.

Antigen detection tests An enzyme immunoassay for the detection of H. capsulatum polysaccharide antigen is available from MiraVista Diagnostics (Indianapolis, IN, USA). Similar tests have been developed by others, although dissimilar results have sometimes been obtained when the tests have been compared.143 The Histoplasma antigen test is particularly useful in immunocompromised individuals with disseminated disease, and can provide a rapid diagnosis. Sensitivity is greatest with urine, but antigen has also been detected in serum, CSF and BAL. Histoplasma antigen has been detected in the urine of 95 to 100% and in the serum of 80% of AIDS patients with disseminated histoplasmosis. Antigen detection in the urine is

also useful for the rapid diagnosis of acute pulmonary histoplasmosis. In this group, antigenuria can be detected in about 75% of cases, provided samples are obtained within 2 weeks after exposure. The amount of Histoplasma antigen in the urine can be used to monitor an individual’s response to antifungal treatment. Levels decline with effective treatment, becoming undetectable in many patients. Failure of antigen concentrations to decline during treatment is suggestive of therapeutic failure. Antigen levels decline more rapidly in serum than urine. In patients with antigenuria, levels increase in 90% of those who relapse. The Histoplasma antigen test may give false-positive reactions with urine samples from patients with blastomycosis, coccidioidomycosis, paracoccidioidomycosis or penicilliosis. False-positive reactions in the urine EIA for B. dermatitidis antigen occur in patients with histoplasmosis. This is important because the endemic regions for the two diseases overlap. The Platelia Aspergillus galactomannan EIA may give falsepositive results in disseminated histoplasmosis, particularly those with high concentrations of circulating Histoplasma antigen. Patients with aspergillosis, however, do not give false-positive results in the Histoplasma antigen test. Patients with African histoplasmosis also give positive results in the Histoplasma antigen test.

Differential diagnosis H. capsulatum var. capsulatum cells can be confused with other pathogens, such as Candida glabrata and Penicillium marneffei, as well as with atypical small yeast cells of Blastomyces dermatitidis and small, capsule-deficient cells of Cryptococcus neoformans. H. capsulatum var. duboisii is also a consideration, although more in name than in either the clinical or morphological sense (see below). The most difficult distinctions are with Candida glabrata and Penicillium marneffei. C. glabrata is of similar size but is amphophilic and stains with H&E stains. P. marneffei is also of similar size and may form short septate hyphal or “sausage” forms that are up to 20 μm. However, it does not bud. Capsuledeficient cryptococci may resemble H. capsulatum, but are larger, ranging in size from 2 to 20 μm. In addition, these stain faintly for mucin by a mucicarmine stain. Leishmanial amastigotes should be considered in the differential if the patient has traveled to warmer parts of the globe such as the Mediterranean, the Orient or Africa. However, these organisms have dot-like basal bodies or kinetoplasts and do not stain with GMS. Similarly, Toxoplasma gondii may be confused with H. capsulatum, but stain entirely with H&E, do not bud, and do not stain with GMS (see Chapter 8). Histoplasma capsulatum var. duboisii, the etiological agent of African histoplasmosis, occurs in African regions where the annual rainfall ranges from 40 to 80 inches. Lesions are found predominantly in skin and bone and less commonly in the lung, gastrointestinal tract, lymph nodes, liver and spleen. Descriptions of pulmonary disease are unusual. Necrotizing

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granulomas contain large numbers of organisms within histiocyte and giant cell cytoplasm. Histoplasma duboisii yeast forms are round to oval and 8 to 15 μm in diameter. They have double-contoured walls and form buds with narrow bases. The distinction from Blastomyces dermatitidis may be difficult. The cells of the latter are smaller with broader-based buds, and are found only within uninucleate rather than multinucleate giant cells.

Treatment Treatment for asymptomatic pulmonary nodules or adenopathy is not indicated.139 Similarly neither broncholithiasis nor fibrosing mediastinitis requires antifungal therapy.134 For the other infections liposomal amphotericin B may be used or patients with milder manifestations may be treated with itraconazole alone.119 Histoplasmosis can be difficult to diagnose and failure to make the diagnosis in immunosuppressed patients can be fatal.

Pulmonary cryptococcosis Introduction

Cryptococcus was first described in 1894.144 Despite current treatment, patients continue to die of cryptococcosis.145,146

Organism C. neoformans and C. gattii are in the phylum Basidiomycota and produce progeny through asexual and sexual means. Asexual reproduction occurs through simple budding and by haploid fruiting. Simple budding is the reproductive mechanism that occurs within the human host. Sexual reproduction increases genetic diversity and has the potential to promote hypervirulence and antifungal resistance.147,148 Cryptococcus yeast or the small basidiospores are inhaled and cause the initial pulmonary cryptococcal infection.149,150 Unfortunately, in many individuals, Cryptococcus also disseminates by an unknown mechanism throughout the body.148,151 Pulmonary cryptococcosis is caused by the encapsulated yeast Cryptococcus neoformans.6,152,153 This pathogenic fungus produces a thick extracellular polysaccharide capsule. There are five serotypes (A, B, C, D and AD). Most infections are caused by serotypes A and D.154 In H&E-stained sections, the yeasts stain pinkish-blue with a diameter ranging from 2 to 20 μm (Figure 28a). Organisms vary from round or oval to crescent-shaped. The capsule appears as an unstained halo around the cell, 2 to 10 μm wide. Mayer’s mucicarmine stains the capsule bright red (Figure 28b). Fungal stains, such as PAS and GMS, give greater detail and show single or multiple budding of the yeasts in active lesions (Figure 28c). In larger mucoid solitary lesions, the cryptococci are encapsulated and readily identified. In small solid primary lesions, the organisms may be intracellular, small and poorly encapsulated (“dry” variants). Fontana Masson stain is helpful, since capsule-deficient cryptococci stain black or brown due

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to melanin-like pigment present in the fungal cell wall (Figure 28d). This stain can be capricious.

Epidemiology Infection is worldwide. C. neoformans infections occur mostly in immunodeficient individuals. It can be associated with less severe levels of immunosuppression, including splenectomy,155 crescentic glomerulonephritis,156 cirrhosis,157 necrotizing fasciitis,158 Mycobacterium tuberculosis159 and even pregnancy.160 The infective particles of Cryptococcus are acquired from the environment rather than from person-to-person transmission.161,162 Serotypes A and D are commonly found in bird excreta and contaminated soil throughout the world. Pigeons carry large amounts of C. neoformans in their crops and the yeast is most abundant in pigeon habitats. Serotypes B and C (var. gattii) are associated with Eucalyptus camaldulensis in Australia and elsewhere.163 Other eucalyptus species are involved, as well as carob trees in the Mediterranean. C. neoformans preferentially infects immunocompromised individuals.164 C. gattii has recently come to public attention owing to an outbreak of devastating illness in immunocompetent individuals in the Northwest region of the United States and Canada.10,165,166 In India, the prevalence of CNS cryptococcal infection in an HIV-seropositive cohort was 46%.167 In Thailand, 24% of HIV-positive patients with fever of unknown origin had cryptococcosis.168 However, pulmonary involvement was more frequent in HIV-negative patients.169 It is suggested that dispersal of basidiospores (reproductive spores produced by Basidiomycete fungi) occurs with the flowering of the plants in late spring or early summer, when airborne basidiospores are infectious. After dissemination, basidiospores synthesize capsular material and transform into encapsulated yeast cells. Cell-mediated immunity is the major defense against cryptococcal infection. Encapsulation allows the organisms to resist phagocytosis as the capsular polysaccharide induces T suppressor cells, thus impairing both cell-mediated and antibody responses. Cryptococcus also evades the innate immune system through the production of antioxidants, such as melanin, superoxide dismutase, thioredoxin reductase and mannitol, that neutralize specific innate host effector molecules.148,170–173 C. neoformans penetrates the alveolus, as it adheres to and is internalized by pulmonary epithelial cells. These cells internalize both unencapsulated and encapsulated strains, as well as purified glucuronoxylomannan in the capsule of the organism.174,175 A recent review discusses cryptococcal interactions with the host immune system.176

Clinical features The host reaction depends on the immunological status of the host, the presence of underlying disease and whether the

Chapter 7: Pulmonary mycotic infections

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(b)

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(d)

Figure 28. Cryptococcus neoformans. (a) Blue-pink organisms vary in size and shape (H&E stain) (B) Mayer’s mucicarmine demonstrates the halo. (c) Grocott methenamine silver stain demonstrates the variably sized organisms and budding. (d) Fontana Masson stain highlights a cluster of capsule-deficient organisms.

cryptococci are encapsulated. In anergic individuals the response is paucicellular.177 Some cases are asymptomatic. Pulmonary disease often remains asymptomatic even when marked radiographic changes are present. Sporadic cases without predisposing conditions occur, but high-risk groups include patients with hematological malignancies, connective tissue disease, AIDS and sarcoidosis as well as transplant recipients and those on steroid therapy.151,178–180 The primary portal of entry is the lung and the initial infection is invariably pulmonary, with subsequent dissemination. Meningitis is often the first evidence of infection, and other manifestations of the disease include cutaneous, mucocutaneous, osseous and visceral forms. The organism’s predilection for the CNS remains unexplained but CSF lacks

immunoglobulins and complement and is a good medium for growth of the organisms. The clinical spectrum of pulmonary disease includes selflimited, localized and disseminated forms. Immunocompetent patients usually present with localized, self-limiting disease. These individuals are often asymptomatic or mildly symptomatic with fever, cough and/or shortness of breath. Progressive disease presents with nonspecific respiratory or constitutional symptoms, including fever, chest discomfort, weight loss, dyspnea and night sweats. Severe respiratory failure has been reported in both immunocompromised and immunocompetent patients.181 Hemoptysis is rare, but cough is present in nearly half the cases. Dissemination of the infection leads to CNS manifestations, such as headache, lethargy and

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Figure 30. Cryptococcal coin lesion. This solitary subpleural mass features central cystic change and obvious necrosis.

Figure 29. Pulmonary cryptococcal infection. This computed tomogram demonstrates right upper lobe nodules with subpleural consolidation.

personality changes. Disseminated pulmonary cryptococcosis is usually recognized after death in profoundly immunodeficient patients. Radiological presentations are varied and nonspecific, and are also influenced by the underlying immune status of the patient. These include normal radiographs, nodular circumscribed infiltrates, pleural effusions and lobar consolidation (Figure 29). Localized granulomatous lesions, so-called cryptococcomas or torulomas, present radiographically as circumscribed, round shadows and are commonly mistaken for malignancy. They may be several centimeters in diameter and are usually subpleural, solitary and non-encapsulated. When multiple, they may involve one lobe or both lungs. Central necrosis and cavitation may occur, but calcification is rare. The lesions heal with fibrosis and scarring. Blood-borne infection can lead to a single large lung lesion, multiple translucent miliary nodules or diffuse intravascular fungemia. Diffuse interstitial, peribronchial or nodular radiographic infiltrates are seen. Interstitial infiltrates usually indicate the presence of another coexisting opportunistic lung infection.

Pathology The bronchial tree is usually normal, but endobronchial abnormalities include circumferential narrowing, red or white thrush-like plaques and mucosal granularity. Primary complex disease resembles primary tuberculosis and produces small subpleural cryptococcal scars with associated draining lymph node lesions. These, usually less than 1 cm in diameter, subclinical lesions involve any lung lobe and are often an incidental post mortem finding. Minute necrotic foci are surrounded by dense fibrosis. While lymph nodes often calcify, lung lesions rarely do. In the fatal cases of

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cryptococcal meningitis, a careful search at post mortem will often reveal a dormant and apparently healed pulmonary cryptococcal nodule. Nodules are firm and well-defined, with a white or gray cut surface that may appear gelatinous (Figure 30). Disease progression may lead to segmental or lobar gelatinous consolidation. Disseminated disease often manifests with diffuse pulmonary edema, necrosis and hemorrhage resembling the red hepatization phase of pneumonia with a gelatinous consistency. Lung parenchyma may be dissolved, form an abscess and be practically replaced by organisms. The tissue reaction to Cryptococcus varies widely from a minimal inflammatory response to a granulomatous reaction with necrosis or diffuse alveolar damage (Figure 31). Massive numbers of organisms are seen in immunodeficient individuals, especially those with AIDS. Yeasts may displace, compress or destroy underlying lung. In other instances, there is a mixed purulent and granulomatous reaction and the cryptococci tend to be less well encapsulated. Multinucleate foreign-body giant cells may contain large numbers of yeasts and are mixed with plasma cells and lymphocytes. Neutrophils may be numerous.

Diagnosis The diagnosis of pulmonary cryptococcosis depends on demonstrating the organism in sputum, bronchial washings, BAL, brushings or biopsy material. Approximately 50% of HIV-negative and more than 80% of HIV-positive patients have a positive India ink examination of the CSF. In persons with AIDS, Cryptococcus cells are usually plentiful in the CSF, although the capsules can be small, making recognition difficult.

Culture The identity of Cryptococcus should be confirmed by sputum, blood, CSF, urine, prostatic fluid or fresh tissue culture. The organism grows well on standard microbiological media. Positive

Chapter 7: Pulmonary mycotic infections

(a)

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Figure 31. Varying morphological appearances of cryptococcal pneumonia. (a) In severely immunocompromised individuals organisms grow unopposed. (b) This PAS-stained section is from an AIDS patient. (c) Overwhelming infection may cause acute lung disease. Diffuse alveolar damage is seen here. (d) Community acquired infection almost always demonstrates a granulomatous response. Airway disease features submucosal granulomatous inflammation and overlying squamous metaplasia. (e) Yeast forms may be identified within giant cells. (f) Granulomatous infarcts suggest a brisk host response to a large number of organisms.

(b)

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blood cultures are obtained in up to 70% of HIV-infected persons with cryptococcosis. The lysis-centrifugation method (Isolator, Wampole Laboratories, Cranbury, NJ, USA) is the most sensitive procedure for recovering Cryptococcus species from blood. Because of the greater load of organisms, microscopic examination and culture of other specimens is more often positive in untreated patients with HIV infection than in other individuals.

Serological tests Antibodies to Cryptococcus can often be detected in patients with localized or past infection, but are rare in patients with untreated meningeal or disseminated disease, in whom tests for antigen are much more helpful.

Antigen detection tests Testing for Cryptococcus capsular antigen is one of the most reliable methods for the diagnosis of cryptococcosis. Several latex particle agglutination (LPA) and ELISA tests are marketed for the detection of antigen in serum, CSF, urine and BAL fluid specimens. These tests are sensitive and highly specific, provided that rheumatoid and other interfering factors are removed. False-negative results can occur if the organism load is low or if the organisms are not well encapsulated. Different manufacturers’ products can give different titers with the same clinical specimen. A serum antigen titer of 1:8 or greater is considered strong presumptive evidence of Cryptococcus infection. A negative serum antigen test result

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does not exclude the diagnosis of cryptococcosis, particularly if only a single specimen has been tested and the patient has symptoms consistent with the infection. In HIV-negative individuals, high levels of serum and CSF antigen prior to treatment are often predictive of death during treatment.182

Differential diagnosis Intact lymphocytes can be confused with the organism. Cryptococci may be difficult to differentiate from other yeasts, notably Histoplasma capsulatum, microforms of Blastomyces dermatitidis, Sporothrix schenckii, Candida glabrata, blastoconidia of Candida species and immature spherules of Coccidioides immitis. The mucicarmine-positive capsule discerns this organism from others.

Treatment and prognosis Amphotericin B is the mainstay of treatment but is limited by its side effects. Without treatment there is a high mortality.183 Despite antifungal therapy, the mortality remains between 10 and 25% in AIDS patients, and at least one-third of patients with cryptococcal meningitis experience mycological or clinical failure.10

Pulmonary blastomycosis (North American blastomycosis) Introduction Blastomycosis is an endemic pulmonary mycosis of the USA and Canada but occurs in many parts of the world. It is caused by the dimorphic fungus Blastomyces dermatitidis.

Organism B. dermatitidis is dimorphic, growing in a mycelial form as a fluffy white mold at 25 C and as a brown, wrinkled, folded yeast form at 37 C.184 Conidiophores arise from the hyphae and produce single terminal conidia, thought to be infectious for humans and animals, particularly dogs, when the mycelia are disturbed. Histologically the branching hyphae are 2 to 4 μm in diameter and have right-angled conidiophores with single terminal conidia. These conidia are round or oval and vary from 2 to 10 μm in diameter. The conidia become airborne and infectious when the mycelia are disturbed. The mycelial form has no morphologically unique characteristics allowing definitive identification.185 In tissue sections, B. dermatitidis appears as round to oval yeasts with thick, sharply defined, refractile walls (Figure 32a). The cells are usually between 8 and 15 μm in diameter, but they may show greater variation, from small forms 2 to 4 μm diameter to giant forms 40 μm in diameter (Figure 32b). In H&E stains, the protoplasm is basophilic and separated from the wall by a clear space. Several nuclei may be visible. The yeasts reproduce by single budding and the bud is

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characteristically attached by a broad base (Figure 32c). Fungal stains highlight organisms.

Epidemiology B. dermatitidis exists in nature as a mold that produces conidia (mycelial phase) and converts to a broad-based budding yeast (yeast phase) at body temperature.185 Lack of a specific skin test and difficulty isolating the fungus in the environment has hampered epidemiological studies. The disease is found predominantly in North America, in the states surrounding the Mississippi and Ohio rivers.186 Sporadic cases in humans and dogs as well as clusters and epidemics are also seen. It also occurs in Midwestern states, the Canadian provinces bordering on the Great Lakes, and along the St. Lawrence River.187 Cases are documented in areas ranging from Venezuela, Mexico, Australasia and the Middle East. Some cases have been reported from Africa. It has been suggested the disease originated in Africa and was carried by the slave trade to America. The probable mechanism for infection is inhalation of spores from the soil. Cellular immunity is considered to be the major protective factor in preventing progressive disease secondary to B. dermatitidis. The male: female ratio ranges from 4:1 to 15:1 in series of endemic cases.185

Clinical features The lung is the probable portal of entry for blastomycosis, even in those cases where disease only manifests in other organs.185,188–191 Primary cutaneous infections have been described in the immunosuppressed, resulting from laboratory or autopsy accidents and dog bites. The incubation period is uncertain. In one outbreak, a mean of 45 days was established, but other estimates range from 21 to 106 days. Infection may be inapparent or there may be an acute systemic illness with pyrexia, cough, arthralgia, weight loss, malaise and myalgia. The cough is initially nonproductive but mucopurulent sputum follows, sometimes with hemoptysis. Acute respiratory distress syndrome has been reported.192 A pet dog may have been ill with the disease.185 Radiographic appearances vary and range from one or more pleural-based rounded opacities to lobar consolidation.193 Miliary or reticulonodular patterns on radiographs are the next most frequent radiological manifestation. Mass lesions that mimic carcinoma are not uncommon. Cavitation and calcification are less common than in histoplasmosis or tuberculosis. Pleural thickening, pleural effusions and pneumothorax may occur. Hematogenous spread often leads to miliary disease, with acute respiratory failure. Endobronchial spread can precipitate diffuse parenchymal consolidation. In some the disease is self-limiting, while in others resolution of pulmonary symptoms is followed by extrapulmonary disease. Skin disease occurs in 40 to 80% of cases and other involved organ sites include bones and joints, the genitourinary tract and the central nervous system.

Chapter 7: Pulmonary mycotic infections

(a)

(b)

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Figure 32. Blastomycosis. (a) Blastomyces dermatitidis with thick, refractile wall and internal structures. (b) Great variations in size are obvious in this Grocott methenamine silver-stained tissue section. Broad-based single buds are noted. (c) A classic broad-based bud is apparent (Grocott methenamine silver stain).

Pathology Following inhalation of conidiophores, which lodge predominantly in the lower lobes, budding yeast forms develop. From the alveoli, yeasts enter the interstitium and cause acute inflammation. Parenchymal necrosis, an influx of eosinophils, giant cells, lymphocytes and fibroblasts follow. The characteristic feature of the disease is the mixture of pyogenic and granulomatous inflammation, forming a suppurating granuloma. Pulmonary lesions vary in extent from small granulomatous nodules to extensive necrotic areas containing multiple abscesses and involving much of the lung (Figure 33). Small lesions may eventually heal and calcify. In chronic infections,

cavity formation may occur but is uncommon. In severe cases, a dense fibrocaseous mass may replace parts or the whole of a lobe. The granulomatous response is rarely as conspicuous as in histoplasmosis or tuberculosis (Figure 34a,b). Extensive tracheal involvement may be seen with numerous white, streaky, nodular and vesicular lesions. Bronchial lesions are common and cause mucosal destruction with spread into the surrounding lung. Bronchial stenosis may occur. Amyloid has been reported as a late complication. Clusters of yeasts may be found in severe infections (Figure 35) but usually occur singly and may reside only within giant cells. They may be very scanty in chronic lesions. Infection of the regional nodes seldom results in the same degree of necrosis as seen in tuberculosis. Hilar nodes are not

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often involved and primary complex lesions are said to be rare. In widespread disease, pleural invasion leads to chest wall involvement.192

Diagnosis Examination of fresh material, after digestion with 10% KOH, has a high yield in experienced hands. Sputum or lung material stained by Papanicolaou may also provide the diagnosis.194

an isolate as B. dermatitidis requires conversion to the yeast form. If rapid identification is required, molecular testing of the mold culture with the AccuProbe test (GeneProbe Inc., San Diego, CA, USA) for identification of B. dermatitidis can be completed within a few hours. The AccuProbe test also produces a positive result with P. brasiliensis. Slow growing, non-sporulating isolates are more characteristic of P. brasiliensis than B. dermatitidis.

Culture

Serological tests

The definitive diagnosis of blastomycosis depends on isolation of the fungus in culture from sputum, pus or biopsy material. Identifiable mold colonies, which are white to brown in color, can be obtained after incubation at 25–30 C for 1–3 weeks on Sabouraud’s dextrose agar supplemented with cycloheximide (actidione). Unequivocal identification of

The most useful serological test is immunodiffusion. Improvements in the performance of these tests have been achieved by the use of a purified surface antigen of B. dermatitidis, termed the A or WI-1 antigen. A positive reaction in an ID test with the A antigen of B. dermatitidis is specific and diagnostic for blastomycosis. However, negative ID reactions have been obtained in 10% of patients with disseminated disease and over 60% with localized infection. CF tests lack specificity because of cross-reactions with antigens of other fungi, particularly H. capsulatum and Coccidioides species.

Antigen detection tests

Figure 33. Pulmonary blastomycosis. Multiple pulmonary abscesses from a patient with acute progressive blastomycosis.

(a)

An enzyme immunoassay for the detection of B. dermatitidis antigen is available from MiraVista Diagnostics (Indianapolis, IN, USA).195 Sensitivity is greatest with urine, but antigen has also been detected in serum, CSF and BAL.195 Antigen levels decline with treatment and increase with treatment failure or relapse. The test gives false-positive reactions with 96% of urine samples from patients with histoplasmosis, the disease from which blastomycosis is in most need of being differentiated, and 100% of urine samples from patients with paracoccidioidomycosis. Direct immunofluorescence is useful for rapid and definite identification of the fungus in smears and formalin-fixed tissue (b)

Figure 34. Pulmonary blastomycosis. (a) Suppurative abscess with a loosely formed granuloma. (b) Yeast forms of Blastomyces dermatitidis in the center of a lesion with a weak granulomatous response.

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(a)

(b)

Figure 35. Pulmonary blastomycosis. (a) Immunocompromised patients may present with overwhelming infection and no host response. Alveolar space filling is striking. (b) The absence of an immune response rivals that seen in AIDS patients with cryptococcal pneumonia (Grocott methenamine silver stain).

sections. Molecular methods are possible using DNA-based assays, and can help identify the organism.

Differential diagnosis Mucicarmine may be useful to distinguish B. dermatitidis from lightly encapsulated forms of C. neoformans. Empty, single cells of B. dermatitidis may be mistaken for the empty spherules of C. immitis. Paracoccidioides brasiliensis may be mistaken for B. dermatitidis, if only single buds are seen. Small intracellular forms of B. dermatitis may be confused with H. capsulatum var. duboisii and var. capsulatum, but the fungi can be distinguished by the morphology of budding forms.

Treatment and prognosis Acute primary blastomycosis is often self-limited and does not usually require treatment. However, follow-up is necessary since arrest and healing of the primary infection may be followed by recurrence. Untreated blastomycosis can be associated with mortality rates approaching 60%.196 Amphotericin B and other antifungals, especially the azoles, have been used in treatment.

Pulmonary coccidioidomycosis Introduction Coccidioidomycosis was identified more than 100 years ago, first in Argentina in 1892 by a medical student and subsequently in California.197,198

Organism Coccidioidomycosis is caused by the dimorphic fungi Coccidioides immitis and Coccidioides posadasii.199,200 The fungi live in soil in the mycelial phase. The hyphae fragment into arthroconidia, which when airborne either return to soil to restart the cycle or are inhaled by an animal host. In the host, the spores swell, become spherical and develop a thickwalled spherule. Cleavage occurs within the spherule to form up to several hundred endospores. When the spherule bursts, endospores are released, and each can develop into a new spherule (the parasitic spherule-endospore cycle). If endospores return to soil, they develop into hyphae and the cycle begins again. Spherules are most often between 10 and 60 μm but approach 200 μm in diameter and have a thick, doubly refractile wall. Mature spherules contain endospores, while empty spherules may be collapsed. As the spherule enlarges and forms endospores, the wall becomes thinner. Routine H&E staining is often adequate, as spherules and endospores are basophilic (Figure 36a). Endospores and spherules are stained by silver techniques (Figure 36b,c). PAS stains endospores but not the walls of spherules (Figure 36d).

Epidemiology Coccidioidomycosis is endemic in certain areas of North, Central and South America.201 The endemic area stretches from the San Joaquin Valley in central California, south to Mexico and east to central Texas.202,203 Over the last decade,

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(a)

(b)

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Figure 36. Coccidioides spp. (a) Spherules, some with endospores, measuring up to 60 μm are obvious in this H&E stained-tissue section. (b) This Coccidioides immitis spherule contains many endospores (Grocott methenamine silver stain). (c) Collapsed spherules may have bizarre shapes (Grocott methenamine silver stain). (d) Endospores within spherules stain brightly with PAS. The external spherule walls do not.

incidence rates have increased, at least in two regions. In Arizona, the highest incidence of active disease is seen in the south-central counties of Maricopa, Pinal and Pima, with incidences as high as 75 cases per 100 000. These areas include Tucson and Phoenix.204 The incidence in the southern portion of the San Joaquin Valley reaches 150 cases per 100 000, while the incidence in the rest of California is generally less than 5 per 100 000.205 Unlike Arizona, most cases of coccidioidomycosis in California occur outside the major metropolitan regions. The reason for the current increase in cases may be the influx of susceptible individuals into the endemic area.

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Sporadic cases are increasingly recognized outside endemic areas. These arise in travelers who have visited an endemic area or are exposed to fomites, such as fruit and cotton, from endemic regions. Infection is widespread in wild rodents, dogs, cattle and sheep, but there is no evidence animals spread the disease directly to humans. Accidental laboratory infections have been recorded, and great care should be exercised in handling culture samples. Interhuman infection occurs only in very exceptional circumstances. Drainage of pus from a coccidioidal osteomyelitis sinus beneath a plaster cast has led to mycelial growth beneath the cast and shedding of arthrospores when the cast was opened.

Chapter 7: Pulmonary mycotic infections

Figure 37. Pulmonary coccidioidomycosis. This computed tomogram demonstrates bilateral nodular infiltrates with focal cavitation and consolidation. (Image courtesy of Professor Antônio de Deus Filho, Teresina/Piauí, Brazil.)

Semiarid climates with brief intense rainy seasons are ideal for mycelial growth. With the onset of dry weather, the arthroconidia break off and become airborne when soil is disturbed. Outbreaks occur at construction sites and archeological digs. Agricultural workers, military personnel and immunocompromised patients are at greater risk of developing the disease, as well as women in the third trimester of pregnancy and early postpartum. Certain ethnic groups are also at high risk of suffering disseminated disease. Persons of Filipino or African-American descent have a 10- to 175-fold higher risk of disseminated infection.206

Clinical and pathological features Primary pulmonary infection

About 60% of infections are asymptomatic. Symptoms of pulmonary disease usually develop 1 to 3 weeks after inhalation of the arthroconidia and include cough, fever, pleuritic chest pain and myalgia. Approximately half of patients have a fine erythematous rash and some develop erythema nodosum or erythema multiforme. Most patients suffer little or no inconvenience, but rarely symptoms are severe with acute respiratory insufficiency. Chest radiographs show nodules or multiple patchy infiltrates that may coalesce (Figure 37). Hilar lymph nodes may be prominent. As the inhaled arthrospores develop into spherules, the initial host response involves the mobilization of macrophages and neutrophils, accompanied by edema. Complement is activated and T cells are stimulated, but the fungus, particularly the spherules, remains resistant to killing. If lymph nodes are involved, the primary complex resembles tuberculosis or histoplasmosis. Necrosis is common (Figure 38). A localized acute inflammatory response is seen

Figure 38. Pulmonary coccidioidomycosis. Multiple necrotizing granulomas are seen.

when spherules rupture and release endospores. As spherules mature, they are typically surrounded by epithelioid histiocytes and giant cells. Organisms may lie within giant cell cytoplasm (Figure 39). Occasionally spherules may become coated with eosinophilic clubbing material and form an asteroid or actinomycetoid form. Most primary lesions heal at this early stage, leaving a small peripheral scar that may later calcify.

Chronic pulmonary infection Coccidioidal spherules may lie dormant for many years in apparently healed primary lung lesions. In fact, positive cultures from autopsy tissue are reported. Reactivation can occur years after the patient has left the endemic area. Reactivation also occasionally occurs in old age, particularly among Native Americans, as immunity wanes. Approximately 5% of those infected develop chronic lung disease, a pulmonary nodule or a cavity.

Coccidioidoma Chronic granulomatous consolidation may result in one or more localized lesions known as coccidioidomas. These 0.5– 3.5 cm in diameter, round lesions feature central necrosis and sometimes calcify. If a bronchus is eroded, cavitation results. Coccidioidomas are sharply demarcated from the surrounding lung by fibrous tissue, as well as numerous chronic inflammatory cells, histiocytes and giant cells (Figure 39a). Microscopic satellite lesions may be seen. Coccidioidomycosis is sometimes diagnosed only when a nodule is resected to exclude malignancy. Spherules are often difficult to find in longstanding lesions. When a cavity communicates with airways,

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(b)

Figure 39. Pulmonary coccidioidomycosis. (a) In immunocompetent individuals organisms are in large part ingested by multinucleated giant cells. (b) Sometimes one only finds an empty spherule (see Figure 36c).

arthrospores may be seen. Many cavities and granulomas contain unusual degenerate and mycelial forms of the fungus, appreciated only on special stains.

Progressive pulmonary disease In this other form of chronic pulmonary coccidioidomycosis, the primary infection persists or is reactivated.202 Diabetics and immunocompromised patients are particularly prone to develop progressive disease. Chest radiographs demonstrate persistent extensive infiltrates, sometimes associated with pleural effusions. Cavities rarely form. Patients may develop fatal acute pneumonia with neutrophils, edema and extensive parenchymal necrosis. Blood eosinophilia greater than 20% and the presence of eosinophilic microabscesses carry a poor diagnosis. Macroscopically, the lung resembles tuberculous bronchopneumonia with irregular areas of gelatinous consolidation (Figure 40). In surviving patients, this process results in chronic granulomatous bronchitis, bronchiectasis, abscess cavities and extensive parenchymal fibrosis. Cavities develop in less than 2% of patients and most are solitary, peripheral and in the upper lobes. Twenty-seven to 50% of lesions resolve spontaneously over 1 to 4 years.207 Smaller lesions (< 2.5 cm) seem to have a better outcome, while cavities larger than 5 cm will most likely persist.208 Cavities probably form secondary to parenchymal necrosis and bronchitis-induced bronchiectasis and appear as thin-wall cystic spaces lined by a fine layer of fibrous tissue. Classically there is no air-fluid level. Unlike tuberculous cavities, they

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Figure 40. Progressive pulmonary coccidioidomycosis. Diffuse consolidation and “gelatinous” abscesses are commonly seen in autopsy specimens.

provide no source of infection, though hemorrhage may occur from their walls, causing hemoptysis. They may rupture into the pleura causing pneumothorax and/or bronchopleural fistula. The cavities fluctuate in size due to air trapping and, very rarely, a fungus ball containing the mycelial form of C. immitis colonizes the cavity.

Chapter 7: Pulmonary mycotic infections Figure 41. Disseminated coccidioidomycosis. Multiple parenchymal lesions bespeak hematogenous spread.

soft tissue lesions. Blood cultures are usually positive in patients with diffuse pneumonia. Cultures are hazardous for laboratory staff and should be performed inside a biological cabinet under conditions of biosafety level (BSL) 3 containment.210 Coccidioides spp. are fast growing and identifiable white to gray or buff mold colonies. Results can usually be obtained after incubation at 25–30 C for 2–7 days on Sabouraud’s dextrose agar supplemented with cycloheximide (actidione). Large numbers of mature arthroconidia are usually present after 7 to 10 days. For confirmation, molecular testing of cultures with the AccuProbe test (Gen-Probe Inc., San Diego, CA, USA) can be completed within a few hours. This test is sensitive and specific, although pre-treatment of isolates with formaldehyde leads to false-negative results. The AccuProbe test does not distinguish between the two Coccidioides spp. It is useful for identifying atypical isolates that fail to form arthroconidia.

Skin tests

Disseminated coccidioidomycosis About 0.5% of patients develop disseminated disease with extrapulmonary spread to involve almost any organ.206,209 Rarely, widespread rapid dissemination is fatal. Dissemination is more common in men, pregnant women, immunocompromised patients, and very young or elderly hosts. Dark-skinned races are more susceptible, particularly Filipinos and Mexicans. Miliary pulmonary coccidioidomycosis is a serious form of the disease, believed to reflect hematogenous dissemination of the organisms to all lung fields, as well as other body sites (Figure 41). It occurs early in the course of primary pulmonary infection in high-risk patients. In immunosuppressed patients, the illness leads to respiratory failure and death. This form of the disease requires early diagnosis and immediate therapy.

Diagnosis Coccidioides immitis can be identified in sputum or other fluids in wet-mount preparations with the addition of 10% potassium hydroxide. The spherules are relatively large and their walls are doubly refractile, if the light is reduced. Papanicolaou-stained smears are superior to KOH preparations. The histological recognition of coccidioidal infection rests on the identification of the spherules.

Culture Cultures from sputum and specimens obtained by bronchoscopy are more helpful than those from pulmonary nodules. Coccidioides spp. can also be isolated from suppurative cutaneous and

A positive coccidioidin (or spherulin) skin test result does not distinguish present from past infection. However, conversion from a negative to a positive result is a sign of recent infection, because it occurs within 4 weeks of the onset of symptoms in 90–95% of cases. False-negative results are common in anergic patients with disseminated coccidioidomycosis. Unlike the histoplasmin skin test, coccidioidin does not interfere with the results of subsequent serological tests. The coccidioidin skin testing reagent is not currently available in the USA.

Serological tests The immunodiffusion and complement fixation tests are the most reliable methods. The principal antigen used in these tests is a mycelial-phase culture filtrate (termed coccidioidin). The ID test is useful for initial screening of specimens, and can be followed by other tests, if positive. With heated coccidioidin as antigen, the test detects IgM antibodies against Coccidioides and is most useful for diagnosing recent infections. These antibodies can be found in 75% of patients within 1 week after the onset of symptoms, and 90% are positive within 3 weeks. IgM antibodies disappear within a few months in persons with acute pulmonary disease. The ID test detects IgG antibodies and gives results comparable to the CF test (see below). IgG antibodies can usually be detected within 2 to 6 weeks after onset of symptoms. The CF test is a sensitive quantitative method in which unheated coccidioidin is used to measure IgG antibodies against Coccidioides. These antibodies do not appear until 4 to 12 weeks after infection, but may persist for long periods in individuals with chronic pulmonary or disseminated disease. Testing of serial samples to detect rising or falling titers can reveal progression or regression of illness and the response to treatment. The failure of the CF titer to fall during treatment of disseminated disease is an ominous sign. Titers of CF antibodies > 1:16 are consistent with spread of Coccidioides infection beyond the respiratory tract. More than 60% of patients with disseminated coccidioidomycosis have CF titers of 1:32 or greater.

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False-negative results occur in immunocompromised individuals and CF titers alone should not be used as the basis for diagnosis of dissemination. Patients with clinical presentations consistent with coccidioidomycosis, but with negative or low CF titers, should be retested at 3 to 4 week intervals. The CF test is more sensitive but less specific than the ID test. If the CF test is positive, but the ID test negative, patients should be investigated for histoplasmosis or blastomycosis. The CF test can be performed with serum, CSF, pleural or joint fluid samples. As with serum, CF titers diminish with successful treatment. A qualitative latex particle agglutination (LPA) test, with heat-treated coccidioidin as antigen, is available from several commercial sources (ImmunoMycologics Inc., Norman, OK, USA; Meridian Bioscience, Cincinnati, OH, USA) for the detection of IgM antibodies. This test is simpler and faster than the ID test, and is more sensitive in detecting early infection. It has a 5–10% false-positive rate, and positive results must be confirmed by the ID and/or CF methods.

Antigen detection tests A quantitative EIA for the detection of Coccidioides antigen is available. Sensitivity is greatest with urine (about 70% in moderately severe to severe disease), but antigen has also been detected in serum, CSF and BAL fluid specimens. Antigen levels decline with treatment. False-positive reactions have been obtained with samples from about 10% of patients with other endemic fungal infections and a positive antigen test result should be verified by other methods.

Differential diagnosis Empty spherules of C. immitis may be mistaken for B. dermatitidis. It is best to examine multiple sections to demonstrate both spherules and endospores. Free endospores may also be confused with H. capsulatum or C. neoformans. The large size and absence of budding are discriminating features.

Treatment and prognosis The treatment consists of amphotericin B deoxycholate or azoles. Coccidioidal fungemia portends high mortality. Respiratory failure is reported in all patients with fatal outcomes. Features were consistent with the systemic inflammatory response syndrome (SIRS) and acute respiratory distress syndrome.211 Miliary disease in immunosuppressed patients may lead to terminal respiratory failure.

Pulmonary paracoccidioidomycosis (South American blastomycosis) Introduction South American blastomycosis, Paracoccidioides brasiliensis, was first described by Lutz in 1908, who recognized the causative organism differed from C. immitis.

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Figure 42. Paracoccidioides brasiliensis. Large yeast cells can be identified by the multiple peripheral buds (Grocott methenamine silver stain). (Image courtesy of Joseph F. Tomashefski, Jr., MD, Cleveland, OH, USA.)

Organism

P. brasiliensis is a dimorphic fungus. At 37 C in tissues and in cultures, it forms oval to round yeasts with multiple buds. At lower temperatures, it grows as a mycelium and forms arthroconidia. The fungus is a white mold composed of thin, septate hyphae that produce chlamydoconidia, and when cultured produces < 5 μm asexual propagules known as microconidia; the latter are believed to be the infectious particles.212 Characteristically large, oval to round yeast cells with translucent walls and multiple peripheral buds are seen. In tissue sections, the organisms are round to oval and 10 to 60 μm in diameter. They have thin, refractile walls. The contents may be basophilic or amphophilic with H&E and tend to retract from the wall. They reproduce by multiple buds, attached to the parent by narrow necks. Buds may be roughly equal or may vary in size (Figure 42). Hyphae are rarely formed in tissues. In longstanding fibrogranulomatous lesions, the yeasts may be fragmented, distorted and unevenly stained by fungal stains.

Epidemiology Paracoccidioidomycosis has a very restricted geographic distribution confined to Central and South America.213 Brazil is the center of the endemic area, which is characterized by forests with high temperatures and humidity. Sporadic cases have been reported in North America, Europe and elsewhere in patients who had previously resided in endemic areas.214 The natural habitat of P. brasiliensis, although difficult to isolate, is thought to be soil. The route of infection has not been determined, although inhalation seems more likely than direct inoculation. No animal reservoir has been identified, but the disease has been reported in armadillos. The disease is rare in children and young people; most patients are over 30 years of age.215 It affects males far more

Chapter 7: Pulmonary mycotic infections

often than females, except in prepubertal children. This may be related to an inhibitory action of estrogens on the transition from mycelium to yeast form. Agricultural workers are particularly vulnerable to the disease. Up to 30% of patients with paracoccidioidomycosis also have tuberculosis.

Clinical and pathological features Acute and subacute pulmonary disease

Symptoms of paracoccidioidomycosis often develop many years after exposure.213,212 This long latent period may be due to the fungus lying dormant in lymph nodes. Disease severity is related to host immunity. In individuals with a normal immune system, it may be asymptomatic. If cell-mediated immunity becomes depressed, the disease manifests itself. Immunosuppressed patients, including those with AIDS, may develop severe disease.216 The lungs are considered the primary site of infection, and most patients have the usual respiratory symptoms. A small number of infections probably remain subclinical. Acute lung infection rapidly disseminates to lymph nodes, liver and spleen. Fungemia may also be detected. At the other end of the spectrum, calcified lung nodules containing P. brasiliensis have been described as an incidental autopsy finding. Radiographs are not specific and may demonstrate micronodular infiltrates, foci of consolidation, cavities, fibrosis or, rarely, calcifications.217 Histopathology ranges from neutrophilic microabscesses or scattered interstitial necrotizing granulomas to consolidative granulomatous pneumonia. Interstitial fibrosis with arterial intimal proliferations may be striking.

Progressive pulmonary disease This form of disease is far more common than acute and subacute presentations. It affects older patients who probably experience reactivation many years after initial infection. Almost half of patients have disease confined to their lungs, while they may also experience either limited or widespread dissemination. Oropharyngeal spread is common. Pulmonary symptoms are dyspnea, cough, hemoptysis and pyrexia. Chest radiographs show patchy or confluent nodular infiltrates, often bilateral and symmetrical, in the mid- or basal lung fields. Macroscopically, the lesions resemble blastomycosis and tuberculosis, with which the disease may coexist. Lung scarring leaves a coarse, hobnail appearance. Cavities may result from the breakdown of necrotic lesions, but calcification is unusual. Extensive fibrosis may cause honeycomb change with cor pulmonale. Small airways become extensively damaged and macroscopically resemble areas of tuberculous caseation. Involvement of larger airways causes ulceration, with resulting bronchiectasis. Localized necrotizing lesions with surrounding fibrosis are called paracoccidioidomas. Pleural involvement is common and results in dense fibrous adhesions and, less commonly, pleural effusions. Hilar lymph nodes are involved

in 50% of cases. Other pulmonary complications include pneumothorax and pulmonary arterial thrombosis. A mixed pyogenic and granulomatous inflammatory response, similar to blastomycosis, is typical of the disease. Neutrophils may predominate in some areas, whereas others contain poorly defined epithelioid cells and giant cell granulomas. Numerous organisms are usually present within giant cells, in the abscesses, or scattered throughout the tissue. In progressively enlarging lesions, necrosis with many free organisms is accompanied by fibrosis. A more rapidly spreading form of lung infection results in a widespread confluent granulomatous and fibrinous intra-alveolar exudate with microabscesses, called paracoccidioidal pneumonia. When the lungs are involved in a blood-borne infection, there are numerous, fungi-containing miliary granulomas. Dissemination from a primary lung infection may involve multiple organs including the liver, spleen, gastrointestinal tract, adrenals, bones and CNS. Mucocutaneous-lymphangitis paracoccidioidomycosis might be the result of direct inoculation. It is probably the result of dissemination from a pulmonary infection. Oral, nasal and anal mucosa are most often involved and spread to regional nodes causes lymphadenopathy.

Diagnosis Microscopic examination of potassium hydroxide preparations of sputum, BAL, exudates, pus from draining lymph nodes and other clinical material permits the diagnosis of paracoccidioidomycosis. The diagnosis is most readily made by demonstrating the budding yeast forms in sputum or lavage. If the submitted material is warmed with 5% KOH, the yeasts are doubly refractile. Cell-block preparations or smears stained with GMS are also sensitive techniques.

Culture The definitive diagnosis of paracoccidioidomycosis depends on isolation of the fungus in culture. Mold colonies can be obtained after incubation in Sabouraud’s dextrose agar supplemented with cycloheximide (actidione). Mycelial cultures seldom sporulate. If more rapid identification is required, the initial mold culture can be subjected to an exoantigen test. There is no commercial DNA probe test for P. brasiliensis. The AccuProbe test for identification of B. dermatitidis crossreacts with P. brasiliensis.

Serological tests Serological tests are useful for the rapid presumptive diagnosis of paracoccidioidomycosis, particularly in untreated disease.218 These tests may also be used to monitor the response to treatment. Antibody detection is less useful for diagnosis of paracoccidioidomycosis in persons with AIDS. The ID test is performed with mycelial-phase culture filtrate antigen. It is highly specific and is positive in 65–100% of patients with paracoccidioidomycosis. Cross-reactions can

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occur in patients with histoplasmosis, but are uncommon. The CF test is positive in 70–100% of patients. A CF titer of at least 1:8 is presumptive evidence of paracoccidioidomycosis. Of patients with active disease 85–95% have CF titers of 1:32 or greater. Cross-reactions can occur with serum from patients with other mycotic infections, particularly histoplasmosis. Indirect enzyme immunoassay measures IgG antibodies against a species-specific 43 kDa glycoprotein antigen of P. brasiliensis. It is simpler to perform, and more sensitive and specific than the CF test, provided that the antigen is deglycosylated. Cross-reactions with histoplasmosis are problematic. A rapid dot immunobinding method for detection of P. brasiliensis antibodies to the 43 kDa antigen has been developed.

Antigen detection tests Tests for the detection of P. brasiliensis antigen appear to be useful, particularly in immunocompromised patients. They may be helpful in evaluating the response to treatment. No validated commercial tests are currently available. Antigen tests for diagnosis of blastomycosis and histoplasmosis give false-positive reactions with urine and other body fluid samples from patients with paracoccidioidomycosis.

Differential diagnosis There may be confusion with empty C. immitis spherules. Blastomyces dermatitidis occasionally forms multiple buds and must therefore be distinguished from P. brasiliensis. Sometimes yeast cells with single buds or small yeast cells may be seen that cannot be differentiated from other fungal pathogens, such as Histoplasma capsulatum, Sporothrix schenckii or capsule-deficient Cryptococcus neoformans.

Treatment Therapy includes sulfonamides, amphotericin B and azoles.

Pulmonary candidiasis Introduction Candida, a genus of yeasts, is a human commensal frequently found on the skin and in the mouth, gastrointestinal tract and vagina.219,220 Candida spp. remain the most important cause of fungal disease in humans worldwide, with most infections acquired from an endogenous source.219

Organism Whilst around 200 species are currently recognized within the genus, fewer than 20 have been implicated in human disease. Approximately 65% of species are unable to grow at 37 C, thwarting their ability to cause disease.221 Five species, C. albicans, C. glabrata, C. parapsilosis, C. tropicalis and C. krusei, cause more than 90% of invasive candidal infections.220

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Figure 43. Candida spp. Hyphal, pseudohyphal and yeast forms are apparent in this lung sample from a case of aspiration pneumonia.

As molecular analysis becomes more common, new species have been recognized. One is C. dubliniensis, described in 1995.222 Furthermore, several “species” are species complexes, incorporating a number of morphologically identical species.223 Examples include C. parapsilosis complex, which comprises C. parapsilosis, C. orthopsilosis and C. metapsilosis.224 C. albicans grows as rounded 3 to 5 μm yeast cells with pseudohyphae made of chains of elongated yeast cells, and/or 3 to 5 μm wide septate hyphae (Figure 43).

Epidemiology and pathogenesis Invasive candidiasis has a bimodal distribution, peaking at both ends of the age spectrum: 75 per 100 000 children younger than 1 year of age and 26 per 100 000 adults older than 65 years of age.225 C. albicans is the most prevalent species worldwide, accounting for between 45 and 70% of invasive disease.63 C. dubliniensis may often be misidentified as C. albicans, since both species produce germ tubes, a common test used for yeast identification. Recent studies show a shift towards increasing prevalence of non-albicans Candida, particularly C. glabrata, C. parapsilosis, C. tropicalis and C. krusei.220,226 Increased use of fluconazole prophylaxis may partly account for increased infection rates with less susceptible species, including C. glabrata and C. krusei.227 Intact skin and mucosal surfaces provide an effective barrier against candidal infection, together with maintenance of the resident microbial flora. If these first lines of defense are breached, subsequent host protection mechanisms comprise a spectrum of innate immunity, followed by specific immune responses mediated by T cells (cell-mediated immunity) or B cells (humoral immunity).228 The main cells responsible for pathogen recognition are circulating monocytes, neutrophils and macrophages. Identification is mediated by pattern-recognition receptors (e.g., Toll-like receptors and lectin receptors), which bind pathogen-associated

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molecular patterns (e.g., components of the Candida cell wall).228,229 The main effector mechanism of these cells is phagocytosis of the yeast, causing death through both oxidative and non-oxidative mechanisms.228 Consequently, abnormalities in the number or function of neutrophils and monocytes are associated with systemic candidiasis.230 Specific protective antibodies may also play a role, including prevention of adherence, antibody opsonization and antibodydependent cellular cytotoxicity.231 Candida pneumonia is increasingly recognized in immunocompromised individuals, especially those with acute leukemia and lymphoma, those undergoing HSCT, or those with candidal esophagitis, intravenous drug abusers or AIDS.232,221 A potential source of candidal pneumonia in neonates is embolization from central venous feeding lines.233 Candida pneumonia in patients without apparent immunodeficiency presents with chronic parenchymal lung damage.234 Candidemia is also associated with increased lengths of ICU and hospital stay,235 neutropenia, use of multiple broadspectrum antibiotics, prior colonization with Candida spp., hemodialysis and uremia.221 Candiduria may be a marker for candidemia in neonates.221 Other factors associated with candidal invasion are burns, vascular grafts, intravenous drug abuse, diabetes mellitus, abdominal surgery and low-birthweight infants.236,233 Disseminated candidiasis is one of the main causes of death in cancer patients with neutropenia.221 Invasive candidal infection is the most common fungal infection in all solid organ transplant patients, other than lung recipients.237 In patients undergoing lung transplantation, Candida is the etiological agent in approximately 20% of invasive fungal infections.238 Candidemia is less common among HIV-positive patients since the advent of highly active anti-retroviral therapy, though it remains a serious complication within this setting.239 Species differences are apparent. C. glabrata is seen more often in adults than in children. Independent risk factors associated specifically with C. glabrata candidemia in ICU patients include recent abdominal surgery, less than 7 days in the hospital and use of fluconazole.240 C. tropicalis is more common in neutropenia and underlying malignancy, whilst C. parapsilosis is usually associated with intravascular lines and may be the most frequent species in neonates.226

Clinical features Pulmonary candidiasis can be divided into primary and secondary types, with Candida reaching the lung either by aspiration or via the bloodstream, respectively. Diagnosis is challenging, since the clinical and radiological presentations are nonspecific.241

Endobronchial spread Primary Candida pneumonia, limited to the lungs, is very rare. The true incidence is unknown, since lung biopsies cannot be performed on every patient with suspected Candida

infection. Only 55 unequivocal cases were reported over a 20-year period in cancer patients.242 Recently, not a single case of Candida pneumonia was found at post mortem of more than 200 ICU patients.243,2 The mechanism of entry is via aspiration of contents from the colonized upper respiratory tract (Figure 43). The most common symptoms include fever and tachypnea, while cough, dyspnea and chest pain may also be present.2,242,241 In infants, the development of oral or pharyngeal thrush during the first 10 days of life is common. This is the probable source of pulmonary infection, rather than inhalation of vaginal Candida. A rare case of true congenital candidiasis was described in a child delivered by cesarean section. The infection appears to have developed within apparently intact fetal membranes prior to delivery.244 Sputum colonization with C. albicans in cystic fibrosis patients predicts a greater rate of decline in lung function and increased hospital admissions. It is unclear whether this is directly related to the yeast or whether other co-species are involved. Interactions between Candida and bacteria, primarily Pseudomonas aeruginosa, exist.245 Radiographically, there are very small asymmetric patchy lesions, predominantly in the lower lobes, or diffuse bilateral areas of consolidation, similar to bronchopneumonia of other causes.5,242,241 In an immunosuppressed rabbit model, the most common thin-section CT findings in early pulmonary candidiasis are multiple peripheral areas of lobular consolidation with or without ground-glass opacity.246

Hematogenous spread More commonly, pulmonary lesions are secondary to candidemia, sourced from the skin or gastrointestinal tract. Skin lesions (10%) and endophthalmitis (10–30%) may be seen. The liver, spleen and kidneys may also be involved.221 Systemic candidiasis may occur in patients treated with cimetidine, suggesting that pharmacological suppression of gastric acid secretion allows fungal colonization of the upper gastrointestinal tract.247 The clinical picture of secondary Candida pneumonia is usually overshadowed by the primary systemic disease, and symptoms range from a low-grade fever to septic shock. Severe sepsis and septic shock are seen in approximately 20% and 40% of Candida cases, respectively.248 Often there is little sign of lung involvement. Candida may also appear in adults in areas of pulmonary fibrosis. In one series of HSCT recipients with pulmonary candidiasis, the most common radiographic finding was multiple nodules, varying in size from 3 to 30 mm, often bilateral (88%), followed by air-space consolidation (65%). Consolidation or abscess formation is suggestive of advanced disease (Figure 44).232

Pathology With inhalational disease, large bronchi may be covered with a gray-yellow membrane. Hyphae and conidia are

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Figure 44. Secondary Candida pneumonia. The computed tomogram from a patient with newly diagnosed acute myelogenous leukemia demonstrates a right upper lobe cavity. (Image courtesy of Dr K. Irion, Liverpool, UK.)

endobronchial, and may be associated with fibrin. Bilateral, symmetrical miliary nodules involve parenchyma and pleura (Figure 45). These 2 to 4 mm randomly distributed irregular single or multiple nodules feature neutrophils and fibrin (Figure 46). Granulomatous inflammation and hemorrhage may also be seen. Generally, clusters of blastoconidia and hyphal elements are apparent within central areas.5,241 Most Candida spp. occur in lung lesions in various forms. Budding yeast cells, blastoconidia and/or hyphal forms, either pseudohyphae and/or hyphae, may be seen. Depending on the species, some or all may coexist within the same lesion. Blastoconidia may be spherical, oval or elongate in shape, measuring from 1 to 8  2.5 to 11 μm.249,250 Hyphae represent the virulent phase of the organism and are associated with tissue invasion.233 True hyphae are slender, uniform in thickness, septate, branching, and may form blastoconidia along their length (Figure 43). The pseudohyphal form consists of elongated oval cells joined together, end to end. Candida is poorly stained in H&E sections, but is intensely colored red by PAS or black by silver stains. Blastoconidia and pseudohyphae usually stain Gram-positive.250 C. glabrata, unlike other Candida spp., does not usually show hyphal forms in sections. In vitro studies show pseudohyphal growth.251,252 In H&E sections, C. glabrata blastoconidia show variable staining and tend to be amphophilic.253

Figure 45. Miliary candidiasis. Scattered subcentimeter yellow tan nodules fill peripheral lung (arrow). A dominant lesion is not seen.

Diagnosis Fungal autofluorescence is reportedly effective in recognizing Candida in H&E sections,254 but others suggest that little clinically useful information is obtained.255 Whitening agents, such as Calcofluor white, a nonspecific fluorochrome stain that binds strongly to chitin in the fungal cell wall, can help distinguish organisms in tissue. Immunohistochemical and direct immunofluorescence methods using specific monoclonal antibodies can be performed on paraffin-embedded tissue sections to aid identification.256

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Figure 46. Pulmonary candidiasis. Suppurative pneumonia with neutrophils, fibrin and vascular congestion often mask fungal yeast and hyphal forms.

Since Candida spp. constitute part of the normal human flora and readily colonize mucosal surfaces, the diagnosis of candidiasis can be made conclusively only by direct demonstration of organisms in tissue. Cultures are required for identification.

Chapter 7: Pulmonary mycotic infections

Candida frequently colonizes the upper respiratory tract, particularly in hospitalized patients, and hence the specificity and positive predictive value of sputum cultures are low. The diagnostic value of BAL may also be poor.257,243 In contrast, the negative predictive value of both sputum and BAL may be 90%.257 Respiratory specimens often require digestion before examination. Since Candida can be present in low numbers, centrifugation concentrates organisms.258 Detection of Candida in the bloodstream is vital for diagnosing invasive disease. Blood cultures are insensitive markers of invasive fungal infection, with positivity rates approaching 50% at best in neutropenic patients. Repeated samples are often performed before Candida is isolated, and this may be at a late stage of the infection.258,259 It is important to establish the species involved for both therapeutic and prognostic reasons.258 Traditionally, identification can only be achieved after the organism has grown, which may take up to 48 hours. Candida species grow on standard mycological media at either 30 C or 37 C and appear as smooth or wrinkled, white-tan pasty colonies. C. albicans forms germ tubes and chlamydospores under certain conditions. Definitive identification is conventionally by biochemical (usually commercial kit-based) and morphological (macroscopic and microscopic) characteristics typically taking a further 24 to 36 hours after the organism has been cultured. Speed of identification is important and more rapid tests exist. The most common test used is that of germ tube production within 3 hours, a characteristic seen in 95 to 97% of clinical strains of C. albicans and C. dubliniensis. Commercial monoclonal antibody-based latex agglutination tests are also available for identification of C. albicans, C. dubliniensis and C. krusei. A result is obtained in 5 minutes with sensitivity and specificity both reportedly between 95 and 100% depending on species tested.260–262 GLABRATA RTT is also a commercially available test, based on rapid hydrolysis of trehalose, for the identification of this yeast within 15 to 20 minutes. Sensitivity and specificity ranged from 84 to 100%, depending on initial culture medium used.263

Non-culture-based methods Serological and molecular methods are potentially more rapid and sensitive than conventional culture, and more specific than microscopy. They may allow early diagnosis of invasive disease.

Antibody detection Detection of anti-Candida antibodies has a role in both the diagnosis of invasive disease and monitoring therapy in the immunocompetent host. This is less so in the immunocompromised patient as antibody response may be diminished or non-existent. Discrimination between colonization and disease can also be problematic. Tests are available to measure antibody levels to various Candida antigens, both cell wall and cytoplasmic. These assays often suffer from poor sensitivity and specificity.250

A commercial ELISA is available which detects antibodies to Candida mannan, a major cell wall constituent. Using the updated Candida Ab Plus kit, patients with malignancy or undergoing an HSCT demonstrated sensitivity of 30–64% and specificity of 60–92%, depending on duration of neutropenia.264 A diagnosis of invasive candidiasis should be based on a combination of both antibody and antigen tests (see below).265–267

Antigen detection Detection of circulating antigens has been widely used in the diagnosis of invasive candidiasis. As with antibody testing, difficulties arise in distinguishing between colonization and infection. Mannan is the predominant antigen circulating in invasive disease. It is transient in the bloodstream, hence testing should be frequent. Commercially available kits demonstrate good specificity but low sensitivity.250,264 Sensitivity of the Platelia Candida Ag test differs between species, the highest being C. albicans and lowest C. parapsilosis or C. krusei.

(1,3)-b-D-Glucan detection (1,3)-b-D-Glucan is a cell wall constituent of many fungi, including Candida spp. This polysaccharide circulates in the blood of patients with invasive mycoses, including candidiasis and aspergillosis, but not cryptococcosis or mucormycosis. bD-Glucan can be used as a screening test and may be useful as a diagnostic marker in adult invasive fungal disease. Sensitivity and specificity values for proven and probable invasive candidiasis range from 50 to 83% and 83 to 100%, respectively.

Molecular methods Nucleic acid-based procedures are available for detection and identification of Candida species, both from cultures and directly from clinical specimens.63 DNA-based amplification techniques are the most widely used, including conventional (with post-amplification detection step) and real-time PCR assays. Additional technologies for identifying the causative species have been evaluated, including a commercially available DNA-based microarray kit.268 RNA-based methods have also been reported.269 Molecular testing offers greater speed and enhanced sensitivity. This superior sensitivity is also a major drawback, resulting in difficulty in discriminating between colonization and infection.250 There are many differences in specimen type and DNA extraction protocols, thus clinical sensitivity and specificity vary significantly. Studies in hematological and critically ill patients detail sensitivity rates of 90.9–100%, and a specificity of at least 97%.63 No validated or standardized methods for either specimen type or DNA extraction are currently available.

Differential diagnosis In histological sections, blastoconidia seen in isolation may be confused with Histoplasma capsulatum, Blastomyces dermatitidis or poorly encapsulated Cryptococcus neoformans. None of

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these other fungi produce hyphal elements in tissue. In particular, C. glabrata may be distinguished from H. capsulatum by its larger size, more frequent budding and predominantly extracellular situation.253 Elongated pseudohyphae may give the appearance of branching, presenting the possibility of confusion with Aspergillus or other filamentous fungal hyphae. Careful examination is required to determine whether the hyphae are septate with acute-angled branching, as is seen with Aspergillus.4 Yeast cells are not observed with any of the filamentous fungi.

Prognosis and treatment Prognosis of primary pulmonary candidiasis is uncertain. Due to the difficulties in diagnosis, and the fact that patients may respond to empirical antifungal treatment, true incidence is unknown. However, candidal pneumonia directly contributed to death in 84% of 31 cancer patients over a 20-year period.242 In secondary pulmonary candidiasis, outcome is linked directly to the underlying disease and recovery of the patient’s immune system. Crude mortality rates generally exceed 50%, and certainly reflect the performance status of patients. The attributable mortality rate of candidemia is high, estimated between 25 and 38%.270 In particular, C. glabrata, C. tropicalis and C. krusei infections are associated with poor outcomes in more than 40% of patients.271 Patients are usually treated with fluconazole, sometimes in combination with another azole compound. C. krusei is intrinsically resistant to fluconazole whilst C. glabrata may acquire resistance. Increased use of fluconazole prophylaxis may at least partly account for increased infection rates with these species.227

Pneumocystis jirovecii infections Introduction Pneumocystis was discovered by Chagas in 1909 and Antonio Carinii in 1910 and thought to be a part of the trypanosome’s life cycle. The original name, Pneumocystis carinii, was in honor of Carinii.272 Der Meer and Brug and Vanek and Jirovec recognized the organism as the cause of interstitial pneumonia affecting premature and debilitated infants in central and eastern Europe in 1942.273 In the 1960s, it emerged in adults as a major cause of pneumonia in immunocompromised patients on steroids and with cancers, while the organism became a well-known entity as AIDS spread across the globe. Recently the name was changed to Pneumocystis jirovecii.274

Organism The taxonomy of this organism has been controversial. It was once considered a protozoan, but is now classified in the fungal kingdom. This is partly based on its ultrastructural features. In addition, analysis of ribosomal RNA sequences suggests Pneumocystis is most closely related to Saccharomyces, while the mitochondrial gene sequences of P. jirovecii show fungal homology.

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Three developmental stages are described.275 Trophozoites are 1 to 5 μm in size. Cysts are larger, 3 to 5 μm, and contain up to eight sporozoites. When the cyst reaches maturity, sporozoites are liberated and mature into haploid trophozoites. Trophozoites multiply asexually, possibly by binary fission, but they appear to develop into cysts by a sexual cycle.

Epidemiology Pneumocystis jirovecii normally exists in the lungs of humans and other mammals.276 It has a worldwide distribution. Antibodies to Pneumocystis are virtually absent before 1 year of age but increase rapidly thereafter. By the age of 4 years, more than 80% of children show latent infection, which persists throughout adult life. Pneumocystis DNA has been detected at low levels in the air of hospital corridors and at high levels in the air of rooms occupied by patients with Pneumocystis pneumonia (PCP).277 The only evidence for aerogenous transmission is from animals. Infected animals placed in close proximity to germfree immunosuppressed rats transmit the organism. However, there are structural differences between rat and human forms of Pneumocystis. An interval oligonucleotide PAZ 1–2/L1 differs in the two species. The exact mode of transmission remains to be clarified. An environmental reservoir, such as soil, has not been identified. Reactivation of a latent infection is considered the main mechanism of infection in immunosuppressed patients.278 Evidence from human and animal studies suggests the mammalian host acts as the reservoir for Pneumocystis. In those with HIV infection, PCP develops when the CD4 count falls below 200/mm3. However, not all cases represent reactivation of latent infection as autopsy studies fail to identify Pneumocystis DNA in post mortem lung from immunosuppressed patients. Lavage fluid from immunosuppressed and immunocompetent patients undergoing bronchoscopy detected Pneumocystis DNA only in those patients who had PCP diagnosed by conventional GMS staining. Reactivation of preexisting latent infection is not, therefore, a completely satisfactory explanation for PCP.279 It seems that reinfection is a likely cause in some cases. The origin and method of spread of the organism requires further clarification. Pneumocystis jirovecii pneumonia occurs almost exclusively in immunocompromised hosts. During and after the Second World War outbreaks were reported in malnourished children in European orphanages, and were described as “plasma cell pneumonia”. PCP is infrequently seen in Africa, particularly in Uganda, where the incidence of AIDS is the highest in the world. This suggests an absence of the organism in that environment. Pneumocystis pneumonia was first recognized as a major complication in immunosuppressed patients during the 1960s and 1970s, when chemotherapy for cancer, particularly leukemias and lymphomas, was developed. T-cell immune defects are recognized as the major risk factor, but

Chapter 7: Pulmonary mycotic infections

hypogammaglobulinemia per se is a risk. In transplants, the risk of PCP is greatest when T-cell function is lowest. Infection may occur 2 or 3 years after transplantation in cardiac transplant patients, if there have been multiple episodes of rejection. Since the 1980s, PCP has most frequently been seen in patients with AIDS and at one time affected up to 80% of HIV-positive individuals.280,281 In addition, a group of elderly patients with chronic illnesses but no overt immunosuppression have been described as suffering from the disease.

Clinical features Clinical disease due to Pneumocystis jirovecii usually presents with an insidious onset of dyspnea, fever and non-productive cough. Sputum production and chest pain are uncommon. Physical examination is also nonspecific but may reveal tachycardia and tachypnea. Lung auscultation is typically normal. Laboratory studies are non-diagnostic. Radiology may be normal but often reveals bilateral diffuse infiltrates, which commonly originate in the perihilar region, with peripheral extension as the disease progresses and consolidates.282 Atypical radiographic presentations such as unilateral disease, nodules, cavities, lymphadenopathy and pleural effusions have also been described.283,284 There are significant clinical differences between affected AIDS and non-AIDS patients. In the former, the onset of the disease tends to be more insidious, with a more prolonged course. It responds less well to therapy and has a high recurrence rate. Atypical presentations in AIDS cases may also occur. These include localized disease, usually confined to the upper lobes, cyst formation or cavitation, and spontaneous pneumothorax.

Pathology

Classic (typical) Pneumocystis pneumonia Typically, PCP causes diffuse disease with consolidation.285 Both lungs are bulky and contain firm areas of yellow or pink tissue, with intervening normal lung. Pale consolidated areas may resemble lobular pancreatic tissue. The septal tissues are prominent and thickened. The pleura is usually normal, apart from interstitial emphysema in some cases. In very acute and fatal infantile infections, the lungs may resemble liver. Many alveoli and alveolar ducts are filled with characteristic amorphous, foamy eosinophilic material (Figure 47a,b). Careful examination may reveal small basophilic dots (trophozoite and sporozoite nuclei) within the foamy spaces, but the organisms are not easily visible on H&E-stained material (Figure 47c). There is variable lymphoplasmacytic interstitial pneumonia (Figure 47d). Eosinophils are occasionally a feature. Alveolar walls may appear thickened due to the cellular infiltrate and edema, but fibrosis is not a feature in the early stages of the disease. Damaged and regenerating type II pneumocytes are seen; diffuse alveolar damage is a rare finding (Figure 47e). The amount of intra-alveolar exudate varies, and it may be

only focal. It may be confused with intra-alveolar edema or alveolar proteinosis. A high index of suspicion is required in immunosuppressed patients. One should also be vigilant for synchronous opportunistic infections, including viral and protozoal processes (Figure 47f). On GMS-stained sections organisms are round, oval, or crescent-shaped, ranging from 5 to 7 μm in diameter. The cyst walls are black and a very typical feature is a focal, darkly staining area of capsular thickening, 1 to 23 μm in diameter (Figure 48). In treated patients, degenerative changes occur with fragmentation and blurring of the cyst walls.286

Atypical features of Pneumocystis jirovecii pneumonia Pneumocystis jirovecii pneumonia may be associated with a diffuse lymphoplasmacytic interstitial infiltrate, which is more prominent in children with the endemic form of the disease. In AIDS patients, parenchymal dystrophic calcification may occur, usually associated with cavitation. Interstitial disease is an important complication in some cases. Diffuse alveolar damage may progress to respiratory failure (Figure 47e). Some patients with treated PCP show organization of intra-alveolar exudates. Damage to alveolar epithelium may occur when the trophozoites attach to type I pneumocytes. Fibrosis is generally mild, but occasionally is more severe, with remodeling. Cysts or centrally cavitating nodular lesions are found most frequently in the upper lobes and are frequently bilateral (Figure 49). Intrapulmonary or subpleural cysts vary in size from microscopic lesions to grossly visible. Localized nodular PCP may be unilateral and simulate carcinoma or tuberculosis (Figure 50a). Granulomatous inflammation is seen in approximately 5% of cases and may involve overlying visceral pleura. About half of the patients with nodular PCP show granulomatous necrosis (Figure 50b). This ranges from isolated giant cells related to the alveolar exudate to well-formed compact epithelioid and giant cell granulomas. The necrotic material may calcify (Figure 50c). Occasionally P. jirovecii cysts infiltrate arteries, veins and capillaries. This is associated with a transmural chronic inflammatory infiltrate and vascular necrosis. Vascular spread and vasculitis may be followed by extrapulmonary dissemination (Figure 51).287

Extrapulmonary Pneumocystis jirovecii infection Dissemination to extrapulmonary sites is reported in both AIDS and non-AIDS patients, but is commoner in the former.286 Regional lymph nodes are most affected, followed by liver, spleen and bone marrow. Any organ may be involved, including the brain, pericardium, thymus and palate. Transplacental spread has also been recorded.

Diagnosis Pneumocystis jirovecii pneumonia cannot be cultured and is diagnosed by histology or cytology. Tissue is obtained by

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(a)

(c)

(e)

Figure 47. Pneumocystis jirovecii pneumonia. (a) Classic histology features intra-alveolar pink froth and interstitial lymphoplasmacytic infiltrates. (b) The froth harbors many organisms. It is finely granular, unlike both pulmonary edema and alveolar proteinosis. (c) Faint basophilic dots, trophozoite and sporozoite nuclei, may be seen, depending on the thickness of the tissue section and the laboratory staining process. Type II pneumocytes are reactive. (d) Patchy lymphoplasmacytic interstitial infiltrates are seen in almost all cases. (e) Intra-alveolar froth may be found adjacent to hyaline membranes. (f) Calcifications should alert one to a concomitant cytomegalovirus infection.

(b)

(d)

(f)

transbronchial biopsy, bronchial brushings, percutaneous lung biopsy, open lung biopsy or BAL. Saline-induced sputum analysis is gaining in popularity. The absence of Pneumocystis from an induced sputum sample does not exclude the diagnosis. In AIDS patients, this procedure has a sensitivity of approximately 55% using GMS but sensitivity is increased by immunostaining. The microscopic demonstration of typical P. jirovecii octonucleate cysts or trophozoites in tissues or body fluids are diagnostic. Often the organisms present as large clusters, in

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which it may be difficult to distinguish the different life-cycle stages. Collapsed organisms are usually found intermingled with intact ones. Unlike other pathogenic fungi, P. jirovecii does not bud. Giemsa and Wright stains detect both the cyst and trophozoite forms, but do not stain the cyst wall. GMS, PAS, Calcofluor white and toluidine blue stain the cyst wall. Trophozoites and cysts can also be detected by direct or indirect immunofluorescent staining of BAL or induced sputum smears with fluorescein-conjugated monoclonal anti-P. jirovecii antibodies.

Chapter 7: Pulmonary mycotic infections Figure 49. Pneumocystis jirovecii pneumonia. Multiple cavitary masses vary in size.

Figure 48. Pneumocystis jirovecii. Round and collapsed organisms are highlighted with a GMS stain. Areas of capsular thickening are apparent.

(a)

(b)

(c)

Figure 50. Pneumocystis jirovecii pneumonia. (a) Solitary coin lesions may be encountered in immunocompetent individuals. (b) Necrotizing granulomas have no particular features suggesting this infectious etiology. (c) Calcifications may be seen.

Depending on the monoclonal antibody selected, staining may target only the cysts or all forms of the organism. In Papanicolaou-stained respiratory cytology specimens, the organisms blend into the mucous blue-green background. Monoclonal antibodies reacting specifically with either trophozoites or cyst walls are commercially available and are more sensitive than silver stains in both tissue sections and cytology. In situ hybridization using DNA probes or PCR increases sensitivity and specificity.288–290

Serological tests

Figure 51. Pneumocystis jirovecii pneumonia. Granulomatous vasculitis usually indicates systemic dissemination.

Serological tests for antibodies to P. jirovecii are useful for epidemiological studies, but not for diagnosis. Most humans become seropositive early in their childhood and probably come into contact with the organism many times over their lifetimes. Serological responses to certain fragments of the major surface glycoprotein of P. jirovecii are reportedly predictive for some clinical outcomes, and may be of use in monitoring response to therapy.

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b-D-Glucan detection (1,3)-b-D-Glucan is a major cell wall polysaccharide component of many fungi, including P. jirovecii. The Fungitell assay can be used for the presumptive diagnosis of a number of invasive fungal diseases. (1,3)-b-D-Glucan levels are elevated in patients with PCP. The sensitivity and specificity of the assay for diagnosis of the infection are still undergoing evaluation.291

Molecular diagnostics Polymerase chain reaction offers promise as a rapid diagnostic method, and is being introduced in some centers.292,293 Detection of P. jirovecii can be accomplished by targeting the large subunit of the mitochondrial ribosomal RNA gene or the major surface glycoprotein gene as both these genes are present in multiple copies per organism. Although more sensitive than conventional staining methods, the presence of P. jirovecii DNA has not been strictly correlated with underlying disease, and a positive result may only represent colonization. Standardized quantitative PCR assays are now commercially available in some countries (MycAssay Pneumocystis PCR, Myconostica Ltd., Manchester, UK).

Differential diagnosis P. jirovecii does not bud and this feature can be used to distinguish between this organism and fungi, such as small variants of Blastomyces dermatitidis, Candida glabrata, capsuledeficient Cryptococcus species and Histoplasma capsulatum.

Treatment and prognosis The treatment of choice for PCP remains trimethoprimsulfamethoxazole. Pentamidine is very much a second-line treatment. Corticosteroids are often used as an adjuvant agent in those with moderate or severe pneumonia. The mortality rate for AIDS patients remains 10 to 20%.294 Sixty-five percent of treated patients relapse. Children have a worse prognosis and the initial infection is frequently fatal, regardless of their CD4 counts.

Pulmonary sporotrichosis Introduction Sporotrichosis is a chronic infection of humans and animals by the dimorphic saprophytic fungus, Sporothrix schenckii.295 The disease usually results from inoculation of the fungus into the skin.

Organism S. schenckii is a dimorphic fungus, growing at room temperature as a mycelial form and developing at 37 C in the tissues into a yeast form.296 Yeast forms are round, oval or elongated, “cigar-shaped” with a diameter of 2 to 6 μm (Figure 52a). Budding yeasts are relatively uncommon and budding may be multiple (Figure 52b). Branching hyphae may occasionally be found.

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Epidemiology The organism is found in soil, wild and domesticated plants, timber, moss, straw and other organic matter.297 The disease usually results from inoculation of the fungus into the skin, classically by rosebush thorns, but also associated with zoonotic spread from domestic cats, rodents, parrots, dogs, horses, armadillos and fire ants.298,299 Systemic sporotrichosis may be localized to a single organ system, such as the joints or lower respiratory tract. It may disseminate to involve multiple organs, in some cases from a primary pulmonary focus.300 Most pulmonary cases are due to primary infection of the lung, resulting from inhalation of conidia of S. schenckii.301–303 Although no definitive link has been established between sporotrichosis and cellular or humoral immunodeficiency, extracutaneous disease is associated with relative immunocompromised states, most commonly chronic alcohol abuse, chronic steroid use, AIDS and diabetes mellitus. Pulmonary sporotrichosis is associated with lung diseases, such as chronic obstructive pulmonary disease.304,305

Clinical features After an incubation period of 3 to 12 weeks, the inoculation site develops a painless nodule, which becomes discolored and ulcerates. If untreated, the infection spreads along lymphatics, causing a chronic, slowly progressive lymphocutaneous reaction. Pulmonary sporotrichosis most commonly presents as chronic cavitary fibronodular disease.298 Presenting pulmonary complaints, including hemoptysis, fever, chills, cough, chest pain, dyspnea, malaise and weight loss, are nonspecific.305 Radiographic findings are nonspecific and include bilateral apical and cavitary lesions. Thus, this disease resembles tuberculosis and histoplasmosis. Solitary peripheral pulmonary nodules and reticulonodular infiltrates are rarely described. Fluid levels in the cavities are infrequent and fungal balls have not been documented.

Pathology Macroscopic findings include 0.5 to 6 cm cavitary masses or bilateral peripheral nodules. The former feature thin walls containing nodular gray to yellow tissue peripherally and hemorrhagic necrotic tissue centrally. These cavities communicate with bronchi. The nodules are yellow-tan with necrotic centers and do not communicate with airways. Bilateral diffuse granulomatous nodules are also reported. Cavities are lined by granulation tissue with giant cells. Hilar lymph nodes may be sufficiently enlarged to cause bronchial obstruction. Satellite lesions surrounding small pulmonary vessels consist of macrophages, lymphocytes, plasma cells and a few eosinophils without central necrosis. Less frequently, the pulmonary architecture is effaced by numerous, often confluent, large necrotizing granulomas.

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Figure 52. Sporothrix schenckii complex. (a) Yeast forms are round and elongated in this lung biopsy (Grocott methenamine silver stain). (b) Sporothrix schenckii asteroid body containing a budding yeast cell (Brown and Hopps stain). (c) This organism is surrounded by eosinophilic Splendore-Hoeppli material (so-called asteroid body). (Images courtesy of R. Neafie, Washington DC, USA.)

Eosinophils are infrequent at the periphery and there is a lymphoplasmacytic infiltrate. Smaller granulomas, both necrotizing and non-necrotizing, are seen around bronchi and bronchioles. A similar histology is seen in some of the regional lymph nodes. Hematoxylin & eosin sections rarely show the fungi, PASstained tissue sections demonstrate brilliant red-purple yeasts, and GMS also highlights the yeast in necrotizing granulomas, giant cells and macrophages. The organism may be detected on sputum cytology, where macrophages contain small intracellular eosinophilic yeasts with a faint halo. Extracellular yeast

forms may also be found. The organisms may be surrounded by eosinophilic Splendore-Hoeppli material as a stellate radial corona (so-called asteroid body) (Figure 52c).

Diagnosis Direct examination of clinical material, such as pus or tissue, is less sensitive than culture, because the organisms are usually present in small numbers. Detection of typical round, oval or cigar-shaped cells of S. schenckii helps confirm the diagnosis. Immunofluorescence is a sensitive and specific method for detecting small numbers of S. schenckii.

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Culture A definitive diagnosis of sporotrichosis depends on isolation of the fungus from clinical material. Samples should be inoculated onto several media, including Sabouraud’s agar and brain-heart infusion agar. Identifiable mold colonies should appear in 3 to 5 days. The color of the colonies usually changes from cream or light brown to dark brown or black with age.

Serological tests Serological tests are not yet widely available for the diagnosis of sporotrichosis. Immunodiffusion and agglutination tests can be used to detect antibodies to S. schenckii, and are more helpful in diagnosing extracutaneous forms of sporotrichosis than in detecting cutaneous infection.

Differential diagnosis The morphology of sporotrichosis may not be sufficiently distinctive to allow differentiation from other yeasts, such as Candida, capsule-deficient variants of C. neoformans, or Histoplasma. Hamazaki-Wesenberg bodies, most often seen in lymph nodes, may be mistaken for S. schenckii yeast forms (see Chapters 2 and 13).

Treatment and prognosis Amphotericin B and itraconazole are effective agents but chronic cavitary disease requires surgical resection in addition to chemotherapy. However, the prognosis for those with disseminated disease is poor.

Rare pulmonary fungal infections Adiaspiromycosis Adiaspiromycosis (syn.: adiaspirosis, haplomycosis) is ubiquitous in rodents and small wild mammals throughout the world but rarely affects humans. The first established human case was diagnosed in France in 1964. Further cases have been described in Eastern Europe, Brazil and other countries. The disease is caused by the filamentous fungus Emmonsia parva (previously Chrysosporium parvum var. crescens), first described in Arizona rodents. The term adiaspiromycosis is derived from the conidia of this fungus, the adiaconidia, which show the unique property of progressive enlargement with replication.306 The inhaled conidium, 2 to 4 μm in diameter, can grow to 500 or 600 μm. Proliferation or replication of adiaconidia does not occur in human tissues. The disease remains confined to the lungs.307 Disease depends on the spore burden and immune status of the host. The disease is usually self-limiting, benign and localized, with few if any symptoms. Manifestations range from asymptomatic infection to necrogranulomatous pneumonia, respiratory failure and, rarely, death.308,309 One case was reported in an individual with pulmonary adenocarcinoma.309 Some infections have been discovered during the course of an

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autopsy or examination of the lung for other causes, such as bronchiectasis.310 Macroscopically, multiple or occasionally single up to 2.0 cm pulmonary lesions are gray-white, and firm. The centers of these nodules are glassy or gelatinous and the whole lesion resembles frog spawn. Microscopically, one sees nodular interstitial granulomas throughout the lung resembling miliary tuberculosis (Figure 53a). Up to 600 μm adiaconidia with thick membranes are encircled by granulomatous reaction. Concentric layers of fibrous connective tissue with eosinophils and a variable number of lymphocytes, plasma cells, macrophages and fibroblasts surround the organism (Figure 53b,c). Eosinophilic globules (1 to 3 μm) may be seen along the inner cell wall. A GMS stain highlights the three wall layers (Figure 53d). Of note, all the granulomas are at a similar stage of development, reflecting a single exposure without subsequent replication within the lung. The pulmonary alveoli not affected by the nodular lesions may contain macrophages and neutrophils.311 Adiaspiromycosis is diagnosed histopathologically. Culture of the organism is difficult and sputum and BAL specimens are seldom positive. At 25 C, Emmonsia species grow as mold colonies. E. crescens produces adiaspores in vitro when cultivated at 35 to 37 C. E. parva requires culture at 40 C to produce adiaspores, while E. pasteuriana produces yeast-like cells on brain-heart infusion agar at 37 C. Adiaspores must be distinguished from the tissue forms of Coccidioides spp. or Rhinosporidium seeberi, organisms that produce large spherules in tissue. In contrast to Coccidioides, the adiaspores are much larger, have a thicker wall and do not contain endospores. The sporangia of R. seeberi are distinguished from those of Emmonsia by the zonation of the internal sporangiospores and distinctive eosinophilic globules in mature sporangiospores. E. pasteuriana is a differential consideration but, unlike E. parva and E. crescens, it produces small yeast-like budding cells, larger thick-walled cells and pseudohyphae in tissue. Adiaspiromycosis is usually a self-healing disease. Since the fungus does not multiply in humans, the utility of chemotherapy is not certain. Nevertheless, one case resolved with ketoconazole.312

Malassezia spp. Malassezia is a genus of lipophilic basidiomycetous yeasts. Although they become constituents of normal human skin flora by 6 months of age, organisms are responsible for several mild but recurrent cutaneous human diseases. Less frequently, these organisms cause life-threatening systemic infection in critically ill low-birth-weight infants and other immunocompromised and debilitated individuals.313 Based on molecular analysis, the M. furfur species complex now includes M. sympodialis, and seven new taxa: M. globosa, M. obtusa, M. restricta, M. slooffiae, M. dermatis, M. japonica and M. yamatoensis. Other members of the complex have been reported only from animals.

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Figure 53. Pulmonary adiaspiromycosis. (a) Emmonsia parva var. crescens adiaspores provoke an intense fibrogranulomatous reaction. (Image courtesy of R. Neafie, AFIP, Washington, USA.) (b) The brisk host inflammatory response is apparent. (Image courtesy of Dr J. Denson, Plymouth Hospitals NHS Trust, Crownhill, UK.) (c) The nodular granulomatous lesion leaves surrounding lung unaffected. (Image courtesy of R. Neafie, Washington DC, USA.) (d) The thick-walled adiaspore is strongly Grocott methenamine silver positive. Three layers are not always apparent. (Image courtesy of R Neafie, Washington DC, USA.)

While M. furfur is dimorphic in the skin, only yeast forms are seen in parenchymal lesions. In tissue sections the fungus is round to oval, up to 5 μm, with many unipolar budding forms sprouting through a collarette. PAS and tissue Gram stains highlight the yeast forms, but the organism is best seen on GMS-stained tissue sections (Figure 54a). Exposure to lipid-rich intravenous infusions through a central venous catheter is the single most important risk factor for systemic Malassezia infection in both infants and older patients.313 Among neonates, other risk factors include low birth weight, early gestational age and length of hospitalization. An investigation of one outbreak of M. furfur infection among low-birth-weight infants, most of whom received intravenous lipids, identified duration of antimicrobial treatment as an additional risk factor. Among older immunocompromised children and adults, infection has occurred in conjunction with a range of underlying illnesses, including hematological

malignancies, HIV infection and stem cell and solid organ transplantation. In immunocompromised patients, Malassezia spp. may be associated with several skin conditions and systemic diseases, including folliculitis, seborrhoeic dermatitis, catheter-related fungemia, sepsis and a variety of deeply invasive infections314 as well as catheter-related infections.313,315 Although most infections appear to be sporadic, nosocomial outbreaks of systemic Malassezia infection are reported. The organism can be transmitted from an infected or colonized low-birth-weight infant to other infants via the hands of healthcare workers. Nosocomial outbreaks of M. pachydermatis infection have been reported. In one outbreak, the organism was introduced into the unit on healthcare workers’ hands after being colonized from pet dogs at home. The organism persisted in the unit through patient-to-patient transmission, but the outbreak was interrupted by improving hand-washing practices. Malassezia species can persist for prolonged periods

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Figure 54. Malasseziasis. (a) Intra-alveolar less than 5 μm yeast are monomorphic round to oval with unipolar budding through a collarette (Grocott methenamine silver stain). (Case courtesy of Joseph F. Tomashefski, Jr., MD, Cleveland, OH, USA.) (b) Organizing diffuse alveolar damage in a lung from an infant with a pulmonary infection, due to disseminated M. furfur. (c) An organizing septic thrombus present within the lung infected with M. furfur. (Case courtesy of Joseph F. Tomashefski, Jr., MD, Cleveland, OH, USA.)

on incubator surfaces, providing another source for continued transmission and outbreaks. The signs and symptoms of Malassezia fungemia and sepsis are generally nonspecific. In infants, it usually presents as fever and/or respiratory distress. The latter may manifest as interstitial pneumonia. Leukocytosis or leucopenia and thrombocytopenia are common. Less frequently there is poor feeding, hepatomegaly and splenomegaly. No signs of infection have been noted at catheter insertion sites, nor has a skin rash been evident in infants with systemic infection. Catheter-associated Malassezia fungemia is a sporadic illness in immunocompromised children and adults. The clinical manifestations are different from those described above. Fever is a consistent presenting symptom. Other symptoms and clinical signs include chills, rigors, myalgia, nausea, vomiting, respiratory distress, pneumonia, leucopenia or leukocytosis and thrombocytosis. Malassezia fungemia should be considered in any febrile patient (particularly if there is clinical and radiological evidence of pneumonia)

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receiving lipid-containing parenteral nutrition through an indwelling vascular catheter, especially if routine blood cultures are sterile. Catheter-associated Malassezia fungemia may result in embolic infection to the heart and lungs and, less frequently, dissemination to other organs, such as the skin, kidneys, liver, spleen and brain. Unusual manifestations include thrombophlebitis, meningitis, septic arthritis, soft-tissue abscesses and catheter-associated peritonitis in peritoneal dialysis patients. Lung pathology includes interstitial pneumonia, scattered broncho- and vasocentric necrotizing nodules, so-called mycotic thromboemboli rich with neutrophils, as well as diffuse alveolar damage (Figure 54b). Catheter-associated disease also demonstrates mycotic thrombi around the tips of indwelling catheters, and endocardial vegetations. There may also be acute respiratory distress syndrome and septic thrombi (Figure 54c). While H. capsulatum and C. neoformans may be approximately the same size as M. furfur, both have narrower budding

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Figure 55. Phaeohyphomycosis. (a) A well-circumscribed necrotic dermal lesion is typical. (b) Disease is caused by a great variety of fungi that usually exist in tissue as pigmented oval yeast forms and short hyphae. (Images courtesy of R. Neafie, Washington DC, USA.)

necks, and the latter has a greater size range. Candida yeast forms lack unipolar buds. Malassezia fungemia has sometimes been diagnosed following detection of the organism in stained smears prepared from catheter blood specimens. The diagnosis is most often based on isolation of the organism from blood taken through the catheter. In instances where the catheter can be removed, the tip of the catheter should be inoculated onto Sabouraud’s agar overlaid with sterile olive oil or another lipid-enriched medium. The lipid concentration of conventional broth and agar media is usually insufficient to support the growth of Malassezia. The exception is the non-lipiddependent species, M. pachydermatis. It appears blood from patients receiving parenteral nutrition often contains sufficient lipids to support initial growth of these organisms in culture.

Phaeohyphomycoses Phaeohyphomycoses are cutaneous and systemic infections caused by darkly pigmented opportunistic fungi.316–319 More than 80 genera are common wood and soil saprophytes. Common fungi within the group include Alternaria, Bipolaris, Cladosporium, Curvularia, Exophiala, Exserohilum, Scedosporum prolificans and Thermomyces. Infections are most commonly subcutaneous via wooden splinters or thorns, but systemic disease occurs following fungal mycelial inhalation. Systemic disease usually affects immunocompromised patients, some with a history of “asthma” and bronchiectasis.320 Lung disease is usually asymptomatic, but a review of seven cases of Bipolaris infection and two of Exserohilum infection shows the capability of these two genera to cause invasive as well as typical “allergic” lung disease, i.e. fungus balls, and granulomatous lung disease.

Cerebral disease follows hematogenous spread from the lungs. Respiratory symptoms may include cough, dyspnea and hemoptysis. Subcutaneous phaeohyphomycosis usually manifests as a solitary encapsulated cystic granuloma with a necrotic and organism-filled center (Figure 55a). The 2 to 6 μm wide irregularly shaped septate and branched hyphae and budding yeast cells have brown cell walls on H&E-stained tissue sections (Figure 55b). The hyphae are irregularly swollen with prominent septa that show constrictions. They may also have terminal or intercalated vesicular swellings with thick walls that resemble chlamydoconidia. Grains and muriform cells are not found. Fontana-Masson stain highlights the fungal cell wall melanin while GMS and PAS stains highlight the hyphae. In lung cavities, fungal balls often form. Disseminated disease in immunocompromised patients is characterized by parenchymal granulomatous necrosis and vascular invasion. Infections caused by Bipolaris/Exserohilum and Aspergillus show many clinical and pathological similarities despite the lack of a taxonomic relationship. They cause CNS disease, osteomyelitis and sinusitis and are associated with allergic bronchopulmonary disease. Sinusitis, the most common systemic form of disease caused by these organisms, occurs in otherwise healthy patients with nasal polyposis and allergic rhinitis. The etiological agents of phaeohyphomycosis are frequently isolated from sputum and BAL. In immunocompromised patients their presence may represent contamination or colonization, rather than infection. Thus, the diagnosis of phaeohyphomycosis depends on a combination of histopathological examination of affected tissue and culture. Clinical material should be inoculated on Sabouraud’s agar and incubated at 25 C to 30 C. Identifiable dark brown or olivaceous to

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black mold colonies should form within 1 to 2 weeks. On culture, conidiospores have areas of narrowing and produce typical conidia. Identifying many of the etiological agents of phaeohyphomycosis is difficult, due to the pleomorphic nature of these fungi and their similar colonial and microscopic characteristics. Traditional identification is based on a combination of morphological and physiological tests; however, molecular identification is assuming a greater importance. Often, species

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230. Richardson M, Rautemaa R. How the host fights against Candida infections. Front Biosci 2009;14:4363–75.

242. Haron E, Vartivarian S, Anaissie E, Dekmezian R, Bodey GP. Primary Candida pneumonia. Experience at a large cancer center and review of the literature. Medicine (Baltimore) 1993;72(3):137–42.

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257. Kontoyiannis DP, Reddy BT, Torres HA, et al. Pulmonary candidiasis in patients with cancer: an autopsy study. Clin Infect Dis 2002;34(3):400–3. 258. Richardson MD, Carlson P. Cultureand non-culture-based diagnostics for Candida species. In Calderone RA, ed. Candida and Candidiasis. Washington DC: ASM Press, 2002. pp. 387–94. 259. Ellepola AN, Morrison CJ. Laboratory diagnosis of invasive candidiasis. J Microbiol 2005;43 Spec No:65–84. 260. Sahand IH, Moragues MD, Robert R, Quindos G, Ponton J. Evaluation of Bichro-Dubli Fumouze to distinguish Candida dubliniensis from Candida albicans. Diagn Microbiol Infect Dis 2006;55(2):165–7. 261. Quindos G, San Millan R, Robert R, Bernard C, Ponton J. Evaluation of bichro-latex albicans, a new method for rapid identification of Candida albicans. J Clin Microbiol 1997;35 (5):1263–5. 262. Freydiere AM, Buchaille L, Guinet R, Gille Y. Evaluation of latex reagents for rapid identification of Candida albicans and C. krusei colonies. J Clin Microbiol 1997;35(4):877–80. 263. Willinger B, Wein S, Hirschl AM, Rotter ML, Manafi M. Comparison of a new commercial test, GLABRATA RTT, with a dipstick test for rapid identification of Candida glabrata. J Clin Microbiol 2005;43(1):499–501. 264. Verduyn Lunel FM, Peter Donnelly J, van der Lee HA, Blijlevens NM, Verweij PE. Performance of the new Platelia Candida Plus assays for the diagnosis of invasive Candida infection in patients undergoing myeloablative therapy. Med Mycol 2011;49(8):848–55. 265. Sendid B, Caillot D, BaccouchHumbert B, et al. Contribution of the Platelia Candida-specific antibody and antigen tests to early diagnosis of systemic Candida tropicalis infection in neutropenic adults. J Clin Microbiol 2003;41(10):4551–8.

255. Elston DM. Fluorescence of fungi in superficial and deep fungal infections. BMC Microbiol 2001;1:21.

266. Prella M, Bille J, Pugnale M, et al. Early diagnosis of invasive candidiasis with mannan antigenemia and antimannan antibodies. Diagn Microbiol Infect Dis 2005;51(2):95–101.

256. Marcilla A, Monteagudo C, Mormeneo S, Sentandreu R. Monoclonal antibody 3H8: a useful tool in the diagnosis of candidiasis. Microbiology 1999; 145(Pt 3):695–701.

267. Alam FF, Mustafa AS, Khan ZU. Comparative evaluation of (1, 3)-betaD-glucan, mannan and anti-mannan antibodies, and Candida speciesspecific snPCR in patients with

candidemia. BMC Infect Dis 2007;7:103. 268. Farina C, Russello G, Anderoni S, et al. The Microarray Technology in Yeast Identification Directly from Positive Blood Cultures. Boston: ICAAC, 2010. 269. Loeffler J, Dorn C, Hebart H, et al. Development and evaluation of the nuclisens basic kit NASBA for the detection of RNA from Candida species frequently resistant to antifungal drugs. Diagn Microbiol Infect Dis 2003;45(3):217–20. 270. Peman J, Zaragoza R. Current diagnostic approaches to invasive candidiasis in critical care settings. Mycoses 2010;53(5):424–33. 271. Concia E, Azzini AM, Conti M. Epidemiology, incidence and risk factors for invasive candidiasis in high-risk patients. Drugs 2009;69 Suppl 1:5–14. 272. Catherinot E, Lanternier F, Bougnoux ME, et al. Pneumocystis jirovecii pneumonia. Infect Dis Clin North Am 2010;24(1):107–38. 273. Armengol CE. A historical review of Pneumocystis carinii. JAMA 1995;273 (9):747, 50–1. 274. Stringer JR, Beard CB, Miller RF, Wakefield AE. A new name (Pneumocystis jiroveci) for Pneumocystis from humans. Emerg Infect Dis 2002;8(9):891–6. 275. Cushion MT, Linke MJ, Ashbaugh A, et al. Echinocandin treatment of pneumocystis pneumonia in rodent models depletes cysts leaving trophic burdens that cannot transmit the infection. PloS One 2010;5(1):e8524. 276. Morris A, Beard CB, Huang L. Update on the epidemiology and transmission of Pneumocystis carinii. Microbes Infect 2002;4(1):95–103. 277. Vargas SL, Hughes WT, Santolaya ME, et al. Search for primary infection by Pneumocystis carinii in a cohort of normal, healthy infants. Clin Infect Dis 2001;32(6):855–61. 278. Hughes WT, Feldman S, Aur RJ, et al. Intensity of immunosuppressive therapy and the incidence of Pneumocystis carinii pneumonitis. Cancer 1975;36(6):2004–9. 279. Walzer PD. Immunological features of Pneumocystis carinii infection in humans. Clin Diagn Lab Immunol 1999;6(2):149–55.

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280. Walzer PD, Djawe K, Levin L, et al. Long-term serologic responses to the Pneumocystis jirovecii major surface glycoprotein in HIV-positive individuals with and without P. jirovecii infection. J Infect Dis 2009;199(9):1335–44. 281. Walzer PD, Evans HE, Copas AJ, et al. Early predictors of mortality from Pneumocystis jirovecii pneumonia in HIV-infected patients: 1985–2006. Clin Infect Dis 2008;46(4):625–33. 282. Sandhu JS, Goodman PC. Pulmonary cysts associated with Pneumocystis carinii pneumonia in patients with AIDS. Radiology 1989;173(1):33–5. 283. Thomas CF Jr, Limper AH. Pneumocystis pneumonia. N Engl J Med 2004;350(24):2487–98. 284. Sundar K, Rosado-Santos H, Reimer L, Murray K, Michael J. Unusual presentation of thoracic Pneumocystis carinii infection in a patient with acquired immunodeficiency syndrome. Clin Infect Dis 2001;32(3):498–501. 285. Thomas CF Jr, Limper AH. Current insights into the biology and pathogenesis of Pneumocystis pneumonia. Nat Rev Microbiol 2007;5 (4):298–308. 286. Saldana MJ, Mones JM, Martinez GR. The pathology of treated Pneumocystis carinii pneumonia. Semin Diagn Pathol 1989;6(3):300–12. 287. Travis WD, Pittaluga S, Lipschik GY, et al. Atypical pathologic manifestations of Pneumocystis carinii pneumonia in the acquired immune deficiency syndrome. Review of 123 lung biopsies from 76 patients with emphasis on cysts, vascular invasion, vasculitis, and granulomas. Am J Surg Pathol 1990;14(7):615–25. 288. Khot PD, Fredricks DN. PCR-based diagnosis of human fungal infections. Expert Rev Anti Infect Ther 2009;7 (10):1201–21. 289. Gal AA, Koss MN, Strigle S, Angritt P. Pneumocystis carinii infection in the acquired immune deficiency syndrome. Semin Diagn Pathol 1989;6 (3):287–99. 290. Blumenfeld W, McCook O, Holodniy M, Katzenstein DA. Correlation of morphologic diagnosis of Pneumocystis carinii with the presence of pneumocystis DNA amplified by the polymerase chain reaction. Mod Pathol 1992;5(2):103–6.

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291. Desmet S, Van Wijngaerden E, Maertens J, et al. Serum (1–3)-beta-Dglucan as a tool for diagnosis of Pneumocystis jirovecii pneumonia in patients with human immunodeficiency virus infection or hematological malignancy. J Clin Microbiol 2009;47(12):3871–4. 292. Rohner P, Jacomo V, Studer R, Schrenzel J, Graf JD. Detection of Pneumocystis jirovecii by two staining methods and two quantitative PCR assays. Infection 2009; 37(3):261–5. 293. Hauser PM, Bille J, Lass-Florl C, et al. Multicenter, prospective clinical evaluation of respiratory samples from subjects at risk for Pneumocystis jirovecii infection by use of a commercial real-time PCR assay. J Clin Microbiol 2011;49(5):1872–8. 294. D’Avignon LC, Schofield CM, Hospenthal DR. Pneumocystis pneumonia. Semin Respir Crit Care Med 2008;29(2):132–40. 295. Davis BA. Sporotrichosis. Dermatol Clin 1996;14(1):69–76. 296. Marimon R, Gene J, Cano J, et al. Molecular phylogeny of Sporothrix schenckii. J Clin Microbiol 2006;44 (9):3251–6. 297. Coles FB, Schuchat A, Hibbs JR, et al. A multistate outbreak of sporotrichosis associated with sphagnum moss. Am J Epidemiol 1992;136(4):475–87. 298. Kauffman CA, Hajjeh R, Chapman SW. Practice guidelines for the management of patients with sporotrichosis. For the Mycoses Study Group. Infectious Diseases Society of America. Clin Infect Dis 2000;30 (4):684–7. 299. de Lima Barros MB, de Oliveira Schubach A, Galhardo MC, et al. Sporotrichosis with widespread cutaneous lesions: report of 24 cases related to transmission by domestic cats in Rio de Janeiro, Brazil. Int J Dermatol 2003; 42(9):677–81. 300. Ware AJ, Cockerell CJ, Skiest DJ, Kussman HM. Disseminated sporotrichosis with extensive cutaneous involvement in a patient with AIDS. J Am Acad Dermatol 1999; 40(2 Pt 2):350–5. 301. Hay RJ, Morris-Jones R. Outbreaks of sporotrichosis. Curr Opin Infect Dis 2008;21(2):119–21.

302. Kauffman CA. Sporotrichosis. Clin Infect Dis 1999;29(2):231–6; quiz 237. 303. Ramos-e-Silva M, Vasconcelos C, Carneiro S, Cestari T. Sporotrichosis. Clin Dermatol 2007;25(2):181–7. 304. Zhou CH, Asuncion A, Love GL. Laryngeal and respiratory tract sporotrichosis and steroid inhaler use. Arch Pathol Lab Med 2003;127 (7):893–4. 305. Donabedian H, O’Donnell E, Olszewski C, MacArthur RD, Budd N. Disseminated cutaneous and meningeal sporotrichosis in an AIDS patient. Diagn Microbiol Infect Dis 1994;18(2):111–5. 306. Watts JC, Chandler FW. Adiaspiromycosis. An uncommon disease caused by an unusual pathogen. Chest 1990;97(5):1030–1. 307. England DM, Hochholzer L. Adiaspiromycosis: an unusual fungal infection of the lung. Report of 11 cases. Am J Surg Pathol 1993;17 (9):876–86. 308. Peres LC, Figueiredo F, Peinado M, Soares FA. Fulminant disseminated pulmonary adiaspiromycosis in humans. Am J Trop Med Hyg 1992;46 (2):146–50. 309. Denson JL, Keen CE, Froeschle PO, Toy EW, Borman AM. Adiaspiromycosis mimicking widespread malignancy in a patient with pulmonary adenocarcinoma. J Clin Pathol 2009;62(9):837–9. 310. Watts JC, Callaway CS, Chandler FW, Kaplan W. Human pulmonary adiospiromycosis. Arch Pathol 1975;99 (1):11–5. 311. Moraes MA, de Almeida MC, Raick AN. [A fatal case of human pulmonary adiaspiromycosis]. Rev Inst Med Trop Sao Paulo 1989;31(3):188–94. 312. Martins RL, Santos CG, Franca FR, Moraes MA. [Human adiaspiromycosis. A report of a case treated with ketoconazole]. Rev Soc Bras Med Trop 1997;30(6):507–9. 313. Tragiannidis A, Bisping G, Koehler G, Groll AH. Minireview: Malassezia infections in immunocompromised patients. Mycoses 2010;53(3):187–95. 314. Gidding H, Hawes L, Dwyer B. The isolation of Malassezia furfur from an episode of peritonitis. Med J Aust 1989;151(10):603.

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315. Barber GR, Brown AE, Kiehn TE, Edwards FF, Armstrong D. Catheterrelated Malassezia furfur fungemia in immunocompromised patients. Am J Med 1993;95(4):365–70.

phaeohyphomycosis: a review of 101 cases. Clin Infect Dis 2004; 38(2):206–16.

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318. Ben-Ami R, Lewis RE, Raad, II, Kontoyiannis DP. Phaeohyphomycosis in a tertiary care cancer center. Clin Infect Dis 2009; 48(8):1033–41.

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319. Brandt ME, Warnock DW. Epidemiology, clinical

manifestations, and therapy of infections caused by dematiaceous fungi. J Chemother 2003;15 Suppl 2:36–47. 320. Barron MA, Sutton DA, Veve R, et al. Invasive mycotic infections caused by Chaetomium perlucidum, a new agent of cerebral phaeohyphomycosis. J Clin Microbiol 2003;41(11):5302–7.

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8

Pulmonary parasitic infections Sebastian Lucas, Philip Hasleton, Ann-Marie Nelson and Ronald C. Neafie

Introduction Mankind is afflicted with hundreds of parasitic diseases that affect every organ, including the upper and lower respiratory tracts. For some infections, man is the definitive host, i.e., the sexual or reproductive part of the parasite’s life cycle takes place within the human body. For others, man is accidentally infected, i.e., humans interrupt another animal’s parasitic life cycle. This latter scenario represents a dead end for the parasite, since reproduction is not possible. The main groups of parasites are protozoa (single cell organisms) and helminths (worms). The taxonomic classifications and interrelations of these can be complex and are of little interest to most pathologists. Occasionally pentastome parasites, worm-like animals that inhabit the upper respiratory tract of reptiles, birds and mammals, also obstruct the upper airways. These are mentioned only briefly in this account, which focuses on the commoner lower respiratory tract protozoal and worm infections.

Principles of parasitic infection Several basic principles of lung parasitic infection should be considered. Pathogenetically, the diseases can be described in several categories. The lung can be the main or only infection site, it can be involved along with many other organs, or it may only be accidentally involved by a parasite that normally causes disease elsewhere in the body. In addition, hematogenous infection can secondarily affect the lung. Alternatively the lung and airways may host a transient phase of the parasite life cycle during the migration of pre-adult forms. Finally, pulmonary drug reactions from antiparasitic agents can cause lung disease. Clinical pathologies induced by parasites include spaceoccupying lesions such as coin lesions mimicking malignancies, necrosis or abscesses, vascular obstruction with subsequent ischemic damage, immunologically driven interstitial inflammation, often with eosinophilia, acute lung injury and asthma. A major difference between protozoal and helminthic infections is that the former can multiply and cause disease

in the host indefinitely, whereas most worm infections are limited chronologically by the death of the worm. Although parasites are often equated with “tropical infections”, a large proportion of the parasitic infections described here are acquired in temperate zones or anywhere in the world. As the numbers of persons globally immunosuppressed by human immunodeficiency virus (HIV) infection, congenital immune defects, malignancy and drug treatments for cancer, organ transplantation and autoimmune diseases continue to increase, not to mention the ease of world travel, the incidence of and clinical manifestations associated with parasitic infections continues to rise.1 Diagnosis and treatment of parasitic disease have become more challenging. The parasites in this chapter are considered taxonomically. The clinical, imaging, microbiological, pathogenetic, diagnostic and treatment aspects of parasitic (and all other) infections are well laid out in Mandell, Douglas and Bennett’s Principles and Practice of Infectious Diseases.2 The most complete parasite pathology text is the monograph by Gutierrez.3 The Armed Forces Institute of Pathology (AFIP) published a comprehensive work on helminthiases.4 A brief but comprehensive review of protozoal lung disease is also available from Martinez-Giron et al.5 Another useful guide to parasites in human tissues was authored by Orihel and Ash.6 For accessible information on the diseases, the website of the USA Centers for Disease Control and Prevention (CDC) is a mine of information and downloadable charts (www.cdc.gov).7 The World Health Organization website (www.who.int) also features current epidemiological data on the major parasitic infections.

Diagnosis of parasitic lung disease Technological advances have greatly improved our ability to identify parasitic lung infections. Until recently one relied on clinical history, in particular travel histories, as well as parasite morphology in sputum, bronchoalveolar lavage (BAL), fineneedle aspirate biopsy and tissue biopsy to identify particular parasitic infections. Blood serologies marking parasitic antigens and organism-specific antibodies are now widely available. Imaging modalities, including computed tomography

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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(CT) and magnetic resonance imaging (MRI) greatly aid in diagnosis. For example, hydatid cysts are confidently diagnosed on imaging along with serology findings without morphological confirmation. While immunohistochemical staining of parasite antigens is limited, molecular DNA analysis is expanding at a rapid pace. Polymerase chain reaction (PCR) testing complements and even supersedes traditional morphological methods. It also enables critical review of taxonomic relationships. For example, Pneumocystis jirovecii was considered a protozoon parasite until 20 years ago, when DNA analysis confirmed its status as a fungus (see Chapter 7). All tests have imperfect sensitivity and specificity, and the diagnosis should be considered as a whole. If one test produces a result that does not fit with the other data and the clinical context, it is probably wrong or represents an artifact simulating a parasite infection. Most of the parasitic infections described in this chapter are uncommon or rare in high-income countries. Globally, malaria is the most common, but does not usually induce clinical lung pathology. In non-immunocompromised persons, ascariasis, schistosomiasis, paragonimiasis and hydatid disease are the commonest parasitic agents of clinical lung disease. Tropical pulmonary eosinophilia is an important disease among susceptible persons with lymphatic filariasis. And among the immunocompromised, toxoplasmosis and strongyloidiasis are the most important parasitic causes of pulmonary disease.

Protozoal infections Leishmaniasis Introduction

The leishmaniases include a complex of vector-borne diseases, caused by more than 20 species of the protozoan genus Leishmania, and range from localized skin ulcers to lethal systemic disease. Leishmaniasis is classified as one of the “most neglected diseases”, based on the limited resources invested in diagnosis, treatment and control, and its strong association with poverty.

Epidemiology The leishmaniases are transmitted to humans in sylvatic (relating to certain diseases contracted from wild animals), domestic and peri-domestic cycles, ranging from cities to deserts and rain forests on every continent, except Australia and Antarctica. In the Western Hemisphere, leishmaniasis is found in some parts of Mexico, Central America and South America. It is not detected in Chile or Uruguay. In the Eastern Hemisphere, leishmaniasis is found in parts of Asia, the Middle East, Africa and southern Europe. Leishmaniases, caused by the intracellular protozoan Leishmania infantum, are an endemic zoonosis in the Mediterranean basin. In Portugal the global climate changes associated with a higher density and activity of sand flies during a longer period might enhance the number of

days favorable for transmission of parasites to humans and animals with a concomitant increase of incidence.8 Visceral leishmaniasis was initially a pediatric disease but in recent years the number of cases in children has decreased with an increase of infection in adults, normally associated with HIV/ acquired immune deficiency syndrome (AIDS) in Portugal. The majority of human cases occur in a small number of countries. Ninety percent of visceral leishmaniasis cases occur in parts of India, Bangladesh, Nepal, Sudan, Ethiopia and Brazil, while 90% of cutaneous leishmaniasis cases occur in parts of Afghanistan, Algeria, Iran, Saudi Arabia, Syria, Brazil, Colombia, Peru, Bolivia and East Africa.9 However, climate change and other environmental changes have the potential to expand the geographic range of the vectors and leishmaniasis transmission in the future.7 In sylvatic cycles, such as those in New World rain forests and the deserts of Central Asia, animal reservoir hosts can maintain transmission indefinitely without human disease. Sporadic or epidemic leishmaniasis occurs when humans enter the sylvatic habitat for economic or military purposes, or when human habitation encroaches on the sylvatic setting. In domestic cycles, humans or dogs form the predominant or sole infection reservoir. In the Mediterranean basin and parts of Latin America, visceral leishmaniasis transmission is zoonotic (dog – sand fly – human). The area that accounts for the largest number of human cases, for example, visceral leishmaniasis in South Asia and cutaneous leishmaniasis in Afghanistan, usually reflects anthroponotic (human – sand fly – human) transmission. Leishmaniasis is a vector-borne disease transmitted by phlebotomine sandflies and caused by obligate intracellular protozoa of the genus Leishmania. Human infection is caused by about 21 of 30 species that infect mammals. Leishmaniasis usually involves a mammalian intermediate host. Species of Leishmania are known as hemoflagellates. They are responsible for cutaneous, mucocutaneous and visceral leishmaniasis.6 Particular species of Leishmania are associated with different clinical patterns. Visceral leishmaniasis is usually caused by Leishmania donovani or L. infantum species, while mucocutaneous leishmaniasis (MCL) or espundia is associated with L. (Viannia) braziliensis in South America. That having been said, pulmonary involvement with Leishmania is usually restricted to L. donovani, or its variant L. infantum.

Organism The parasite in man is an intra-macrophage amastigote, 2–3 µm in diameter, with clear refractile cytoplasm, a hematoxyphilic nucleus and a rod-shaped hematoxyphilic kinetoplast (composed of DNA) (Figure 1). The kinetoplast is easier to see in smears than histological sections. The amastigote is the preferred name for the parasite, in place of LeishmanDonovan body. The cell biology and immunology of the disease were recently reviewed.10,11

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Clinical features There are three classic forms of the disease.2,12 The commonest is the cutaneous form, which appears as painless cutaneous ulcers. Mucocutaneous leishmaniasis is a rare form of the disease that may occur months or years after a cutaneous leishmaniasis (CL) ulcer has healed. This form of the disease can affect the upper respiratory tract, i.e., nose, oropharynx, nasopharynx, larynx and upper trachea, with destructive inflammation. Laryngeal involvement is frequent in AIDS patients but may be seen in immunocompetent cases.5 Visceral leishmaniasis (VL, kala azar), the third form of the disease, infects macrophages throughout the body. It is a febrile illness with weight loss, enlargement of the spleen and liver, and decreases in the production of blood cells that can lead to anemia, bleeding and infections with other microorganisms. Without treatment, this form of the disease is nearly always fatal.13 Respiratory tract leishmaniases, with the exception of upper respiratory tract MCL, are rare.5 However, with the advent of the HIV pandemic, as well as increasing numbers of patients on local and systemic immunosuppressive chemotherapy, distinctions between the main clinical types blur, with some species producing several patterns of disease. Isolated upper respiratory tract infections are considered either MCL or laryngeal leishmaniasis. The latter is precipitated by inhaled steroid use in asthmatics. Intrathoracic disease associated with visceral leishmaniasis includes bronchial and mediastinal lymph node infection, pulmonary interstitial infection and pleural infection. A dry cough is reported in Brazilian cases.14 Rare cases of bronchial disease presenting with cough and hemoptysis are recorded.15 HIV-co-infected patients are rarely noted, sometimes with atypical pulmonary symptoms such as pleural effusions and laryngeal involvement.5,16–18

Pathology A Brazilian autopsy series of visceral leishmaniasis noted interstitial pneumonitis. Alveolar septa feature edema, lymphocytes and plasma cells while macrophages contain parasites (Figure 1). Fine septal fibrosis accompanies the inflammation in some cases.5,14 Bronchial leishmaniasis features mucosal non-necrotizing granulomas, which also involve the mediastinal nodes.15 There may be pleural effusions associated with fibrinous pleuritis, which may contain organisms in macrophages (Figure 2).18,19 Organisms can be confirmed with immunohistochemistry. Unpublished cases in HIV-negative persons with visceral leishmaniasis include lung infection associated with B-cell lymphoma, and leishmanial infection of pleural granulation tissue, clinically and radiologically mimicking mesothelioma. Leishmania parasitemia can sometimes seed and proliferate in inflamed tissue, similar to tuberculous bacillemia.

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Figure 1. Leishmaniasis. Leishmanial amastigotes in macrophages. The rod-shaped kinetoplast is seen (Giemsa stain).

Diagnosis Diagnosis of visceral leishmaniasis may require taking a blood sample and/or bone marrow biopsy to show the parasite. Diagnosis of cutaneous leishmaniasis requires a small biopsy or scraping of an ulcer. Diagnosis of mucocutaneous leishmaniasis needs a biopsy of the affected tissues. Biopsy samples are examined by microscopy, culture and other methods to look for the parasite and identify the specific kind of Leishmania causing the ulcer. Some of these methods will give results within a few days, but culture may take 2 to 4 weeks.13 No special stains are required to identify leishmania amastigotes. Hematoxylin and eosin-stained tissue sections highlight the amastigotes better than Giemsa-stained sections (Figure 1). Immunohistochemical stains are available but rarely used, as fresh or fixed tissue PCR molecular diagnostics have become a standard confirmatory method.12 Serology for leishmania antibodies is positive in visceral leishmaniasis, although it is not reliable in HIV-infected patients. In the rare leishmanial pleural effusion, the parasites may be seen in aspirates. Parasitemia levels can be measured by means of quantitative real-time PCR targeting kinetoplast DNA with TaqMan chemistry. Enzyme-linked immunosorbent assay and western blotting (WB) are used for serological screening.20 Diagnosis of VL using PCR and buccal swabs is simple, well tolerated and has good potential for development, showing 83% sensitivity with 90.56% specificity in control groups.21

Differential diagnosis The morphological differential diagnosis includes apoptotic nuclear debris, Trypanosoma cruzi infection and Histoplasma capsulatum yeasts, which are more commonly encountered in diagnostic lung pathology. Histoplasma capsulatum yeasts are also intra-macrophage organisms yet lack a kinetoplast. Their nucleus is also less hematoxyphilic. This fungus also stains with fungal PAS and Grocott silver stains, whilst amastigotes do not (see Chapter 7). Distinguishing T. cruzi from

Chapter 8: Pulmonary parasitic infections

(a)

(b)

(c)

Figure 2. Pleural leishmania. (a) Pleural biopsy from a patient with pleural thickening suspected of having malignant mesothelioma. The inner fibrin exudate and chronic organizing inflammation are apparent. (b) Inflammation is not granulomatous. (c) Oil immersion reveals characteristic amastigotes with eccentric nuclei. A rod-shaped kinetoplast is seen in several organisms (arrow).

Leishmania amastigotes is impossible on morphology, and reliance would be on epidemiology, specific immunohistochemistry and molecular diagnostics.

Treatment Pentavalent antimonial derivatives are one of the treatments of choice in leishmaniasis, although other substances such as paromomycine and liposomal amphotericin B are effective alternatives.22 Eliminating the parasite, and cure, in immunosuppressed patients may be impossible.

Amebiasis Introduction

This disease can affect anyone, although it is more common in poor people who live in tropical areas with poor sanitary conditions. It is also present in temperate countries.23,24 Infected people do not always become sick. There are two

distinct amebic infections of man: the intestinal infection Entamoeba histolytica and the free-living ameba genera, Acanthamoeba and Balamuthia.2 Neither of these infections is primarily located in the lung and pulmonary disease is more common with E. histolytica.

Epidemiology Although no age is exempt, amebiasis commonly occurs in patients aged 20 to 40 years, with an adult male to female ratio of 10:1. Children rarely develop thoracic amebiasis. When it does occur there is an equal sex distribution.25 Malnutrition, advanced age, pregnancy, immune suppression states, alcoholism and anilingus sexual practices are risk factors.26 In the present context, visits to endemic areas (Africa, Asia and Central America) and immigration are additional factors.5 Transmission can also occur through exposure to fecal matter. During sexual contact not only cysts but also trophozoites could prove infective

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(Figure 3). E. histolytica is to be distinguished from the morphologically identical amebic infection E. dispar, which has a vegetative life cycle in the human gut but does not invade bowel mucosa, and cannot cause liver or lung disease.23

Organism

Figure 3. Amebiasis life cycle. Image courtesy of Centers for Disease Control and Prevention (CDC).

The 30 to 60 µm parasitic trophozoite is a single cell but noninvasive forms may be smaller.5,6 Trophozites are found in tissues, whereas cysts are only present in feces. The trophozoite has a granular, vacuolated cytoplasm that may be gray on H&E stains, and contains a round, purplish, single nucleus with a single karyosome dot (Figure 4a). Phagocytosed red blood cells may be evident. It is larger than a macrophage, whose nucleus is larger and more hematoxyphilic than the amebic nucleus. PAS staining is very helpful since amebae contain much glycogen, and stain bright magenta (unlike macrophages) (Figure 4b). A PAS-diastase stain does not stain the organism. Resort to immunohistochemistry or molecular diagnostics is not usually needed given the role of serodiagnostics.27 The mature cyst, 10–20 mm in diameter, may contain chromatoid bodies with rounded ends. Chromatoid bodies are commoner in immature cysts. The invasive and non-invasive forms represent two separate species, respectively E. histolytica and E. dispar. These two species are morphologically indistinguishable unless E. histolytica is observed with ingested red blood cells (erythrophagocystosis). The prime site of E. histolytica infection is the colon; cysts from another person’s feces are ingested from fecally contaminated food, water or hands and exocyst in the cecum, releasing trophozoites. The trophozoites multiply by binary fission and produce cysts. Both stages are passed in the feces. Because of the protection conferred by their walls, the cysts can survive days to weeks in the external environment and are responsible for transmission. Cysts may be excreted for a considerable time (carrier state) and infections can terminate spontaneously.6 Trophozoites passed in the stool are rapidly destroyed once outside the body, and if ingested would not survive exposure to the gastric environment. In many cases, the trophozoites remain confined to the intestinal lumen (noninvasive infection) of individuals who are asymptomatic

(a)

(b)

Figure 4. Pulmonary amebic infection. (a) Entamoeba histolytica trophozoite with engulfed erythrocyte in the lung. Compare its nucleus with that of the adjacent histiocyte. (b) Numerous PAS-positive trophozoites cluster around the edges of fibrin-filled alveolar sacs (Periodic acid-Schiff stain).

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carriers, passing cysts in their stool. Trophozoites invade the colonic mucosa by lysing the epithelium. The preferential site is the cecum, where they form small ulcers.6 As these ulcers spread laterally, they form flask-shaped ulcers. Having breached the lamina propria, the organisms can spread via the mesenteric veins to the liver and induce an amebic liver abscess. The organisms may pass through the bloodstream to extraintestinal sites such as the liver, brain, and lungs (Figure 3). How amebae cause invasive disease is much studied. The current pathogenesis theory relies heavily on the organism’s production of ‘amebapore’. This is a perforin that lethally injures adjacent host cells. Amebic lytic enzyme secretion facilitates passage through tissues.23,24

Clinical features Pleuropulmonary infection by E. histolytica is, after that of the liver, the most frequent form of extraintestinal amebiasis. Thoracic amebic disease is secondary to a liver abscess in virtually all cases.25 However, it may be due to fistula formation, with the right lower lobe most frequently affected.28–30 A hematogenous path has also been described as a cause of pulmonary lesions due to E. histolytica.31 There may have been preceding general malaise, anorexia, weight loss and abdominal discomfort or moderate or profuse bloody diarrhea.6 These gastrointestinal features can delay the diagnosis of hepatic and pulmonary disease. The most frequent symptoms are fever, cough, hemoptysis and pleuritic pain.25,32 On occasion, a hepatic abscess may provoke venous compression and be the cause of respiratory distress and alterations in arterial gases.33 An initial accumulation of serous fluid due to reactive pericarditis followed by intrapericardial rupture may develop either acute onset of severe symptoms with chest pain, dyspnea and cardiac tamponade, shock and death, or progressive effusion with thoracic cage pain, progressive dyspnea and fever.25 Five clinico-pathological pleuropulmonary patterns, namely pleural effusion, empyema, lung abscess, hepatopleurobronchial fistula and superior vena caval syndrome, are noted in decreasing frequency.34–36 The clinical symptoms, in addition to those of liver abscess, include pleuritic pain, cough and hemoptysis. The appearance of a purulent fluid of a chocolate-like color (often described as anchovy paste) following the puncture of an abscess, discharge or through vomiting is highly suggestive of amebiasis.37 Fever and high neutrophil blood count are usual. The radiology indicates inflammation in pleura and lung, and sometimes cavitation in the lung abscess, but is not specific.

Pathology Pleural effusion is a reactive process, induced by subdiaphragmatic inflammation. The effusion has a high protein level and numerous neutrophils, but no amebae. The right side is more commonly affected, because of its proximity to the liver. Amebic empyema has been described as resembling “anchovy sauce”. It is dark red or brown (from hemorrhage), and variably thick or runny (Figure 5). Necrosis and usually a

Figure 5. Aspiration of a liver/pleural amebic abscess. Much brown thick “anchovy sauce” necrotic liquid is removed.

few neutrophils or any other viable inflammatory cells are seen microscopically. Amebae, if present, are at the edges, attacking the surrounding living tissue. In these patients, there will be a communication from the liver to pleura across the diaphragm. Subpleural lung abscesses have a similar consistency to empyema, with coagulative necrosis of lung tissue. There is granulation tissue, but no granulomatous reaction (Figure 4). Secondary bacterial infection may complicate evaluation and treatment. A left lower lobe abscess is less common than right, reflecting the commoner location of infection in the right lobe of the liver. If an amebic lung abscess ruptures into a bronchus, the patient will expectorate necrotic tissue. Amebae may be seen in such material. Sometimes an amebic lung abscess can simulate lung cancer and directly obstruct the superior vena cava.36 Finally, there is the uncommon amebic lung abscess that results from hematogenous spread from the gut or liver.34 Infection spreads via the hemorrhoidal veins, the hepatic vein or the thoracic duct. One sees a solitary lung lesion or concomitant separate liver and lung abscesses.

Diagnosis Diagnosis of liver and lung amebiasis is increasingly made on clinical and imaging criteria, supported by very specific positive serology. Ultrasound is the gold standard technique for diagnosis of amebic liver abscess.38 Immunologically based diagnostic tests for detection of anti-amebic secretory antibodies in feces or detection of intestinal amebic antigens through polyclonal or monoclonal specific antibodies (ELISA) are excellent tests in hospitals where fresh specimens are readily obtained. However, these techniques can be biased when samples are more than 18 hours old.38 Formol ether concentration and PCR techniques are also useful.

Differential diagnosis Diagnosis can be difficult as other parasites can look very similar histologically to E. histolytica. Fine-needle aspiration

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and sputum cytology and tissue biopsy may or may not reveal amebic trophozoites.27 Another species, E. gingivalis, present in the mouth, has the potential to be confused with E. histolytica on cytology.5 But the former does not contain phagocytosed red corpuscles.

Treatment Metronidazole and tinidozole are used in the treatment of amebiasis.39

Free-living amebic infections Acanthamoeba and Balamuthia species are free-living amebae that globally live in water, including water- and air-conditioning plants. They infect man uncommonly, and the major site of disease is the cornea (amebic keratitis) of contact-lenswearing individuals.2 They can also cause local and disseminated necrotizing and granulomatous lesions, as amebae pass

Figure 6. Pulmonary acanthameba infection may cause parenchymal infarction.

(a)

through skin defects or enter an individual through the nasal sinuses. The brain is the commonest site of internal infection, with high mortality. Whilst free-living amebiasis occurs in non-immunosuppressed patients, it is increasingly reported in immunocompromised hosts. The two main groups are those with HIV disease40 and those who have received bone marrow or solid organ transplants. Those with graft-versus-host disease are particularly susceptible.41,42 Via hematogenous spread, amebae infect brain, lungs and liver, and cause widespread organ dysfunction. Whilst pre mortem diagnosis rates are increasing with greater awareness and improved diagnostic techniques, many patients are diagnosed only at autopsy. A recently described bacterial agent of human pneumonia, Parachlamydia acanthamoebae, grows within acanthamebae. It is likely that some outbreaks of this infection associated with humidifiers may be attributed to the ameba as a reservoir site for the bacterium.43 Pulmonary nodules and pleural effusions are noted radiographically. Grossly these lesions are pale and necrotic. Yet often microscopy also indicates foci of infection without any macroscopic findings. Histopathologically, the lung parenchyma shows focal necrosis (Figure 6). Trophozoites and cysts are seen. The trophozoites are 15 to 45 µm across and have a large dense hematoxyphilic or purple nucleus, a prominent intranuclear karyosome and pale bubbly cytoplasm (Figure 7a). Cysts are smaller (15–20 µm) and have a crinkled double cell wall. A PAS stain often highlights the cell walls, but confusion with macrophages can occur, and organisms may be scanty (Figure 7b). Naegleria may be confused with E. histolytica, but the former is most frequently seen in the brain. Like Acanthamoeba and Balamuthia species, this organism has large prominent karyosomes in their nuclei.6 Immunohistochemistry and molecular testing confirm the diagnosis.40 While many morphological subtypes are very

(b)

Figure 7. Pulmonary acanthameba. (a) Acanthameba trophozoites are larger than those of E. histolytica, have more hematoxyphilic nuclei, a prominent intranuclear karyosome, and do not phagocytose erythrocytes. (b) Cyst forms feature crenated double cell walls (PAS stain).

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similar, chemotherapy treatment is the same for all free-living amebic infections.

Trypanosomiasis Introduction

Two Trypanosoma species affect man. T. cruzi in South America is responsible for Chagas disease, and T. brucei species in west and central Africa cause African trypanosomiasis.2 Chagas disease is named after the Brazilian physician Carlos Chagas, who discovered the disease in 1909. Trypanosoma cruzi is transmitted to animals and people by infected insect vectors that are found only in the Americas. Chagas disease is also referred to as American trypanosomiasis. In the United States, Chagas disease is considered a Neglected Infection of Poverty (NIP) since it is found mostly in those with limited resources and limited access to medical care.13 African trypanosomiasis is caused by T. brucei gambiens and T. rhodensiense. These are transmitted by tsetse flies and are restricted to the African continent.

Epidemiology Chagas disease is endemic throughout much of Mexico, Central America and South America, where an estimated 8 to 11 million people are infected. By applying published seroprevalence figures to immigrant populations, the CDC estimates that more than 300 000 persons with T. cruzi infection live in the United States. Most people with Chagas disease in the United States acquired their infections in endemic countries. Although there are triatomine bugs in the USA, only rare vector-borne cases of Chagas disease have been documented. The disease is most commonly acquired through contact with the feces of an infected triatomine bug (or “kissing bug”), a blood-sucking insect that feeds on humans and animals. Infection can also occur from mother-to-baby (congenital), contaminated blood products (transfusions), an organ transplanted from an infected donor, laboratory accident or contaminated food or drink (rare). The triatomine bug thrives under poor housing conditions (for example, mud walls and thatched roofs), so in endemic countries people living in rural areas are at greatest risk of acquiring infection. Public health efforts aimed at preventing transmission have decreased the number of newly infected people and completely halted vector-borne transmission in some areas. Infection acquired from blood products, organ transplantation or congenital transmission continues to pose a threat.13 Initially a parasitamia, T. cruzi can infect any cell, and goes to the heart, gut and brain particularly44 to cause acute inflammatory syndromes (Figure 8).

Organism Trypanomastigotes (denotes the infective stage in trypanosomiasis, where the flagellum arises from a posterior kinetoplast,

with an undulating membrane running along the length of the body) of T. cruzi range from 12 to 22 µm in length. They taper at the ends. The organisms have a large subterminal kinetoplast at the pointed posterior end. There is a long flagellum that courses forward and emerges at the anterior end as a free structure. A large central nucleus typically stains red or violet with Giemsa. On stained sections the organisms assume a “C” shape. Amastigotes are round to ovoid, measuring 1.5 to 4.0 µm in diameter.6

Clinical features The congenital infection causes an interstitial pneumonitis as part of systemic infection, and results in stillbirth or early infant death.45 The acute phase is usually asymptomatic, but can present with fever, anorexia, lymphadenopathy, mild hepatosplenomegaly and myocarditis. Romaña’s sign (unilateral palpebral and periocular swelling) may appear as a result of conjunctival contamination with the vector’s feces. A nodular lesion or furuncle, usually called a “chagoma”, can appear at the site of inoculation. Most acute cases resolve over a period of a few weeks or months into an asymptomatic chronic form of the disease. The symptomatic chronic form may not occur for years or even decades after initial infection. Its manifestations include cardiomyopathy, megaesophagus and megacolon, and weight loss.13 There is no acute Chagasic lung pneumonitis disease in children or adults. The infection may eventually damage the heart and gut, including the esophagus, to produce the “megasyndromes”. Thus the lung can acquire secondary complications of chronic cardiac failure, aspiration pneumonia and lung abscess.

Pathology Pathologically, there is chronic alveolar wall inflammation with endothelialitis, and the parasites are mainly in macrophages (Figure 9a). Pseudocysts may form. They are 3 to 5 µm across, resembling leishmania amastigotes, with a kinetoplast (Figure 9b). Thus this is similar to toxoplasmic lung infection (see below), although T. gondii does not have a kinetoplast.

Diagnosis Diagnostic confirmation includes serology, immunohistochemistry and molecular techniques. The indirect fluorescent antibody (IFA) test is available. IFA antigen slides are prepared from a suspension of epimastigotes. Although IFA is very sensitive, cross-reactivity can occur with sera from patients with leishmaniasis. The CDC also utilizes an FDA-cleared commercial diagnostic enzyme immunoassay (EIA).13

Differential diagnosis Amastigotes of Leishmania species are similar to T. cruzi. Nests of amastigotes are commoner with T. cruzi. Toxoplasma and Histoplasma lack a kinetoplast. Trypanomastigotes of T. cruzi must be distinguished from the organisms causing

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Figure 8. Trypanosoma cruzi life cycle. Image courtesy of CDC.

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(b)

Figure 9. South American trypanosomiasis. (a) Interstitial pneumonitis is obvious, but parasites are not visible. (b) Intracellular amastigotes within heart muscle. Some of the forms have a kinetoplast body within the cell.

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African trypanosomiasis. These (T. brucei gambiens and T. rhodensiense) are not seen in the lung, have small kinetoplasts and divide in the blood.6

African trypanosomiasis African trypanosomiasis causes so-called “sleeping sickness”, primarily parasitemias with secondary immunological effects, encephalitis and interstitial myocarditis. Wasting predisposes to secondary bacterial infections and death. Thus the lungs have no primary pathology, but in fatal cases show pneumonia.

Malaria

Introduction The malarias are the most important global protozoal infections, with 200 to 300 million patients affected in the tropics and approximately 3 million deaths a year, mainly in children. The disease is usually seen in the tropical areas of Asia, Africa and Latin America. It is endemic in over 90 countries.5 About 1500 cases of malaria are diagnosed in the United States each year. Most cases in the United States are in travellers and immigrants returning from countries where malaria transmission occurs, many from sub-Saharan Africa and South Asia. P. falciparum and P. vivax are the species that most frequently affect the lungs, while P. ovale and P. malariae are the rarest.

Epidemiology P. falciparum is found worldwide in tropical and subtropical areas. P. vivax is seen mostly in Asia, Latin America and some parts of Africa and because of the population densities, especially in Asia, it is probably the most prevalent human malaria parasite. P. ovale is found mostly in Africa (especially West Africa) and the islands of the western Pacific. It is biologically and morphologically very similar to P. vivax. P. malariae, found worldwide, is the only human malaria parasite species that has a quartan cycle (3-day cycle). P. knowlesi is seen throughout Southeast Asia as a natural pathogen of long-tailed and pig-tailed macaques. It has recently been shown to be a significant cause of zoonotic malaria in that region, particularly in Malaysia.13 The most virulent species is P. falciparum, accounting for nearly all the deaths.2,5 However, P. vivax, normally considered benign, is increasingly reported to cause lung injury, though there are few fatalities.46 P. knowlesi, with limited geographical presence in Southeast Asia, causes severe and potentially fatal malaria.47 Two genetic factors, both associated with human red blood corpuscles, are epidemiologically important. Persons with the sickle cell trait (heterozygotes for the abnormal hemoglobin gene HbS) are relatively protected against P. falciparum malaria and enjoy a biological advantage. Because P. falciparum malaria has long been a leading cause of death in Africa, the sickle cell trait is now more

Figure 10. Plasmodium falciparum blood smear. The intra-erythrocytic trophozoites are single and multiple (Giemsa stain).

frequent in Africa and in persons of African ancestry than in other population groups. In general, hemoglobin-related disorders and other blood cell dyscrasias, such as hemoglobin C, the thalassemias and glucose-6-phosphate dehydrogenase deficiency (G6PD), are more prevalent in malaria endemic areas and are thought to provide protection from malarial disease. Persons who are negative for the Duffy blood group have red blood corpuscles that are resistant to infection by P. vivax. Since the majority of Africans are Duffy negative, P. vivax is rare in Africa south of the Sahara, especially West Africa. In that area, the niche of P. vivax has been taken over by P. ovale, a very similar parasite that does infect Duffy-negative persons.

Organism Malaria parasites infect successively two types of hosts: humans and female Anopheles mosquitoes. In humans, the parasites grow and multiply first in the liver cells and then in the red corpuscles of the blood (Figure 10). In the blood, successive broods of parasites grow inside red cells and destroy them, releasing daughter parasites (“merozoites”) that continue the cycle by invading other red cells. The blood-stage parasites cause the symptoms of malaria. When certain forms of blood-stage parasites (“gametocytes”) are picked up by a female Anopheles mosquito during a blood meal, they start another cycle of growth and multiplication in the mosquito. After 10–18 days, the parasites, as “sporozoites”, are found in the mosquito’s salivary glands. When the Anopheles mosquito takes a blood meal from another human, the sporozoites

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Chapter 8: Pulmonary parasitic infections Figure 11. Malaria life cycle. Image courtesy of CDC.

are injected with the mosquito’s saliva. The sporozoites start another human infection when they parasitize liver cells (Figure 11).13 Merozoites within erythrocytic schizonts are small, up to 1.5 mm, with a nucleus surrounded by a small amount of cytoplasm. Mature tissue schizonts measure 45 to 80 mm and contain up to 40 000 or more merozoites, depending on the species. The exoerythrocytic schizonts have thin walls and displace the nucleus.6 Malarial pigment can be seen in parasitized red blood corpuscles, free in vessels, and in macrophages.

Clinical features The malarias are intra-erythrocytic parasites that do not invade tissues. Anemia and fever are the common manifestations.48 There are several organ-centered pathologies, of which the most serious is cerebral malaria. The parasites cause disease by obstructing small vessel blood flow (red cell sequestration), and by inducing systemic cytokine activation, endothelial damage and local inflammation. The syndromes of malaria-related lung damage present as breathlessness, cough, sometimes chest pain and, in cases of acute respiratory distress, life-threatening pulmonary failure. Children often present with metabolic acidosis-induced hyperventilation, while adults with pulmonary disease usually have signs of pulmonary edema or acute lung injury (ALI)/acute respiratory distress syndrome (ARDS).49 The radiological features are the same as for any other cause of ARDS. An organizing pneumonia pattern is uncommon and anti-malaria drug toxicity is rare.

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Physiological studies demonstrate pulmonary vascular dilatation in patients with edema. This non-cardiogenic pulmonary edema is a mild and usually transient form of acute lung injury.50 Its frequency in adult malaria is unclear and it usually follows other manifestations of acute systemic malaria, even in the recovery phase. Acute respiratory distress syndrome can present before or after treatment in falciparum malaria and is nearly always part of a systemic sepsis-like syndrome with multi-organ failure. In contrast, when ARDS uncommonly develops in vivax malaria, the lung is nearly always the only organ affected.46 Acute respiratory distress syndrome usually develops 1–4 days after initiation of chloroquine treatment;51 however, Acute respiratory distress syndrome can also be a presenting feature.49,52 The pulmonary edema commonly seen in acute falciparum malaria can result from iatrogenic overhydration, when IV drips are given to deliver chemotherapy. Many patients also have comorbid cardiac disease and hypoalbuminemia. 49

Pathology Very few observations of lung pathology in the malarias are made during life, and most of our knowledge comes from autopsy examinations. This information is likely to be biased or distorted by case selection, variable intervals from initiation of systemic and lung disease to death, and the effects of treatment in intensive care, which has particular impact on lung morphology. It is also likely that variations in host response in different countries, in different age groups and

Chapter 8: Pulmonary parasitic infections

(a)

Figure 12. Lung in falciparum malaria. (a) Alveolar edema is seen. Alveolar wall capillaries have numerous parasitized red cells with the hematoxyphilic trophozoites partly obscured by small brown pigment granules (hemozoin). Clumps of hemozoin are also noted in macrophages. There is no interstitial pneumonia. (b) Following hemozoin pigment removal by pricric acid, amphophilic parasites are more obvious. Pulmonary edema is apparent in this microscopic field.

(b)

Figure 13. Lung in falciparum malaria. High magnification of de-pigmented lung, where the ring-form trophozoites are seen in erythrocytes (arrow).

perhaps also different strains of the parasites all complicate our understanding of this systemic infection. Grossly, the lungs of patients who die acutely of falciparum malaria are heavy, red, congested and edematous. The lung color may be darker than expected because of accumulation of malaria hemozoin pigment in the vessels and macrophages. This pigment is a product of parasite metabolism. A serosanguineous pleural effusion may be seen. Histologically, there is wide variation in appearances. Classically pulmonary capillaries and venules are dilated and distended with parasitized red blood corpuscles (PRBC) and airspaces are filled with pink edema fluid and macrophages containing hemozoin pigment (Figure 12). Hyaline membranes are seen in almost half of cases. Within the RBC, the parasites are small hematoxyphilic dots within a clear round cytoplasm. Usually they are obscured by the brown hemozoin pigment. This pigment, which is confusingly like formalin pigment, can be removed by pre-treatment with picric acid to improve identification of the parasite (Figure 13). The proportion of PRBC in lung vessels is usually less than seen in the peripheral blood and the brain vessels. If there is alveolar hemorrhage it is most likely to be due to resuscitation efforts. In severe cases of falciparum and vivax malariainduced ARDS, typical hyaline membranes are noted (see Chapter 9). Associated with PRBC are variable amounts of hemozoin pigment in intravascular monocytes and interstitial

macrophages. The degree of interstitial lymphocytic, but never neutrophilic, inflammation is variable and usually little. With the electron microscope, the PRBC in falciparum malaria have abundant “knobs” of parasite-derived protein (PfEMP1) on their surfaces. These proteins are critical in organism adherence to each other and endothelial surfaces. Interstitial edema and endothelial cell cytoplasmic swelling are noted. Intravascular coagulopathy may be seen in cases with superimposed bacterial sepsis.53 There are occasional biopsy-proven reports of pulmonary fibrosis following non-cardiogenic pulmonary edema in falciparum malaria.54 Vivax malaria is also reported with bronchiolitis obliterans55,6 although the pathogenesis is obscure, and just might be coincidental.

Pathogenesis The pathogenesis of the malaria syndromes, in the lung and elsewhere, is incompletely understood. Blood infections elicit significant cytokine activation, vascular dilatation and capillary leakage. These responses are predictable and account for much of the pulmonary edema. Adherence of the infected red blood cells to each other and to endothelial cells leads to PRBC sequestration and small vessel obstruction. Upregulation of intercellular adhesion molecules and related ligands plays a central role.6,48 Most of this cascade follows systemic cytokine production, such as tumor necrosis factor alpha (TNFa).48 There is also a variable lymphocytic interstitial infiltrate in some but not all cases.56 The specific contributions of these different mechanisms in causing acute lung injury are still unclear. With vivax-related ARDS, there is no generalized red cell sequestration,46 although there may be pulmonary sequestration.57 Additionally, post-treatment inflammation may be a significant factor in vivax compared with falciparum pathology.

Diagnosis The specific diagnosis of an acute malaria infection is usually made on blood films. A proportion of patients may have more

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than one species of infection. Immunohistochemistry with Falciparum-specific antigens in expert centers is helpful, especially in cases misdiagnosed in life and where blood is no longer available.58 Rapid antigen detection assays are probably comparable to good microscopy. Polymerase chain reaction diagnosis is increasingly applied to clinical cases and can differentiate the four Plasmodium species, but may not be better than light microscopy.59

Differential diagnosis The main differential diagnosis histologically is artifactual formalin pigment and rare cases of babesiosis (see below). Removing the pigment with picric acid is helpful in determining whether these are parasites and not pigment in the red cells.

Treatment and drug toxicity Even with intensive care treatment, mortality rates for those with severe falciparum infection approach 30%. The mortality of uncomplicated acute falciparum malaria is less than 1%. Malarial lung, i.e., pulmonary edema or diffuse alveolar damage, carries a high mortality rate. Those who develop pulmonary disease may die within 24 hours. Treatment consists of antimalarials and adequate ventilation support. Despite vast amounts of malaria chemotherapy administered globally, drug toxicity to the lung is very rare. Maloprim, used as malaria prophylaxis, has been associated with pulmonary eosinophilia on transbronchial biopsy.60 Mefloquine used in the treatment of low-level falciparum malaria can also cause ALI. Glucose-6-phosphate dehydrogenase deficiency might contribute in some mefloquine-related cases.61 Finally, one of the authors (SL) has encountered a fatal case of ALI that developed after parasitemic cure of mild falciparum malaria, attributed to excessive quinine administration.

Babesiosis Introduction

Babesiosis is caused by microscopic parasites that infect red blood corpuscles and are spread by certain ticks. In the United States, tick-borne transmission is most common in particular regions and seasons: it mainly occurs in parts of the Northeast and upper Midwest and usually peaks during the warm months.13

Epidemiology People can be infected with Babesia parasites in several ways. The main route is via an infected tick bite. This is usually during outdoor activities in areas where babesiosis is found. A less common way of becoming infected is by having a transfusion from a blood donor with a silent Babesia infection. Of note, no tests have been licensed yet for screening blood donors for Babesia. A few possible cases of congenital transmission have been reported.

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Many different species (types) of Babesia parasites have been found in animals, only a few of which have been found in people. Babesia microti, which usually infects white-footed mice and other small mammals, is the main species found in patients in the United States. Occasional (sporadic) cases of babesiosis caused by other Babesia species are reported. Babesia microti is transmitted in nature by Ixodes scapularis ticks (also called blacklegged ticks or deer ticks). Tick-borne transmission primarily occurs in the Northeast and upper Midwest, especially in parts of New England, New York, New Jersey, Wisconsin and Minnesota. The parasite is typically spread by the young nymph stage of the tick, which is most apt to be found (seeking or “questing” for a blood meal) during warm months, in areas with woods, brush or grass. Infected people might not recall a tick bite because I. scapularis nymphs are very small (about the size of a poppy seed).

Organism The Babesia organisms are intraerythrocytic protozoan parasites. They range in shape from single, round or pyriform bodies to ameboid or ring forms. Dividing forms may be seen. B. microti is the main species in the USA, and B. divergens in Europe.

Clinical features Although many people infected with Babesia do not have symptoms, disease is characterized by fever and anemia. Asplenic patients, those with HIV disease or iatrogenic immunosuppression, hepatic or renal disease, and the elderly are at particular risk of infection. It is usually acquired in temperate and tropical zones. Because Babesia parasites infect and destroy red blood cells, babesiosis can cause hemolytic anemia, leading to jaundice and dark urine. Complications include hypotension, severe hemolytic anemia, thrombocytopenia, disseminated intravascular coagulation (DIC), renal, hepatic, pulmonary or cardiac failure, and death. While most cases resolve without treatment, quinine and clindamycin are reportedly effective.62

Pathology Severe lung disease is uncommon, but a few cases of non-fatal acute lung injury manifesting as pulmonary edema are reported.5,63 As with malaria, ALI cases feature vascular congestion with parasitized erythrocytes. Hyaline membranes and reparative type II pneumocyte hyperplasia are also seen. The pathogenesis probably relates to cellular damage from local and systemic cytokine production.

Diagnosis and differential diagnosis Diagnosis is by blood film examination and DNA analysis by PCR.64 In symptomatic people, babesiosis is usually diagnosed by examining blood microscopically and seeing Babesia parasites inside red blood corpuscles. The parasites are similar to P. falciparum, but demonstrate a characteristic

Chapter 8: Pulmonary parasitic infections Figure 14. Toxoplasma life cycle. Unsporulated oocysts are shed in cat feces. (1) Intermediate hosts, including birds and rodents, become infected after ingesting contaminated soil, water or plant material. (2) Oocysts transform into tachyzoites shortly after ingestion, and they localize in neural and muscular tissues. Here they develop into tissue cyst bradyzoites. (3) Cats become infected after consuming intermediate hosts harboring tissue cysts or sporulated oocytes. (4) Animals bred for human consumption and wild game are infected with tissue cysts after ingestion of environmental sporulated oocysts. (5) Humans can be infected by eating undercooked meat of animals harboring tissue cysts, (6) consuming food or water contaminated with cat feces or by contaminated environmental samples such as litter box or fecal-contaminated soil, (7) blood transfusion or organ transplantation, (8) or transplacentally from mother to fetus. (9) Image courtesy of CDC.

Maltese cross pattern inside the red cells, and do not produce hemozoin pigment.

Toxoplasmosis Introduction

Toxoplasmosis is considered a leading cause of death attributed to food-borne illness in the United States. More than 60 million men, women and children in the USA carry the Toxoplasma parasite, but very few have symptoms because the immune system usually keeps the parasite from causing illness. Because toxoplasmosis is associated with impoverished people it is considered a Neglected Infection of Poverty (NIP).

Epidemiology The increase in the number of immunocompromised persons, and particularly those with HIV disease, is responsible for the dramatic global increase in cases of toxoplasmosis over the last 20 years. In addition to HIV infection, transplantation, lymphoma, cancer and immunosuppressive chemotherapy are other underlying predispositions. Many of these cases represent reactivation of latent infection. Toxoplasma gondii is a parasite of birds and mammals. Cats are the only definitive host and thus the only source of infective oocysts, but other mammals and birds can develop tissue cysts. Although feline infections are typically asymptomatic, infection during human pregnancy can cause severe disease in the fetus. Cat owners can reduce their pets’ exposure risk by keeping all cats indoors and not feeding them raw meat. Humans usually become infected through ingestion of oocyst-contaminated soil and water, tissue cysts in

undercooked meat, or congenitally. Most infections occur by eating the meat of another intermediate host. Cooking the meat kills the infection. This explains why human toxoplasmosis is more prevalent in countries where eating undercooked and uncooked meat is popular. Direct contact with cats is not thought to be a primary risk for human infection owing to their fastidious nature, the passing of non-infective oocysts and the short duration of oocyst shedding.65 Some human infection follows ingestion of contaminated feces (Figure 14).

Organism Oocysts excreted in feces are spherical, measuring 10 to 12 mm in diameter. When they are fully sporulated, they contain two sporocysts, each with four sporozoites. In smears, tachyzoites are crescent-shaped, taper at both ends and measure approximately 6 by 2 mm. Bradyzoites are smaller and more slender than tachyzoites. The nuclei of both organisms are situated at or slightly posterior to the midbody. Tissue cysts are spherical to subspherical, vary in size from 5 to 100 mm or larger, and contain up to thousands of bradyzoites. Tissue cysts have a relatively thin wall (0.5 to 1.0 mm.) This wall is eosinophilic, only slightly PAS-positive and stains black with silver stain.6 Tachyzoites and bradyzoites are seen when ruptured from cells or cysts. The nucleus stains red with Giemsa. Bradyzoites within cysts are usually PAS-positive, tachyzoites are not.

Clinical findings Congenital toxoplasmosis can damage the eyes and brain, but not the lungs. Primary human infection is usually asymptomatic. Among symptomatic adults there is a mild febrile selflimiting lymphadenopathic disease. Once infected, tissue bradycysts in muscle and brain last for many years and probably

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(a)

for the lifetime of the host. The prevalence of this latent infection ranges from 10 to 90% in different populations, as measured by serological surveys. Rare cases of acute primary self-limited pulmonary toxoplasmosis in immunocompetent persons are documented.66,67 Patients present with fever, dry cough and myalgia. The blood film features an atypical lymphocytosis, and serological conversion is noted. In immunocompromised patients, the situation is different since the infection is fatal if not treated. Lung disease is often part of multi-system infection, and it may be the presenting complaint in previously undiagnosed HIV disease. The pulmonary symptoms are dyspnea and cough. In most of these cases, the development of pulmonary toxoplasmosis represents reactivation of a previously latent infection.68,69 The commonest location of reactivated toxoplasmosis is the brain. Toxoplasma encephalitis is a major opportunistic disease in HIV infection. Heart and lung involvement is not uncommon. Radiographically, acute primary self-limited pulmonary toxoplasmosis shows bilateral interstitial infiltrates. In immunocompromised patients radiological studies can show a variety of patterns, including patchy interstitial infiltrates, pleural effusion, lobar pneumonia, widespread small miliary nodules5 or “white-out” (Figure 15a).

Pathology Grossly the lungs may show consolidation, suggestive of acute lung injury or yellow miliary nodules (Figure 15b).70 Only the yellow color distinguishes these lesions from mycobacterial infection. They may be congested and have petechial hemorrhages.5 Histopathology shows interstitial pneumonitis with lymphocytic septal infiltrates, or diffuse alveolar damage with fibrinous alveolar exudates and hyaline membranes. Numerous alveolar macrophages contain parasite cysts. Necrotizing pneumonia appears as a more advanced lesion, characterized

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Figure 15. Pulmonary toxoplasmosis. (a) Chest radiograph of military toxoplasmosis from an HIV-positive patient. Bilateral infiltrates are not a specific finding. (b) The accompanying autopsy lung features miliary nodules. Lesions are more yellow than usually seen in tuberculosis.

(b)

by extensive areas of parenchymal necrosis and the presence of numerous extra- and intracellular tachyzoites.5 Nodules, if present, feature infarct-like necrosis with ghosts of solid cells and necrotic blood vessels (Figure 16a). Some of these nodules may be related to the pneumonic process described above. The blood vessels often have thickened walls. Interstitial and intra-alveolar parasites can be difficult to see in H&E stains. Tachyzoites appear as small blue dots, similar to apoptotic nuclei (Figure 16b). Using oil immersion, there may be a clear cytoplasmic ring around the nucleus (Figure 16c). If bradycysts are present, these are large host cells containing multiple tightly packed small zoites. Bradycysts resemble raspberries.

Diagnosis Bradycysts may be very evident or the diagnosis can be overlooked. Immmunohistochemistry has transformed the diagnosis of this infection since there is always abundant parasite antigen, even if the lesion has been treated (Figure 17). Bronchoalveolar lavage may reveal the zoites, although its sensitivity is not known. Thus, lung biopsy may be needed in uncertain cases.71 Identifying the zoites may not be easy, and immunohistochemistry should be applied in all such necrotic pulmonary lesions. In immunocompromised patients, serodiagnosis is not as reliable and useful as in immunocompetent individuals. Thus a negative serology does not exclude toxoplasmosis, and one must interpret positive IgG and IgM titers carefully. The presence of IgM antibody or four-fold rise in the IgG antibody is usually indicative of acute infection while stable IgG antibody levels suggest prior infection. Dormant bradyzoite cysts may reactivate in the setting of immunosuppression. Empirical chemotherapy is another means of confirming the diagnosis through therapeutic response. Polymerase chain reaction has a high diagnostic value in the acute disease but, like many

Chapter 8: Pulmonary parasitic infections

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(b)

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Figure 16. Pulmonary toxoplasmosis. (a) Pulmonary infarction is often noted. (b) Bradycysts in the alveolar walls may be a subtle histological finding (arrow). Little inflammation accompanies the parasites in this case. (c) Under oil immersion a bradycyst contains abundant small hematoxyphilic zoites.

“in-house” PCR assays, suffers from lack of standardization and variable performance according to the laboratory. Molecular diagnosis of toxoplasmosis can be improved by performing real-time PCR protocols.72

Differential diagnosis There may be confusion with Histoplasma capsulatum. Periodic acid-Schiff and Grocott silver methods do not stain toxoplasma tachyzoites (see Chapter 7). Unlike Leishmania and Trypanosoma cruzi parasites, there is no kinetoplast. Since pulmonary toxoplasmosis in the immunocompromised host often has such a characteristic gross and imaging appearance, it is helpful to tabulate the other main causes of multiple miliary nodular lesions in the lungs (Table 1).

Treatment Figure 17. Pulmonary toxoplasmosis. An anti-T. gondii immunohistochemical stain highlights mainly single-cell tachyzoites.

While the prognosis for those with disseminated toxoplasmosis is poor, prophylaxis and treatment with pyrimethamine/

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Chapter 8: Pulmonary parasitic infections Table 1 Differential diagnosis of multiple miliary nodular lesions in the lung

Tuberculosis, and other mycobacterioses Bacterial infections Nocardiosis Plague Melioidosis Fungal infections Aspergillus Histoplasmosis Coccidiodomycosis Pneumocystosis Viral infections Herpes simplex and zoster Parasitic infections Toxoplasmosis Schistosomiasis Strongyloidiasis Cysticercosis Visceral larva migrans (Toxocara canis)

Figure 18. Microsporidiosis. Encephalitozoon cuniculi spores feature a dark band across their mid-section (arrow) (Brown and Hopps stain).

Other non-infectious diseases Wegener granulomatosis Tumor – carcinoma, sarcoma, lymphoma, melanoma Langerhan’s cell histiocytosis Foreign injected material (e.g. in IV drug users)

sulfadoxine kills proliferating tachzoites. However, the drug therapy is not active against quiescent cysts and patients remain at risk of future reactivation of disease.39

Microsporidiosis Introduction

Microsporidia are obligate intracellular protozoa belonging to the phylum Microspora. They are increasingly recognized as opportunistic infectious agents worldwide. Cases have been reported in developed as well as in developing countries.

Epidemiology Microsporidia infect mammals, other vertebrates and invertebrates, and until the HIV pandemic73 were known only to veterinary pathologists, with very few human cases. Oral, ocular, sexual and respiratory transmission routes are described; the latter is confirmed by finding microorganisms in the sputum and in the tracheobronchial tree.74,75 Patients with microsporidiosis are usually immunosuppressed, the commonest cause being HIV/AIDS. Other at-risk groups include solid organ transplant,76 acute leukemia77 and allogeneic bone marrow transplant patients.78 The source of infection in man is likely to be from water and the environment.

Organism Four genera of microsporidia affect man, the most common being Enterocytozoon (which normally affects only the gut)73 and Encephalitozoon (which can disseminate from the intestine). The species are now defined by a combination of

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Figure 19. Tracheal microsporidiosis. Semi-thin section clearly demonstrates spores (Toluidine blue stain).

ultrastructural morphology and molecular DNA characteristics.2 Within the host cell, they have a unique life cycle. After sporogenesis, the mature spores are extruded from the cell via the uncoiling of an intracellular tubule, and they are injected directly into an adjacent cell. Microsporidia produce resistant spores that vary in size, depending on the species. They possess a unique organelle, the polar tubule or polar filament, which is coiled inside the spore. The 1 to 5 µm microsporidia spores of species associated with human infection are oval, pyriform or elongated and thick-walled.13 Hematoxylin & eosin-stained sections show spores in tissue but special stains, such as Brown-Hopps, Grocott and Ziehl-Neelsen, are often more useful in diagnosis (Figure 18). A PAS-positive granule at the anterior end of mature spores is diagnostic of microsporidia. Electron microscopy aids study of this small organism (Figure 19).6

Clinical features Microsporidiosis is primarily a diarrhea-causing gut pathogen. Most patients with lung infection have disseminated extraintestinal disease also affecting the kidneys, eyes and biliary

Chapter 8: Pulmonary parasitic infections

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Figure 20. Tracheal microsporidiosis. (a) Ulcerative tracheitis in an HIV-positive patient with disseminated disease. (b) High magnification demonstrates Encephalitozoon cuniculi within metaplastic respiratory epithelium (arrows).

tree.79 Pulmonary symptoms include fever, cough, rhinorrhea, dyspnea and respiratory failure in severe cases. Radiology shows bilateral infiltrates and bilateral pulmonary opacities, along with areas of extensive consolidation. Species that affect the lung in man are Encephalitozoon cuniculi and E. hellem, and rarely Enterocytozoon bieneusi. Occasionally asymptomatic HIV-infected patients are documented with microsporidia in bronchial secretions.80 Microsporidia have also been found in the lining of a lung cavity caused by the bacterial infection Rhodococcus.81

Pathology Microsporidia are seen in bronchiolar and alveolar duct epithelial cells associated with lymphoplasmacytic infiltrates.82 In some areas the organisms are associated with an erosive tracheitis, bronchitis and/or bronchiolitis with or without pneumonia (Figure 20a). In well-oriented sections of the tracheobronchial epithelium large numbers of 2 µm in diameter Gram-positive ovoid spores are seen in the supranuclear or subapical regions of the host cells. On H&E stain, they have a hematoxyphilic asymmetric nucleus and clear cytoplasm; sometimes they are refractile. Infected cells desquamate and regenerative epithelial hyperplasia and atypia are noted (Figure 20b). Speciation requires electron microscopy or PCR examination. Because of the propensity for systemic dissemination of Encephalitozoon infections, all patients diagnosed as having E. hellem or E. intestinalis should be examined for respiratory tract involvement. This can be done by sputum cytology or BAL. Cytological preparations should be stained with Gram’s stain or Weber’s modified chromotrope stain to identify the spores.

Differential diagnosis On light microscopy, the “small blue dot” infections that may be confused with microsporidia include Cryptosporidium,

Toxoplasma gondii, Leishmania spp., bacteria, Histoplasma capsulatum and neuroendocrine granules. Standard fungal silver stains highlight Histoplasma well, but do not stain the other infections. Cryptosporidia reside at the surface of epithelial cells; leishmania amastigotes and fungi are found within macrophages while toxoplasma is seen within alveolar cells.

Treatment Albendazole improves patient gastrointestinal symptoms but may not decrease the organism burden.

Cryptosporidiosis Introduction

Cryptosporidium is a parasite that causes the diarrheal disease cryptosporidiosis. Both the parasite and the disease are commonly known as “Crypto”. Many Cryptosporidium species infect humans and animals. The parasite is protected by an outer shell that allows it to survive outside the body for long periods of time and makes it very tolerant to chlorine disinfection.13

Epidemiology Cryptosporidium parasites are found throughout the world. Travelers to developing countries may be at greater risk of infection because of poorer water treatment and food sanitation. In the United States, an estimated 748 000 cases of cryptosporidiosis occur each year, and it is one of the most frequent causes of water-borne disease among humans in that country. While this parasite can be spread in several different ways, water (drinking water and recreational water) is one of the commonest methods of transmission. For human infection, cows are the main source, with fecal contamination of water supplies. Contaminated water may include water that has not been boiled or filtered, as well as contaminated recreational

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Figure 21. Fecal detection of Cryptosporidium. Feces with characteristic small round oocysts (modified Ziehl-Neelsen stain).

water sources. Several community-wide outbreaks of cryptosporidiosis have been linked to drinking municipal water or recreational water contaminated with Cryptosporidium. Person-to-person transmission probably also occurs.

Organism Many Cryptosporidium species infect humans and a wide range of animals. Although C. parvum and C. hominis (formerly known as C. parvum anthroponotic genotype or genotype 1) are the most prevalent species causing disease in humans, infections by C. felis, C. meleagridis, C. canis and C. muris have been reported. The various forms of the parasite, non-cyst and oocyst, attach themselves to the surface of airway epithelial cells and elicit a mild lymphoplasmacytic infiltrate. Sporulated oocysts are 4 to 6 µm in diameter and are the resistant stage excreted in the feces. Oocysts stain pink to bright red and typically contain several prominent black or hematoxyphilic dots with a small amount of clear cytoplasm. Oocyst identification in aspirated material or fecal samples is facilitated with the Kinyoun modified Ziehl-Neelsen stain (Figure 21). Immunohistochemistry, available in some units, may be helpful. Electron microscopy and PCR molecular testing are diagnostically useful (Figure 22).83

Clinical features C. hominis and C. parvum cause self-limiting watery diarrhea in normal persons but a chronic and potentially fatal diarrhea occurs in undiagnosed HIV-infected patients. An infected person or animal sheds millions of parasites in the stool. Shedding of parasites starts when the symptoms begin and can last for weeks after the symptoms stop. They may be found in soil, food, water or surfaces that have been contaminated with the feces from infected humans or animals. It is not spread by contact with blood but can be spread by eating contaminated uncooked food, especially fruits and vegetables, and by touching one’s mouth with contaminated hands.

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Figure 22. Electron photomicrograph of a Cryptosporidium parasite undergoing schizogony. There are eight merozoites awaiting release. Note how the parasite is just within the host cell membrane.

Once infected, people with decreased immunity are at risk of severe disease. The risk of developing severe disease may differ depending on each person’s degree of immune suppression. Respiratory tract infection in man is virtually restricted to the immunocompromised, and mainly HIV-infected,2,84 but also occurs in children with immunoglobulin deficiency,85 severe combined immunodeficiency84 or CD40 ligand deficiency,86 and in occasional patients with malignant lymphoma.87 The route of infection is probably aspiration of infected gastric secretions Most patients with pulmonary cryptosporidiosis also have intestinal infection as well as other pulmonary infections, such as mycobacteria, cytomegalovirus and pneumocystosis.88 Isolated pulmonary disease is rare. Patients present with cough, tachypnea and excessive bronchial secretions. Imaging shows interstitial pneumonia.5 It is not always clear whether Cryptosporidium is a true pulmonary pathogen, since these patients have other ongoing infective and immune-pathogenic inflammatory processes to explain their bronchitic and pneumonic symptoms.88 In most cases, the parasites are seen only at the apex of the airway respiratory epithelium, and rarely in the lung parenchyma proper.

Pathology Cryptosporidium lives in the intestine of infected humans or animals, especially in the jejunum, where they are seen on the brush border.5 The organisms lie on the bronchial epithelium and may be confused with mucus or cellular debris because of their small size and indistinct structure (Figure 23). There may be some associated metaplasia but no inflammation. In AIDS patients organisms may be found in parenchymal inflammatory exudates.

Chapter 8: Pulmonary parasitic infections

and pleural effusions. Such is probably the result of a gut and lung fistula tract.94 The organism is a pear-shaped flagellate protozoan with an undulating membrane. The trophozoite of T. tenax is up to 12 µm in length and has five flagellae.

Lophomonas

Figure 23. Bronchial cryptosporidiosis. The zoites appear as small hematoxyphilic dots on the surface of the cell, although they are actually intracellular (arrows). Mild submucosal chronic inflammation is seen.

Diagnosis

These flagellated protozoa are symbionts of arthropods. A small number of renal transplant patients are reported with lung infections due to Lophomonas blattarum.95 Interestingly, similar protozoa are found in the intestines of dust mites and cockroaches. This finding raises the possibility that these vectors may be environmental causes of asthma5 and allergic rhinitis. They are also found in the sputum of AIDS patients.5

Cyclospora

Not surprisingly, there are several single or small series case reports of very rare protozoal infection in the lung or upper respiratory tract. Some of the species are common in other parts of the body and are included here for completeness.

Cyclospora species are related to Cryptosporidium, and are a cause of diarrhea from bowel infection. Of the various species belonging to this genus, only Cyclospora cayetanensis is pathogenic for man. The microorganism is acquired following the ingestion of sporulated oocysts through contaminated food and water.96 In immunocompetent individuals, depending on their immune state, infection may be asymptomatic or provoke a self-limited diarrhea episode. Cases frequently occur following visits to tropical countries. In immunosuppressed patients, especially in AIDS cases, the intestinal episode is more serious and tends to become chronic.5 C. cayetanensis has an oocyst of 8–10 µm in diameter and when infective contains two sporocysts, each with two sporozoites. These organisms are found within a vacuole at the luminal end of the enterocyte cytoplasm. These developmental stages measure 6–8 µm in length and 1–4 µm in width.6 There does not appear to be a primary lung disease, but two patients with pulmonary tuberculosis were reported to have C. cayetanensis oocysts in their sputum.97

Trichomonas

Balantidium

Trichomonads have worldwide distribution. Three species, T. vaginalis, familiar to gynecological cytopathologists, T. tenax and T. hominis are pathogenic in humans. Pulmonary trichomoniasis is usually associated with T. tenax acquired from the oral cavity. Newborns may acquire T. vaginalis infection from their mothers, causing immediate respiratory distress. It is also reported as a co-infection with Pneumocystis in immunocompromised patients.89 T. tenax is an oral commensal, but patients already debilitated with alcohol, lung cancer, poor dental hygiene or malnutrition may develop lung abscess or bronchiectasis.90,91 Organisms may also be found in the pleura.92,93 T. hominis is an intestinal tract commensal, reported in pulmonary abscess

Balandtidium coli is a large ciliated protozoon that normally resides in porcine intestine and is worldwide in distribution. The host most often acquires the cyst through ingestion of contaminated food or water. Trophozoites measure from 50 to 200 µm in length and 40–70 µm in width. They are ciliated and have two nuclei with prominent kidney-bean-shaped macronucleoli. The characteristic mouth opening is called a cytostome. Cysts are usually spherical, thick-walled and measure between 50 and 70 µm with a stainable macronucleus.6 Human colitis is uncommon and lung disease is even rarer and only affects immunocompromised patients.5 Lower lobe pneumonias are seen. Aspiration or BAL samples may contain characteristic trophozoites. It is probable the organism accesses the lung via the diaphragm, after perforating the colon.98

Diagnosis can be made on sputum, BAL, bronchial brushings and lung biopsy samples.

Differential diagnosis As mentioned above, the organisms may be confused with mucus or cellular debris because of their small size and indistinct structure.

Treatment Treatment with paromomycin and azithromycin combined with standard combination anti-HIV therapy is often effective.2

Very rare protozoal lung infections

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Helminths Worms have a complex taxonomy, but are conveniently categorized into nematodes (roundworms) and platyhelminths (flatworms). The nematodes are subdivided into filariae and soil-transmitted intestinal worms (which includes visceral larva migrans). Platyhelminths are subdivided into trematodes and cestodes. Cestodes do not have an intestinal tract.

Pulmonary eosinophilia syndromes Local and systemic eosinophil reactions are classic immunological T-cell-mediated responses to many parasitic infections, particularly worms. There are a number of clinical syndromes characterized by intermittent or chronic pulmonary disease featuring cough, dyspnea and asthma, eosinophilic pneumonitis or bronchitis, and a raised eosinophil blood count. Some of these syndromes are due to helminthic parasites.99 The terminology is confusing, with several historical eponyms. Distinguishing the various entities may require serology for infections and autoimmune diseases, molecular diagnostics, lung cytology and tissue biopsy, in addition to geographical history and basic chest imaging. The pathogenesis of parasitic pulmonary eosinophilia syndromes (PES) includes transient passage of larvae through the lung as part of the normal life cycle (Löffler syndrome, Katayama syndrome), lung as the definitive host phase of the parasite’s life cycle, disseminated infection during hyperinfection syndromes, hyperreactivity in the lung due to parasites in the blood (“tropical pulmonary eosinophilia” syndrome), ectopic location of a parasite in the lung, and involvement of the lung by non-human parasites (visceral larva migrans syndromes). Several infections can cause lung disease in more than one way. A practical way of approaching the worm infections is a morphological sieve-by-size. One should consider the diagnostic possibilities based on the morphological findings in the respiratory tract including eggs, adult reproducing worms, pre-adult immature and sterile non-reproducing worms and small larvae. Table 2 indicates which parasites cause a particular syndrome, with some diagnostic clues, and differential diagnoses, including non-parasitic diseases. These are the commoner infections. Many animal helminthic diseases (zoonoses) can affect man accidentally, and in some of these the lung can be involved.

Filarial infections Lymphatic filariases and tropical pulmonary eosinophilia syndrome Introduction

Filariasis is caused by nematodes that inhabit the lymphatics and subcutaneous tissues. Eight main species infect humans. Three species are responsible for most of the morbidity.

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Wuchereria bancrofti and Brugia malayi cause lymphatic filariasis, and Onchocerca volvulus causes onchocerciasis (river blindness). The other five species are Loa loa, Mansonella perstans, M. streptocerca, M. ozzardi and B. timori. B. timori also causes lymphatic filariasis.13

Epidemiology Among the agents of lymphatic filariasis, W. bancrofti is encountered in tropical areas worldwide, B. malayi is limited to Asia, and B. timori is restricted to Indonesian islands. Onchocerca volvulus occurs mainly in Africa, with additional locations in Latin America and the Middle East. Among the other species, Loa loa and M. streptocerca are found in Africa; M. perstans occurs in both Africa and South America; and M. ozzardi occurs only in the American continent. The filarial worms W. bancrofti and B. malayi are widespread in the tropics, the former being prevalent in Africa, India and South America, and the latter in Southeast Asia. They are transmitted by an infected mosquito bite. The larvae migrate to the appropriate site of the host’s body, where they develop into microfilariae-producing adults. The adults dwell in various human tissues, where they can live for several years (Figure 24). Their main clinical pathology is lymphatic filariasis, as adult worms reside in lymph node sinuses. Depending on the host response to this infection, the worms can induce an acute lymphadenitis which progresses to fibrosis.100 This causes lymphatic obstruction and dependent lymphedema. Adult worms are not found in lung tissues. As part of the life cycle, the adult worms release larval microfilariae into the blood at night. In susceptible individuals, these microfilaria cause the tropical pulmonary eosinophilia (TPE) syndrome. The term “TPE” when used without qualification indicates this disease. Most reports of TPE are from India.

Organism W. bancrofti adult worms are found typically in lymphatics or lymph nodes, where they may be coiled up. The body features are important for identification (Figure 25). Depending on its age, the female worm may measure up to 10 cm in length with a maximum diameter of 300 mm. The cuticle appears smooth, but fine 1 to 2 mm thick transverse striations are present, except in the lateral fields. The prominent lateral chords occupy up to two-fifths of the body circumference. Hypodermal nuclei are seen in most sections, typically near the base and toward the periphery of the lateral cords. One to three muscle cells are present in each quadrant. At most levels, transverse sections through the female worm contain paired uterine tubes filled with eggs, developing into microfilaria. The male is smaller than the female with a similar structure. The testis lies in the anterior end of the worm. The remaining tube is straight, cylindrical and contains spermatozoa. The morphological structure of the reproductive system gives no clue to the identification of the species.6 Worms that die in situ degenerate quickly and calcify.

Chapter 8: Pulmonary parasitic infections Table 2 Pulmonary eosinophilia syndromes

Pathogenesis

Infection

Pattern of lung disease

Raised IgE

Specific infection antibodies

Lung migration phase

Ascaris Hookworms Strongyloides

Transient

Variable

Lung migration phase

Schistosoma

Definitive host phase

Response to diethylcarbamazine (DEC)

Syndrome name

Yes

Variable

Löffler syndrome

Transient

Yes

No

Katayama syndrome

Paragonimus

Chronic

Yes

Hyperinfection syndrome

Strongyloides

Acute severe

Variable

No

Hyperreactivity in lung

Wuchereria Brugia

Recurrent at night

Yes

Yes

Yes

Tropical pulmonary eosinophilia (TPE)

Ectopic location

Schistosoma Echinococcus Taenia solium Enterobius Fasciola

Chronic

Variable

Yes

Visceral larva migrans

Toxocara canis Gnathostoma Angiostrongylus Dirofilaria

Recurrent Irregular or chronic

Yes

Yes

Non-parasitic infection syndromes

Aspergillus

Chronic with exacerbations

Yes

Yes

No

Bronchopulmonary aspergillosis

Moderate

No

No

Churg-Strauss syndrome

Variable

No

No

Idiopathic hypereosinophilia syndrome

Non-infection syndromes

The adult B. malayi worms resemble those of W. bancrofti but are smaller. Female worms measure 43 to 55 mm in length by 130 to 170 mm in width, and males 13 to 23 mm in length by 70 to 80 mm in width. Adults produce microfilariae, measuring 177 to 230 mm in length and 5 to 7 mm in width. The microfilariae are sheathed and have nocturnal periodicity.13

Clinical features Men are affected more than women, in a ratio of 4:1, and patients are usually aged 20–40 years.101 At night, there are attacks of cough, wheezing, dyspnea, fever and fatigue. Pulmonary function tests show a restrictive defect with diminished residual volume and total lung capacity. In chronic disease, these changes may be irreversible, indicative of pulmonary fibrosis. The blood eosinophilia count is usually > 3000/µl, and may be > 50 000/µl. The total blood IgE concentration is high. If patients are not treated, lung function declines irreversibly.

Other serology

ANCA

Chest radiographs and CT scans show increased bronchovascular markings and diffuse interstitial 1 to 3 mm lesions, or mottled opacities involving mainly the medial and basal lung regions. Up to a fifth of patients with active TPE may have a normal chest radiograph. Less common manifestations include focal consolidation resembling lobar pneumonia and bronchiectasis.102,103 The radiological picture can mimic miliary tuberculosis.

Pathology Mortality is low in TPE, so the pathological descriptions come from lung biopsies.104 Grossly, the visceral pleura is thickened with a pale nodular appearance. Hemorrhagic parenchymal lung lesions are also noted. The bronchioles contain muco-pus. Histologically, adult worms are rarely seen, and if identified are usually dead (Figure 26). Both adult worms and microfilariae elicit an eosinophilic alveolitis. The organisms

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Chapter 8: Pulmonary parasitic infections Figure 24. Wuchereria bancrofti life cycle. Image courtesy of CDC.

Figure 25. Wuchereria bancrofti microfilaria in a thick blood film demonstrating cephalic space, caudal space and sheath (Delafield’shematoxylin stain).

are usually degenerate and fragmented; where measurable they are 3–7 µm in diameter (Figure 27). There may be surrounding eosinophilic debris, sometimes termed a Meyers-Kouwenaar body, which is analogous to the Splendore-Hoeppli reaction around schistosome eggs (see below). Old lesions become granulomatous, and are surrounded by many lymphocytes and plasma cells. Hemosiderin-laden macrophages are also present and interstitial fibrosis follows. The lung is not the only organ affected in this syndrome, as similar lesions are found in lymph nodes, the liver and spleen.104 These findings reflect a heightened immune response to circulating microfilariae.

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Figure 26. Eosinophilic pneumonia rarely contains a necrotic adult worm.

Diagnosis The diagnosis of TPE is usually made on clinical, radiological and immunological criteria, along with patient response to diethylcarbamazine (DEC) treatment.105 Lung biopsies are rarely undertaken. Cytopathology of bronchial brushings, lavage and fine needle aspiration may identify the microfilariae as well as numerous eosinophils.106,107

Differential diagnosis The differential diagnosis includes the other larval PES described in this chapter. In addition, other conditions with

Chapter 8: Pulmonary parasitic infections

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Figure 27. Lung biopsy from a patient with TPE syndrome. (a) A microfilarial fragment is surrounded by an eosinophil-rich inflammatory infiltrate (arrow). (b) Higher magnification demonstrates the internal nuclei of the microfilaria (arrow).

(b)

pulmonary eosinophilia should be considered (see Chapter 15). Vasculitic diseases such as Churg-Strauss syndrome and Wegener granulomatosis must be excluded (see Chapter 19). Morphology restricts possible infective etiologies to a certain extent, but clinical and immunological features are critical in making a definite diagnosis. In practice, a precise diagnosis is not always possible in cases of pulmonary microfilariae.

Pathogenesis The TPE syndrome was first described in the 1940s, but it was not until the next decade that an epidemiological link with lymphatic filariasis was made. In 1960 the microfilarial fragments were first reported in the lung.104 The adult worms in the lymph nodes release their microfilariae at night, hence the predominantly nocturnal symptoms of TPE. Most patients with lymphatic filariasis do not suffer from TPE. The current concept suggests that immunological processes involved in the clearance of microfilaria in the bloodstream are responsible for the manifestations of the disease.108 It is the heightened immune response in certain susceptible individuals that causes the protean manifestations of TPE. These patients have exceptionally high levels of specific IgE and IgG antibodies which opsonize microfilariae in lung vessels. This contrasts with the minimal immunological reactions in most persons with chronic filarial infections in endemic regions, who do not develop the pulmonary or other manifestations of TPE. Recent work has identified complex interleukin secretion patterns that contribute to airways hyperresponsiveness in TPE. It is intriguing that human pulmonary epithelium harbors a g-glutamyl transpeptidase, similar to that found in B. malayi larvae.109

Dirofilariasis Introduction

The Dirofilaria genus is an animal infection (zoonosis) and paratenic, so man is infected accidentally. There are 24 species,

which can be divided into two groups; those that inhabit the heart and vascular systems of their host, and those that live in the subcutaneous tissues.6

Epidemiology Most human infections following an infected mosquito bite occur in the USA, but cases are also reported in Europe, Africa and Asia.105 Pulmonary (D. immitis) and subcutaneous (D. repens and D. tenuis) organisms affect humans. D. immitis is found in dogs and has a worldwide distribution, D. repens is common in dogs in Europe. D. tenuis is found in racoons in the southern and southeast United States. Canines are the definitive host of D. immitis (“dog heart worm”) but cats, foxes, sea lions, wolves and otters can be affected.4 This parasite occurs in most states of the USA and transmission has been reported in Europe, the Mediterranean, Japan and Australia. Larvae are injected by mosquitoes and mature into adult female and male worms that reside in the heart and pulmonary artery. The Dirofilaria require several months to reach a sexually mature stage. The female releases microfilariae, which are taken up by biting mosquitoes (Figure 28).

Organism The larva develops to an immature 1–2 mm long and 100–350 µm wide worm in the pulmonary artery. They have a smooth, thick cuticle (5–25 µm) with three distinct layers. The cuticle projects inward at the lateral chords forming two prominent internal longitudinal ridges. Somatic muscle is often prominent. Transverse sections may reveal two large uteri and a smaller intestine in female worms. Male worms have a single reproductive tube and intestine.

Clinical features More than half of infected patients are asymptomatic and lesions may only be detected on routine chest radiographs. Pulmonary manifestations are most common and include paroxysmal cough (worse at night), breathlessness and wheezing.

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Chapter 8: Pulmonary parasitic infections Figure 28. Dirofilaria immitis life cycle. Image courtesy of CDC.

Figure 29. Pulmonary dirofilariasis. Computed tomogram demonstrating a right lower lobe subpleural nodule (arrow) along with a small loculated pleural effusion.

Nonspecific features of fever, malaise, weight loss and nonpulmonary manifestations such as vomiting and diarrhea may also occur. Chest pains, hemoptysis or acute joint pains are also reported.108 Peripheral eosinophilia occurs in no more than 15% of patients. Radiographs demonstrate solitary non-calcified nodules. Reticulonodular opacities, particularly in the middle and lower zones, and miliary mottling with prominent hila are also noted.108 In 5% of cases the nodules may be multiple.110 Recent CT imaging descriptions indicate that the nodules are connected to pulmonary artery branches and sometimes to venous branches. The homogeneous low-

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Figure 30. Pulmonary dirofilariasis. This resected coin lesion features a round light tan subpleural infarct with a fibrous capsule. The color and fibrous capsule distinguish the lesion from a typical vascular infarct.

attenuation centers of the nodules on contrast enhancement correlate with the necrosis seen on histopathology.111 Pleural effusion near a subpleural nodule has been reported (Figure 29).112

Pathology The typical round well-circumscribed 1 to 4 cm in diameter, firm gray-yellow nodule is usually, but not always, pleuralbased (Figure 30).113–115 Wedge-shaped nodules are uncommon. Microscopically one sees a degenerate worm coiled within a necrotic pulmonary arteriole or artery or within

Chapter 8: Pulmonary parasitic infections

(a)

(b)

Figure 31. Pulmonary dirofilariasis. (a) The well-circumscribed necrotizing lesion has an eosinophilic hue. (b) The granulomatous infarct is surrounded by lymphoeosinophilic inflammation while a dead worm sits in a thrombosed blood vessel (arrow).

(a)

(b)

Figure 32. Pulmonary dirofilariasis. (a) Fragments of dead worm feature a thick multilayered and smooth cuticle with a suggestion of longitudinal ridges. (b) Dead parasites often calcify.

necrotic material in the center of the lesion. The necrosis is typical of a so-called helminthoma, i.e., very fibrinoid and eosinophilic, including Charcot-Leyden crystals (Figure 31a). A fibrous wall contains histiocytes, lymphocytes, eosinophils and occasional multinucleated giant cells (Figure 31b). Worm morphology, though degenerate, is characteristic (see above). In practice, finding a worm of this diameter within a pulmonary artery surrounded by necrosis is diagnostic of dirofilariasis (Figure 32). Reactive squamous metaplasia around the necrosis is often seen.116 Other findings in adjacent lung include a desquamative interstitial pneumonia-like reaction, follicular bronchiolitis and patchy organizing pneumonia.117 Overlying pleura often demonstrates chronic fibrosis but only rarely eosinophilic or necrotizing granulomatous inflammation.

Diagnosis Serology is available in special centers but is of uncertain utility. Fine-needle aspiration cytology of suspicious lung lesions has the potential for identifying more cases pre-operatively.110 Polymerase chain reaction DNA diagnostics for Dirofilaria sp. in formalin-fixed paraffin-embedded tissues are also emerging.114

Differential diagnosis Other nematodes that may lodge in pulmonary arteries include Angiostrongylus, Brugia and Wuchereria.117 Distinction of these lesions requires both a travel history and morphological knowledge. The differential diagnosis of lung helminthoma is given in Table 3. Squamous metaplasia lining the infarct cavity may cause confusion with squamous cell carcinoma.

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Chapter 8: Pulmonary parasitic infections Table 3 Differential diagnosis of a solitary necrotic nodule simulating cancer

Protozoa

Entamoeba histolytica

Helminths – relatively common

Dirofilaria Paragonimus

Helminths – relatively uncommon

Enterobius vermicularis Gnathostoma Capillaria Taenia solium cysticercus Fasciola Sparganum Pentastome

Mycoses

Histoplasma capsulatum Coccidioides immitis

Mycobacterial

Tuberculoma and other species

Non-infectious

Wegener granulomatosis

Organism

Thromboembolic infarction

Ascaris lumbricoides is the largest nematode parasitizing the human intestine. Adult females may reach 60 cm while adult males are only 15 to 30 cm (Figure 34).13 The adult worms are cylindrical and tapered at their extremities (Figure 35). The anterior end has three fleshy lips. In sections the worm is recognized by its large size, thick body wall with multilayered cuticle and many muscle cells in each quadrant of the body.6

Primary and metastatic carcinoma

Treatment Pulmonary dirofilaria is diagnosed and treated with surgical excision.

Soil-transmitted helminth infections Nematode infections of the intestine, acquired from eggs or larvae in warm moist soil, are major global causes of human disease.118 They are widely distributed across the tropics and subtropics. Several, Ascaris lumbricoides, the hookworms Necator americanus, Ancylostoma duodenale and Strongyloides stercoralis, have an obligatory larval migration phase via the respiratory tract and lungs. This larval migration may be clinically silent or symptomatic. Since larvae moult in the lungs, they shed antigens that can induce an allergic reaction. In strongyloidiasis, an additional pulmonary syndrome can develop from hyperinfection. The other main soil-transmitted intestinal nematode infections, Trichuris and Enterobius, do not have a pulmonary phase to their life cycle.

Ascariasis

Introduction Of the soil-transmitted helminth group, Ascaris lumbricoides is responsible for most clinical lung disease. It may also be more severe on reinfection. Humans are essential for completion of the life cycle.

Epidemiology Almost one-quarter of the world’s population is infected with A. lumbricoides (sometimes called just “Ascaris”). It is

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especially prevalent in tropical regions, including Latin America and parts of Africa. Ascaris lives in the small intestine, mainly the jejunum, and eggs are passed in the feces of infected persons. If the infected person defecates outside or if the feces of an infected person are used as fertilizer, eggs are deposited on soil. They can then mature into an infective form. Ascariasis is caused by ingesting eggs. This can happen when hands or fingers that have contaminated dirt on them are put in the mouth or by consuming vegetables or fruits that have not been carefully cooked, washed or peeled. After ingestion the eggs hatch into larvae in the duodenum, invade the bowel wall and gain access to lymphatics or veins. Organisms reach the lungs through these vascular channels. They mature and moult in the lungs, ascend the airways, are swallowed and then migrate to the intestines, where the adults reside and excrete eggs (Figure 33).

Clinical features Intestinal disease may cause no symptoms. Pulmonary symptoms are commonest in previously exposed patients, who are hypersensitive to the Ascaris antigens. Symptoms last 7 to 10 days and resolve when the parasites migrate from the lung. The pulmonary phase manifests with a non-productive cough, wheeze or dyspnea. Several different patterns of pulmonary ascariasis are noted, while blood-tinged sputum indicates a severe infection.118 Patients may present with acute self-limiting Löffler syndrome (see Chapter 15). This presentation lasts up to 2 weeks. Occasionally it develops into a chronic eosinophilic pneumonia.119 In endemic areas “Löffler syndrome” is frequently seen in emergency rooms.120 This process can clinically mimic bacterial pneumonia.121 HIV-infected persons are also described with Löffler syndrome.122 Other patients less frequently present with pleural effusion, chronic eosinophilic pneumonia (see Chapter 15) and calcified granulomas. Status asthmaticus is epidemiologically associated with ascariasis, suggesting it is an occult environmental cause of asthma.56,118 Smoke inhalation injury exacerbates infections. Limited experience from pediatric burn centers suggests pulmonary ascariasis, superimposed on smoke inhalation injury, reduces burn patient survival rates.123 It is postulated that host stress stimulates worm migration into the respiratory tract. Worms can obstruct bronchi and endotracheal tubes.124 If there is a bronchopleural fistula, a mature worm can migrate into a pyopneumothorax.125 Also,

Chapter 8: Pulmonary parasitic infections Figure 33. Ascaris lumbricoides life cycle. Image courtesy of CDC.

Figure 34. Bolus of Ascaris lumbricoides from a fatal case. Over 800 worms were removed from the small intestine of a 2-year-old South African girl.

ascariasis obstructs bile ducts, and resulting liver abscesses can rupture into the right chest cavity. Löffler syndrome presents with migratory patchy consolidation or subpleural nodules on CT scan.126 The radiology may resemble miliary tuberculosis or viral pneumonia.

Pathology Opportunities to see the histopathology of pulmonary ascariasis are few. Adult worms usually measure up to 20 cm in

Figure 35. Ascaris lumbricoides. This female is round and tapered at the ends. Adult females may grow up to 60 cm in length.

length and 1 cm in diameter, while larvae are 1–2 mm long and 75 µm in diameter. The large larvae cause alveolar hemorrhage when exiting pulmonary capillaries. Pulmonary edema, an allergic eosinophilic bronchitis/pneumonitis and desquamation of alveolar lining cells ensues. Charcot-Leyden crystals may be seen in the alveoli, reflecting the intensity of the eosinophil chemotactic response. Larvae can also be seen in

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Figure 36. Ascaris larvae elicit an eosinophilic reaction within a bronchiolar lumen (arrows). Lateral alae are noted (arrowheads).

Figure 37. Part of a hookworm larva in an airspace. These organisms are smaller than Ascaris larvae.

small airways (Figure 36). Granulomas develop where larvae die and sometimes organisms undergo dystrophic calcification.127 The lungs can thus be indirectly damaged. Worms can also obstruct the bile ducts and cause a liver abscess, which may result in an empyema.

Strongyloidiasis

Diagnosis The diagnosis of pulmonary ascariasis is clinical, radiological and immunological. Blood eosinophilia, eosinophils in sputum and bronchoalveolar lavage fluid, identification of eggs in feces, and positive serology combine to make a diagnosis. Sometimes the eggs, larvae or adult worms can be identified in biopsy or autopsy specimen.4 The CDC only recommends examination of feces for diagnosis.13

Treatment Anthelminthic medications, such as albendazole and mebendazole, are the drugs of choice for treatment of Ascaris infections. Infections are generally treated for 1–3 days. The drugs are effective and appear to have few side effects.13 Of note, A. suum is related to A. lumbricoides. This zoonosis in pigs can cause a visceral larva migrans, i.e., the larvae migrate to the lungs but do not mature into adults in man. The infection comes from eating uncooked pig liver that contains adult worms. Imaging and histopathology are similar to those of A. lumbricoides infection.128

Hookworm infection The migration phases of Necator americanus and Ankylostoma duodenale hookworm infections also cause Löffler syndrome, but are less common and less severe than those seen with ascariasis.2,4 It is often impossible to identify the worm in histological sections; however, the larvae are smaller than Ascaris, measuring 300 µm by 20 µm, and may cause a localized eosinophilic pneumonia (Figure 37).

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Introduction

Strongyloides stercoralis is almost unique as a human parasite, as it features both an auto-infection and a more typical soiltransmitted life cycle. This nematode infection is globally distributed across tropical and subtropical zones, where sanitation and hygiene are poor. Infections in other areas, including northwest Europe, represent imported cases.129 Other Strongyloides include S. fülleborni, which infects chimpanzees and baboons and may produce limited infections in humans.

Epidemiology Strongyloides stercoralis is common in warm, wet regions with fecal contamination of soil or water. The disease is endemic in Indochina and Africa, while control programs in regions of Japan have eliminated the problem. The parasite is unusual in that there is no male adult worm in the human life cycle. Female worms reside in the human intestinal crypts, where they release their eggs. These rapidly hatch into rhabditiform larvae and are excreted in feces. In the soil, the larvae either mature into free-living adult male and female worms or transform into filariform larvae, which can invade skin and access the dermal veins. These larvae exit the circulation, when they invade the lungs, migrate up to the pharynx, and are swallowed. In the duodenum the larvae mature into adult female worms. Unlike nearly all other worm diseases, the infection can continue once the adult worms die, because the rhabditiform larvae in the lower intestine and anus transform directly into filariform larvae and these reinvade the mucosa or skin. This process perpetuates the infection for decades, even in the absence of a recent environmental exposure. The free-living soil cycle is perpetuated as long as there is a suitable environment. Temperature and humidity are important factors.4

Chapter 8: Pulmonary parasitic infections

The auto-infection loop is accelerated by several immunocompromised states, most notably old age, immunosuppressive therapy for autoimmune diseases, transplantation patients, anti-neoplastic chemotherapy, non-Hodgkin lymphoma, malnutrition and HTLV-1 infection.2,56,129 In such persons, a hyperinfection syndrome may develop with rapidly increasing larval infective loads, reflecting a breakdown in normal host defenses. Oddly, HIV infection is not a major risk factor for hyperinfection. This is probably because HIV affects Th-1-related immune defenses, rather than Th-2, which is important in strongyloides immunity. Of note, the Th-2related immune defenses are particularly depleted by HTLV-1 infection.2,129

Pathology

The female Strongyloides stercoralis measures up to 2.7 mm in length by 30 to 40 mm in width. The anterior one-third of the body contains the esophagus, while the reproductive tube fills the posterior two-thirds. Most sections contain the tubular intestine and two reproductive tubes. There are usually many eggs in the process of development adjacent to sections of adult worms. Rhabditoid larvae measure 180 to 380 mm by 14 to 20 mm. Key morphological features include a long esophagus (about half the body length) and a notched tail. These features are easy to see, especially in sputum samples. The morphological features of the adult female are difficult to discern in human tissue but the minute size of the worm and the presence of developing eggs in the vicinity aids diagnosis. They are seen in the lung on rare occasions.

Nearly all the descriptive histopathological accounts of pulmonary strongyloidiasis come from HS patients. Rhabditoid larvae in tissues may cause no tissue reaction. Pathological patterns include eosinophilic pneumonitis with larvae, acute lung injury patterns (Figure 38a,b), alveolar hemorrhage (Figure 38c), granulomas around larvae, septal fibrosis, bronchitis with mucus plugging, secondary bacterial pneumonia and hyperinfection-associated pathology, such as lymphoma. The acute infection Löffler syndrome is similar to that described in ascariasis and features focal eosinophilic pneumonitis with alveolar hemorrhage (see Chapter 15). The alveolar hemorrhage is partly ascribed to larvae breaking out of the pulmonary capillaries into the alveoli, and has been noted as an initial presentation of HS after stem cell transplantation131 and in HIV disease.132 There may be a mixed inflammatory infiltrate with macrophages, giant cells, lymphocytes, plasma cells, neutrophils and eosinophils. Larvae in the bronchioles also induce mucus secretion, provoking secondary pneumonia.133 If the larvae do not exit the lung, they die and are phagocytosed by histiocytes. Granulomas form and may result in parenchymal fibrosis.134 Acute lung injury occurs in HS-associated septic shock. But it has also been described following apparently successful treatment.135 Perhaps paradoxical reaction to abundant antigen released from killed larvae triggers severe lung injury. This reaction has also been described in successfully treated tuberculosis.

Clinical features

Differential diagnosis

Five clinical patterns of strongyloidiasis may involve the lungs.129 The initial migration phase can induce a transient Löffler syndrome (see above). Symptomatic chronic intestinal infection, asymptomatic auto-infection, symptomatic autoinfection or hyperinfection syndrome (HS) with dissemination are also recognized. Pulmonary disease in non-hyperinfection cases is similar to the presentation encountered in ascariasis. Individuals experience cough, hemoptysis, dyspnea and/or bronchospasm. The hyperinfection syndrome, an acceleration of a chronic infection, results in large numbers of invasive larvae in the intestine, lungs and elsewhere, including liver, lymph nodes and brain. In addition to this damaging pathological process, a Gramnegative bacteremia (e.g., E. coli) may develop, since invasive filariform larvae carry fecal bacteria with them from the intestinal lumen. These patients are very ill with multi-organ failure, and unless the diagnosis is suspected and treated promptly, mortality is high. Disease is characterized radiographically by nonspecific infiltrates and nodular lesions. Occasionally there may be a mass lesion on chest radiograph that simulates a malignant tumor.130

The morphological differential diagnosis of Strongyloides larvae includes the larvae of Ascaris, the hookworms, Toxocara and rare zoonotic infections such as Baylisacaris. Morphological distinction may require an expert parasitologist. The clinical context usually suggests the correct diagnosis, and serology is very helpful and increasingly specific.

Organism

Diagnosis Low-grade strongyloidiasis is difficult to diagnose as the intestinal parasite load is small. In hyperinfection, however, larvae can be found in large numbers in the feces, sputum, bronchoalveolar lavage and urine.56,130 Unfortunately, the diagnosis is often made at autopsy. Immunodiagnostic tests for strongyloidiasis are indicated when the infection is suspected and the organism cannot be demonstrated by duodenal aspiration, string tests or repeated examinations of stool. Antibody detection tests should use antigens derived from S. stercoralis filariform larvae for the highest sensitivity and specificity. Enzyme immunoassay (EIA) is currently recommended because of its greater sensitivity (90%). Immunocompromised persons

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(a)

(b)

(c)

Figure 38. Pulmonary strongyloidiasis. (a) Hyperinfection often manifests with necrotizing inflammation and diffuse alveolar damage. (b) Small filariform larvae may be difficult to identify within the intra-alveolar exudate (arrow). (c) Larval forms may be easier to recognize in instances of pulmonary hemorrhage.

with disseminated strongyloidiasis usually have detectable IgG antibodies despite their immunosuppression. Crossreactions in patients with filariasis and other nematode infections may occur. Antibody test results cannot be used to differentiate between past and current infection. A positive test warrants continuing efforts to establish a parasitological diagnosis followed by antihelminthic treatment. Serological monitoring may be useful in the follow-up of immunocompetent treated patients since antibody levels decrease markedly within 6 months after successful chemotherapy.13 S. stercoralis can be differentially detected in infected human fecal samples with PCR technology. The detection limit of the method is as little as four S. stercoralis larvae in a 100 mg fecal sample.135

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Treatment Ivermectin is a standard treatment for strongyloidiasis, and is sometimes used empirically as a prophylaxis against chronic strongyloidiasis in high-risk individuals.13

Visceral larva migrans Introduction

These syndromes are infections by parasites that cannot mature into the next phase of the life cycle or adult worms in man. The larvae wander through many tissues, causing variable inflammatory lesions before dying. Implicated organisms include Toxocara canis, Angiostrongylus sp., Gnathostoma sp. and D. immitis. The term “visceral larva migrans” (VLM), when used without qualification, is taken to indicate T. canis infection. Dirofilariasis is discussed under the filarial worms

Chapter 8: Pulmonary parasitic infections Figure 39. Toxocara canis life cycle. Image courtesy of CDC.

above. Other infections are considered under “Uncommon nematode lung infections” below.

Toxocariasis Introduction

Infected dogs and cats shed Toxocara eggs in their feces and contaminate the environment. Humans or other animals can become infected if they ingest soil containing Toxocara eggs. Although rare, people can be infected by eating undercooked or raw meat from an infected animal such as raw lamb or calf’s liver.13

whereas T. cati has a diameter of 15 to 17 µm in the living state.6 In transverse section the body wall has a thin cuticle and inconspicuous single lateral alae. There are a few muscle cells of the coelomyarian type in each body quadrant. These nuclei are large and are often seen in transverse sections. The diagnostic features of Toxocara larvae include the diameter, prominent single lateral alae, and well-defined excretory columns that occupy a greater portion of the body cavity than the intestine.

Clinical features

Worms are globally endemic in the guts of dogs (T. canis) and cats (T. cati). Adult worms play no part in human toxocariasis. The worm excretes eggs in the feces. When man ingests these eggs from contaminated soil or food, the Toxocara eggs hatch and roundworm larvae can travel in the bloodstream to several parts of the body, including the liver, heart, lungs, brain, muscles and eyes. However, the larvae cannot mature (i.e., the infection is paratenic) (Figure 39). Children are the main victims of this visceral larva migrans syndrome (VLM), and the disease can continue for many years following the initial infection.2

Most infected people do not have any symptoms. However, in heavy infections, the larvae can cause damage.6 The VLM usually affects the eye, brain and liver, but respiratory symptoms may predominate.105 Pulmonary presentations include a Löffler syndrome with fever, cough, dyspnea, wheezing and (in children) failure to thrive. Toxocara infection is also an occult environmental cause of asthma.118 Chest imaging shows migratory infiltrates. Blood eosinophilia is usual, and the serum IgE level is high. Pleural effusion is another presentation, and there may also be pericardial effusion with tamponade.136 Whilst the usual cadence is a self-limiting disease, there are reports of chronic eosinophilic pneumonitis lasting 7 weeks.137 Severe lung infection may produce nonspecific infiltrates from a pneumonitis with subpleural nodules on CT.138,126

Organism

Pathology

Adult T. cati and T. canis are similar in their gross appearance. The infective larvae of T. cati and T. canis are morphologically identical, except for their diameters. The larvae measure about 400 µm in length but T. canis has diameter of 18 to 21 µm,

The typical VLM lesion demonstrates necrotizing eosinophilic granulomas. Granulomas may be surrounded by alveolitis (Figure 40a). However, in lung tissue and effusions, as in the liver, it is very uncommon to find actual parasites since they

Epidemiology

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(a)

migrate and only a small number of them are required to induce a large inflammatory response. Sampled lesions may have empty tracks with necrosis and residual eosinophils, macrophages and lymphocytes. If identified, the 400 µm in length and 20 µm in diameter larvae feature intestinal and excretory columns with nuclei. However, they have often degenerated and direct morphological confirmation is impossible (Figure 40b).

Diagnosis The constellation of clinical features, imaging, eosinophilia, high serum IgE and positive specific serology of anti-T. canis antibodies is usually diagnostic. Enzyme-linked immunoabsorbent assays are 85–90% specific.139 Western blot is more specific and diminishes cross-reactivity problems.140 A high eosinophil count in BAL and pleural aspirates is characteristic. Biopsy identification is less common, and histology may only suggest the diagnosis.

Differential diagnosis Baylisascaris is another ascarid infection, found in racoons in the USA and elsewhere. The larvae of Baylisascaris, unlike those of Toxocara, grow in the human host. They are larger than Toxocara.

Uncommon nematode lung infections Given the large number of human and zoonotic parasitic nematode infections, it is not surprising that there are occasional cases of airway, lung and pleural involvement by unexpected species. In general, finding worms, larvae or eggs in lung tissue pathology will be resolved diagnostically by focusing on the commoner infections. But identifying some of the entities briefly mentioned often requires consultation with a parasite reference center, where morphological help as well as serodiagnostics are available.

Enterobiasis Among the commonest worm infections, Enterobius vermicularis is usually restricted to the large intestine and anus.

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Figure 40. Pulmonary toxocariasis. (a) Larvae are rarely seen within typical necrotizing granulomas (arrow). As illustrated here, not all cases feature prominent eosinophilia. (b) While not diagnostic of T. canis, size and body contents suggest the diagnosis. The single ala is not seen in this case.

(b)

Occasionally an adult female accidentally ascends the female genital tract into the pelvis and abdomen or worms penetrate through pre-existing defects in the gut wall into the abdomen. Rare 1.0 to 2.0 cm localized solitary pulmonary coin lesions are noted radiographically. How the worm reaches the lung is unclear. Perhaps it is accidentally inhaled or ingested, or it might embolize to the lung through an abdominal vein.141 The granulomatous nodule, a typical helminthoma, features an eosinophilic necrotic center about the remains of an adult worm and its eggs (Figure 41a). The E. vermicularis adult has a characteristic structure with lateral longitudinal ala ridges (Figure 41b). The eggs have a flat side and a convex side, a colorless shell (unlike the brown shell of Schistosoma eggs) and recognizable internal contents.4

Anisakiasis Anisakis worms are parasites of sea mammals, with fish as the intermediate host. Thus eating raw or undercooked fish can transmit larvae, which burrow into the stomach or small bowel mucosa. This produces a localized eosinophilic gastroenteritis. Less commonly the worms enter the abdominal cavity or subcutis. A few patients are described presenting with pleural effusions and blood eosinophilia, where serum and the effusion react most strongly to Anisakis antigens with in vitro tests.142,143 Presumably the worms cross the diaphragm into the pleural cavity and initiate a brisk allergic reaction. Workers who process raw fish may also develop asthma-like symptoms and dermatitis.144

Capillariasis Capillariasis is a parasitic disease in humans caused by two different species of capillarids: Capillaria hepatica and Capillaria philippinensis. C. hepatica is transferred through the fecal matter of infected animals and can lead to hepatitis. C. philippinensis is transferred through ingesting infected small freshwater fish and can lead to diarrhea and emaciation.7 The organism can live in soil. The disease was first seen in the Philippines but has extended from that location and has been

Chapter 8: Pulmonary parasitic infections

(a)

(b)

Figure 41. Pulmonary enterobiasis. (a) Gravid Enterobius vermicularis centered in necrotizing granuloma (arrow). (b) Higher magnification of worm demonstrates green lateral alae and several eggs (Movat stain).

(a)

(b)

Figure 42. Pulmonary angiostrongyliasis. (a) Sections of immature adult worms within a pulmonary artery are difficult to diagnose as anything other than a nematodal infection. (b) Two cross sections of A. cantonensis within a pulmonary arteriole. The thin cuticle and intestinal lumen are well seen (Movat stain).

reported as far west as Egypt.6 A case report from Iran described a peribronchial infection in a child.145

Angiostrongyliasis Angiostrongylus cantonensis is the rat lungworm in the Far East tropics. The primary host is the rat, and the first-stage larvae reach the soil and may be ingested by snails or slugs. The third stage develops in these molluscs but crabs, crayfish and fish may all be infected. The migration is from the central nervous system to the pulmonary arteries. As a zoonosis in man, the usual manifestation is eosinophilic meningoencephalitis.

There are rare reports of migrating larval worms in thrombosed pulmonary arteries (Figure 42) Clinically this presents with dry cough and lower zone opacities on lung imaging.146

Gnathostomiasis Gnathostoma spinigerum is a parasite encountered in Asia, especially Thailand and Japan, yet more recently emerged as an important human parasite in Mexico.7 In the natural definitive host, such as pigs, cats, dogs and wild animals, the adult worms reside in the gastric wall. Humans become infected by eating undercooked fish or poultry containing third-stage

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larvae, or reportedly by drinking water containing infective second-stage larvae. If man is infected, the larvae burrow into the bowel wall to produce a space-occupying lesion. Rarely worms migrate into the lung and/or brain. Patients are described with solitary lung coin lesions or pleural effusions.147 The 1 mm in diameter worms induce a large necrotic eosinophilic reaction.148

Mammomonogamiasis Mammomonogamiasis (or syngamosis) is an upper respiratory tract inflammatory lesion caused by Mammomonogamus laryngeus. Domestic ruminant mammals are the usual host, where the male and female worms attach to the respiratory mucosa. Man is infected accidentally by ingesting larvaecontaining tissue of the intermediate host. Male and female worms attach to the respiratory tract, causing much nasopharyngeal discomfort (similar to linguatulosis – see below) and cough. The diagnosis is usually made by laryngoscopy or bronchoscopy, at which time the worms may be removed. Alternatively they may be spontaneously expectorated. The adult worms are paired, red, and are up to 20 mm long. Patients also swallow the eggs of this worm, so fecal examination can also be diagnostic.149,150

Loiasis Loiasis is caused by Loa loa, found in west and central Africa, and transmitted by Chrysops flies. The usual syndromes are migrating edematous swellings in the subcutis (“calabar” swelling) that follows the migrating adult worm. These worms also wander across the conjunctiva, causing a characteristic conjunctivitis with a visible superficial worm. Because of demographic migration, increasing numbers of such cases are now seen in Europe. The lung is not involved in the adult worm migration, but occasionally the microfilaremia spills into the pleura. Patients are described with chest pain, blood-tinged cough and a loculated pleural effusion. Microfilariae can be seen on effusion or even aspiration samples.151,152

Onchocerciasis Onchocerca volvulus is a tropical filarial infection seen predominantly in parts of tropical Africa and to a lesser extent in Latin America. The disease is often called “river blindness” since there is a close association between fast-flowing rivers and streams, where the blackfly vector breeds. Adult worms and larval microfilaria usually cause subcutaneous nodules with subsequent itching.2,100 Since microfilariae do not normally access the circulation, lung or pleural lesions are rare. However, autopsy may reveal microfilariae in the pulmonary circulation.153 The adult female is a large worm, up to 70 cm in length, has cuticular ridges, striae, a highly developed hypodermis and reduced muscle development.6

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Subcutaneous dirofilarasis In addition to the commoner D. immitis lung lesion, other Dirofilaria species may occasionally cause solitary lung lesions. D. repens is a common mosquito-borne zoonosis of canines found in southern Europe, especially Italy. When man is infected, it normally causes only subcutaneous or breast inflammatory nodules, in which a sterile adult worm with characteristic morphology is found.2 Similar nodules have been reported in the lung. In these cases the filarial worm is surrounded by infarcted lung.115

Trematode infections There are four major and numerous rarer trematode infections of man. The main ones are schistosomiasis, paragonimiasis, opisthorciasis/clonorchiasis and fascioliasis. Of these only the first two are important intrathoracic parasites, while the latter two are hepatic.4 Schistosomiasis differs from the others in that the parasites are paired male and female, rather than hermaphrodite, and are transmitted by water contact. The remainder are acquired by ingestion; hence the term foodborne trematode infections.154

Schistosomiasis Introduction

Schistosomiasis affects more than 200 million people in more than 70 countries, and mainly in places where socioeconomic conditions are substandard.155 There are three main species of schistosomes, namely S. mansoni, S. haematobium and S. japonicum.2,156 S. haematobium predominantly affects the bladder, pelvic organs and distal large bowel, whilst S. mansoni and S. japonicum produce hepato-splenic and intestinal schistosomal disease.157

Epidemiology Schistosomiasis is an important cause of disease in many parts of the world, most commonly in places with poor sanitation. People who live in, or travel to, areas where schistosomiasis is found, and are exposed to contaminated freshwater, are at risk.7 School-age children who live in these areas are often most at risk because they tend to spend time swimming or bathing in water containing infectious cercariae. Schistosoma mansoni is distributed throughout Africa. There is risk of infection in freshwater in southern and subSaharan Africa, including the great lakes and rivers, as well as smaller bodies of water. Transmission also occurs in the Nile River valley in Sudan and Egypt, as well as in Brazil, Suriname and Venezuela. S. haematobium is distributed throughout Africa, including the Nile River valley in Egypt and the Mahgreb region of North Africa. S. japonicum is found in Indonesia and parts of China and Southeast Asia.7 The life cycle depends on the species, but all involve excretion of eggs in the urine or feces (Figure 43). Infection occurs by the miracidium parasite within the egg of an appropriate

Chapter 8: Pulmonary parasitic infections Figure 43. Schistosomal life cycles. Image courtesy of CDC.

through the blood to the abdominal veins, and then to their preferred sites, as adults, within the vascular system. S. haematobium migrates to the perivesical veins, while S. mansoni and S. japonicum settle in mesenteric veins. Egglaying commences 4 to 6 weeks after skin infection. The adult paired male and female worms, 6 to 26 mm long and up to 1 mm wide, live for 3 to 5 years, although occasionally for more than 20 years. The adult worms coat themselves with host proteins and are thus immunologically hidden. Adults release hundreds of eggs a day into the veins and these eggs live for 23 days. Using the host immune system, about half of the eggs are passed through the bladder or bowel mucosae and excreted from the body. Retained eggs cause virtually all disease syndromes.

Organism

Figure 44. Schistosoma mansoni. This intrapulmonary egg has an unmistakable large lateral spine.

species of water snail. This is followed by maturation in the snail into cercaricae. The cercariae cause transcutaneous infection of man after water contact. The cercariae lose their tail, become small schistosomula, and migrate to the lung via the venous circulation. Maturation continues as worms pass

The S. mansoni egg is the part of the cycle most often found in tissues, though adult male and female fluke may be detected in pulmonary arteries. Schistosome eggs are ovoid and range in size from 70 to 100 µm in length by 50 to 80 µm in width (S. japonicum) to 114 to175 µm in length and 45 to 70 µm wide (S. mansoni and haematobium). They have a brown shell and contain a miracidium. When viable, the internal nuclei of the eggs are intensely hematoxyphilic, while the cytoplasm is eosinophilic. S. mansoni has a lateral spine (Figure 44), while Schistosoma haematobium eggs have one terminal spine and S. japonicum has only a minimal spine on the eggshell (Figure 45). These spines are rarely seen histologically, as they are often out of the plane of section. More useful is the fact that

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Figure 46. Cross section of a pair of schistosomal worms in copula within a pulmonary vessel. The smaller female is tucked into the gynecophoric canal of the male. The brown pigment well seen in the female is hemazoin, excreted following digestion of host erythrocytes. Host reaction is absent.

Clinical features Figure 45. Schistosoma japonicum. While mostly calficied, these eggs lack a prominent spine and thus represent S. japonicum.

Figure 47. Pair of schistosomal worms. The male is the thicker and appears larger, the female is partially coiled. Both measure approximately 1.0 cm in length.

the shells of S. mansoni and S. japonicum retain carbol fuchsin in a Ziehl-Neelsen stain, whilst those of S. haematobium remain unstained. In adult S. mansoni and S. haematobium males the tegument has tuberculations, while the adult S. japonicum does not. Adult worms of S. mansoni are usually found in copula (Figure 46). Male worms measure 6.4 to 12 mm in length, while the females are 7.2 to 17 mm long (Figure 47). Males have two small suckers on the anterior part of the body and up to nine testes, slightly posterior to the ventral sucker. Female worms have a single ovary near the ventral sucker and a short uterus with one or more lateral-spined eggs.

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Clinically the disease is divided into acute and chronic stages, since the acute host reaction matures over time from an antibody-antigen type to cell-mediated immunity.56 The main disease pathogeneses are local reactions to eggs (mainly) or schistosomula, and systemic reaction to antigen.157 The basic reaction to schistosome eggs in tissues is triphasic, and is a classic example of T-cell-mediated immunopathology. Rapid eosinophil, plasma cell and macrophage reaction around the egg often features antigen-antibody complex deposition (the Splendore-Hoeppli reaction). When the egg dies, macrophages break through the eggshell to digest the contents, although the shells often remain. Around the granuloma, eosinophilia declines with time. The central peri-egg zone may be necrotic, and the granulomas can remain for years. The granulomas also generate fibrosis. Vascular obliteration also contributes to mass lesions. Acute schistosomiasis (Katayama syndrome) The clinical pathology of pulmonary schistosomiasis encompasses many entities.158 This is a systemic hypersensitivity reaction against the migrating schistosomula or newly laid eggs, occurring 14 to 84 days after a primary infection.159 It is seen in non-immune, first-time infected persons, and rarely in those chronically infected. Disease onset is sudden with fever, rigors, headache, abdominal pain, malaise, urticaria, dry cough and eosinophilia. The syndrome subsides over a period of 2 months. In one series the interval between exposure and the beginning of symptoms was 3 to 6 weeks, although the diagnosis was not confirmed for at least 1 month in all of the patients.160 Acute pulmonary schistosomiasis features pneumonitis and hilar lymphadenopathy. Radiologically these patients have miliary mottling, reticulonodular infiltrates, or diffuse interstitial ground-glass pattern with ill-defined nodules.160

Chapter 8: Pulmonary parasitic infections

Figure 48. Computed tomogram from a patient with chronic pulmonary schistosomiasis. Bilateral nodules and pruning of peripheral pulmonary arteries are evident.

Chronic schistosomiasis The chronic syndromes are clinically the most important. They are usually due to eggs carried from adult worms in the abdomen or pelvic veins. Schistosoma haematobium eggs can theoretically get to the pulmonary circulation more readily than S. mansoni or S. japonicum, since the organs where they are laid drain directly into the returning venous circulation. However, pulmonary syndromes are more commonly seen in patients with hepatosplenic schistosomiasis, mostly due to S. mansoni.157 Portal hypertension results from progressive fibrosis in the liver, and the eggs arriving in the liver then bypass that organ and, via porto-systemic venous anastomoses, end up in the lungs. Hypoxemia from arteriovenous shunting and cor pulmonale may develop (see Chapter 18). Chronic schistosomiasis passes through sequential stages. Initially patients are asymptomatic but radiology shows lower zone widened proximal arteries, thicker peripheral arteries and focal nodules, greater than 1 mm in diameter, near the peripheral vessels. There is peripheral pruning of lung vessels and in advanced cases vascular imaging demonstrates pulmonary hypertension (Figure 48). The peripheral blood vessels are blocked, partly by the eggs and mainly by the host inflammatory reaction within and around the vessel. In a few cases, worms embolize to lung. Later symptoms are breathlessness and fatigue, cyanosis and right ventricular failure. At this stage the radiology shows right ventricular hypertrophy, aneurysmal dilatation of the main pulmonary artery and its branches, as well as fine mottling throughout the lung fields. The nodular lung lesions are due either to eggs in the pulmonary artery or to ectopic adult worms, egg-related vasculopathy with subsequent pulmonary hypertension and cor pulmonale, bronchiectasis, or hepatopulmonary syndrome from liver portal fibrosis. The hypoxemic hepatopulmonary syndrome is reported from Brazil, in patients who have severe hepatosplenic schistosomiasis and portal hypertension.161 It is

Figure 49. Pulmonary schistosomiasis. Necrotizing granulomas often contain dead eggs (arrow).

not directly due to eggs or worms in the lung, but right-left shunting of blood, similar to that seen in hepatic cirrhosis.

Pathology The pathology of the nodular and vascular lesions is granulomatous with eosinophilic inflammation around eggs trapped in small arterioles. The pathology of the vascular disease is well described in Chapter 18. Additionally, in the acute phase of infection, there may be a Splendore-Hoeppli reaction around eggs. S. mansoni eggs measure 150
50 µm and so they arrest in 50 to 100 µm diameter arterioles. The granulomas are both intravascular and perivascular, with endarteritis and intimal obliteration. Fibrosis develops around the lesion. Eggs may also impact in larger vessels, measuring up to 250 µm in diameter. Granulomas and an acute necrotizing vasculitis with luminal thrombosis ensue, with eventual obliteration of vascular lumina. Plexiform lesions can develop. If this process is widespread, the pruning of the pulmonary artery tree leads to pulmonary hypertension with medial thickening. In the nodular lesions, the vessel center is usually invisible and there are granulomas around eggs (Figure 49).162,163 Sometimes, adult schistosome worms arrive in lung arteries. If paired and fertile, they can deposit large numbers of eggs in a small area of lung, and so produce a tumor-like lesion full of eggs, granulomas and fibrosis – a “pulmonary bilharzioma”. Egg-related granulomas are seen in the bronchial wall. Bronchiectasis is occasionally reported due to schistosomiasis.164 The pathogenesis is presumably vasculopathy of the intra-bronchial arterioles with destruction of airway elastica and muscle.

Diagnosis Examination of stool and/or urine for ova is the primary method of diagnosis. The choice of sample to diagnose schistosomiasis depends on the species of parasite likely to be

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causing the infection. Adult stages of S. mansoni, S. japonicum, S. mekongi and S. intercalatum reside in the mesenteric vein plexus of infected hosts and eggs are shed in feces; S. haematobium adult worms are found in the vein plexus of the lower urinary tract and eggs are shed in urine. Testing of stool or urine can be of limited sensitivity, particularly for travelers who may have lighter infection burdens. The eggs are shed intermittently and in low amounts in light-intensity infections. Serological testing for anti-schistosomal antibody is indicated for diagnosis of patients who may have lighter-intensity infections or who have not been treated appropriately for the disease in the past. Serological testing may not be appropriate for diagnosis of current infection in patients who have been repeatedly infected and treated in the past. This is because antibody persists despite cure. In these patients, serological testing cannot distinguish resolved infection from active infection.

Treatment Following effective treatment for acute or chronic schistosomiasis with praziquantel, some patients suffer recurrence or a new syndrome of pulmonary symptoms and infiltrates on radiology. Post-treatment reactions can be seen in either acute or chronic disease. The tissue pathology is an eosinophilic pneumonia, and eggs are not found on tissue samples. It is presumed this is a local reaction to antigens released by the chemotherapy, similar pathogenetically to the “paradoxical reactions” seen in some patients treated for tuberculosis.39

Paragonimiasis Introduction

Paragonimus is a platyhelminth that infects the lungs of humans after they eat infected raw or undercooked crab or crayfish. Less frequent but more serious cases of paragonimiasis occur when the parasite travels to the central nervous system instead of to the lungs. More than 30 species of trematodes of the genus Paragonimus infect animals and humans. Several species of Paragonimus cause most infections. The most important is P. westermani.

Epidemiology The organism is endemic in Southeast Asia, China, South America and many parts of sub-Saharan Africa.2 P. westermani occurs primarily in Asia, including China, the Philippines, Japan, Vietnam, South Korea, Taiwan and Thailand. P. africanus causes infection in Africa, and P. mexicanus is seen in Central and South America. Specialty dishes in which shellfish are consumed raw or prepared only in vinegar, brine or wine without cooking play a key role in transmission. Raw crabs or crayfish are also used in traditional medicine practices in Korea, Japan and some parts of Africa.7 Although rare, human paragonimiasis from P. kellicotti has been reported in the United States, with multiple cases from the Midwest. Several cases have been associated with ingestion of uncooked crawfish during river raft float trips in Missouri.

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Figure 50. Whole mount of a stained Paragonimus worm. The paired intestine and central genital apparatus are obvious.

The life cycle is complicated, requiring three hosts. Eggs from the definitive host (man) enter the aquatic environment from sputum or feces and the miracidium penetrates the appropriate snail species. Cercariae from the snail enter crayfish or crabs. Metacercariae within such animals are ingested by man, and the larvae are released into the duodenum. They penetrate the bowel wall, wander through the abdominal cavity, and cross the diaphragm. They attach themselves to the pleural surfaces with their suckers and usually enter and invade the lung. They take up a final position near bronchioles and release eggs into the airways. The total incubation period is about 70 days, and adult worms can live up to 20 years in man. Since this is a migrating larva, worms can end up in ectopic non-pulmonary locations. The commonest sites for adult worms are the subcutis, liver, abdominal cavity and brain.4

Organism P. westermani worms are hermaphrodite, flattened, measure 10
6 mm and 0.5 mm thick, have two suckers and a tegument with small spines (Figure 50). The eggs are goldenbrown, birefringent, oval, with one side slightly flattened. They have a single operculum and the shell at the opposite end is thicker. With the Ziehl-Neelsen stain, the eggshells retain carbol fuchsin.6 Occasionally the ova may be covered by eosinophilic material and may resemble corpora amylacea. Speciation is performed by identifying subtle morphological differences in the adult worms and the eggs. DNA sequencing of parasitic material may play a supportive role.

Clinical features Infected individuals may present with chronic cough, hemoptysis or shortness of breath. The infection can mimic tuberculosis and lung cancer.165,166 Chest pain often presents and there may be night sweats in the early stages. Signs of pleuropulmonary paragonimiasis include pleural effusion, pneumothorax, empyema, pleural fibrosis, solid or cavitary lung nodules and bronchiectasis. Radiologically, there are several stages of the infection, ranging from acute to chronic.167 Many patients have a spectrum

Chapter 8: Pulmonary parasitic infections

Figure 51. Paragonimus westermani adult in thickened visceral pleura.

Figure 53. Pulmonary paragonimiasis. Partially calcified eggs rarely demonstrate morphological features such as the operculum.

of abnormalities on chest radiographs such as lobar infiltrates, coin lesions, cavities, calcified nodules, hilar enlargement, pleural thickening and effusions. Ring-shaped opacities of contiguous cavities giving the characteristic appearance of a bunch of grapes are highly suggestive of pulmonary paragonimiasis.7 Pneumothorax and hydrothorax are seen as larvae invade the pleural cavity, while focal consolidation and linear opacities are caused by the juvenile worms migrating within lung. The cavities, nodules and masses develop as the adult worm generates a surrounding inflammatory reaction. Bronchiectasis develops if the brisk host response damages airways.

Pathology The often benign and chronic nature of this disease gives few opportunities for histology study. Pleural fibrosis may be

Figure 52. Pulmonary paragonimiasis. Two worms within a bronchus. The intestines are pigmented. Central dilated structures in the right worm are the genital apparatus.

observed (Figure 51). Upper lung zones are more affected than lower fields. Lung lesions consist of nodules and cavities containing necrotic material and the worms. These abscesses may measure up to 5 mm in diameter. They are gray-white in the early stages and contain adult worms. Inflammation may involve vessels, leading to parenchymal infarction. In addition the inflammation may erode into airways, causing squamous metaplasia, atelectasis and/or chronic inflammation (Figure 52). The inflammation comprises neutrophils, macrophages and abundant eosinophils with Charcot-Leyden crystals, i.e., typical helminthoma histopathology. Worms generate inflammation through the release of toxic secretions, the irritation from spiny cuticles and by the granulomatous response to the eggs deposited around the worm. In later lesions the worms disintegrate, leaving eosinophilic amorphous debris or an empty fluid- filled cystic cavity (Figure 53). Adjacent lesions may fuse to form multicystic cavities. Secondary bacterial infections may develop. Eggrelated destructive granulomatous inflammation in the bronchial walls may cause bronchiectasis. Peripheral fibrosis develops and in this layer there is progressive egg deposition, causing a granulomatous reaction. The peripheral lung is collapsed and the overlying pleura thickened. Subpleural lesions can cause a prominent effusion. Old lesions may calcify and focal scarring is prominent.

Diagnosis Classically, the diagnosis of paragonimiasis is made by observing expectorated oval 50 µm
100 µm eggs with a single operculum in the brown-colored shell. Sputum may also contain eosinophils and Charcot-Leyden crystals.165 Since they are also swallowed, fecal examination for eggs is also helpful. Fineneedle aspiration of a pulmonary nodule can provide eggs for examination.168 Specific serology has been available for many years, and many diagnoses are now made on this basis, alongside the clinical and imaging appearances. Serological tests can be especially useful for early infections or for ectopic infections

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where eggs are not passed in stool.7 Molecular diagnostics are increasingly applied to tissue or cytology samples.169

stage of Echinococcus multilocularis. E. oligarthrus is an extremely rare cause of human echinococcosis.

Differential diagnosis

Echinococcus granulosus hydatid lung disease

Other rare trematode infections, such as Achillurbainia and Poikilorchis, can be found in human tissues and their eggs may be difficult to distinguish from Paragonimus.

Treatment Praziquantel is the drug of choice. Alternatives are triclabendazole (not available in the USA) or bithionol.

Other uncommon food-borne trematode infections of lung and pleura Fascioliasis The liver fluke, Fasciola hepatica, has a worldwide distribution, is associated with watercress fields, and can be acquired in northwest Europe.154 The estimated number of infected people is at least 2.4 million and might be as high as 17 million. Fasciola hepatica is found in more than 50 countries in all continents except Antarctica. Fasciola gigantica is less widespread. Human cases have been reported in the tropics, in parts of Africa and Asia and also in Hawaii. In some areas where fascioliasis is found, human cases are uncommon (sporadic). In other areas, human fascioliasis is very common (hyperendemic). For example, the areas with the highest known rates of human infection are in the Andean highlands of Bolivia and Peru. Humans are infected by ingesting encysted metacercariae on raw vegetation. The parasites invade the duodenum and the liver capsule, and migrate through liver parenchyma to reach the bile ducts, where they reside. This produces an eosinophilic hepatitis and helminthomas. Occasionally, the migrating worms cross the diaphragm and enter the pleura and lung.170 In the chest, the worms do not mature, die, and induce necrotizing eosinophilic granulomas and often calcify. As in the liver, the identification of the worm in the lung may be difficult.4

Alaria Alaria species are found in the Americas and Europe, and produce a paratenic (non-maturing) infection in man as they invade tissues. The lung may occasionally be involved, with local hemorrhage around the larvae.171

Cestode lung infections Hydatid cyst – echinococcosis Introduction

Cystic echinococcosis (CE) is caused by an infection from the larval stage of Echinococcus granulosus. Alveolar echinococcosis (AE) disease results from being infected with the larval

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Epidemiology

The annual incidence of CE at the turn of this century ranged from less than 1 to 200 per 100 000 inhabitants in various endemic areas.172 Echinococcus granulosus is the main cestode species to cause hydatid cyst. Alveolar echinococcosis is less common, with an annual incidence in most endemic areas of 0.03–1.2 per 100 000 inhabitants.172 Cystic echinococcosis is a gut parasite of canines found in temperate as well as tropical and subtropical regions, including the UK.2,173 It is found especially in Africa, Europe, Asia, the Middle East, Central and South America and, in rare cases, North America.7 In the above countries, excepting the UK, it may be said that a tumor-like lesion in any part of the body could be a hydatid cyst until proven otherwise. South America is affected, and an exceptional number of reports come from Turkey. The life cycle has a definitive and an intermediate host. The adult E. granulosus worm resides in the small bowel of canines, which excrete eggs in the feces. Ingested by ruminants (cattle, sheep, giraffes, etc.), larval hydatid cyst develops in their tissues. The eggs hatch in the animal’s duodenum and the larvae pass to the liver via the portal vein, where a cyst is commonly found. Within the cyst, a parasite germinal membrane develops, giving rise to a myriad scolices, the head segments of future worms. When the animal and its cyst are eaten by a canine, these scolices latch onto gut mucosa and grow into a new adult worm. Thus, man acquires hydatid cyst disease from accidentally ingesting eggs from canine feces. Since man is rarely eaten by canines, humans represent a dead-end in the life cycle (Figure 54).

Organism Adult E. granulosus are found only in the small intestine of canids, never in humans. The hermaphroditic adult worms are 3 to 6 mm in length. They have a scolex that has a rostellum with approximately 30 to 36 taeniid-type hooklets, a short neck region and only three proglottids – one immature, one mature and one gravid. The eggs are morphologically identical to the eggs of all Taenia species, having a prismatic shell surrounding the six-hooked embryo (oncosphere) and measuring 30 to 43 mm in diameter.

Clinical features In CE in man, the liver is most commonly affected. The rank order of other organ involvement is lung, peritoneum, soft tissues, spleen, kidney, brain and bone. The lung has a single parenchymal cyst in 25% of cases but there may be multiple, uni- or bi- lateral lung cysts. The cysts may reach up to 20 cm in diameter. Size depends on the age of the cyst, its location and the type of intermediate host.

Chapter 8: Pulmonary parasitic infections Figure 54. Echinococcus granulosus life cycle. Image courtesy of CDC. Humans become infected by ingesting eggs 2 , with resulting release of oncospheres 3 in the intestine and the development of cysts 4 , 4 , 4 , 4 , 4 , 4 in various organs.

Hydatid cysts grow steadily for many years, at about 1 to 2 cm diameter per year, followed by senescence and death.174 On death, the cyst collapses and the internal daughter cysts fold up. The host fibrous rim thickens and the structure often undergoes dystrophic calcification. This form of self-cure is common. Lung cysts are particularly seen in young adults and children.174 Essentially the cysts behave as space-occupying lesions, with compression of local structures. Unlike nematode helminthomas, they do not induce a massive local host reaction, and are frequently seen with only a thin host fibrous rim around the cyst. Three-quarters of hydatid cysts are solitary.175 Giant cysts may fill the entire hemithorax, compressing the lung or mediastinum.176 In the early stages, the cysts are asymptomatic.174 The clinical presentation, if the cyst is not first identified by screening, is with chest pain, cough and hemoptysis.177 Horner syndrome and brachial plexus compression can result from an apical lesion.178 Cysts can compress bronchi and induce an infective pneumonia or they can either spontaneously or posttraumatically rupture into a bronchus, with expectoration of cyst contents. Secondary bacterial infection may produce a lung abscess. Anaphylaxis may develop following the spontaneous or iatrogenic rupture of a lesion. Pleural and chest wall hydatid cysts usually develop from a ruptured pulmonary cyst rather than extension of a hepatic lesion.179 A pleural cyst causes an effusion. Cysts within a rib can mimic a neoplasm.180 Blood eosinophilia is uncommon, affecting no more than 20% of patients, and reflects the lack of host cellular reactivity to the infection. When hepatic lesions

erode into the inferior vena cava, pulmonary parasitic thromboemboli may result.174 Radiologically, the cysts are well-defined round lesions with fluffy centers on plain X-ray (Figure 55a). The appearance may mimic an abscess, and some have a peri-cystic pneumocyst.175 On CT scan they are homogeneous with a thin enhancing rim (Figure 55b).174 Pleural effusion may be seen if the cyst is in the pleural space or impinges on the visceral pleura.

Pathology Lesions are grossly cystic (Figure 55c). Microscopically, E. granulosus hydatid cysts have a characteristic structure (Figure 56). The outer part is the 1–2 mm thick white, friable and satin-like membrane. Microscopically it is laminated and is recognizable in expectorated fragments. Inside this is the germinal membrane, a unicellular layer of cells 10–25 µm thick, which gives rise to the scolices and to smaller daughter cysts (Figure 57). Protoscolices contain several proglottids and a rostellum with hooklets (Figure 58). Gomori methenamine silver (GMS) stains the concentric laminar layers black and the rest of the tissue light green. This stain distinguishes the hydatid cyst from surrounding host tissue. In surgical and autopsy samples, the germinal membrane may be dead and scolices may be difficult to find or absent, as these cysts die naturally. Finding them indicates the cyst is alive and can potentially spread if ruptured. But their absence does not mean that the cyst is necessarily dead as portions of the cyst wall with scolices may not have been sampled. Degenerate cysts break down and fragment. This generates an eosinophilic host reaction resplendent with macrophages and

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(a)

(b)

(c)

Figure 55. Pulmonary hydatid disease. (a) Chest X-ray demonstrates a large left lung mass obscuring the mediastinal borders. (b) The corresponding computed tomogram shows a subpleural left upper lobe cystic mass abutting the anterior mediastinum. (c) The lobectomy specimen contains a subpleural bulging mass.

Figure 56. Pulmonary hydatid cyst. This fragmented cyst is well demarcated from lung parenchyma (arrows).

giant cells phagocytosing fragments of laminated membrane, and fibrosis (Figure 59).

Diagnosis Most hydatid cysts are diagnosed on clinical suspicion, imaging and serology. Most patients with established cysts have highly specific antibodies, which can be measured in specialist centers. Antibodies may persist for years after treatment.173 Occasionally, a lung hydatid cyst is an operative surprise at thoracotomy for a tumor-like lesion.180 Conversely, there are

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case reports of presumed lung and chest wall hydatid disease that, on histopathology, turns out to be carcinoma.181 Immunodiagnostic tests can be very helpful in the diagnosis of echinococcal disease and should be used before invasive methods. The clinician must have some knowledge of the characteristics of the available tests and the patient and parasite factors associated with false results. False-positive reactions may occur in persons with other helminthic infections, cancer and chronic immune disorders. Negative test results do not rule out echinococcosis because some cyst carriers do not have detectable antibodies. Whether the patient has detectable antibodies depends on the physical location, integrity and vitality of the larval cyst. Cysts in the liver are more likely to elicit antibody response than cysts in the lungs, and, regardless of localization, antibody detection tests are least sensitive in patients with intact hyaline cysts. Cysts in the lungs, brain and spleen are associated with lowered serodiagnostic reactivity, whereas those in bone appear to more regularly stimulate detectable antibody. Fissuration or rupture of a cyst is followed by an abrupt stimulation of antibodies. A patient with senescent, calcified or dead cysts is generally seronegative. Indirect hemagglutination (IHA), indirect fluorescent antibody (IFA) tests and enzyme immunoassays (EIA) are sensitive tests for detecting antibodies in serum of patients with cystic disease. Crude hydatid cyst fluid is generally employed as antigen.7 Antibody responses have also been monitored as a way of evaluating the results of treatment, but with mixed results. Following successful radical surgery, antibody titers decline and sometimes disappear; titers rise again if secondary cysts develop. Tests for Arc 5 or IgE antibodies appear to reflect antibody decline during the first 24 months post-surgery, whereas the IHA and other tests remain positive for at least 4 years.7 Polymerase chain reaction analyses show promising results.182

Chapter 8: Pulmonary parasitic infections

(a)

(b)

Figure 57. Hydatid cyst germinal membrane. (a) A viable germinal membrane features protoscolices. (b) The nucleated germinal membrane produces the scolices.

(a)

Figure 58. Echinococcus granulosus protoscolices. (a) Protoscolices contain a rostellum with hooklets. (b) Hooklets are acid-fast (Ziehl-Neelsen stain).

(b)

Laminated membrane and protoscolices can be expectorated in sputum when a cyst ruptures into a bronchus. Alternatively, FNA cytology of aspirated cyst contents can enable morphological identification and serology studies on the fluid. Scolices have a characteristic appearance cytologically, and the hooklets can be highlighted with a Ziehl-Neelsen stain (Figure 60). Fragments of laminated membrane are recognizable, with only artifactual perioperative material or unusual plasma condensation as differential considerations. The oncefeared risk of anaphylactic reaction from needling a hydatid cyst and inadvertently spilling antigen-laden fluid has been overestimated; fine-needle sampling is safe. Seeding of a cyst into the pleura is, however, a potential complication.

Differential diagnosis

Figure 59. Degenerate pulmonary hydatid cyst. The laminated membrane elicits a giant cell and neutrophilic reaction.

The morphology of the unilocular hydatid cyst is unique, so it is unlikely to be confused with other cestode larvae such as cysticercus and coenurus. In some cases the unilocular hydatid disease may be confused with the alveolar form or other rare species.

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Figure 61. Alveolar echinococcosis. A hepatic multilocular hydatid cyst demonstrates a laminated membrane without scolices. Note the invasive pattern of spread, rather than the space-occupying lesions seen in granulosus-type hydatid cysts. Figure 60. Hooklets may be encountered in bronchoalveolar lavage or fine-needle aspirate samples (Ziehl-Neelsen stain).

Echinococcus multilocularis hydatid lung disease Epidemiology

E. multilocularis causes alveolar hydatid cyst or AE. This is a more severe disease than E. granulosus disease. E. multilocularis occurs in central and eastern Europe, Russia and Asia and is currently spreading into China and Japan.173 In the USA, the distribution ranges from Alaska southward to the upper Midwest. The definitive hosts are foxes, and to a lesser extent dogs, cats, coyotes and wolves. Small rodents are the intermediate hosts. Although human cases are rare, infection in humans causes parasitic tumors to form in the liver and, less commonly, the lungs, brain and other organs. If left untreated, infection can be fatal as the complex organism behaves more aggressively than the passive space-occupying E. granulosus.4 Larval growth in the liver remains indefinitely in the proliferative stage, resulting in invasion of the surrounding tissues.7

Lung cysts may be several centimeters in diameter, and have necrotic centers and margins. The membrane is thinner than with E. granulosus cysts, and proliferates by external budding with invasion of host tissue (Figure 61). This produces an alveolar pattern of small vesicles. There is much fibrinoid necrosis around the cyst wall, and internally it is unusual to find scolices.4

Diagnosis

E. multilocularis is similar to E. granulosus with a scolex with an armed rostellum, a neck region and three proglottids. The worms of E. multilocularis are shorter than E. granulosus, being only 1.2 to 3.7 mm in length.6

Most patients with alveolar disease have detectable antibodies in serological tests using heterologous E. granulosus or homologous E. multilocularis antigens. With crude Echinococcus antigens, nonspecific reactions create the same difficulties as described above; however, immunoaffinity-purified E. multilocularis antigens (Em2) used in EIA allow the detection of positive antibody reactions in more than 95% of alveolar cases. Comparing serological reactivity to Em2 antigen with antigens containing components of both E. multilocularis and E. granulosus permits discrimination of patients with alveolar from those with cystic disease. In seronegative patients with hepatic image findings compatible with echinococcosis, ultrasoundguided fine-needle biopsy may be useful for confirmation of diagnosis; during such procedures precautions must be taken to control allergic reactions or prevent secondary recurrence in the event of leakage of hydatid fluid or protoscolices.7 Polymerase chain reaction may prove even more specific.

Clinical features

Treatment of hydatid disease

E. multilocularis affects the liver as a slow-growing, destructive tumor, with abdominal pain, biliary obstruction and occasionally metastatic lesions into the lungs and brain.7 Chest radiographs and CT scans demonstrate multiple, lobulated, wellcircumscribed nodules of varying shapes and sizes.183 Lesions may also be located between lung segments.

Ideally, complete surgical removal of granulosus cysts is required, which is realistic for single and small numbers of cysts only.184 Cysts may be shelled out if there is not much surrounding collapse or fibrosis. Cyst rupture during surgery may lead to local spread. Oftentimes lobectomy is required to ensure the laminated membrane with germinal membrane is

Organism

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Pathology

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(a)

(b)

Figure 62. Pulmonary cysticercosis. (a) The bladder wall completely surrounds the parenchymal component and the folded spiral canal is apparent at low magnification. Hooklets on the rostellum are also seen (arrow). (b) Higher magnification clearly demonstrates the well-developed protoscolex with refractile hooklets. Note the parenchymal portion of the parasite with dark staining nuclei and loose connective tissue.

entirely removed. Albendazole has an adjunctive role.173 Mebendazole may be also used for E. multilocularis infections. Neoadjuvant praziquantel (a scolicide) therapy is commonly undertaken. Postoperative chemotherapy is continued for an undefined period.185 If the lung cysts are not operable, or cannot be entirely removed, indefinite albendazole chemotherapy (which is toxic to germinal membrane) is given. There is no direct lung toxicity from this regime.

Cysticercosis Introduction

Cysticercosis is a parasitic tissue infection caused by larval cysts of the pork tapeworm Taenia solium. These larval cysts infect brain, muscle or other tissue, and are a major cause of adult-onset seizures in most low-income countries.7

Epidemiology Cysticercosis is found worldwide, especially in areas where pork tapeworm is common. Both pork tapeworm and cysticercosis are most often found in rural developing countries with poor sanitation, where pigs are allowed to roam freely and eat human feces. Taeniasis and cysticercosis are rare among persons who live in countries where pigs are not commonly raised for food, or countries where pigs raised for food do not have contact with human feces. Although uncommon, cysticercosis can occur in people who have never travelled outside the United States. Infected people with poor hygiene, with or without symptoms, will shed tapeworm eggs in their feces and might accidentally contaminate their environment. This can lead to transmission of cysticercosis to themselves or others unknowingly.7

The adult worm resides in the human gut excreting eggs but causing little direct pathology. If eggs are ingested from feces, via contaminated food, the eggs hatch in the duodenum, and the oncospheres invade the bowel wall and migrate. The commonest locations are skeletal muscle, subcutis and brain.186 In susceptible tissues they grow slowly into 1–2 cm diameter cysticerci, i.e., space-occupying lesions.2

Organism The larval stage consists of a 0.5 to 1.5 cm fluid-filled bladder into which a single protoscolex is inverted. Microscopically the moderately folded spiral canal is often seen leading to a welldeveloped protoscolex (Figure 62a). This has suckers, rostellum and hooklets. The surface of the spiral canal is folded, with the underlying parenchyma containing fibrous and muscle tissue and numerous calcareous corpuscles (Figure 62b). The bladder wall is moderately thick and has warty protrusions on its surface.

Clinical features Occasionally lung infection develops. This is usually asymptomatic in that lung symptoms are minor, compared with those of neurocysticercosis and subcutaneous nodules.187 A solitary lung nodule comprising a granuloma around a cysticercus may be the only evidence of pulmonary involvement.188 Another case report from a heavily infected patient noted multiple bilateral nodules on chest X-ray and CT scans. Since these are rarely biopsied, but may be seen at autopsy, the usual diagnostic proof is resolution of the abnormal imaging with appropriate chemotherapy (praziquantel).189

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(a)

(b)

(c)

Figure 63. Pentastomiasis. (a) An encapsulated worm-like Armillifer armillatus nymph expands a peribronchial lymph node sinus. The simple intestinal tract is noted. (b) Striated muscle is unique to this organism and eliminates all helminths from the differential diagnosis. (c) Sclerotized openings are black and confined to the cuticle. (Movat stain).

Pathology Cysticerci generate little host reaction apart from a fibrous capsule while alive. When they die, they often calcify and remain evident on imaging as small calcific deposits. In the meninges, a proliferative form of cysticercus (racemose) is a morphological variant of this infection, but does not appear to occur in the lung or pleura.

Diagnosis Magnetic resonance imaging or CT of the brain is often used for diagnosis. Diagnosis of Taenia tapeworm infection is made by examination of stool samples. Individuals should also be asked if they have passed tapeworm segments. Stool specimens should be collected on three different days and examined in the lab for eggs. Tapeworm eggs can be detected in the stool 2 to 3 months after the tapeworm infection is established.7 Serology

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is an important diagnostic modality as a negative result usually excludes cysticercosis.

Sparganosis Sparganum mansoni is a cestode zoonosis, with the adult worm residing in the intestine of dogs and cats in the tropics and subtropics. Few human cases have been reported. The life cycle is complex with several intermediate hosts.4 If man is accidentally infected, the larval worm is a spaghetti-like thread that migrates through subcutaneous tissues. The host generates a local eosinophilic reaction with edema. Two recorded cases in the lung presented with either a single or multiple nodule(s).190,191 Morphologically the parasite is 1 mm thick, with a cestode larval tegument and spongy-solid interior that includes thin muscle fibers and calcareous corpuscles. When it dies, the

Chapter 8: Pulmonary parasitic infections

typical host eosinophilic granuloma with fibrosis develops. Treatment with standard anti-helminthic drugs is unsatisfactory. A variant parasite, S. proliferum, causes disseminated infection in man. Very few cases are recorded, but the lung and pleura have been involved in two patients.192,193 In one of the cases lung imaging showed patchy infiltrates, and the CT scan demonstrated a nodule. A transbronchial biopsy showed an eosinophilic vasculitis, indicative of the typical host reaction to a parasite. The multiple cystic and solid parasites are likened to bizarre cysticercus.

Pentastome infections Pentastomes are a group of strange reptilian and mammalian parasites that are part helminth, annelid and arthropod. They have complicated life cycles with several intermediate hosts. Man is an accidental host. Armillifer and Linguatula are two genera that cause human disease. These organisms are found most frequently in Southeast and Southwest Asia and tropical Africa. Linguatula may infect North American, South American and European humans. Adult females may be up to 10 cm long while males are only 2 to 3 cm. Both sexes have four anterior crescentic hooks. Unlike most arthropods, pentastomes lack respiratory and circulatory systems. Armillifer have external annulations and resemble a sheathed coil while Linguatula looks like a tongue as the anterior end of the organism flattens broadly in the dorsoventral aspect and tapers to a narrow posterior end. Histologically, these organisms have a simple intestinal tract, striated muscle and the cuticle features sclerotized openings (Figure 63). These latter two features exclude all other parasitic helminths from diagnostic consideration. Such an organism with numerous spines is probably a species of Linguatula, while no spines suggest a species of Armillifer.

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Armillifer pentastomiasis Armillifer live in the respiratory tracts of snakes and lizards. Humans are infected by eating the uncooked meat of an intermediate host that contains larvae. The larvae migrate through the body, and the liver and mesentery are the commonest final locations. Small nodules eventually calcify. In Africa, these are said to be the commonest causes of calcified nodules in the liver and abdomen. A similar lesion may occasionally be seen in the lung and features necrotizing eosinophilic granulomas.194 Clinically, pneumonitis and lobar collapse are noted in the migrating phase. Active pulmonary infection lacks a morphological description aside from degenerate and calcifying lesions.195

Linguatulosis Linguatula serrata adults inhabit the nasal cavities of dogs, cats and foxes. They are most common in the Middle East but are found worldwide. Humans usually acquire the organisms by ingesting eggs from either nasal secretions or feces. Nasopharyngeal infection (the “halzoun” [suffocation] syndrome) is the commonest version of human infection with Linguatula serrata. This develops usually if undercooked or raw larvaecontaining sheep liver or lymph nodes are ingested. The larvae ascend the esophagus and latch onto the nasopharyngeal mucosa. This produces edema and inflammation, with itching, discomfort and sneezing. The larvae then migrate in the body and, as with Armiliffer, rarely cause disease. Others may be infected through ingestion of contaminated water. This infection can present as a pulmonary coin lesion or characteristic comma-shaped shadow on imaging.196 In such a case report with histopathology of the necrotic nodule, it was possible to identify the morphology as Linguatula despite degenerative changes.197 The larval parasite presumably dropped off the nasopharynx and was aspirated into the lung.

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J Comput Assist Tomogr 2004;28(6): 796–800. 112. Kido A, Ishida T, Oka T, et al. Pulmonary dirofilariasis causing a solitary lung mass and pleural effusion. Thorax 1991;46(8):608–9. 113. Asimacopoulos PJ, Katras A, Christie B. Pulmonary dirofilariasis. The largest single-hospital experience. Chest 1992;102(3):851–5. 114. Milanez de Campos JR, Barbas CS, Filomeno LT, et al. Human pulmonary dirofilariasis: analysis of 24 cases from Sao Paulo, Brazil. Chest 1997;112(3):729–33. 115. Pampiglione S, Rivasi F, Paolino S. Human pulmonary dirofilariasis. Histopathology 1996;29(1):69–72. 116. Mizrachi HH, Lieberman PH, Tolui SS, Sun T. Pulmonary dirofilariasis: mimicry of well-differentiated squamous carcinoma. Hum Pathol 1989;20(8):818–9. 117. Flieder DB, Moran CA. Pulmonary dirofilariasis: a clinicopathologic study of 41 lesions in 39 patients. Hum Pathol 1999;30(3):251–6. 118. Bethony J, Brooker S, Albonico M, et al. Soil-transmitted helminth infections: ascariasis, trichuriasis, and hookworm. Lancet 2006; 367(9521):1521–32. 119. Rexroth G, Keller C. [Chronic course of eosinophilic pneumonia in infection with ascaris lumbricoides]. Pneumologie 1995;49(2):77–83. 120. Valentine CC, Hoffner RJ, Henderson SO. Three common presentations of ascariasis infection in an urban Emergency Department. J Emerg Med 2001;20(2):135–9. 121. Acar A, Oncul O, Cavuslu S, Okutan O, Kartaloglu Z. [Case report: Loffler’s syndrome due to Ascaris lumbricoides mimicking acute bacterial community – acquired pneumonia]. Turkiye Parazitol Derg 2009;33(3):239–41. 122. Lau SK, Woo PC, Wong SS, Ma ES, Yuen KY. Ascaris-induced eosinophilic pneumonitis in an HIV-infected patient. J Clin Pathol 2007;60(2):202–3. 123. Heggers JP, Muller MJ, Elwood E, Herndon DN. Ascariasis pneumonitis: a potentially fatal complication in smoke inhalation injury. Burns 1995;21(2):149–51.

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124. Bailey JK, Warner P. Respiratory arrest from Ascaris lumbricoides. Pediatrics 2010;126(3):e712–5. 125. Sen MK, Chakrabarti S, Ojha UC, Daima SR, Gupta R, Suri JC. Ectopic ascariasis: an unusual case of pyopneumothorax. Indian J Chest Dis Allied Sci 1998;40(2):131–3. 126. Sakai S, Shida Y, Takahashi N, et al. Pulmonary lesions associated with visceral larva migrans due to Ascaris suum or Toxocara canis: imaging of six cases. AJR Am J Roentgenol 2006;186(6):1697–702. 127. Baar HS, Galindo J. Ossifying pulmonary granulomatosis due to larvae of ascaris. J Clin Pathol 1965; 18(6):737–42. 128. Okada F, Ono A, Ando Y, et al. Pulmonary computed tomography findings of visceral larva migrans caused by Ascaris suum. J Comput Assist Tomogr 2007;31(3):402–8. 129. Marcos LA, Terashima A, Dupont HL, Gotuzzo E. Strongyloides hyperinfection syndrome: an emerging global infectious disease. Trans R Soc Trop Med Hyg 2008;102(4):314–8. 130. Mayayo E, Gomez-Aracil V, Azua-Blanco J, et al. Strongyloides stercolaris infection mimicking a malignant tumour in a nonimmunocompromised patient. Diagnosis by bronchoalveolar cytology. J Clin Pathol 2005; 58(4):420–2. 131. Gupta S, Jain A, Fanning TV, et al. An unusual cause of alveolar hemorrhage post hematopoietic stem cell transplantation: a case report. BMC Cancer 2006;6:87. 132. Agarwal VK, Khurana HS, Le HX, Mathisen G, Kamangar N. 30-yearold HIV-positive female with diffuse alveolar hemorrhage. J Intensive Care Med 2009;24(3):200–4. 133. Chu E, Whitlock WL, Dietrich RA. Pulmonary hyperinfection syndrome with Strongyloides stercoralis. Chest 1990;97(6):1475–7. 134. Lin AL, Kessimian N, Benditt JO. Restrictive pulmonary disease due to interlobular septal fibrosis associated with disseminated infection by Strongyloides stercoralis. Am J Respir Crit Care Med 1995;151(1):205–9. 135. Thompson JR, Berger R. Fatal adult respiratory distress syndrome

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136. Herry I, Philippe B, Hennequin C, et al. Acute life-threatening toxocaral tamponade. Chest 1997;112(6): 1692–3.

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137. Inoue K, Inoue Y, Arai T, et al. Chronic eosinophilic pneumonia due to visceral larva migrans. Intern Med 2002;41(6):478–82.

149. de Lara TdeA, Barbosa MA, de Oliveira MR, de Godoy I, Queluz TT. Human syngamosis. Two cases of chronic cough caused by Mammomonogamus laryngeus. Chest 1993;103(1):264–5.

138. Roig J, Romeu J, Riera C, et al. Acute eosinophilic pneumonia due to toxocariasis with bronchoalveolar lavage findings. Chest 1992; 102(1):294–6. 139. Glickman L, Schantz P, Dombroske R, Cypess R. Evaluation of serodiagnostic tests for visceral larva migrans. Am J Trop Med Hyg 1978; 27(3):492–8. 140. Magnaval JF, Fabre R, Maurieres P, Charlet JP, de Larrard B. Application of the western blotting procedure for the immunodiagnosis of human toxocariasis. Parasitol Res 1991; 77(8):697–702. 141. Sinniah B, Leopairut J, Neafie RC, Connor DH, Voge M. Enterobiasis: a histopathological study of 259 patients. Ann Trop Med Parasitol 1991;85(6):625–35. 142. Saito W, Kawakami K, Kuroki R, et al. Pulmonary anisakiasis presenting as eosinophilic pleural effusion. Respirology 2005;10(2):261–2. 143. Matsuoka H, Nakama T, Kisanuki H, et al. A case report of serologically diagnosed pulmonary anisakiasis with pleural effusion and multiple lesions. Am J Trop Med Hyg 1994; 51(6):819–22. 144. Nieuwenhuizen N, Lopata AL, Jeebhay MF, et al. Exposure to the fish parasite Anisakis causes allergic airway hyperreactivity and dermatitis. J Allergy Clin Immunol 2006; 117(5):1098–105. 145. Aftandelians R, Raafat F, Taffazoli M, Beaver PC. Pulmonary capillariasis in a child in Iran. Am J Trop Med Hyg 1977;26(1):64–71. 146. Yii C-J, et al. Human angiostrongyliasis involving the lungs. Chin J Microbiol 1968;1:148–50. 147. Intapan PM, Morakote N, Chansung K, Maleewong W. Hypereosinophilia and abdominopulmonary

150. Severo LC, Conci LM, Camargo JJ, Andre-Alves MR, Palombini BC. Syngamosis: two new Brazilian cases and evidence of a possible pulmonary cycle. Trans R Soc Trop Med Hyg 1988;82(3):467–8. 151. Klion AD, Eisenstein EM, Smirniotopoulos TT, Neumann MP, Nutman TB. Pulmonary involvement in loiasis. Am Rev Respir Dis 1992;145 (4 Pt 1):961–3. 152. Cambanis A. Pulmonary loiasis and HIV coinfection in rural Cameroon. PLoS Negl Trop Dis 2010;4(3):e572. 153. Meyers WM, Neafie RC, Connor DH. Onchocerciasis: invasion of deep organs by Onchocerca volvulus. Am J Trop Med Hyg 1977;26(4):650–7. 154. Fried B, Abruzzi A. Food-borne trematode infections of humans in the United States of America. Parasitol Res 2010;106(6):1263–80. 155. Mahmouod AAF, Abdel Wahab MF. Schistosomiasis. In Warren KS, Mahmoud AAF, eds. Tropical and Geographical Medicine, 2nd ed. New York: McGraw-Hill, 1990. pp. 470–1. 156. Gryseels B, Polman K, Clerinx J, Kestens L. Human schistosomiasis. Lancet 2006;368(9541):1106–18. 157. Mahmoud AAF. Schistosomiasis. London: Imperial College Press, 2001. 158. Bethlem EP, Schettino GP, Carhalho CR. Pulmonary schistosomiasis. Curr Opin Pulm Med 2010;3:361–5. 159. Ross AG, Vickers D, Olds GR, Shah SM, McManus DP. Katayama syndrome. Lancet Infect Dis 2007; 7(3):218–24. 160. Schwartz E, Rozenman J, Perelman M. Pulmonary manifestations of early schistosome infection among nonimmune travelers. Am J Med 2000;109(9):718–22.

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161. Ferreira Rde C, Domingues AL, Markman Filho B, et al. Hepatopulmonary syndrome in patients with Schistosoma mansoni periportal fibrosis. Acta Trop 2009;111(2):119–24. 162. al-Fawaz IM, al-Rasheed SA, al-Majed SA, Ashour M. Schistosomiasis associated with a mediastinal mass: case report and review of the literature. Ann Trop Paediatr 1990; 10(3):293–7. 163. Rodrigues GC, Lacerda DC, Gusmao Eda S, Colares FA, Mota VT. Pseudotumoral presentation of chronic pulmonary schistosomiasis without pulmonary hypertension. J Bras Pneumol 2009;35(5):484–8. 164. Grillo IA. Bronchiectasis with pulmonary schistosomal granuloma. East Afr Med J 1971; 48(5):204–8. 165. Nagakura K, Oouchi M, Abe K, Araki K. Pulmonary paragonimiasis misdiagnosed as tuberculosis: with special references on paragonimiasis. Tokai J Exp Clin Med 2002;27 (4):97–100. 166. Osaki T, Takama T, Nakagawa M, Oyama T. Pulmonary Paragonimus westermani with false-positive fluorodeoxyglucose positron emission tomography mimicking primary lung cancer. Gen Thorac Cardiovasc Surg 2007;55(11):470–2. 167. Im JG, Chang KH, Reeder MM. Current diagnostic imaging of pulmonary and cerebral paragonimiasis, with pathological correlation. Semin Roentgenol 1997;32 (4):301–24. 168. Zarrin-Khameh N, Citron DR, Stager CE, Laucirica R. Pulmonary paragonimiasis diagnosed by fineneedle aspiration biopsy. J Clin Microbiol 2008;46(6):2137–40.

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171. Freeman RS, Stuart PF, Cullen SJ, et al. Fatal human infection with mesocercariae of the trematode Alaria americana. Am J Trop Med Hyg 1976;25(6):803–7. 172. Pawlowski Z, Eckert J, Vuitton D. Echinococcosis in humans: clinical aspects, diagnosis and treatment. In Eckert J, Gemmel MA, Meslin FX, Pawlowski ZS, eds. WHO/OIE Manual on Echinococcosis in Humans and Animals: A Public Health Problem of Global Concern. Paris: World Health Organization and World Organization for Animal Health, 2001. 173. McManus DP, Zhang W, Li J, Bartley PB. Echinococcosis. Lancet 2003; 362(9392):1295–304. 174. Morar R, Feldman C. Pulmonary echinococcosis. Eur Respir J 2003; 21(6):1069–77. 175. Jerray M, Benzarti M, Garrouche A, Klabi N, Hayouni A. Hydatid disease of the lungs. Study of 386 cases. Am Rev Respir Dis 1992; 146(1):185–9. 176. Halezeroglu S, Celik M, Uysal A, et al. Giant hydatid cysts of the lung. J Thorac Cardiovasc Surg 1997; 113(4):712–7. 177. Tekinbas C, Turedi S, Gunduz A, Erol MM. Hydatid cyst disease of the lung as an unusual cause of massive hemoptysis: a case report. J Med Case Reports 2009;3:21. 178. Gotterer N, Lossos I, Breuer R. Pancoast’s syndrome caused by primary pulmonary hydatid cyst. Respir Med 1990;84(2): 169–70. 179. Ozdemir N, Akal M, Kutlay H, Yavuzer S. Chest wall echinococcosis. Chest 1994;105(4):1277–9. 180. Demir HA, Demir S, Emir S, Kacar A, Tiryaki T. Primary hydatid cyst of the rib mimicking chest wall tumor: a case report. J Pediatr Surg 2010;45(11): 2247–9.

169. Devi KR, Narain K, Bhattacharya S, et al. Pleuropulmonary paragonimiasis due to Paragonimus heterotremus: molecular diagnosis, prevalence of infection and clinicoradiological features in an endemic area of northeastern India. Trans R Soc Trop Med Hyg 2007; 101(8):786–92.

181. Singh N, Srinivas R, Bal A, Aggarwal AN. Lung carcinoma mimicking hydatid cyst: a case report and review of the literature. Med Oncol 2009; 26(4):424–8.

170. Chen MG, Mott KE. Progress in assessment of morbidity due to Fasciola hepatica infection. Trop Dis Bull 1990;87:R1–R38.

182. Abushhewa MH, Abushhiwa MH, Nolan MJ, et al. Genetic classification of Echinococcus granulosus cysts from humans, cattle and camels in

Libya using mutation scanning-based analysis of mitochondrial loci. Mol Cell Probes 2010;24(6):346–51. 183. Ohsaki Y, Sasaki T, Shibukawa K, Takahashi T, Osanai S. Radiological findings of alveolar hydatid disease of the lung caused by Echinococcus multilocularis. Respirology 2007; 12(3):458–61. 184. Wilson ID. Multiple hyphenation of liquid chromatography with nuclear magnetic resonance spectroscopy, mass spectrometry and beyond. J Chromatogr A 2000;892(1–2): 315–27. 185. Junghanss T, da Silva AM, Horton J, Chiodini PL, Brunetti E. Clinical management of cystic echinococcosis: state of the art, problems, and perspectives. Am J Trop Med Hyg 2008;79(3):301–11. 186. Garcia HH, Gonzalez AE, Evans CA, Gilman RH. Taenia solium cysticercosis. Lancet 2003;362(9383): 547–56. 187. Choi JH, Chung SI, Whang YS, et al. A case of pulmonary cysticercosis. Korean J Intern Med 1991;6(1):38–43. 188. Walts AE, Nivatpumin T, Epstein A. Pulmonary cysticercus. Mod Pathol 1995;8(3):299–302. 189. Scholtz L, Mentis H. Pulmonary cysticercosis. A case report. S Afr Med J 1987;72(8):573–4. 190. Iwatani K, Kubota I, Hirotsu Y, et al. Sparganum mansoni parasitic infection in the lung showing a nodule. Pathol Int 2006;56(11): 674–7. 191. Phunmanee A, Boonsawat W, Indharapoka B, Tuntisirin C, Kularbkeaw J. Pulmonary sparganosis: a case report with five years follow-up. J Med Assoc Thai 2001;84(1):130–5. 192. Beaver PC, Rolon FA. Proliferating larval cestode in a man in Paraguay. A case report and review. Am J Trop Med Hyg 1981;30(3):625–37. 193. Aoshima M, Nakata K, Matsuoka M, Kawabata M, Nakamura T. [A case of proliferative sparganosis associated with PIE syndrome and pulmonary embolism]. Nihon Kyobu Shikkan Gakkai Zasshi 1989; 27(12):1521–7. 194. Azinge NO, Ogidi-Gbegbaje EG, Osunde JA, Oduah D. Armillifer

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armillatus in Bendel State (Midwest) Nigeria (a village study in Ayogwiri Village, near Auchi, 120 kilometres from Benin City) phase I. J Trop Med Hyg 1978; 81(5):76–9.

195. Guardia SN, Sepp H, Scholten T, Morava-Protzner I. Pentastomiasis in Canada. Arch Pathol Lab Med 1991;115(5):515–7. 196. Mulder K. [Porocephalosis]. Dtsch Med Wochenschr 1989;114(49):1921–3.

197. Pampiglione S, Gentile A, Maggi P, Scattone A, Sollitto F. A nodular pulmonary lesion due to Linguatula serrata in an HIVpositive man. Parassitologia 2001; 43(3):105–8.

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9

Acute lung injury William A.H. Wallace, A. John Simpson and Nik Hirani

Introduction Acute respiratory distress syndrome (ARDS) was first described by Ashbaugh and colleagues in 19671 and has been the focus of intense clinical and academic scrutiny ever since. ARDS is generally recognized as a catastrophic lung injury developing rapidly in response to a variety of pulmonary and/or extra-pulmonary insults, and is associated with significant mortality. The diverse etiologies leading to ARDS and the broad spectrum of clinical severity associated with the syndrome stimulated the need for universally accepted clinical definitions. As a result, a landmark American-European consensus conference (AECC) provided a clear, pragmatic definition of ARDS, while recognizing that the syndrome is a severe form of acute lung injury (ALI).2 In this scheme, ALI is a condition characterized by:
acute onset
the development of bilateral infiltrates on a chest X-ray
impaired oxygenation (a PaO2:FiO2 ratio of  300 mmHg) and
the absence of left atrial hypertension (either on clinical grounds or by a measured pulmonary arterial wedge pressure of 18 mmHg). The requirement for acute onset precludes confusion with preexisting lung disease, while the requirement for a normal left atrial pressure excludes cardiogenic pulmonary edema as a cause of lung infiltrates. Reflecting the fact that ALI and ARDS represent ends of a clinical continuum, ARDS is defined in exactly the same way, except that the required PaO2:FiO2 ratio is  200 mmHg.2 While it is widely accepted that these working definitions of ALI and ARDS are not perfect,3,4 they have served two important and related purposes. Firstly, clinicians in different centers and countries can use internationally accepted criteria when dealing with patients with ALI/ARDS. Secondly, clear definitions have allowed the design of randomized controlled trials (RCTs) with the aim of improving the management of ALI/ ARDS.

Pathologists interested in ALI/ARDS are faced with a curious paradox. While pathology has been instrumental in characterizing the pathogenesis of ALI/ARDS (largely based on post-mortem specimens), diagnostic material is rarely submitted from patients during life. This reflects the fact that patients with ARDS are usually critically ill, mechanically ventilated, and that diagnosis is based on clinical criteria. Therefore, while diffuse alveolar damage (DAD) is regarded as the histological hallmark of ALI/ARDS, it is rarely demonstrated on histology during life. As a consequence, pathologists generally only receive ante-mortem lung tissue from the most complicated and difficult cases of suspected ALI/ARDS. An understanding of the definitions and pathogenesis of ALI/ARDS is essential if the pathologist is to interpret the context, usefulness and limitations of tissue submitted from patients. This chapter therefore begins with an outline of the etiology, epidemiology and radiology of ALI/ARDS. This is followed by a detailed description of the macroscopic and microscopic pathology. The clinical setting in which diagnostic tissue and/or bronchoalveolar lavage (BAL) fluid may be obtained and submitted will then be considered. Thereafter, the pathophysiology of ALI/ARDS is discussed in detail. The chapter ends with the principles of management and an evaluation of how improved understanding of pathogenesis has informed the design of important clinical trials. Throughout, we aim to use the terms ALI and ARDS specifically in the context of the AECC definitions provided above.

Etiology of ALI/ARDS The clinical syndrome of ALI/ARDS develops secondary to a wide range of pathological processes.5,6 For convenience, the primary insults are divided into those arising in the lung and those that are extra-pulmonary (Table 1). Pulmonary causes include pneumonia, significant aspiration of gastrointestinal contents, inhaled toxins (e.g. smoke, illicit drugs, such as heroin, and sustained high concentrations of oxygen), near drowning, and pulmonary contusion. Due vigilance is clearly required in identifying rapidly emergent

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 9: Acute lung injury Table 1 Etiologies of ALI/ARDS

Direct lung injury

Indirect lung injury

Pneumonia (bacterial, viral, fungal)

Systemic sepsis especially with Gram-negative bacilli

Inhalation injury, e.g. gastric contents, smoke, corrosive vapors, such as ammonia

Major extrathoracic trauma with or without fat emboli

Therapeutic drug reactions, e.g. bleomycin and methotrexate

Acute pancreatitis

Pulmonary contusion/blast injury

Drug overdose, e.g. opiates and barbiturates

Fat emboli

Transfusion of blood products

Near-drowning

Disseminated intravascular coagulation

Reperfusion/re-expansion lung injury (including post lung transplant)

Systemic poisoning, e.g. paraquat

Radiation injury

Eclampsia

Altitude (acute mountain sickness)

pulmonary causes of ALI/ARDS, as exemplified by recent outbreaks of avian H5N1 and swine-origin H1N1 influenza.7–11 Extra-pulmonary causes are as diverse as sepsis, severe extra-thoracic trauma (including burns), iatrogenic exposures (e.g. drugs, irradiation, massive blood transfusion, vigorous resuscitation, cardiopulmonary by-pass), systemic poisoning (e.g. with paraquat or illicit drugs), embolization of fat/air/ amniotic fluid, pancreatitis, uremia, malignancy, and eclampsia. Three points are worth considering in relation to the multiple etiologies resulting in ALI/ARDS. Firstly, ALI/ARDS represents a relatively uniform pathological response to such a diverse range of insults. Considerable debate surrounds the issue of whether the pathology/pathogenesis of ALI/ARDS is truly identical in the face of very different primary stimuli.12,13 The legitimacy of designing clinical trials made up of patients with diverse primary pathologies is questioned in certain quarters. For the purpose of this chapter, however, the pathological entity of ALI/ARDS is considered a sufficiently uniform “endpoint” arising from the processes above. Secondly, infection (systemic sepsis and/or pneumonia) emerges as the predominant trigger for ALI/ARDS. Significant advances in the management of sepsis have arisen in recent years,14 and prompt recognition and treatment of this condition will impact upon the incidence of ALI/ARDS. A practical implication of the link between infection and ALI/ARDS is that a mechanically ventilated patient developing lung infiltrates in the intensive care unit (ICU) could have ALI/ ARDS, a pneumonic process quite distinct from ALI/ARDS,

or co-existence of both. As discussed later in this chapter, the decision to obtain BAL fluid from such patients is usually aimed at excluding infection, and the decision to obtain tissue is driven by concerns that a process other than ALI/ARDS may explain lung infiltrates in the absence of clear evidence for infection. Thirdly, it is clear that only a minority of patients with each of the diverse etiologies described above develop ALI/ARDS. Not all patients with ALI are alike. The pathogenesis is influenced by the host genotype, the environment and the nature of the initiating insult.15–17 This has prompted careful studies of genetic susceptibility. Candidate genes are generally identified because their products are considered to be involved in disease pathogenesis or through their consistent emergence in powerful screening tools such as high-throughput expression profiling.18–22 Thereafter clear demonstration that polymorphisms in the candidate gene(s) are over-represented in carefully characterized ALI/ARDS provides firmer evidence of genetic association. This topic has been the subject of an excellent review,18 but novel associations are emerging continually.19 The associated polymorphisms can be broadly categorized according to the function of the gene product and its relevance to ALI/ARDS.18,19 These include genes encoding
cytokines (interleukins, IL-6, IL-8, IL-10, tumor necrosis factor a (TNFa), macrophage migration inhibitory factor (MIF), lymphotoxin a, and pre-B cell colony-enhancing factor 1)
proteases and antiproteases (urokinase, angiotensin I converting enzyme and elafin)
oxidants and antioxidants (superoxide dismutase 3 and NAD(P)H:quinine oxidoreductase I)
endothelial barrier function (myosin light chain kinase)
surfactant proteins (surfactant protein B)
innate immune regulators (mannose-binding lectin (protein C) 2)
coagulation factors (factor V)
iron hemostasis (ferritin light polypeptide)
transcriptional regulators (nuclear factor of k light polypeptide gene enhancer in B cells inhibitor, a and 1 and nuclear factor of k light polypeptide gene enhancer in B cells 1)
Toll-like receptors. Furthermore, microRNAs (miRNAs) have emerged as a novel class of gene regulators that play critical roles in complex diseases, including ALI. Comparisons of global miRNA profiles in animal models of ALI and ventilator-induced lung injury identified several miRNAs (e.g. miR-146a and miR155) previously implicated in immune response and inflammatory pathways. By regulation of target genes in these biological processes and pathways, miRNAs potentially contribute to the development of ALI.23 Frequent reference to the molecules listed above, and the processes they regulate, will be made when considering the

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Chapter 9: Acute lung injury Table 2 Short- and long-term complications of ALI/ARDS

Pulmonary complications

Non-pulmonary complications

Short term

Long term

Short term

Long term

Pneumothorax and pneumomediastinum (3–20%)

Mild impairment in lung function (30–50%)

Multi-organ dysfunction (> 80%)

Neuromuscular weakness (up to 60%)

Nosocomial pneumonia (> 35%)

Severe lung function impairment (rare)

pathogenesis of ALI/ARDS later in this chapter. Future dissection of genetic associations is expected to shed further light on disease pathogenesis and suggest logical therapeutic candidates.

Epidemiology The true incidence of ALI remains controversial. A recent study estimated that approximately 190 000 patients develop ALI each year in the United States.24 In European studies, it has been estimated that approximately 10% of patients admitted to ICUs develop ALI.25–27 The mortality of ALI/ARDS varies considerably between published studies, but more recent estimates are broadly in keeping with an approximate in-hospital mortality of 40%.28–30 Little is known about the influence of race and ethnicity on mortality from ALI, but recent work is intriguing, as genetic susceptibility probably plays a significant role. For example, single nucleotide polymorphisms (SNP) of the TIR domaincontaining adaptor protein (TIRAP) gene contribute to increased risk of sepsis-associated ALI and mortality in Han Chinese individuals.22 A retrospective cohort study of patients enrolled in the Acute Respiratory Distress Syndrome (ARDS) Network trials showed that African-American and Hispanic patients with ALI had a significantly higher risk of death, compared with white patients. The increased risk was associated with increasing severity of illness at presentation for AfricanAmericans, but this association was not observed among Hispanics.31 Evidence suggests the mortality associated with ALI/ARDS is decreasing over time.32 Outcomes for patients who survive to leave the ICU have been the focus of increasing research. The picture evolving suggests these patients continue to have a poor prognosis.33–35 For example, it has been suggested that half of patients discharged from hospital require further medical input and about a quarter of these patients are re-admitted to a hospital within 6 months. In most survivors, while lung function tests, in particular diffusing capacity, may be impaired, the functional consequences of this are generally mild.36 On the other hand, data suggest that survivors very commonly suffer from extra-pulmonary sequelae. For example, health-related quality of life is significantly impaired when compared with age- and sex-matched controls. Exercise capacity is reduced in the majority due to neuromuscular abnormalities. Cognitive impairment, commonly in the form of memory loss, is

344

Post-traumatic stress syndrome (up to 30%)

common in survivors of ALI/ARDS and severe depression is overrepresented in this population (Table 2).36

Radiological findings in ALI/ARDS Chest radiographs taken at the time of presentation with ALI/ ARDS usually show bilateral alveolar infiltrates, though it is recognized that radiographic changes may lag behind profound hypoxemia by a few hours (Figure 1).37 The alveolar infiltrates reflect disruption of the barrier function of the alveolar-capillary membrane, as discussed in more detail below. The resultant congestion and depletion of surfactant lead to impaired lung compliance and alveolar atelectasis. As such, “recruitment” of congested/collapsed alveoli is fundamental to mechanical ventilation strategies aiming to improve ventilation-perfusion matching. While the example shown is relatively typical for ALI/ ARDS, in practice interpretation may be more difficult. The extent of alveolar infiltration may vary greatly between patients, a wide variety of coexistent pathologies may be represented on the X-ray (including effects of barotrauma, such as pneumothorax), and the position of the patient (often supine) may make interpretation more difficult.37 There is considerable interobserver variability in the interpretation of chest radiographs from patients in the ICU generally, and from patients with ALI/ARDS specifically.38–41 Computed tomography (CT) scanning can add considerable information regarding the nature and cause of alveolar infiltrates seen on a chest radiograph (Figure 1). However, CT involves moving sick, mechanically ventilated patients from the ICU to the scanner and back. All the logistic considerations have to be weighed against the likely diagnostic benefit. CT in ALI/ARDS generally reveals homogeneous and gravitationally dependent alveolar shadowing, often in a rather patchy distribution.41 The variable extent of parenchymal involvement and the gravitational dependence are worth keeping in mind when considering the practicalities of tissue biopsy, discussed later in the chapter. CT scanning has recently provided valuable insights into the extremely variable degree to which patients’ lung tissue can be successfully recruited for better oxygenation during mechanical ventilation.41,42 CT studies have also suggested that the structure and function of the lung varies markedly over time in ARDS, with progressive evolution of a more restrictive lung defect, development of microcystic bullae

Chapter 9: Acute lung injury

Figure 1. Chest X-ray and high-resolution CT changes in ALI/ARDS. Serial images over a 1 month period (a–d) of evolving ALI/ARDS in a patient undergoing bowel surgery complicated by postoperative sepsis. The developing radiographic changes, whilst typical of ARDS, are not diagnostic and are not readily distinguishable from infective pneumonia or pulmonary edema. The classic CT features of ARDS are seen in lower panel c. There is a heterogeneous pattern of disease with relatively normal lung anteriorly (black arrow), dense air-space opacification in the most dependent areas (double arrow) and ground-glass changes in the intervening lung (white arrow).

and a greater risk of pneumothorax once the syndrome has been present for over 2 weeks.42

Pathological features of acute lung injury It is important to remember that ALI and ARDS are clinical syndromes and that the corresponding pathology is DAD.43–48 Distinction between ALI and ARDS is not possible on pathological grounds, although at the ultrastructural level there may be some features that suggest either direct pulmonary or systemic secondary injury. In routine pathological practice the diagnosis of DAD is most likely to be encountered at autopsy, particularly in patients who have been in ICUs. On occasion lung biopsies may be performed on patients with the clinical features of ALI or ARDS to confirm the nature of the process and in an attempt to identify precipitating causes, e.g. infection, aspiration, vasculitis, recurrent tumor.48–50 Acute lung injury may be superimposed on a background of chronic lung disease, such as usual interstitial pneumonitis.43 This may be a terminal event in these patients or in some cases can be the presenting clinical picture. Pathologically DAD can be divided into different phases. These represent a dynamic, continuous process rather than discrete, pathological steps. The early events are described as the “exudative” phase, followed by the “proliferative” phase. If the patient survives long enough the proliferative phase may be followed by organization with the development of established fibrosis. Alternatively resolution of the proliferative phase may occur with a return to normal or near normal lung architecture. These two possibilities are, however, not mutually exclusive and may overlap histologically in different areas of the lung.

Exudative phase In the very earliest stages of this phase the lungs may appear macroscopically normal. As the process develops the lungs show increasing degrees of edema. At autopsy they are heavy, often weighing in excess of 1 kilogram each and the cut surface is red and beefy in appearance with the lung slices being rather firm (Figure 2). The earliest changes identifiable in the lung are only detectable by electron microscopy.44,51,52 At the ultrastructural level there is evidence of injury and necrosis of types I and II pneumocytes, leaving a denuded and in some cases damaged basement membrane (Figures 3 and 4). The alveolar capillaries are typically described as showing increased numbers of marginated neutrophils, although this is not a universal finding, and there is interstitial edema. Small fibrin thrombi may be identified. The endothelial cells may also show injury but this is variable, and there may also be some evidence of capillary proliferation. Some groups have suggested that endothelial injury may be more marked in patients developing ALI secondary to extra-pulmonary causes.53 Two to three days following injury intra-alveolar edema will be apparent at the light microscopic level (Figure 5). The sometimes hemorrhagic edema fluid is an exudate; rich in fibrin due to the “leaky” alveolar walls. This exudate, mixed with necrotic cellular debris, condenses to form hyaline membranes. These membranes are often regarded as the characteristic histological feature of the exudative phase (Figure 6). In the early stages hyaline membranes may be relatively focal but as the injury develops they may be more widespread. These are characterized on H&E staining as intensely eosinophilic relatively dense bands of proteinaceous material lining alveolar

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(a)

(b)

Figure 2. Macroscopic views of a lung slice from a patient with recent-onset ALI. (a) The cut surface of the lung is red and beefy. The lungs are heavy and have a firm consistency. (b) The process may be localized or diffuse depending on the nature and extent of the disease process. Subpleural lung is spared. (Image courtesy of Dr D. Flieder, MD, Philadelphia, PA, USA.)

Figure 4. Electron micrograph from a case of ALI showing disruption and a break in the basement membrane between the capillary space and the adjacent alveolar space. This reflects significant alveolar injury and, if extensive, may be associated with a reduced likelihood of resolution. (Image courtesy of Dr P. Hasleton, Manchester, UK.)

Proliferative phase Figure 3. Electron micrograph from a case of ALI demonstrating evidence of papillary processes on the surface of type I epithelial cells, edema of the basement membrane and electron lucency of the endothelial cells consistent with early alveolar injury. (Image courtesy of Dr P. Hasleton, Manchester, UK.)

airspaces and ducts. Hyaline membranes are associated with a rather variable and often patchy increase in interstitial chronic inflammatory cells. Small fibrin thrombi may also be identified (Figure 7). Although neutrophils are believed to be important in the pathogenesis of ALI and are detectable in BAL samples early in the disease process, they are often inconspicuous on light microscopy in the airspaces. In cases where significant numbers of neutrophils are evident associated with fibrinous exudates, the alternative diagnosis of pneumonia needs to be considered. However, both processes can co-exist, with the pneumonia precipitating the ALI or arising as a secondary complication.

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The proliferative phase is usually evident by 5–7 days from the onset of the process. Macroscopically the lungs remain heavy and firm but by this stage begin to develop a grayer consolidated appearance (Figure 8). Histologically this phase is characterized by organization of the exudative fluid with the appearance of myofibroblasts and the development of granulation tissue polyps within alveolar spaces and ducts (Figure 9). This process may be variable in its distribution and foci of organization may be seen admixed with persisting hyaline membranes and interstitial inflammatory cells. In association with this picture, there is marked proliferation of type II alveolar cells along the alveolar walls (Figure 10). These cuboidal cells have a “hob-nail” appearance and usually feature a considerable degree of cytological atypia with pleomorphic nuclei and prominent nucleoli. There may also be squamous metaplasia, which can be widespread (Figure 11). Care needs to be taken not to make an erroneous diagnosis of viral infection or malignancy in such patients, particularly in frozen

Chapter 9: Acute lung injury

sections or cytological samples that may be taken primarily to look for infective agents. At the ultrastructural level, the basement membrane may be disrupted (Figure 4) and there may be further evidence of endothelial injury and microthrombi within alveolar capillaries. Myofibroblasts both proliferate within the interstitium and also migrate through the breaks in the basement membrane into the exudates within the alveolar spaces and ducts.54,55 This process results in the development of airspace granulation tissue polyps. This is associated with secretion of extracellular matrix components, such as fibronectin and tenascin.

Increasing interstitial fibrosis, collapse of the alveolar architecture and progressive distortion of the lung architecture often follow (Figure 12 and 13). In patients surviving several weeks a variety of patterns of established fibrosis may be observed. In some, there may be a honeycomb pattern although care needs to be taken to exclude a pre-existing usual interstitial pneumonia-pattern in such cases (Figure 14).43,56 Others show extensive nodular fibrosis with residual cleft-like spaces lined by alveolar epithelial cells. Some develop a more diffuse pattern of alveolar wall thickening with better architectural preservation, the appearance of which may resemble the

Figure 5. Lung from a patient with early ALI. The alveolar capillaries are congested and edematous. Neutrophils can be prominent. (Image courtesy of Dr P. Hasleton, Manchester, UK.)

Figure 7. DAD with thrombus. This small fibrin thrombus has pathogenetic implications but no great clinical ramifications.

(a)

(b)

Figure 6. Hyaline membranes. (a) Note the prominent hyaline membranes. Adjacent lung has an inflammatory cell infiltrate with focally scattered neutrophils. (b) Electron micrograph demonstrating the ultrastructural features of the alveolar wall in ALI. The hyaline membrane is the dark material at the top of the image, below which is the bare basement membrane. The focal lucent area beneath the basement membrane is focal damage in an endothelial cell. Note the red blood cell in a capillary lumen. (Image courtesy of Dr P. Hasleton, Manchester, UK.)

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(a)

Figure 9. Fibroproliferative phase of DAD. The airspaces are filled with plugs of organizing immature fibroblastic tissue comprising myofibroblasts and loose matrix elements.

(a)

Figure 8. Macroscopic views of a lung slice from a patient with ALI. (a) The cut surface of the lung features geographic gray consolidation. This appearance represents the proliferative phase of ALI. (b) Note the incomplete involvement of lung (Images courtesy of Dr D. Flieder, MD, Philadelphia, PA, USA.)

(b)

Figure 11. DAD. Squamous metaplasia is a common histologic finding.

(b)

Figure 10. Type II alveolar epithelial cell hyperplasia in the lung of a patient with ALI. (a) Marked cytological atypia raises the possibility of viral infection or even malignancy, especially in cytology specimens. (b) Higher magnification of these hyperplastic cells.

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Figure 12. Organizing DAD. Fibroblastic exudates are incorporated into adjacent alveolar walls. This leads to pulmonary fibrosis. Figure 14. DAD superimposed on IPF. The majority of the lung tissue shows a red beefy pattern typical of the exudative phase of DAD while adjacent subpleural lung tissue features pale fibrous tissue. (Image courtesy of Dr K. Kerr, Aberdeen, UK.)

Figure 13. Organizing DAD. Re-epitheialization of the alveolar spaces by type II epithelial cells with permanent apposition of the collapsed alveoli. Alveolar collapse represents a critical step in the development of fibrosis. (Reproduced from Katzenstein and Askin’s Surgical Pathology of Non-Neoplastic Lung Disease. 4th edition, 2006, by kind permission of Saunders Elsevier)

Figure 15. Lung fibrosis in a patient who had died following a prolonged ICU admission. A nonspecific pneumonia-like pattern is seen.

fibrotic nonspecific interstitial pneumonitis pattern (NSIP) (Figure 15). An appropriate clinical history of previous severe lung injury is likely to distinguish these cases from idiopathic NSIP. There may be extensive squamous metaplasia and secondary acute inflammatory changes with neutrophils in cystic spaces. In the latter stages there is usually vascular remodeling with extensive medial hypertrophy of muscular pulmonary arteries, irregular intimal fibrosis in both arteries and veins, and arteriolization of pulmonary arterioles. Pulmonary veins also show marked intimal fibrosis, most likely secondary to the pulmonary fibrosis. Destruction of the pulmonary vascular bed leads to secondary pulmonary hypertension.44

Resolution and patient survival may follow the proliferative phase. The factors responsible for the clearance of the immature organizing exudative process are poorly understood. Apoptosis of myofibroblasts combined with phagocytosis of the debris and immature extracellular matrix elements are suggested mechanisms. If resolution occurs early, before the development of fibrosis, then the lung architecture may return to near normal. Patients with more protracted courses are often left with variable degrees of established lung fibrosis and subsequent loss of respiratory function (Table 2).

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Acute fibrinous organizing pneumonia This is a relatively recently described histological pattern of lung injury, which may clinically follow a similar clinical pattern to DAD.57 The morphology is entirely nonspecific and seen in a variety of clinical settings, including connective tissue disorders, infections and drug effects. Intra-alveolar fibrin without hyaline membranes is seen along with scattered foci of organizing pneumonia (see Chapter 10).

Samples submitted to pathology departments from patients with suspected ALI/ARDS This section will briefly consider the accuracy of the clinical diagnosis of ALI/ARDS, the principal reasons for pursuing BAL and/or biopsies in ALI/ARDS patients, and the relative clinical usefulness of BAL/transbronchial biopsy/open lung biopsy (OLB). A number of studies have compared post-mortem pathological diagnosis with ante-mortem clinical diagnosis in the ICU. Taking DAD to be the pathological correlate, these studies found only moderate accuracy for the clinical diagnosis of ARDS (sensitivity 71–83%, specificity 51–84%).3,58–60 Infection was the commonest pathological diagnosis when ARDS was not accompanied by DAD. Some caution must be exercised in interpreting post-mortem studies as, by definition, only the most severely ill patients are included. With this caveat in mind, it appears that the clinical syndrome of ARDS is not always accompanied by DAD. Considered another way, if DAD is accepted as the histological gold standard for ARDS, the clinical diagnosis is wrong much of the time. However, as discussed above, recent studies have suggested that other patterns of lung injury may be seen in some of these patients.57 While the AECC definitions for the diagnosis of ALI/ ARDS are immensely beneficial, they are imperfect. A further clinical implication is that the principal differential diagnosis for ALI/ARDS is infection. Clearly many patients with ALI/ ARDS have no infection. Some patients may have ALI/ARDS because of pulmonary or extra-pulmonary infection, some may develop pneumonia complicating ALI/ARDS, and others may have pneumonia in the absence of, but closely mimicking, ALI/ARDS. Because bacterial and fungal infections are potentially reversible, and since pulmonary infection is common in the ICU, the decision to perform BAL is usually driven by the desire to exclude or confirm pneumonia. The decision to obtain lung tissue is usually reserved for cases in which treatment for ALI/ARDS has been unsuccessful and where significant diagnostic doubt remains after a vigorous search for infection. In cases of suspected ALI/ARDS, BAL is usually performed under direct vision using a flexible fiberoptic bronchoscope passed through the endotracheal tube. Risk factors for adverse events during bronchoscopy in mechanically ventilated patients are well recognized and include hemorrhage, arrythmias, hypoxia and pneumothorax.61 As long as these relative

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contraindications are remembered, BAL is generally safe and well tolerated. The procedure must be performed using a standardized technique.62 Samples should be submitted fresh to expert cytology and microbiology labs, which are made aware in advance that the sample is being submitted and provided with full clinical data. Most cells in normal human BAL fluid are alveolar macrophages, with neutrophils comprising less than 5% of the total population. In ALI/ARDS, regardless of whether pulmonary infection co-exists, BAL fluid contains a clear predominance of neutrophils.63 The fluid aspirated may be tinged with blood, reflecting the “leaky” alveolar-capillary membrane. As the proliferative phase of ALI/ARDS proceeds, macrophages begin to repopulate BAL fluid. Rarely bronchoscopy may reveal an unexpected finding (e.g. a tumor), or BAL fluid may reveal an unexpected cell population (e.g. adenocarcinoma, eosinophils in florid eosinophilic pneumonia, lymphocytes in hypersensitivity pneumonitis or macrophages in desquamative interstitial pneumonia, etc.). The isolation of potential pathogens in an adequate sample of BAL fluid is helpful in directing management. The concentration of pathogens required in BAL fluid to make a confident diagnosis of pneumonia is controversial.64 More than 104 colony forming units (cfu) per ml of BAL fluid is generally accepted as indicative of pneumonia.62 Therefore, quantitative culture of BAL fluid is recommended where possible. Although BAL may be extremely useful, potential limitations must be considered. Firstly, patchy ALI/ARDS lung distribution means BAL may not always sample representative regions. Secondly, the procedure is dependent on the availability of operators who perform BAL to a high standard, and on the accessibility of appropriate microbiological and pathological expertise to maximize diagnostic information. Thirdly, although the procedure is regarded as safe, it usually demands additional oxygen and transient neuromuscular blockade in already very sick patients. Transient hypoxia during the procedure is common. In view of these considerations, tracheal aspirates are sometimes used for the diagnosis of infection in the ICU. In general, tracheal aspirates yield a high false-positive rate for pneumonia, largely due to frequent colonization of the proximal airways.65–68 Other techniques used to sample the alveolar region of the lung include use of a protected specimen brush, thus avoiding the requirement for instillation of fluid using lavage, or the use of a blind (non-bronchoscopic) lavage.69,70 Transbronchial biopsy is occasionally used when diagnostic doubt surrounds a difficult case of possible ALI/ARDS. Few studies have specifically addressed the utility of transbronchial biopsy in this setting. In mechanically ventilated patients with respiratory failure caused by various diagnoses, transbronchial biopsy seems to provide diagnostic information about half of the time, with an appreciable risk of complications such as pneumothorax, bleeding, hypotension and hypoxia.71–74 In a separate study, transbronchial biopsy showed a poor level of agreement with post-mortem diagnosis,

Chapter 9: Acute lung injury

although interpretation of such data is difficult given the variable differences in time between biopsy and autopsy.75 In the specific context of suspected ARDS, transbronchial biopsy seems able to confirm the diagnosis, but does not drive important changes in management.74 One study showed that transbronchial biopsy provided moderately useful prognostic information in ARDS. For example, alveolar interstitial fibrosis was associated with poor outcome.76 The alternative to transbronchial biopsy is open lung biopsy (OLB). A larger body of evidence exists for this procedure, generally performed in cases refractory to treatment or in which diagnostic uncertainty remains. Remarkably similar findings emerge whether the studies dealt exclusively with ARDS (as determined by AECC criteria),77–82 a mix of ALI and ARDS,83 or patients with respiratory failure and diffuse pulmonary infiltrates, where ALI/ARDS is well represented.84–88 In these studies OLB, which was frequently performed as a “bedside” procedure in the ICU, almost always yielded diagnostic information. A specific diagnosis was suggested in a little over 50% of cases. Importantly, a diagnosis other than DAD was also suggested in over 50% of cases, with infection again being prominent. OLB typically resulted in a change of management in 60–80% of patients. Despite this, the available data suggest that 50–60% of patients with clinical ARDS proceeding to OLB die in hospital. Few data exist to predict whether this figure would be higher or lower in similar patients not having OLB. Complications were reported in 20–40% of patients, with air leak being the most prominent. Deaths directly attributable to the procedure were rare, approximately 1%, although one study reported a rate of 8.4%.84 Finally, one group considered only cases of OLB in which DAD was confirmed, and showed broadly comparable findings, with complications in 22% of cases and an overall cohort mortality of 53%.89 Interpretation of these studies is hampered by the fact that the timing of, and indication for, biopsy was not uniform across the retrospective studies. The case-mix in different centers also varied. It seems likely that publications are generated by active proponents of OLB, and results for unpublished series may differ from those above. Nevertheless, if the risk of complications is deemed justifiable and if appropriate surgical expertise exists locally, OLB often yields diagnostic information and alters management in the small proportion of carefully selected patients who fail to respond to treatment and have no readily identified “treatable” lung condition. There is currently no evidence to suggest that OLB favorably alters outcomes. In summary, pathologists and microbiologists are likely to receive BAL samples relatively often, principally to exclude infection and to assess the predominant host cell type. Biopsy will usually be pursued in the rare setting where patients are not responding to treatment and where infection has been excluded by BAL.

Pathogenesis of ALI The pathogenetic processes in ALI are complex and much is not understood. The principal process is one of excessive inflammatory damage to the lung involving complex interactions between inflammatory cells, principally neutrophils, resident lung cells, pro-inflammatory cytokines, oxidants and proteases (Figure 16). Given the broad range of potential etiologies, it is difficult to formulate a single pathogenetic pathway for ALI and the aim of this section is to describe the roles of different cell types and molecular events that are believed to play a critical role in this process. The study of the pathogenetic and prognostic value of plasma and airspace protein biomarkers for ALI and sepsis has become an area of active research over the past decade.90

Exudative phase Inflammatory cells

Neutrophils A sustained inflammatory insult principally mediated by neutrophils is considered responsible for ALI.91 Radiolabeled neutrophil trapping in the pulmonary circulation can be demonstrated at a very early stage of the disease process.92 Histological and ultrastructural studies generally show increased numbers of neutrophils within the pulmonary vasculature and interstitium,93,94 and they can be recovered in BAL fluid from patients with ALI.95,96 Although these cells are thought to play a significant contribution to ALI, they are not essential and the process can occur in neutropenic patients.97 Mechanisms of neutrophil recruitment have been most extensively studied in the systemic circulation (Figure 17). IL-8 is one of the most important chemoattractants for neutrophils to the lung in ALI,91,96,98 although other members of this chemokine family are also upregulated.98,99 Activated neutrophils are probably retained in the lung via two potential mechanisms. The first, and probably most important, is through cells becoming less compliant and “stiffer” when stimulated by activating factors, such as C5a, leukotrienes, IL-8 and bacterial endotoxin.100–102 This inhibits their passage through the pulmonary microcirculation and promotes contact with the endothelium. In addition, the pulmonary capillary endothelial cells are activated by the pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNFa), and IL-1. Endothelial cells then express adhesion molecules, such as selectins and intercellular adhesion molecule 1 (ICAM-1).103–106 These adhesion molecules facilitate neutrophil margination, rolling attachment and firm attachment to the endothelium via b2 integrins on the neutrophil cell surface. In addition, IL-1 induces neuregulin-1-human epidermal receptor-1 shedding, leading to HER2 activation. This pathway disrupts the epithelial cell barrier.107 Although these mechanisms are thought to play roles in ALI, experimental animal studies suggest this may not be as important as the loss of deformability described above.91,103,105,106

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Figure 16. Pathogenesis of ALI: acute-phase. Diagram illustrating the changes that occur in the alveolus during the acute phase of ALI (right-hand side) compared with the normal state (left-hand side). Neutrophils adhere to the injured capillary endothelium and marginate through the interstitium into the airspace (see also Figure 18). The alveolus is filled with protein-rich edema fluid. Macrophages secrete cytokines, which act locally to stimulate chemotaxis and activate neutrophils. Neutrophils release oxidants, proteases and leukotrienes and pro-inflammatory molecules, which promote tissue damage. Anti-inflammatory molecules are also present. The influx of protein-rich fluid also inhibits the function of surfactant and is associated with activation of the coagulation cascade. (Reproduced from Ware LB, Matthay MA. The acute respiratory distress syndrome. N Engl J Med 2000;342:1339 by kind permission of the Massachusetts Medical Society.)

The activated neutrophils migrate through the alveolar septal interstitium and into alveoli.98 Simultaneously, biologically active molecules including proteolytic enzymes, reactive oxygen species, leukotrienes and platelet activating factor are

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released.108 The principal proteolytic enzyme is neutrophil elastase109,110 although collagenases111 and gelatinases112 also play important roles. These molecules damage alveolar epithelial cells, as well as the rest of the alveolus (Table 3).

Chapter 9: Acute lung injury

Figure 17. Schematic illustration of the changes that occur in the interaction of normal endothelium (upper row) and activated endothelial cells (lower row) which result in interactions with neutrophils and their exit from the capillaries into adjacent tissues. LPS, lipopolysaccharide, NO, nitric oxide; ET-1, endothelin-1; PGI2, prostacyclin; TxA2, thromboxane A2; t-PA, tissue plasminogen activator; ROS, reactive oxygen species; PSGL-1, P-selecting glycoprotein-1; ICAM-1, intercellular adhesion molecule-1; PECAM-1, platelet-endothelial cell adhesion molecule-1. Underlined = adhesion molecules, red arrows = actions, black arrows = synthesis. (Reproduced from Orfanos SE et al. Pulmonary endothelium in acute lung injury: from basis science to critically ill. Intensive Care Med 2004;30:1702 by kind permission of Springer.)

Neutrophil turnover and survival in the lung in ALI may also be dysregulated. Normally, neutrophils recruited by inflammatory signals undergo apoptosis and are cleared by phagocytosis.113 This limits the damage to tissue during acute inflammation. In ALI there is some evidence to suggest that neutrophils in the lung are more resistant to apoptosis, perhaps due to increased levels of granulocyte-macrophage colony-stimulating factor (GM-CSF).114 This abnormality is further suggested by the fact that increased numbers of apoptotic neutrophils are identified as ALI resolves.115 Macrophages Resident tissue macrophages are present in both the alveolar spaces (alveolar macrophages) and in the interstitium. These cells produce many biologically active molecules (Table 4). Macrophages are activated by exposure to bacterial products, such as lipopolysaccharide (LPS)116 and cytokines, such as macrophage migration inhibitory factor (MIF).117 Although the neutrophil is believed to be the principal effector cell

responsible for much of the damage in ALI, there are increased numbers of pulmonary macrophages, on both histology 117 and BAL.118 Phenotypic studies have suggested these are largely tissue-derived. High levels of monocyte chemoattractant protein 1 (MCP-1) and activated transforming growth factor beta (TGFb), chemotactic for monocytes, have been demonstrated.119 Activated macrophages release primary inflammatory cytokines, such as IL-1 TNFa, IL-8 and MIF, all of which amplify the inflammatory response and promote endothelial and epithelial injury.116 These cells may also contribute directly to tissue damage through the production of both reactive oxygen species and nitric oxide (NO), generated through activation of inducible NO synthetase (iNOS).120 In experimental models of ventilator-induced ALI, depletion of alveolar macrophages is associated with decreased indices of lung injury. This suggests that in some settings, such as neutropenia, these cells are important regulatory cells in the acute phase of injury.121

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Chapter 9: Acute lung injury Table 3 Biologically active molecules released by neutrophils believed to contribute to lung damage in ALI/ARDS

Table 4 Biologically active molecules released by macrophages implicated in the pathogenesis of ALI/ARDS

Proteolytic enzymes Neutrophil elastase Collagenases Gelatinases

Pro-inflammatory cytokines

Reactive oxygen species Leukotrienes

TNFa IL8 Macrophage inhibitory factor Tissue-damaging factors

Endothelial cells As already described, ultrastructural evidence of endothelial cell changes occur at an early stage in ALI.94 The endothelial population of the pulmonary microvasculature is highly specialized and is important in regulating the movement of fluid into the pulmonary interstitium, as well as transporting macromolecules.124,125 Increases in lung vascular permeability result in the movement of fluid with a high protein content into the alveolar interstitium and then into the alveolar space.126 Increased circulating concentrations of endothelin-1 and von Willebrand factor (vWF) indicate endothelial injury.127–129 There is a correlation between vWF levels and survival, suggesting the extent of endothelial injury/activation is an important factor in determining the severity of ALI.107,129,130 Endothelial cell activation and injury at a functional or even structural level in the lung can be mediated by a wide range of triggers, reflecting the heterogeneous conditions which may drive ALI. Among the most important are cytokines, especially TNFa and IL-1, vascular endothelial growth factor (VEGF), bacterial products, such as LPS, immune complexes, radiation and ischemia/reperfusion.124 The cytokine TGFb has traditionally been thought of as an anti-inflammatory, pro-fibrotic cytokine. However, recent studies suggest it may also have a role in promoting endothelial activation.130 Experimental animals lacking the integrin avb6, essential for conversion of latent to active TGFb, are protected against bleomycin-induced lung injury. This suggests a role for this cytokine in early and late fibrotic events in ALI.132 Injury/activation of endothelial cells promotes changes in cell morphology and biology, which in conjunction with epithelial injury described above are central to the development of

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Reactive oxygen species Nitrous oxide

PAF

Platelets Platelets can directly interact with neutrophils and monocytes and are themselves a source of proinflammatory cytokines. Platelet depletion markedly reduces lung injury in several murine models.122 Platelet sequestration in the lung appears to be neutrophil dependent, although neutrophil sequestration is not platelet dependent. Neutrophils integrate signals received from the endothelium, and there seems to be communication between the vessel wall and platelets. These interactions can cause vascular injury.123

IL-1

Collagenases Pro-fibrotic factors

TGFb PDGF IGF bFGF

Pro-angiogenic factors

C-X-C cytokines VEGF

ALI. The earliest morphological changes involve the contraction of endothelial cells due to cytoskeletal changes. This results in the development of gaps132,133 through which fluid and macromolecules can escape the vascular space.108,134 Endothelial injury in the absence of intercellular gaps is insufficient to cause ALI in experimental animals. The change in endothelial morphology is probably triggered by GTPase activation of myosin light chain kinase.135 In murine models, deletion or inhibition of this enzyme protects against experimental pulmonary edema, lung inflammation and death.136 This morphological change is driven by calcium ion influx into endothelial cells, via vanilloid channels. The latter are a subset of transient receptor potential channels found in pulmonary endothelial cells.137,138 Pro-inflammatory cytokines, such as TNFa, may contribute to breakdown in endothelial impermeability by promoting phosphorylation of endothelial cadherin and b-catenin at endothelial junctions.139 Activation of endothelial cells switches them to a prothrombotic, pro-inflammatory phenotype with increased expression of inflammatory cell adhesion molecules (Table 5).124 There is increased production of thromboxaneA2, platelet activating factor and endothelin-1 and a reduction in prostaglandin I2. Simultaneously chemokines, such as IL-8 and other pro-inflammatory cytokines, including, IL-1, IL-6 and MIF, are released.124,140,141 These effects are at least in part mediated through nuclear factor kappa B (NF-kB). This is a transcriptional “master switch” that can be directly induced by pro-inflammatory cytokines, such as IL-1 and TNFa, as well as bacterial products and reactive oxygen species.142 Activation also results in cell surface upregulation of the adhesion molecules involved in pulmonary neutrophil trapping. E-selectin and P-selectin slow neutrophils in the circulation by allowing a so-called “rolling” pattern of interaction with the endothelial cells.124 The upregulation of the selectins may be signaled by the same changes in calcium ion concentration that activate

Chapter 9: Acute lung injury Table 5 Biologically active molecules produced by endothelial cells implicated in the pathogenesis of ALI/ARDS

Cytokines

IL1 IL6 IL8 and other chemokines Macrophage inhibiting factor PAF

Adhesion molecules

ICAM-1 E-selectin P-selectin

Others

Endothelin-1 von Willibrand factor

myosin light chain kinases and alter endothelial morphology (Figure 17).

Alveolar epithelial cells At the ultrastructural level the alveolar epithelial cell population is damaged during the development of ALI.132 Type I pneumocytes form a thin layer covering over 95% of the alveolar surface area and a tight barrier, which in health prevents the passive movement of fluid from the interstitium into the alveolar space or organisms into the tissues.143 Type II alveolar epithelial cells function as stem cells for the alveolar epithelium and also secrete a range of substances, including surfactant, that are important in maintaining alveolar integrity and function.144 A further important function in type II and probably type I cells is the critical maintenance of fluid balance in the lung and the prevention of fluid accumulation within the alveolar spaces.145 These cells possess active ion transport channels, which pump Naþ and Kþ ions from the lumina into the tissue. This pulls water out of the airspace into the interstitium.146,147 Aquaporins also allow movement of water across these cells independently of ionic pumping mechanisms.148 In ALI there is damage to the alveolar epithelium with morphological evidence of injury, necrosis and denudation of the basement membrane.132 The mechanisms of epithelial injury in ALI are believed to be the result of neutrophil activation and the release of injurious proteases, as well as reactive oxygen species. In sepsis, bacterial-derived products and toxins are also involved.91,140,148,149 In some instances, direct alveolar injury occurs due to mechanical factors in the lung, e.g. artificial ventilation150 or exposure to either systemic or inhaled toxic agents.151,152 There may be extensive alveolar epithelial apoptosis driven by Fas ligand binding.153 The significance of this is unclear but the effect may be to further reduce the number of epithelial cells and possibly delay epithelial healing. Experimental studies show that in the initial stages of ALI endothelial permeability increases, allowing fluid to move into the interstitium and, if there is associated epithelial injury,

into the alveolar spaces. In the short term this increase in fluid within the alveoli may be counteracted by an increase in Naþ/Kþ pumping, stimulated by increased concentrations of catecholamines.154,155 This requires functional epithelium156 but as the degree of epithelial injury increases, this capacity is lost and the alveoli are flooded with protein-rich edema fluid. Oxidative stress increases epithelial permeability and reduces the ability of the cells to pump fluid out of the alveoli.157 In addition, injury, particularly to the type II cells, results in changes in the lipid and protein content of the alveolus.158 Resultant surfactant is less effective at reducing surface tension. This promotes alveolar collapse and increases the work required for breathing.159

Coagulation system The net effect of the inflammatory cell milieu with endothelial and epithelial activation/injury is that protein-rich edema forms in the alveolar spaces. The normal alveolus is resistant to the formation of fibrin, due to a predominance of antithrombotic molecules, such as activated protein C, antithrombin and tissue factor pathway inhibitor.146 With lung inflammation the balance shifts with tissue factor expression by alveolar macrophages, activated neutrophils and alveolar epithelial cells160,161 and decreased concentrations of protein C.162 In addition, the fibrinolytic system appears to be inhibited, with increased levels of plasminogen activator inhibitor-1 detected in BAL fluid.163,164 The net effect is deposition of fibrin within the alveoli. Simultaneously, micro-thrombi develop within the capillary network of the lung. Activated components of coagulation cascades, especially thrombin and fibrin, further promote recruitment and activation of neutrophils and platelets promoting additional tissue damage. Disseminated intravascular coagulation and multi-organ failure may follow.

Fibroproliferative phase The histological hallmark of this phase is the migration of fibroblasts/myofibroblasts from the interstitium into alveolar spaces with subsequent deposition of extracellular matrix proteins and angiogenesis. Loose granulation tissue forms. Type II pneumocytes also proliferate. At the histological level, this process is not usually appreciable until 5–7 days from the onset of injury, but there is some evidence that it may start much earlier. BAL fluid obtained from patients 1 day after injury shows increased mitogenic activity for fibroblasts and epithelial cells in vitro and increased fluid levels of pro-collagen peptides.165 Macrophage-derived mediators including platelet-derived growth factor (PDGF),166 insulin-like growth factor-1 (IGF-1),167 basic fibroblast growth factor (bFGF)168 and TGFb199 are thought to play a central role in the fibroproliferative phase. The combined effect of these factors is to promote (a) proliferation of resident fibroblasts169 in the alveolar spaces, (b) development of a myoepithelial phenotype,170 (c) migration

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through the damaged basement membranes into the fibrinrich intra-alveolar coagulum, and (d) secretion of extracellular matrix proteins, such as fibronectin, tenascin and collagen III.163,171,172 Deposition of these molecules is associated with accumulations of mucopolysaccharides, including large quantities of hyaluronic acid. This substance gives rise to the loose myxoid appearance that characterizes these areas in the lung.173 Endothelial proliferation and angiogenesis in response to injury is less well understood, but IL-8 may play a role.174 Other studies suggest that some monocyte-derived CXC chemokines are pro-angiogenic, while others inhibit neovascularization.175 Similar patterns of pro-angiogenic CXC expression have been demonstrated in both ALI and idiopathic pulmonary fibrosis, suggesting this may be an important signaling mechanism. Levels of these cytokines correlate with procollagen levels, supporting the concept that neovascularization is a critical step in the development of granulation tissue and the laying down of new extracellular matrix. The role of VEGF is less clear. As discussed above, levels are increased in the exudative phase, where it is believed to play a role in increasing vascular permeability. Levels are lower by 7 days, when the proliferative phase is developing.176,177 Regulation of florid epithelial proliferation is complex. It involves signaling between epithelial cells in autocrine and paracrine fashions, as well as through direct interaction between these cells and elements in the extracellular matrix.107,178 One recognized effect of basement membrane re-epithelialization is the inhibition of fibroblast proliferation, a crucial step in lung healing.179,180 Pulmonary edema fluid obtained from patients with ALI promotes epithelial proliferation in vitro.178 This phenomenon is mediated, at least in part, by IL-1b. These factors bind to epidermal growth factor (EGF) and TGFa, associated with epidermal growth factor receptor (EGFR) and mitogen-activated protein (MAP) kinases.178,181 Keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) are additional mitogens.182–184 KGF is produced predominantly by fibroblasts and serum levels correlate with epithelial proliferation in animal models of acute lung injury.185 This molecule may also signal through the EGFR, suggesting the presence of a common pathway for stimulating epithelial proliferation.178

Organization and fibrosis versus resolution Regulation of the reparative processes remains poorly understood. In normal wound healing at non-pulmonary sites such as the skin, granulation tissue organizes into mature fibrous tissue. The extracellular matrix remodels and surface epithelium regenerates. The area of injury eventually heals and a scar develops.186 In many ARDS patients this occurs. Accretion of the organizing fibro-proliferative process into the adjacent alveolar walls by the proliferating type II epithelial cells leads to fibrotic lungs.140,187 In some ALI patients, the fibro-proliferative component may either in part or wholly resolve, leaving the lung

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architecture largely intact (Figure 18).91,140,187 The mechanisms regulating this process are uncertain but clarification could potentially reduce disease morbidity and mortality. This process stops the inflammatory stimulus, removes edema fluid along with fibro-proliferative granulation tissue from the alveolar spaces, re-epithelializes the alveoli, and repairs endothelial damage. Some authors have suggested that this process represents “reversal of fibrosis” in the lung, implying that scarred lung remodels back to healthy alveolated lung tissue.187,188 This terminology is misleading since the dissipation of fibroproliferative granulation tissue is not to be equated with dissolution of collagen type fibrosis.189 The time course for resolution is highly variable and some patients continue to show improvements in lung function for many months following ALI. This suggests remodeling of the immature fibroblastic tissue may take some time.190 The inflammatory process driving ALI must cease before resolution can occur.191 Removal of the initial triggering event is often responsible. For example, in patients with Gram-negative septicemia, treatment with antibiotics and the subsequent clearance of LPS results in decreased neutrophil and macrophage activation in the lung, increased neutrophil apoptosis and downregulation of macrophages.114,192 There is a possible further contribution from the effects of cytokines, such as IFNg, produced by T lymphocytes.193 Most of the pro-inflammatory agents discussed above have natural tissue inhibitors within lung tissue. The balance in concentration of these factors may shift from pro-injury and fibrosis to pro-resolution. Edema fluid is cleared by the epithelial membrane ion pumps discussed above.145–147 This clearance is reduced by epithelial loss during the exudative phase but subsequent proliferation of type II epithelial cells probably increases the capacity for fluid transfer.194 Increased lymphatic drainage may also play a role but confirmatory evidence is lacking. Removal of intra-alveolar granulation tissue requires removal of both cellular components, i.e. myofibroblasts and the endothelial cells, as well as the extracellular matrix proteins. The cellular component is probably removed by apoptosis.195 BAL fluid from patients with resolving ALI induces apoptosis in fibroblasts in vitro but the nature of the agent(s) responsible is unclear. The mechanism whereby matrix proteins are removed is also uncertain but is likely to involve digestion by matrix metalloproteases (MMPs).196 MMP-2 and MMP-9 are increased in the lungs of patients with ALI, as are their specific inhibitors. The relative balance and activity of these enzymes in relation to disease progression are unknown.196 Re-epithelialization of the damaged alveoli begins during the fibro-proliferative phase and if resolution occurs, the hyperplastic type II epithelial cells flatten out and differentiate into type I cells.197 This requires an intact basement membrane. The creation of an intact epithelial layer inhibits further fibroblast proliferation and in conjunction with endothelial

Chapter 9: Acute lung injury Figure 18. Pathogenesis of ALI: organizing phase and resolution. Resorption of alveolar edema fluid is shown. The neutrophil population clears by apoptosis with subsequent phagocytosis by alveolar macrophages. Type II alveolar epithelial cells proliferate and differentiate into type I cells. This restores the epithelial integrity of the alveolus. There is gradual resorption of the intraluminal fibroblastic material with apoptosis of myofibroblasts and removal of matrix by metalloproteinases. (Reproduced from Ware LB and Matthay MA, The acute respiratory distress syndrome. N Engl J Med 2000;342:1339 by kind permission of the Massachusetts Medical Society.)

recovery starts to recreate a normal tissue barrier between the circulation and the alveolar space. Little is known about endothelial repair while capillaries occluded by fibrin thrombi are remodeled and re-canalized by activation of the fibrinolytic system. The critical events that dictate whether the disease will resolve or progress to pulmonary fibrosis are poorly understood. At a simplistic level, persistence of the initiating insult and inflammatory damage inflicted on the lung are probably important considerations. Numerous studies indicate that markers of inflammatory damage correlate with clinical outcome.107,127,128 Unlike some tissues, such as liver and bone, there is no evidence the lung can reconstitute itself following extensive tissue destruction. If the elastic and collagen scaffolding of the lung remain intact, and if there is considerable alveolar basement membrane preservation, resolution should be possible.189 If the degree of tissue injury is such that the alveolar structure is lost, there is no possibility of resolution and healing. This leads to fibrosis replacing the lost tissue.

Management of ALI/ARDS The management of ALI/ARDS remains largely supportive, with concomitant treatment of the underlying cause where possible. The principles of mechanical ventilation are to recruit

alveoli and improve oxygenation without further damaging lung.198,199 Throughout this chapter the critical interaction between infection and ALI/ARDS has been apparent. Pneumonia and sepsis are major causes of ALI/ARDS, and ALI/ARDS is commonly complicated by hospital-acquired infection.200,201 Significant advances have been made in the management of sepsis with well-designed randomized controlled trials (RCTs).14,15 The Surviving Sepsis Campaign has consolidated a raft of evidence-based proposals in recent years.202 This is a constantly evolving area, exemplified by the ongoing controversy over the relative benefits and risks of activated protein C in sepsis.203,204 Furthermore, detailed guidelines exist for the management of both community- and hospital-acquired pneumonia.205–209 The ARDSnet study is a significant advance in patient management. This large RCT demonstrates a clear survival advantage for a ventilation strategy based on relatively low tidal volume and plateau pressure.210 This low tidal volume (“lung protective”) strategy is now standard practice in ALI/ ARDS. To date, pharmacological therapy for ALI/ARDS has proved disappointing.211 Given the key role of inflammation in ALI/ARDS, corticosteroids have received considerable attention.212–218 Interpretation of the available evidence remains controversial, with some authorities concluding that

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there is insufficient evidence for efficacy and others advocating administration of corticosteroids early in the course of ARDS.216–218 Leaving aside the controversies surrounding corticosteroids, pharmacological therapies have only had demonstrable effects in highly selected small groups of patients.219 The pathogenetic complexity of ALI/ARDS hinders the development of successful pharmacological therapies. The concept that targeting specific mediators can alter outcome may be flawed given the abundant mechanisms contributing to pathogenesis. In addition, the heterogeneous etiologies leading to ALI/ARDS raise the possibility that pathogenesis may be subtly different in each patient. Therefore, increasing attention is focusing on differences between ALI/ARDS arising from pulmonary and extra-pulmonary etiologies. While AECC criteria have been instrumental in driving improved understanding of ALI/ARDS, the underlying pathology may not be DAD in a significant minority of cases. Finally, clinical trials of pharmacological therapies for ALI/ARDS are at a relatively early stage of evolution, and defining the optimal dose, timing, duration and combination of therapies is likely to prove complex. Despite these obstacles, valuable information has been gleaned from well-designed RCTs testing hypotheses based on our current understanding of disease pathogenesis. Trials have studied the effectiveness of surfactant,220,221 the pleiotropic molecule NO,222 prostaglandins,223 and the antifungal ketoconazole.224 Smaller trials have specifically targeted antioxidants,225,226 key effectors of neutrophil-mediated damage,227 mechanisms controlling lung edema formation,228 and coagulation pathways.229 Advances in the understanding of pathogenesis are likely to suggest further innovative trials,

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for example assessing the use of statins, or regulating clearance of cells and fibrin from the alveolar space.230–232 RCTs have also suggested advances in management related to non-pharmacological interventions. Clear evidence has shown that central venous catheters are as effective and safer than pulmonary artery catheters in ALI/ARDS.233,234 A further trial suggested that a conservative fluid management protocol was at least as effective as a liberal protocol.235 Managing patients with ARDS in the prone position may improve oxygenation but this has not yet translated into improved survival.236–238 Finally, the optimal level of positive endexpiratory pressure (PEEP) required to optimize and maintain alveolar recruitment has been the focus of debate.239,240 A recent RCT has suggested that PEEP levels guided by esophageal pressure significantly improve oxygenation and compliance.241

Summary ALI represents a complex clinical problem with a poor outlook. While our understanding of ALI pathophysiology has grown, much remains unclear. ALI presents an interesting “model” of lung injury, remodeling and repair. While we have learnt much about the mechanisms of lung injury, we still know very little about the processes regulating remodeling and in particular factors favoring resolution rather than progression to fibrosis. An understanding of these may in time allow for the development of therapeutic options to treat other progressive fibrotic lung diseases, as well as improving the outlook for seriously ill patients in a wide range of clinical settings.

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10

Interstitial lung diseases Andrew G. Nicholson and Alexandra J. Rice

Introduction The diffuse parenchymal lung diseases (DPLD) comprise a large number of inflammatory and fibrosing pulmonary conditions, in which the pathological changes predominantly involve the alveolar parenchyma, alveolar spaces and, to a lesser degree, the peripheral airways. They can show acute, subacute or chronic presentations and can be subclassified into various groups (Figure 1). Of these, the idiopathic interstitial pneumonias are among the most problematic to diagnose for a number of reasons. The basic pathological features of the idiopathic interstitial pneumonias (IIPs) are inflammation and/or fibrosis of varying degrees and distribution. Few cases show typical specific histological features and one pattern may evolve into another over time, so there are inevitable overlaps. Furthermore, the lung parenchyma may be so scarred in advanced disease that the requisite histopathological features are only focal, or not present at all. The main role of the pathologist in diagnosis is to classify the histological patterns of disease. He/she must then integrate this information with the clinical and imaging data to provide a final clinicopathological diagnosis, typically through multidisciplinary team (MDT) reviews.1,2 Some pathologists prefer to integrate such MDT data in their report, thereby providing a final clinicopathological diagnosis, rather than a histological

DPLD of known cause e.g. drugs, CTD

Idiopathic

IPF

NSIP DIP RBILD AIP LIP OP

Granulomatous

pattern. A surgical lung biopsy (SLB) is generally required to provide suitable diagnostic material. However, in practical terms, where clinical and radiological features are typical of a specific interstitial pneumonia, pathological confirmation is not required for diagnosis and SLB is therefore only performed in a minority (10–20%) of cases. Patients that come to biopsy are generally those with atypical clinical findings and/or imaging at presentation, or those patients in whom there is unexpected longitudinal behavior. As histological patterns of disease may vary within and between lobes, most centers undertaking biopsies now take at least two samples from different sites, normally from different lobes. Biopsies can be from the same lobe but from areas showing different degrees of severity or different high resolution computed tomography (HRCT) appearances. This practice is based on papers showing so-called “discordance”, where different histological patterns may be present in different areas.3 Furthermore, it reduces the chance of sampling error and obtaining either only normal lung or only end-stage fibrotic lung. This is important as in cases with wholly end-stage fibrosis on biopsy, a diagnosis of a histological pattern of interstitial pneumonia cannot be made with confidence. Ideally, liaison between surgeons, physicians and radiologists preoperatively will reduce such occurrences in the future.

Other forms of DPLD e.g. HX, LAM

Figure 1. ATS/ERS classification of diffuse parenchymal lung disease. DPLD, diffuse pulmonary lung disease; CTD, connective tissue disease; HX, pulmonary Langerhans cell histiocytosis; LAM, lymphangioleiomyomatosis; IPF, idiopathic pulmonary fibrosis; NSIP, nonspecific interstitial pneumonia; DIP, desquamative interstitial pneumonia; RBILD, respiratory bronchiolitis interstitial lung disease; AIP, acute interstitial pneumonia; LIP, lymphoid interstitial pneumonia; OP, organizing pneumonia.

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 10: Interstitial lung diseases Table 1 ATS/ERS classification of histopathological patterns of interstitial pneumonias with clinicopathological counterparts in an idiopathic setting

Histological pattern

Clinicopathological diagnosis

Usual interstitial pneumonia (UIP)

Idiopathic pulmonary fibrosis (IPF) / cryptogenic fibrosing alveolitis (CFA)

Nonspecific interstitial pneumonia (NSIP)

Nonspecific interstitial pneumonia*

Organizing pneumonia (OP)

Cryptogenic organizing pneumonia

Diffuse alveolar damage (DAD)

Acute interstitial pneumonia

Desquamative interstitial pneumonia (DIP)

Desquamative interstitial pneumonia

Respiratory bronchiolitis

Respiratory bronchiolitis-interstitial lung disease (RB-ILD)

Lymphocytic interstitial pneumonia (LIP)

Lymphocytic interstitial pneumonia

* Provisional term for clinicopathologic diagnosis.

The use of ancillary histochemical stains for connective tissue, such as Elastin Van Gieson, are recommended for histopathological assessment, with additional ancillary stains (e.g., Perls’), as required. There is a limited role for transbronchial biopsy in the diagnosis of interstitial pneumonias, although it may be of value in excluding sarcoidosis. In addition this technique may aid in the distinction of specific differential considerations, such as respiratory bronchiolitis-associated interstitial lung disease (RB-ILD) versus hypersensitivity pneumonia (HP). Identification of foci of organizing pneumonia may be sufficient in some cases to provide a diagnosis of cryptogenic organizing pneumonia (COP), in the appropriate clinical setting. Some groups proposed a diagnosis of usual interstitial pneumonia (UIP)/idiopathic pulmonary fibrosis (IPF) can also be made in certain cases.4 This last suggestion is not universally accepted.5 In a small number of cases, further classification beyond “chronic fibrosing lung disease” is not possible. This may be due to sampling error or sample size, or the presence of a mixture of histological patterns. Correlation with clinical and radiological appearances may be helpful in further classifying such cases.

History of classification Liebow and Carrington initially classified the interstitial pneumonias into a number of different subtypes.6 Additional classifications incorporated new patterns and discounted others. Confusion followed until an international workshop, comprising specialist clinicians, radiologists and pathologists, published the American Thoracic Society/European Respiratory

Society (ATS/ERS) consensus classification of idiopathic interstitial pneumonias in 2002 (Table 1).7 This classification defines distinct clinicopathological entities on the basis of combined clinical, radiological and histological criteria. Pathologists are encouraged to diagnose histological “patterns”, rather than disease entities, as it was recognized the same pattern could be seen in a variety of conditions. The consensus classification also emphasizes the need for a multidisciplinary approach to the diagnosis of interstitial lung disease. Such an approach results in greater concordance,1 particularly within specialist centers.8 Interobserver studies amongst radiologists and pathologists demonstrate moderate to good reproducibility in relation to routine diagnostic application, with most disagreements centering on the diagnosis of nonspecific interstitial pneumonia (NSIP).9,10 In a small number of cases of chronic fibrosing lung disease it may be difficult to assign a specific histopathological diagnosis; in such instances the term unclassifiable interstitial fibrosis may be appropriate. Correlation with clinical and radiological appearances can be helpful in classifying such cases. The most important feature of this classification is that it provides robust prognostic data. The rate of clinical progression and response to treatment vary dramatically between the different IIPs. Stricter criteria in relation to both histological patterns and clinico-pathological entities allow more research into better-defined cohorts of patients. This has led to a greater understanding of pathogenesis in some of these hitherto idiopathic disorders. Minor criticisms of the ATS/ERS classification include the use of the term “interstitial” in diseases with intra-alveolar pathology (e.g., organizing pneumonia and diffuse alveolar damage (DAD)). Others note the inclusion of entities that are rarely idiopathic, such as respiratory bronchiolitis-interstitial lung disease (RB-ILD)/desquamative interstitial pneumonia (DIP) (which are largely smoking-related) and lymphoid interstitial pneumonia (LIP) (which is usually associated with an underlying connective tissue disorder or infection). Given that these disorders enter the differential diagnosis of UIP/IPF, they are included in the classification.

Epidemiology The idiopathic interstitial pneumonias occur worldwide and constitute up to 15% of a respiratory physician’s practice. UIP/ IPF is by far the commonest form, accounting for approximately 60% of cases, the remainder consisting of NSIP (14– 36%), DIP/RB-ILD (10–17%), COP (4–12%), acute interstitial pneumonia (AIP) (< 2%) and LIP (1%).7 Although smoking and underlying connective tissue disease (CTD)/infection play key roles in the etiology of RB-ILD/DIP and LIP respectively, the cause of the remaining patterns of IIP is not clear. They are probably triggered by a variety of environmental agents and modified by an underlying genetic susceptibility. The nature of the trigger remains unknown, but the role of genetics in the development of these diseases is beginning to be elucidated. The term “cryptogenic fibrosing alveolitis” is no longer

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recommended for patients with a pattern of usual interstitial pneumonia; “idiopathic pulmonary fibrosis” is preferred. However, “cryptogenic fibrosing alveolitis” continues to be used from an epidemiological perspective to encompass patients presenting with unexplained interstitial pneumonias.11

Genetics Familial and twin studies have suggested an underlying genetic component to the development of at least some interstitial pneumonias. Estimates of numbers of familial cases vary but it is thought to be in the range of 5%, though some authors suggest the figure may be higher, possibly up to 20%.12 The commonest pattern of interstitial pneumonia is UIP, but NSIP, organizing pneumonia and HP have also been reported. Up to 20% of asymptomatic patients in familial IP kindreds may show abnormal pulmonary function tests (PFTs) and subtle changes of IP on HRCT scanning13 along with histological features of IP on biopsy.14 Interestingly, 45% of families showed phenotypic heterogeneity with a variety of histological subtypes present in the same family,13 and similar heterogeneity was seen in a study of asymptomatic relatives.14 Cases of NSIP have been associated with surfactant protein C mutations in infants (see Chapter 3).15 The inheritance pattern is most often autosomal dominant with variable penetrance. Gene expression profiling has shown familial clustering of genes involved in chemokine production, extracellular matrix and growth regulation.16 Candidate gene studies suggest pulmonary fibrosis is associated with polymorphisms and/ or increased expression of a variety of growth factors involved in fibrogenesis. These include transforming growth factor B1 (TGFb1)17 and platelet-derived growth factor (PDGF),18 extracellular matrix proteins such as fibronectin19 and other molecules, including alpha-1-antitrypsin.20 Telomerase and telomere length have also been implicated in the development of both familial and sporadic IP.21 The prognosis of familial IP is reported to be the same as for sporadic IP. Outside the setting of familial IPF, there are some data on genetic susceptibility. In a small number of non-familial adult cases of fibrosing interstitial lung disease, mutations of surfactant protein C have been found. Accumulation of surfactant protein C within alveolar epithelial cells interferes with protein folding and results in epithelial dysfunction and apoptosis.22,23 Microarray analysis of human tissue from patients with IPF identified increased expression of genes associated with smooth muscle differentiation and contractility,24 extracellular matrix proteins and metalloproteases,25 and some specific proteins including osteopontin.26 Osteopontin is a protein required for development of bleomycin-induced lung fibrosis in rat models. There is also some evidence from microarray analysis that different histological patterns of interstitial pneumonia are associated with increased expression of different groups of genes.27,28 Analysis of tissue from patients with HP shows a predominance of genes involved in regulation of the inflammatory response and T cell activation. In contrast, in

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patients with IPF and a histological pattern of UIP, genes involved in remodeling of the extracellular matrix and myofibroblast differentiation and contraction are more often upregulated. Cases of NSIP studied to date show a mixture of gene expression profiles, some resembling HP, others UIP and various cases having a unique pattern.

Idiopathic pulmonary fibrosis and usual interstitial pneumonia Idiopathic pulmonary fibrosis (previously termed cryptogenic fibrosing alveolitis (see above)) is the clinical term for a relatively uncommon fibrosing lung disease, with an estimated prevalence of 7–20/100 000.29 It is found worldwide and shows no ethnic predilection. It is twice as common in men, and most patients are over the age of 50.

Etiology The etiology of IPF is unknown, but genetic and environmental factors are suspected. Familial IPF is rare, accounting for no more than 2% of cases. It is associated with a variety of gene mutations and polymorphisms (discussed below), and has recently been described in association with bone marrow hypoplasia and hepatic nodular regeneration.30 Reported environmental risk factors include smoking, gastroesophageal reflux,31 drugs,32 and occupational exposures.33 Several occupational and environmental factors, adjusted for age and smoking in conditional multivariate logistic regression analyses, are significantly associated with IPF. These include farming (odds ratio (OR) ¼ 1.6, 95% confidence interval (CI): 1.0, 2.5); livestock (OR ¼ 2.7, 95% CI: 1.3, 5.5); hairdressing (OR ¼ 4.4, 95% CI: 1.2, 16.3); metal dust (OR ¼ 2.0, 95% CI: 1.0, 4.0); raising birds (OR ¼ 4.7, 95% CI: 1.6, 14.1); stone cutting/polishing (OR ¼ 3.9, 95% CI: 1.2, 12.7); and vegetable dust/animal dust (OR ¼ 4.7, 95% CI: 2.1, 10.4). An interaction between smoking and exposure to livestock (P ¼ 0.06) and farming (P ¼ 0.08) is also noted.33 Viral infections, such as hepatitis C,34 adenovirus35 and Epstein-Barr virus (EBV), have also been implicated in the pathogenesis of this disease.7 UIP is the most common of the histological patterns of IIP and is the typical histopathological correlate of the clinical entity of IPF. However, it should be noted that a UIP pattern may be seen in a minority of patients with other diseases including chronic HP,36 adverse drug reactions,37 underlying CTD,38,39 asbestosis40 and Hermansky-Pudlak syndrome (Table 2).41

Clinical presentation Patients typically present with gradual onset of symptoms for greater than 6 months. These include dyspnea, cough and weight loss but systemic symptoms are rare. Clubbing may be seen in up to 50% of patients and inspiratory basal crackles are heard on chest auscultation. Pulmonary hypertension is

Chapter 10: Interstitial lung diseases Table 2 Conditions associated with UIP pattern

Idiopathic pulmonary fibrosis / cryptogenic fibrosing alveolitis Chronic hypersensitivity pneumonitis Connective tissue disease Chronic infection Chronic aspiration Drug toxicity Asbestosis Hermansky-Pudlak syndrome Familial IPF

reported in 32–85% of patients, the variation probably reflecting local diagnostic protocols and criteria. Patients may have an elevated erythrocyte sedimentation rate (ESR) and up to a third show serological abnormalities with low levels of antinuclear antibodies and rheumatoid factor.42,43 However, the titers of these antibodies are much lower than those seen in patients with pulmonary fibrosis associated with CTD. Pulmonary function tests most commonly show restrictive abnormalities with a reduction of both total lung capacity and vital capacity. There is also impaired gas exchange with a reduced DLCO. Any obstructive airflow limitation usually reflects concomitant chronic obstructive pulmonary disease (COPD) or asthma, and background emphysema may alter the pattern of PFT abnormalities seen with relative preservation of lung volumes.44–46 Bronchoalveolar lavage (BAL) typically shows increased numbers of neutrophils with a lesser increase of eosinophils. While BAL plays a limited role in the diagnosis of IPF, the levels of both eosinophils and neutrophils correlate with a poorer prognosis in patients with UIP pattern of fibrosis.47 Some data suggest that neutrophil levels reflect disease severity, and eosinophil levels predict future disease progression (see below).48 Lymphocytosis is not a typical finding and if present, other causes of pulmonary fibrosis, such as chronic HP, should be considered.49

Radiological findings Chest X-ray shows small lung fields with irregular reticulonodular or nodular shadows at the periphery of the lung fields and lung bases. Reduced lung volumes, honeycomb change and features of pulmonary hypertension may be seen in advanced disease. High-resolution computed tomography (HRCT) features include bilateral peripheral, subpleural and basal coarse reticular opacities with traction bronchiectasis and honeycomb change (Figure 2). In some cases a “ground-glass” pattern may also be seen. This finding corresponds to an active alveolitis, and predicts some response to steroids.50 When typical, HRCT appearances are up to 90% specific for UIP pattern on biopsy. Up to 50% of patients with IPF show classical HRCT features. Thus, in most instances, a diagnosis

Figure 2. HRCT scan of IPF showing predominantly peripheral and basal reticular change with traction bronchiectasis and honeycomb change. (Image courtesy of Professor D. Hansell, London, UK.)

of IPF can be made without recourse to a surgical lung biopsy. However, in a number of cases UIP can mimic a variety of other conditions on HRCT including NSIP, chronic HP and sarcoid (see Chapters 12 and 13).51

Pathology The UIP pattern is characterized macroscopically and microscopically by mainly peripheral lower lobe interstitial fibrosis. In the late stage of disease, the lungs appear shrunken with a cobblestone pleural surface. The cut surface shows varying degrees of subpleural fibrosis and honeycomb change, adjacent to areas of relatively normal lung (Figure 3). The hallmark of UIP on histology is patchy established interstitial fibrosis with typically quite sharp demarcation between abnormal areas and normal/nearly normal lung, together with the presence of so-called “fibroblastic foci”. The presence of this variation between dense established fibrosis and younger fibroblastic tissue is termed “temporal heterogeneity” (Figures 4 and 5). The distribution of fibrosis is predominantly subpleural and paraseptal, although this can be difficult to appreciate on small surgical lung biopsies. The fibroblastic foci comprise loose fibroblastic tissue apposed to the areas of established fibrosis, the latter characterized by dense hypocellular collagen deposition. These “fibroblastic foci” are thought to represent the areas of recent lung injury and are often overlain by atypical reactive alveolar epithelium (Figure 6). They have a loose myxoid stroma containing active fibroblasts and myofibroblasts but only occasional chronic inflammatory cells. The number of fibroblastic foci varies considerably from case to case. Most studies show that

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Chapter 10: Interstitial lung diseases Figure 3. Lungs with IPF showing peripheral and basal honeycomb change.

Figure 5. Subpleural fibrosis with microscopic honeycomb change.

Figure 4. UIP pattern showing patchy fibrosis with a subpleural and paraseptal distribution and intervening areas of preserved alveolar parenchyma. There is little associated chronic inflammation.

increased numbers of fibroblastic foci are associated with an increased mortality and rate of disease progression (Figure 7).52–54 There is usually a minor degree of chronic inflammation, that is limited to areas of established fibrosis and does not extend into areas of normal lung. Neutrophils may accumulate in areas of mucostasis, associated with honeycomb change (Figure 8a), but interstitial neutrophils and eosinophils are usually inconspicuous. Over time, there is increased loss of alveolar architecture due to remodeling of the alveolar interstitium, with eventual end-stage fibrosis, traction bronchiectasis and macro- as well as microscopic

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honeycomb change (Figure 5). The latter is characterized by enlarged airspaces showing bronchiolization and varying degrees of goblet cell metaplasia. Minor histological features seen in UIP include smooth muscle hypertrophy (Figure 8b), endarteritis obliterans, mild chronic pleuritis and pleural fibrosis, osseous and squamous metaplasia (Figure 8c) and type II pneumocyte hyperplasia. Desquamative interstitial pneumonia-like areas and eosinophil-rich areas have also been described in some cases, although these have no prognostic significance.55 The histological features of UIP are summarized in Table 3.

Differential diagnosis When the full constellation of histological features is present, then a diagnosis of UIP can be made with confidence. There are areas of diagnostic difficulty. The commonest is the distinction between UIP and fibrotic NSIP. Patchy disease

Chapter 10: Interstitial lung diseases

Figure 7. IPF with UIP pattern showing prominent activity characterized by numerous fibroblastic foci (arrows).

Figure 6. Fibroblastic focus comprising loose fibromyxoid connective tissue apposed to an area of established collagen fibrosis. Note reactive and degenerative change of the overlying alveolar epithelium, consistent with alveolar epithelial injury.

distribution, fibroblastic foci and honeycomb change are typical of UIP. Rare fibroblastic foci may be seen in other lung disorders but in shows UIP they are seen in association with established fibrosis and architectural distortion. A minor degree of microscopic honeycomb change is sometimes seen in fibrotic NSIP, but the presence of radiological, macroscopic or extensive microscopic honeycomb change again favors UIP. In patients who have had multiple biopsies, there are occasions when one biopsy shows fibrotic NSIP and the other biopsy shows UIP. In the correct clinical context of IPF, such discordant cases should be viewed overall as showing a histological pattern of UIP.3,56 Some patients show discordance between radiological and pathological diagnoses of UIP and fibrotic NSIP.57,58 Again, it is the presence of a UIP pattern, either radiologically or histologically, which predicts a poorer prognosis and dictates the final classification. Usual interstitial pneumonia-like areas have been described in some patients with chronic HP36,59 and underlying CTD (particularly rheumatoid arthritis) (see Chapters 12 and 21).38 Chronic HP exhibits a more bronchocentric pattern of fibrosis and shows prominent chronic inflammation in the interstitium (Figure 9a,b). Granulomas, if seen, are helpful in suggesting chronic HP. Occasionally rare, incidental microgranulomas may be found in lungs removed for a variety of conditions and alone are not specific for HP. Some cases of chronic HP also show discordant histology, with a UIP pattern in one lobe and an HP pattern in another, so examination of multiple biopsies is often helpful.59 Patients with chronic HP, who show either a fibrotic NSIP or UIP pattern on biopsy, have a poorer prognosis than

those with a cellular/inflammatory pattern.36,59–62 The studies are too small to determine whether there is any significant difference between fibrotic NSIP and UIP within this group of patients, or as compared to idiopathic cases of NSIP and UIP. Patients with an underlying CTD often show more chronic inflammation and fewer fibroblastic foci than usually seen in IPF.53 In addition biopsies from patients with an underlying CTD more often show a mixture of histological patterns, as well as involvement of other anatomic lung compartments, such as co-existent vasculitis or pleural thickening.38,39 It is important to recognize cases of UIP associated with CTD, as some studies suggest they have a better prognosis than those associated with IPF.53,63 This is not a universal finding and may depend on the type of underlying CTD.64,65 In rare cases, the underlying features of UIP may be masked by other superimposed histological patterns of lung disease, such as DIP-like areas with abundant intra-alveolar macrophages, or areas of DAD in the context of acute exacerbations (see below). In these instances careful attention to the background pattern of interstitial fibrosis confirms the underlying pattern of UIP, with patchy subpleural fibrosis, honeycomb change and fibroblastic foci. Occasionally organizing pneumonia may progress to interstitial fibrosis,66 and foci of organizing pneumonia can be indistinguishable from the fibroblastic foci seen in UIP as they become incorporated into the interstitium. More typical areas of organizing pneumonia may be seen elsewhere in the biopsy, but clinical correlation to ensure the features do not represent an acute exacerbation is additionally required. Ultimately, in these difficult cases, knowledge of the longitudinal behavior proves to be the best discriminant between idiopathic pulmonary fibrosis and a fibrosing organizing pneumonia. Asbestosis can show a UIP pattern of fibrosis, but asbestos bodies are almost always readily identified.40 If there are markedly increased numbers of eosinophils or foci of

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(a)

(b)

(c)

Figure 8. Secondary features seen in UIP pattern. (a) Honeycomb change with mucostasis and neutrophil accumulation. (b) Myoid metaplasia. (c) Squamous metaplasia.

Table 3 Histological features of UIP

Key features Dense fibrosis causing remodeling of lung architecture with frequent “honeycomb” fibrosis Fibroblastic foci typically scattered at the edges of dense scars Patchy lung involvement Subpleural and paraseptal distribution Pertinent negative findings Lack of active lesions of other interstitial diseases (e.g., sarcoidosis or Langerhans cell histiocytosis) Lack of marked interstitial chronic inflammation Inconspicuous or absent granulomas Lack of substantial inorganic dust deposits Lack of marked eosinophilia 7

Source: modified from .

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eosinophilic pneumonia, then a drug reaction should be considered. Drugs associated with a UIP pattern include nitrofurantoin, cyclophosphamide and chorambucil (see Chapter 16).67 Patients with IPF are at increased risk of developing carcinoma of all types, particularly squamous cell carcinoma and adenocarcinoma.68–72 In some cases of UIP it may be difficult to distinguish florid reactive type II pneumocyte hyperplasia from well-differentiated adenocarcinoma. It is important to assess these areas for the presence of stromal invasion, complex papillary or micropapillary architecture, proliferations of mucinous cells and an absence of underlying inflammation, which all point towards malignancy.

Pathogenesis The cause of IPF is not known but is thought to involve a combination of genetic and environmental factors. The temporal heterogeneity seen in UIP/IPF suggests the disease

Chapter 10: Interstitial lung diseases

(a)

(b)

(c)

(d)

Figure 9. Chronic hypersensitivity pneumonitis with a UIP-like pattern. (a) Subpleural fibrosis. (b) Rare fibroblastic focus present with chronic inflammation. (c) Bronchocentric pattern of fibrosis elsewhere in same biopsy. (d) Microgranuloma with cholesterol clefts.

progresses as a result of multiple episodes of lung injury.52,73 The stepwise decline in lung function in these patients and their acute exacerbations likewise support a multistep process. It is currently hypothesized that following an initiating insult to the lung, the disease progresses by repeated episodes of microscopic alveolar epithelial injury followed by abnormal tissue repair, resulting in eventual fibrosis and irreversible remodeling of lung architecture. The nature of the initiating trigger is unknown, but it is likely to be multifactorial and, as described above, viral infection, drug-induced injury and aspiration injury have all been implicated. Though inflammation is not a prominent feature in UIP/ IPF, the immune system probably plays a role. Antibodies against alveolar epithelial cells and immune complex deposition within pulmonary capillaries are possible initiators.74,75 There is an imbalance in the T helper lymphocyte subsets and cytokine milieu, both in animal models and in patients with

IPF,76 with an increase in Th2 cells and cytokines, including interleukin-4, 5, 10 and 1377 leading to a pro-fibrotic environment. Neutrophils and mast cells may contribute to local injury and fibrosis, through release of oxidants, proteases and fibrogenic cytokines. The degree of BAL neutrophilia has been shown to predict poorer prognosis.78 However, inflammation is not a prominent histological feature in most cases of IPF/UIP and anti-inflammatory therapies, including modulators of the Th1 vs. Th2 response, are relatively ineffective. This disappointing finding has led to a recent shift from an inflammatory model to one of abnormal tissue repair, in which inflammation is secondary to repeated episodes of epithelial injury, resulting in failure of re-epithelialization, and abnormal tissue repair. Thus, the fibroblast/myofibroblast regulators of extracellular matrix remodeling and fibrotic mediators assume a central role in the pathogenesis of this disease.79

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The integral role of abnormal tissue repair is supported by studies of the fibroblastic focus, the presumed site of damage. The number of fibroblastic foci present in a biopsy is predictive of the rate of disease progression.52,54,80 Fibroblasts in these areas demonstrate an activated phenotype with expression of extracellular matrix proteins and cytokines.81 Damage to the alveolar epithelial cells exposes the underlying basement membrane to oxidative damage and degradation. Cell and matrix injury results in macrophage activation, release of a variety of cytokines and growth factors, including TGFb, TNFa, PDGF and IL10. These promote fibroblast recruitment and activation as well as tissue remodeling.82,83 Gene microarray analysis of both familial and sporadic cases of IPF confirms upregulation of these genes.16,24–28 Fibroblasts may also be recruited from circulating stem cells and can arise from epithelial cells in a process known as epithelial-mesenchymal transition (EMT).84,85 Immunohistochemistry has shown evidence of EMT in tissue sections from cases of UIP.86 Data from several studies suggest that gene polymorphisms of some cytokines, including TGFb1, IL-1, TNFa, a-1-antitrypsin and type-2 cytokines are associated with the development and/or progression of IPF.20,87–90 Particular attention has been focused on TGFb1, a profibrotic cytokine, which shows increased expression in fibroblastic foci and surrounding macrophages in IPF lungs.91 Polymorphisms of codon 25 of the TGFb1 gene have been associated with allograft fibrosis following lung transplantation for a variety of diseases.17,92,93 Recently an association has been demonstrated between polymorphisms of codon 10 and the rate of deterioration of gas exchange in patients with IPF.90,94 Microsatellite instability of the TGFb1 receptor gene is reported in alveolar epithelium, suggesting an important role for this signaling pathway in IPF.95 There is also evidence that abnormal apoptosis is important, with increased epithelial apoptosis preventing effective reepithelialization and decreased myofibroblast apoptosis, resulting in persistence of a fibrogenic environment.96 As well as increased epithelial apoptosis, the presence of short telomeres and telomerase mutations in some familial and sporadic cases of IPF suggests tissue renewal/regeneration in this disease is limited.21,97,98 Abnormalities of coagulation and vascular remodeling have also been demonstrated. Local procoagulant activity is increased in IPF,99,100 with tissue factor and fibrin deposition noted at fibroblastic foci.101 Tissue factor plays a key role in initiation of local coagulation but also activates proteinase-activated receptors, specifically PAR-1. This receptor plays an important role in tissue injury and repair and increased expression levels are reported in the lungs of patients with UIP/IPF.102,103 A high level of interconnection of fibroblastic foci has recently been demonstrated on 3-D tissue reconstruction. The intriguing hypothesis is proposed that these foci represent part of a continuous network of proliferative fibroblastic tissue extending from the pleura into the lung, possibly in reaction to local environmental stimuli (Figure 10).104 The preferential

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basal and subpleural distribution of disease is as yet unexplained.

Prognosis and natural history There is currently no optimal treatment for IPF, but as a firstline treatment most patients receive steroid therapy supplemented with immunosuppressive agents, such as cyclophosphamide or azathioprine. Newer drugs, of uncertain benefit, include antioxidants such as N-acetyl cysteine,105 and antifibrotic agents, such as bosentan,106 etanercept107 and imatinib.108 Single lung transplantation is performed in some patients with a reported 5-year survival rate of 44%.109 Despite treatment, the overall prognosis for patients with IPF is poor, with a median survival of 2–4 years following diagnosis and a 5-year survival rate between 20 and 40%.3,42,110–121 Most patients show progressive deterioration of lung function, with death commonly due to respiratory failure, infection, pulmonary embolism and/or cor-pulmonale. There is also a 14-fold increase in the risk of lung cancer of all types in patients with IPF (see above),122 more commonly arising in the periphery and in the lower lobes, where fibrosis is most marked. Many are resectable but the operative mortality is higher than usual.123 A percentage of patients show one or more episodes of severe acute deterioration, characterized pathologically by features of DAD superimposed on fibrosis of UIP type.124 These episodes are termed acute exacerbation of IPF (AE-IPF) and are frequently fatal (see below). Clinical variables associated with poorer prognosis include age > 50 years at diagnosis, male gender, symptomatic periods of more than 1 year, severe symptoms, reduced pulmonary function and poor response to steroids. Smoking does not appear to have a protective effect.125 Longitudinal changes in lung function are also prognostic and a composite physiological index has been proposed.126 Baseline BAL neutrophilia is an independent predictor of early mortality.78 In terms of histopathological features, most but not all studies have shown that increased numbers of fibroblastic foci correlate significantly with disease progression and mortality.52–54 This fits well with the increasingly held view that the main pathogenic mechanism in IPF is one of epithelial injury and dysfunctional repair. There is some evidence that molecular phenotypes and circulating biomarkers of disease, such as numbers of circulating fibrocytes and levels of alpha-defensins, may provide prognostic information.127–130

Nonspecific interstitial pneumonia The term NSIP was originally coined for a pattern of parenchymal inflammation seen in relation to HIV infection,131–133 but Katzenstein and Fiorelli134 first suggested NSIP was a distinct pattern of interstitial pneumonia, separate from UIP/ IPF. They described three subtypes of NSIP: cellular, fibrotic and mixed cellular-fibrotic, depending on the degree of interstitial lymphoplasmacytic infiltrate and fibrosis. Although accepted as a histological pattern, it was only designated a

Chapter 10: Interstitial lung diseases Figure 10. Computer-generated three-dimensional reconstructions of fibroblastic foci in UIP. (a) The pleural surface is yellow and blood vessels are red. (b). Fibroblast foci are added in green. (Reproduced with permission from Cool CD, Groshong SD, Rai PR, Henson PM, Stewart JS and Brown KK. Fibroblast foci are not discrete sites of lung injury or repair: the fibroblast reticulum. Am J Respir Crit Care Med 2006;15:654–8, Official Journal of the American Thoracic Society. Copyright © American Thoracic Society.)

provisional entity in the ATS/ERS classification of 2002. This was because it was recognized that as well as showing a spectrum of histological features from cellular to fibrotic types, it was a pattern not uncommonly seen in other interstitial lung diseases, and that idiopathic cases were poorly characterized. Despite these uncertainties, it was clinically useful to recognize this pattern as it was found to confer a better prognosis, compared to patients with UIP/IPF.56,111,114–116 An NSIP pattern is reported in a number of non-idiopathic forms of ILD, including chronic HP, connective tissue and autoimmune diseases, resolving DAD and drug reactions (Table 4). The finding of a histological pattern of NSIP should trigger thorough clinical investigations for such associations. Until recently little was understood about true idiopathic cases, but since 2002 data from an international workshop on NSIP have shown that, although comparatively rare, idiopathic cases do exist as a distinct clinical entity with characteristic clinical, imaging and pathological features. With careful definition, an NSIP pattern accounts for between 14 and 35% of cases of IIP in the literature.135

Clinical presentation There is a female predominance, and patients generally present at a slightly younger age than those with UIP/IPF, the average being 52 years.135 Symptoms are, however, similar and include chronic dyspnea, chronic cough and fever. Auscultation may reveal basal crackles and about 10% of patients show clubbing. Pulmonary function tests usually show a restrictive pattern with reduced gas transfer. Bronchoalveolar lavage may feature a lymphocytosis. Serological abnormalities are reported with positive rheumatoid factor and anti-nuclear antibodies identified in one study in 43% and 23% of patients, respectively.135

Radiological findings High-resolution computed tomography features include groundglass opacification and a fine reticular pattern (Figure 11).

Figure 11. HRCT scan of NSIP showing extensive ground-glass opacification. (Image courtesy of Professor D. Hansell, London, UK.)

A recent report of the ATS NSIP project found predominant involvement of the lower lobes, with about 50% showing a peripheral distribution and 50% showing diffuse changes.135 In addition traction bronchiectasis and volume loss may also be seen.135

Pathology Macroscopic examination of lungs with NSIP shows a pattern of fine fibrosis, without the extensive honeycomb change seen in IPF. There may be some degree of traction bronchiectasis, as seen on radiology. Histologically NSIP is characterized by a temporally and spatially uniform pattern of interstitial lung disease with

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(a)

(b)

Figure 12. Mixed cellular and fibrotic NSIP. (a) Diffuse uniform alveolar wall expansion by a mild chronic inflammatory cell infiltrate and loose connective tissue is noted. No fibroblastic foci are present. (b). An elastic von Gieson stain highlights the fibrosis.

(a)

(b)

Figure 13. NSIP. (a) The cellular pattern features a predominantly interstitial chronic inflammatory cell infiltrate. (b) The fibrotic pattern has diffuse alveolar expansion by hyalinized fibroconnective tissue and small numbers of chronic inflammatory cells.

Table 4 Conditions commonly associated with NSIP pattern

Idiopathic NSIP Connective tissue disease Drugs Hypersensivity pneumonitis HIV Previous acute lung injury Chronic infection Chronic aspiration

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expansion of the alveolar walls by varying degrees of chronic inflammation and interstitial fibrosis (Figure 12). The lung architecture is relatively preserved. There is no evidence of either bronchocentric or subpleural/paraseptal distribution. Katzenstein and Fiorelli initially classified the disease into three subtypes, cellular, fibrotic and mixed cellular-fibrotic.134 Subsequent studies showed a much better prognosis for the cellular group, compared to the fibrotic and mixed cellularfibrotic groups, which showed similar survivals.114,115 The ATS/ERS consensus classification therefore collapses this three-tier classification into two groups, namely cellular and fibrotic NSIP.7 Cellular NSIP (cNSIP) is characterized by a mild-moderate diffuse lymphoplasmacytic infiltrate within the alveolar walls (Figure 13a), though there may be some variation in intensity

Chapter 10: Interstitial lung diseases Table 5 Histological features of NSIP

Key features Cellular pattern Preservation of parenchymal architecture Mild to moderate interstitial chronic inflammation Type II pneumocyte hyperplasia in areas of inflammation Fibrosing pattern Variable degree of dense or loose interstitial fibrosis Interstitial inflammation mild or moderate Temporal uniformity of affected areas Minor features Mild follicular hyperplasia, occasional foci of organizing pneumonia, mild chronic pleuritis, focal alveolar fibrin, mild alveolar macrophage accumulation Pertinent negative findings Cellular pattern Absent dense interstitial fibrosis Not prominent organizing pneumonia Lack of diffuse severe alveolar septal inflammation Fibrosing pattern Absent temporal heterogeneity pattern Absent or inconspicuous fibroblastic foci with dense fibrosis (especially in cases of NSIP with patchy involvement) Both patterns Absent acute lung injury pattern, especially hyaline membranes Inconspicuous or absent eosinophils Lack of granulomas, inorganic dusts, Langerhans cells Lack of viral inclusions and organisms on special stains for organisms Source: modified from7.

of the infiltrate. Rare neutrophils, eosinophils and histiocytes may be seen. The lymphoid population comprises mainly CD3-positive T lymphocytes, with CD20-positive B lymphocytes confined to small reactive lymphoid aggregates. The degree of infiltration is less than in LIP, although it is recognized that there may be overlap. Fibrotic NSIP (fNSIP) features mild diffuse expansion of the alveolar walls by fibrous tissue, which may be fibroblastic or collagenous in nature (Figure 13b). It is temporally and spatially uniform across affected areas of lung, although the distribution can be patchy. There may be occasional buds of organizing pneumonia but true fibroblastic foci are either absent or very infrequent. There is less smooth muscle metaplasia and especially less honeycomb change, compared to UIP. Smokers may show co-existent respiratory bronchiolitis and emphysema.136 Both types of NSIP can show reactive type II pneumocyte hyperplasia and foci of organizing pneumonia, but hyaline membranes and other features of acute lung injury are absent. Recently proposed refinements to the histological definition of NSIP pattern by the NSIP working group include an emphasis on the preservation of lung architecture with inconspicuous or absent honeycombing, absence of granulomas and absence of dominant airway disease, such as extensive

peribronchial metaplasia. Cases of NSIP with organizing pneumonia involving up to 20% of the biopsy were included in their series, although they accepted this was a somewhat arbitrary figure.135 The histological features of NSIP are summarized in Table 5.

Differential diagnosis As stated earlier, NSIP is viewed as a histological pattern of interstitial pneumonia with a number of clinical correlates including CTD, environmental allergens, drug reaction, immunodeficiency states (including HIV), prior acute lung injury, as well as an idiopathic form. Therefore clinicopathological correlation is essential to reach the correct final diagnosis. Since NSIP is a diagnosis of exclusion, this pattern is the source of much of the interobserver variation between pathologists. Discerning fibrotic NSIP from UIP, especially in the setting of emphysema, is quite difficult.10 In this context, a patchy subpleural and paraseptal distribution of fibrosis, honeycomb change and more than an occasional fibroblastic focus favor UIP, although these features may be sparse, especially on small single biopsies. If there is discordance between

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multiple biopsies, with fibrotic NSIP at one site and UIP at another, the biopsy should be classified as UIP for management purposes.3,56 It is uncertain in such cases whether the areas showing fibrotic NSIP represent an early phase of the fibrosing process or simply an area lacking disease activity. Though correlation with HRCT findings may be helpful, concurrent emphysematous change can also make the radiological distinction between NSIP and UIP difficult.137 An NSIP pattern, both cellular and fibrotic, can be seen as the major histological pattern in some cases of HP.36,58 Useful pointers suggesting HP are the presence of occasional illdefined granulomas and bronchocentricity of the inflammation/fibrosis. In chronic cases without recent antigenic stimulation, granulomas may be absent and a final diagnosis of HP may only be rendered after clinical review of the imaging, lavage findings and/or clinical data. Differences between NSIP, UIP and HP on gene microarray profiles have been demonstrated. 28 These laborious and costly tests may soon play a clinical role but the mainstay of diagnosis is likely to remain a multidisciplinary team approach.

Figure 14. Mixed cellular and fibrotic NSIP with follicular bronchiolitis raising the possibility of an underlying connective tissue disease.

Connective tissue diseases are associated with several patterns of interstitial pneumonia and series have shown that an NSIP pattern is common in rheumatoid arthritis, scleroderma, dermatomyositis/polymyositis, mixed connective tissue disease, Sjögren syndrome and systemic lupus erythematosus.39 Involvement of several compartments within the biopsy with a mixture of histological patterns is suggestive of an underlying connective tissue disease (e.g. follicular bronchiolitis with NSIP) (Figure 14). The histological patterns of interstitial lung disease in CTD are briefly summarized in Table 6 and are discussed in detail in Chapter 21. An NSIP-like pattern may be present as a background minor component of more easily identifiable histological patterns, such as organizing pneumonia (OP), or at one end of the spectrum of histologically similar disorders, such as LIP. In OP, the distinction of less than 10%134 or 20%135 of the biopsy showing intra-alveolar organization represent subjective cut-offs. Correlation with the HRCT data may help in deciding whether OP or cellular NSIP predominates. Some cases of organizing pneumonia may also progress to a pattern of fibrotic NSIP rather than show resolution. This is both in the context of chronic disease and also in relation to late stages of DAD. In this last instance, a clinical history of acute lung injury may aid in this distinction. In LIP the lymphoplasmacytic infiltrate is generally significantly denser. Furthermore LIP may also show cystic changes on HRCT and histology, which is not a feature of NSIP. Most patients with a histological pattern of DIP show a mild degree of diffuse, so-called “NSIP-like”, interstitial fibrosis. It is a subjective distinction as to whether the fibrosis or the accumulation of intra-alveolar macrophages represents the predominant feature. In these instances, the presence of lymphoid aggregates favors a diagnosis of DIP. In the clinical setting of a positive smoking history with typical HRCT findings, an overall classification as “smoking-related interstitial lung disease” may be appropriate.138,139 Finally, the presence of significant numbers of interstitial eosinophils raises the possibility of a drug reaction.

Table 6 Patterns of ILD in connective tissue disease

CTD

UIP

NSIP

DAD

OP

LIP

Other

RA

þþþ

þþ

þ

þþ

þ

FB

PM/DM

þþ

þþ

þ

þþ

þ

PSS

þþ

þþþ

þ

þ

þ/

Sjögrens

þ

þþ

þ/

þ

þþþ

PHBP

SLE

þ

þþ

þþ

þ

þ/

Alveolitis, hemorrhage

MCTD

þþþ

þþ

þ

þ

þ

PHBP

CTD, connective tissue disease; RA, rheumatoid arthritis; PM/DM, polymyositis/dermatomyositis; PSS, progressive systemic sclerosis; SLE, systemic lupus erythematosus; MCTD, mixed connective tissue disease; OP, organizing pneumonia; LIP, lymphoid interstitial pneumonia; FB, follicular bronchiolitis; PHBP, pulmonary hypertension.

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Pathogenesis The NSIP pattern may be associated with a fairly wide spectrum of clinical conditions, and it is reasonable to assume there are a number of pathogenic mechanisms that lead to the histological pattern. The etiology of true “idiopathic” NSIP is unclear, though recent studies of patients classified as “idiopathic” NSIP found a subset with clinical and serological abnormalities, suggestive of an underlying or occult CTD.140–142 It is well recognized that in some patients with CTD, lung involvement may precede development of systemic symptoms by several years. These data have led to the hypothesis that some cases of idiopathic NSIP may represent a unique/undefined connective tissue disease restricted to the lungs.140 Other studies report that a percentage of idiopathic cases have gene expression profiles suggestive of underlying HP. A clear antigenic trigger has not been identified.28 Recent data suggest that at least some cases of idiopathic NSIP may represent pulmonary involvement by IgG4-related autoimmune disease.143

Prognosis and natural history Non-specific interstitial pneumonia, in particular the cellular type, responds well to corticosteroid therapy, although this is often augmented with immunosuppressive agents, such as azathioprine or cyclophosphamide. The main factor that determines prognosis is the presence of fibrosis. Fibrotic NSIP has a poorer overall survival than cellular NSIP. Patients with cellular NSIP have a 5-year survival of almost 100%.113–115 In contrast, the 5- and 10-year survival rates for fibrotic NSIP are approximately 85% and 35%, respectively. This 35% 10-year survival rate is better than that noted for UIP patients (15% at 10 years).114,115 In a recent international collaboration with a well-defined cohort of cases with idiopathic NSIP, the overall 5-year survival was reported as 82%.135

Diffuse alveolar damage and acute interstitial pneumonia The term acute interstitial pneumonia (AIP) describes a distinct clinicopathological entity characterized clinically by the rapid onset of respiratory failure in a previously healthy individual in the absence of an identifiable cause. Diffuse alveolar damage is the histological finding (see Chapter 9). Acute interstitial pneumonia can only be distinguished from other causes of acute respiratory distress syndrome (ARDS) after any underlying etiology has been excluded by extensive clinical and microbiological investigations. The term AIP was first used by Katzenstein et al., who described eight cases of acute onset interstitial pneumonia in which an underlying cause could not be found.144 The entity is now considered synonymous with many of the cases of acute respiratory failure first recognized by Hamman and Rich in 1935 and subsequently coined “Hamman-Rich” syndrome.145,146 The condition is included in the ATS/ERS classification of IIPs and correlates with the histological

Figure 15. HRCT scan showing widespread patchy ground-glass opacification with denser areas in the more dependent lung in a patient with diffuse alveolar damage. (Image courtesy of Professor D. Hansell, London, UK.)

pattern of DAD. This pattern is nonspecific and is most commonly seen in the context of the acute respiratory distress syndrome (ARDS) (see Chapter 9). It is also seen in the context of acute exacerbation in patients with a pre-existing fibrotic interstitial pneumonia, most often UIP/IPF (see below). Therefore careful correlation with clinical, radiological and microbiological results is necessary to exclude an underlying etiology before diagnosing AIP.

Clinical presentation AIP is a rare condition with relatively few case series in the literature.144,147–151 Thus, the incidence and prevalence are unknown. It shows an equal sex distribution with a mean age of 49 years at presentation (range 7–83 yr). Patients typically present with a fairly acute onset over 1–3 weeks of dyspnea and cough, rapidly progressing to respiratory failure requiring mechanical ventilation. Most patients give a history of prodromal flu-like symptoms prior to respiratory deterioration and up to 50% are febrile on admission. By definition all known causes of acute respiratory failure, such as infection, pulmonary embolism, trauma, drug reaction, etc., should be excluded by exhaustive clinical, chemical and microbiological investigations. Bronchoalveolar lavage findings are nonspecific, but a neutrophilia and sometimes type II pneumocytes with reactive atypia may be seen.

Radiological findings The imaging findings are similar to those of ARDS. In the early stages HRCT commonly shows bilateral ground-glass opacification and dependent consolidation in the absence of traction bronchiectasis, cystic change or architectural distortion (Figure 15). Rare cases show diffuse or upper lobe predominance. These early HRCT findings are thought to correlate with

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Chapter 10: Interstitial lung diseases Figure 16. Lungs showing diffuse consolidation with hemorrhage. (Image courtesy of Dr M Burke, Uxbridge, UK.)

Figure 17. Exudative-phase DAD in a patient with AIP showing hyaline membrane formation with mild interstitial inflammation and edema.

Figure 18. AIP. Florid type II pneumocyte hyperplasia with mild reactive cytological atypia. (Image courtesy of Dr M. Burke, Uxbridge, UK.)

the exudative phase of DAD seen on histology. In the later stages the HRCT may show some architectural distortion with traction bronchiectasis, as well as cyst formation, reflecting damage to the lung parenchyma. This correlates histologically with the organizing phase of DAD. The pattern of HRCT changes may provide information to guide treatment and prognosis.147 Patients who recover show progressive resolution of groundglass changes and consolidation, though some are left with reticular shadowing, cysts and hypoattenuation, reflecting late stage interstitial fibrosis.152

Pathology Macroscopic and histological findings are those of DAD, identical to those seen in ARDS. They are characterized by several

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phases: an early (exudative) and a late (organizing) phase, depending on when the biopsy is performed in the course of disease. Grossly in the early phases the lungs are heavy and congested with a deep red (“plum”-like) cut surface and pulmonary edema (Figure 16). Later in the course of the disease the lungs are firm and consolidated, and may have a spongy texture with microcystic and cystic change or fibrosis. This spectrum of changes is reflected in the histological findings. In the earliest stage of disease, neutrophil congestion and interstitial edema may be the only identifiable features. These changes progress over the first week into the exudative phase, characterized by alveolar and interstitial edema, hyaline membranes and fibrinous exudates within alveolar airspaces (Figure 17). The former are derived from a combination of degenerate alveolar epithelial cells and secretory and serum proteins. In addition, some alveolar hemorrhage, interstitial chronic inflammation and early type II pneumocyte hyperplasia may be seen (Figure 18). The proliferative phase begins around the second week and is characterized by resorption and organization of the alveolar exudates and hyaline membranes by intra-alveolar buds of fibroblastic tissue (Masson bodies/organizing pneumonia) (Figure 19a). Hyaline membranes may become incorporated into the alveolar walls. There is often florid type II pneumocyte hyperplasia and squamous metaplasia, both of which can show quite striking cytological atypia (Figure 19b). Intravascular thrombi are also often present. Patients who recover may show restoration of normal lung architecture, but a percentage will be left with a degree of residual inactive interstitial fibrosis with an NSIP-like pattern (Figure 20) and microcystic change.

Chapter 10: Interstitial lung diseases

(a)

(b)

Figure 19. Organizing phase DAD. (a) Intraluminal buds of organizing pneumonia. (b) Squamous metaplasia with atypia. Table 7 Histological features of DAD/AIP

Key features Diffuse distribution Uniform temporal appearance Early Alveolar septal edema Hyaline membranes (patchy or diffuse) Late Airspace organization (patchy or diffuse) Alveolar thickening secondary to organizing fibrosis Alveolar septal fibrosis and microcyst formation (some) Pertinent negative findings Lack of granulomas, necrosis or abscesses Lack of infectious agents Figure 20. Late-stage DAD, resembling NSIP with uniform fine interstitial fibrosis.

These findings are associated with chronic respiratory compromise (see Chapter 9). The histological features of DAD/AIP are summarized in Table 7.

Variants

Acute fibrinous organizing pneumonia (AFOP) This is a relatively recently described pattern of acute lung injury, first reported in 17 patients with acute respiratory failure.153 The principal radiological findings on HRCT are bilateral basal opacities and areas of consolidation. The dominant histological pattern is one of patchy, air-space consolidation with intra-alveolar fibrin deposition, organizing pneumonia, and some secondary type II pneumocyte hyperplasia (Figure 21). Classic hyaline membranes are not a

Lack of prominent eosinophils or neutrophils Negative cultures Source: modified from7.

feature. An associated underlying disorder, such as CTD, drug reaction and infection, appears to be common; however, idiopathic cases are also noted. The mortality rate is 50%, similar to that seen in DAD (50–60%), but only 30% of the patients with AFOP apparently require ventilatory support, which is almost always required in patients with AIP/DAD. Cases have also been described in the pediatric population and one case of AFOP presenting initially as a localized mass has been reported.154–159 It is uncertain whether this represents a distinct entity or simply a more rapidly progressive variant of organizing pneumonia.160 The histological pattern of AFOP is not specific and can be seen in the context of underlying infection, vasculitis and eosinophilic pneumonia, as well as a nonspecific reaction surrounding an infarct or abscess.

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Chapter 10: Interstitial lung diseases Table 8 Histological features of acute fibrinous organizing pneumonia

Major features Dominant finding of organizing intra-alveolar fibrin Organizing pneumonia Patchy distribution Minor features Associated interstitial changes Acute/chronic inflammation Type II pneumocyte hyperplasia Alveolar septal expansion with myxoid connective tissue Interstitial inflammation and expansion mild to moderate Interstitial changes confined to areas adjacent to intra-alveolar fibrin with intervening lung showing minimal changes

Figure 21. Acute fibrinous organizing pneumonia showing prominent intraalveolar fibrin and some admixed inflammatory cells, without hyaline membranes.

Therefore, correlation with clinical and radiological findings and extensive microbiological investigation is essential. The histological features are summarized in Table 8.

Regional alveolar damage In some patients DAD appears to present as a patchy relatively discrete process within the lung. Yazdy et al. described a pattern of localized alveolar damage in 10 cases from a large autopsy series.161 These were characterized by one or more macroscopically visible areas of DAD, unilateral in six cases and mainly seen in the upper lobes. The underlying cause of the DAD was multifactorial and included shock, septicemia and pancreatitis. The reason for the regional nature of the changes is not certain. It is unclear whether these cases represent a localized form of DAD or simply a localized phase in the evolution of more typical disease.

Differential diagnosis Known causes of acute lung injury must be excluded by the pathologist. The presence of granulomas, neutrophil microabscesses and necrosis suggests underlying infection or Wegener granulomatosis. Special stains for infectious agents should be performed and fresh tissue sent for microbiological and virological investigations. Careful attention must be paid to the pulmonary vasculature to exclude an underlying vasculitis and ANCA serology may be helpful. If an alveolar hemorrhage syndrome is suspected clinically, frozen sections for immunofluorescence studies to exclude Goodpasture syndrome may be required. The relationship of the biopsy to the time course of the disease must be considered, as there may be considerable histological overlap between OP and NSIP patterns, as sequelae of acute lung injury, and their more chronic counterparts. The presence of residual hyaline membranes, the diffuse nature of the changes and myxoid rather than collagenous

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Pertinent negative findings Absent hyaline membranes Inconspicuous or absent eosinophils Absent extensive bronchopneumonia/abscesses Lack of granulomas Source: modified from153.

fibrosis all favor a late phase of acute lung injury. At autopsy, it is also worth assessing the lungs for any evidence of background UIP/IPF. These features represent an acute exacerbation of IPF undiagnosed during life.

Pathogenesis The pathogenesis of AIP is unclear, but it is probably triggered by a temporally restricted global insult to the lung, as evidenced by rapid clinical progression and histological temporal homogeneity. Given the histological similarity to DAD, the sequence of events that follow is probably the same as seen in ARDS, with epithelial and/or endothelial injury triggering a cytokine cascade resulting in activation of inflammatory and thrombotic pathways leading to lung injury (see Chapter 9). Unlike ARDS, the nature of the initiating event in AIP is, by definition, unknown. The possibility of an underlying genetic susceptibility is raised by a single case report of AIP presenting synchronously in two of three children from the same family, with asymptomatic radiological changes compatible with AIP seen in the third sibling.162 It is equally possible that this clustering represented common exposure to an environmental trigger. One recent study has suggested a relationship between at least some cases of AIP and IPF. In a series of 14 autopsy cases diagnosed as AIP, 50% were found to have early subpleural fibrosis resembling UIP/IPF.163 These patients were indistinguishable from those with AIP alone, on both clinical and radiological grounds, but the study raises the possibility that this group of patients may somehow be related to those with acute exacerbations of subclinical IPF.164

Chapter 10: Interstitial lung diseases Table 9 Proposed diagnostic criteria for AE-IPF

Previous or concurrent diagnosis of idiopathic pulmonary fibrosis Unexplained worsening or development of dyspnea within 30 days High-resolution computed tomography with new bilateral ground-glass abnormality and/or consolidation superimposed on a background reticular or honeycomb pattern consistent with usual interstitial pneumonia pattern No evidence of pulmonary infection by endotracheal aspirate or bronchoalveolar lavage Exclusion of alternative causes, including left heart failure, pulmonary embolism and other identifiable causes of acute lung injury Source: modified from167.

Treatment and prognosis Treatment is largely supportive, with almost all patients requiring mechanical ventilation often supplemented with antimicrobials. Corticosteroids are also commonly used and although their efficacy in the early stages is unclear they may have a beneficial role in later stages in reducing progression to fibrosis.149,165 The mortality rate approaches 70%. Survivors are often left with varying degrees of interstitial fibrosis. Repeated episodes and chronic progressive fibrosis may also occur in some patients who survive the initial presentation.

Acute exacerbation of idiopathic pulmonary fibrosis IPF is a progressive disease, with many patients dying of endstage respiratory failure. Up to 57% of patients with established IPF undergo acute deterioration of respiratory function during the course of disease. This decline in lung function is a not due to infection, pulmonary embolism or heart failure. In 1993 Kondoh et al.166 described three patients with IPF who developed acute deterioration of lung function with flu-like symptoms, cough, fever, leukocytosis and hypoxia in the absence of an identified infectious etiology. It has since been described by a number of terms in the literature, but the consensus terminology is acute exacerbation of IPF (AE-IPF). The overall incidence is uncertain and varies between studies, ranging from 8 to 57% of IPF patients.167–169 Acute exacerbation of IPF can occur at any time during the course of disease and there is no association with age or pulmonary function, although it appears more common in men. Some cases have occurred following surgical lung biopsy,170 bronchoalveolar lavage171 or etanercept therapy.172–174 Rare cases have also been reported following treatment with interferon, but larger trials of this drug in IPF failed to show any difference in the incidence of AE-IPF.175,176 Despite several case series and case reports, this entity remains poorly understood. A set of consensus diagnostic criteria were

Figure 22. HRCT of a patient with AE-IPF showing consolidation and ground-glass opacification on a background of honeycomb change, fibrosis and traction bronchiectasis. (Image courtesy of Professor D. Hansell, London, UK.)

published in 2007 with the aim of better defining the entity (Table 9).167,177 Of note, those patients with idiopathic clinical worsening who fail to meet all five criteria due to missing data should be termed “suspected acute exacerbations”. Establishing this diagnosis can be difficult, as the clinical and radiological features are relatively nonspecific, and may be seen with infection. Furthermore it is often not possible to perform an open lung biopsy due to the poor clinical status of these patients. Finally, there is a subset of patients who present with acute respiratory failure and are found to have IPF with superimposed DAD on either biopsy or autopsy material.164,168

Clinical presentation Patients typically present with acute worsening of shortness of breath, cough and flu-like symptoms within 30 days of hospital admission, often progress to respiratory failure and require mechanical ventilation. There are abnormalities of gas exchange on lung function tests and BAL often shows a neutrophilia,167 in association with negative cultures.

Radiological findings HRCT shows new bilateral ground-glass opacification and/or consolidation superimposed on the subpleural reticular and honeycomb change of IPF (Figure 22). Three radiographic patterns of distribution are described: peripheral, multifocal and diffuse.178,179 Peripheral opacities correspond to fibroblastic foci, while multifocal and diffuse changes correspond to DAD. The radiological pattern is predictive of response to treatment and prognosis. The best response to treatment is seen in patients with peripheral opacities, and the worst outcome is in patients with diffuse HRCT changes.

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Chapter 10: Interstitial lung diseases

(a)

(b)

Figure 23. Alveolar parenchyma showing UIP pattern and superimposed diffuse alveolar damage with hyaline membranes.

Pathology

Pathogenesis

The pathological features are those of DAD superimposed on underlying changes of a fibrotic interstitial pneumonia, most commonly UIP (Figure 23). The amount of DAD does not appear to correlate with clinical severity.180 Recently Churg et al. reported a superimposed organizing pneumonia pattern (perhaps representing an organizing phase of DAD) and a pattern characterized by abundant fibroblastic foci, associated with better survival.181 Acute exacerbations have also been reported in patients with other fibrosing interstitial pneumonias, including fibrotic NSIP, chronic HP and CTD-associated IP. The prognosis in all instances is poor.181–184 In BAL and surgical lung biopsies from acutely ill patients it is important to exclude known and treatable causes of acute lung injury, especially infection. Special stains for organisms and submission of tissue for microbiological investigations are essential. In reality, many of these patients are too sick to undergo biopsy and the diagnosis is often only confirmed at autopsy.

The pathogenesis of AE-IPF is unknown, and it is not clear whether the process represents a specific manifestation of IPF or is secondary to a distinct but undetected trigger, such as aspiration or infection. The presence of DAD histologically suggests an episode of acute lung injury, although no specific agent has as yet been identified. While flu-like symptoms and neutrophilia on BAL raise the possibility of an unidentified infectious trigger,166 the answer may lie in the pattern of progression of IPF itself. Katzenstein suggested that rather than showing a smooth progression of disease, IPF progresses in a discontinuous fashion with multiple episodes of acute lung injury, represented pathologically by fibroblastic foci, resulting in a stepwise decline in lung function.185 This “multi-hit” hypothesis raises the possibility that acute exacerbations may represent a similar but more global and severe episode of acute lung injury in the form of DAD, with rapid decline in lung function.186 However, it does not readily explain the reports of acute exacerbation of NSIP and chronic HP, in which an unrelated trigger may play a role. Cases have been reported following BAL,187 surgical lung biopsy188 and resection of an associated lung cancer.123 Rare cases are reported following institution of interferon-g 1b.189 Several randomized double blind trials failed to confirm any association of AE-IPF with treatment.175,176 Nevertheless, disturbances of epithelial and fibroblast function, the extracellular matrix and coagulation system play pathogenetic roles. Recent gene expression studies of AE-IPF showed a similar profibrotic gene expression profile, as found in stable IPF without evidence of either an infectious or significant inflammatory component. The data suggest a role for epithelial injury, apoptosis and proliferation.129

Differential diagnosis Infection and drug reactions are the most common differential considerations. As these patients are often on immunosuppressive therapy, atypical infectious agents must be excluded. Features suggestive or diagnostic of infective etiologies may be evident on histology (e.g., granulomas, viral inclusions, fungal hyphae, etc.) but microbiological examination is necessary. If a surgical biopsy is received fresh it is important to ensure some tissue is sent for culture and PCR, as appropriate. Prominent numbers of eosinophils may suggest a drug reaction, but close clinicopathological correlation is usually required in such cases.

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Prognosis and natural history Patients frequently require ventilatory support and outcome is poor, with up to a 70% mortality rate, despite the use of broadspectrum antibiotics and anti-inflammatory and immunosuppressive regimes.124,164,168 Newer potential therapeutic strategies currently under investigation include the use of anticoagulation and antifibrotic agents.190,191 Lung transplantation has been performed in some young patients with AEIPF.192 Those who survive the first episode of acute exacerbation may develop further episodes, to which they often succumb.

Smoking-related interstitial lung disease The spectrum of non-neoplastic lung disorders associated with smoking comprises two main groups; chronic obstructive pulmonary disease (COPD) and the smoking-related interstitial lung diseases. The latter include respiratory bronchiolitis (RB), respiratory bronchiolitis-interstitial lung disease (RB-ILD), desquamative interstitial lung disease (DIP), and pulmonary Langerhans cell histiocytosis (LCH) (see Chapters 17 and 34). Patients may show a combination of changes and there is some evidence to suggest that RB may be a precursor of centrilobular emphysema.193–196 Currently RB-ILD and DIP are included in the ATS/ERS classification of idiopathic interstitial pneumonias. The association with smoking, particularly for RB-ILD, is well established. Although some argue that RB/RBILD and DIP represent ends of a spectrum of smoking-related lung damage,138,197 they are currently regarded as separate clinicopathological entities. This is because their presenting features, clinical course, histological features and treatment approach differ. Desquamative interstitial lung disease is also seen in a number of conditions unrelated to smoking. The pathogenesis of smoking-related interstitial lung disease is poorly understood and, as it only develops in a percentage of smokers, is likely to be multifactorial. Aside from a genetic component, multiple pathogenic pathways are probably triggered by tobacco smoke components, which accumulate in alveolar macrophages.198 The persistence and fluctuation in disease after smoking cessation suggest continued macrophage activation and inflammation. Cigarette smoke contains high concentrations of oxidants, which induce a pro-inflammatory state in alveolar macrophages and increase macrophage production of matrix metalloproteases and osteopontin, resulting in local tissue destruction.199 It may also trigger the release of ferritin-bound iron within alveolar macrophages, further promoting oxidative injury.200

Respiratory bronchiolitis/respiratory bronchiolitis-interstitial lung disease Respiratory bronchiolitis and RB-ILD are closely related entities, characterized histologically by bronchiolar and peribronchiolar airspace accumulation of lightly pigmented alveolar macrophages (see below). These conditions show a very

close association with cigarette smoking,139 affecting both current and former smokers. There is a single case report of RBILD in association with passive smoking201 and rare reports of RB/RB-ILD in non-smokers exposed to other inhaled fumes.202,203 Niewoehner et al. first described RB as an incidental autopsy finding in the lungs of young male smokers.193 It is now recognized as an almost universal finding in smokers, most of whom are asymptomatic with no evidence of impaired lung function, although minor radiological abnormalities may be seen in a minority of cases. A small percentage of patients have mild respiratory dysfunction including respiratory symptoms, abnormal pulmonary function tests and/or imaging abnormalities.204 The term RB-ILD is used for this group of patients, although the pathological features are indistinguishable from those seen in RB.202,204–208

Clinical presentation Most patients with RB and RB-ILD are current smokers or exsmokers between 30 and 40 years of age; a slight male predominance is reported in some studies.202,204,205 By definition patients with RB are asymptomatic with normal PFTs. Patients with RB-ILD may present with mild chronic dyspnea and persistent cough, but chest pain, weight loss and hemoptysis are rarely described.209 A few cases presenting with acute symptoms210 or hemoptysis209 have also been reported. On clinical examination, bilateral basal crackles are found in approximately 50% of patients, but clubbing is rare.139,202,204–208 Pulmonary function tests commonly show a mixed obstructive-restrictive pattern and a reduction in carbon monoxide diffusing capacity (DLCO). Some patients with RBILD may be asymptomatic but show abnormal imaging or PFTs, and likewise some symptomatic patients may have normal PFTs. On BAL increased numbers of pigmented macrophages may be counted, although with smoking cessation these may fall to levels seen in lifelong non-smokers.211

Radiological findings Chest X-ray may be normal in up to 20% of patients, but often shows bilateral reticulonodular opacities. High-resolution computed tomography findings include centrilobular nodules, ground-glass attenuation and peripheral bronchial wall thickening (Figure 24).207 The changes are more prominent in the upper lobes. There may be mosaic attenuation, reflecting bronchiolar obstruction and air-trapping. The features may be more extensive in RB-ILD compared with RB. In contrast honeycomb change and traction bronchiectasis are not present. The features may show considerable overlap with DIP and similar changes are also described in asymptomatic smokers.212,213

Pathology Respiratory bronchiolitis and RB-ILD are identical histologically. At low-power magnification there is a bronchiolocentric

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Chapter 10: Interstitial lung diseases Figure 24. HRCT showing small indistinct ground-glass density nodules randomly distributed (but mainly in upper lobes) in a patient with RB-ILD. (Image courtesy of Professor D. Hansell, London, UK.)

Figure 25. Respiratory bronchiolitis showing bronchiolar and peribronchiolar accumulation of lightly pigmented alveolar macrophages.

pattern comprising alveolar macrophages within terminal and respiratory bronchioles and adjacent alveolar spaces (Figure 25). The macrophages have a characteristic glassy cytoplasm, which contains fine granular light brown pigment. This represents a mix of cigarette smoke constituents (Figure 26a). Perls’ stain shows variable, often faint, positivity for iron within the cytoplasmic pigment (Figure 26b), with one study showing that the degree of pigmentation was proportional to the pack year smoking history.139 Other studies have shown no difference between RB and DIP in the amount of iron present.138 By contrast, large amounts of often globular hemosiderin are seen following pulmonary hemorrhage, with strong Perls’ positivity (Figure 26c). A mild chronic inflammatory cell infiltrate, including eosinophils, both in the interstitium and admixed with the alveolar macrophages may be present. A mild degree of fibrous expansion of the alveolar walls adjacent to the bronchioles can be seen, sometimes associated with peribronchiolar metaplasia (Figure 27). The background alveolar architecture is generally preserved, although there may be mild coexistent emphysema.194–196 Granulomas, organizing pneumonia, honeycomb change and fibroblastic foci are not features of this disease. If present, a clinicopathological review should ensure the case represents RB-ILD and not incidental RB superimposed upon another disorder. The severity of changes is not significantly different between current and former smokers.139 Some authors have described a “variant RB” in non-smokers, where the cytoplasmic contents of the macrophages are more eosinophilic.139 As with cases associated with dust exposures, these are not included in the definition of RB/RB-ILD. While peribronchiolar and patchy subpleural fibrosis are recognized in up to 50% of patients with RB/RB-ILD,139 cases have been described where there is quite striking patchy hyaline subpleural fibrosis, often in association with some degree of emphysema.196 It remains unclear whether this NSIP-like

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pattern of fibrosis is a specific feature of RB-ILD or whether it represents either unrelated fNSIP occurring in a smoker, or another manifestation of smoking, namely alveolar septal fibrosis in areas of emphysema.214–216 Indeed, an association between smoking and interstitial fibrosis was noted by Hammond et al. in their experimental studies of smoking in dogs.217 It is further complicated by results of HRCT studies, in which rare cases of RB-ILD show gradual development and progression of emphysematous changes.218 Whatever the relationship of the subpleural fibrosis and emphysema to RB/RBILD, the difficulty for pathologists in limited biopsy material is to make the distinction from fNSIP in a smoker or a “macrophage-poor” pattern of DIP.134,205 RB and RB-ILD are histologically indistinguishable as similar degrees of macrophage accumulation, inflammation and fibrosis are described in both conditions. Distinction between the two depends on assessment of clinical symptoms, pulmonary function abnormalities and HRCT changes, and should not be made on the basis of biopsy alone.219 Furthermore in patients with features of RB on biopsy it is important to determine whether the finding truly represents the cause of their symptoms (i.e., RB-ILD) or whether it is merely an incidental feature in a smoker with another disease process (including other smoking-related disorders, such as Langerhans cell histiocytosis). The histological features are summarized in Table 10.

Differential diagnosis Hemosiderin-laden macrophages may be seen following alveolar hemorrhage of any cause. However the extent of iron deposition within the macrophages is usually far greater than seen in RB/RB-ILD. Furthermore, the pigmentation is often coarser, with large irregular iron granules within the

Chapter 10: Interstitial lung diseases

(a)

(b)

(c)

Figure 26. Alveolar (“smokers”) macrophages. (a) Macrophages contain fine light brown cytoplasmic pigment. (b) Perls’ stain highlights fine intracytoplasmic iron. (c) Perls’ stain highlights coarse intracytoplasmic iron granules secondary to pulmonary hemorrhage.

Figure 27. Mild nonspecific peribronchiolar interstitial fibrosis in a patient with respiratory bronchiolitis.

cytoplasm, rather than a finer granularity in RB/RB-ILD (best appreciated on Perls’ stain) where there is also an admixture of granular material that does not stain for iron. Other minerals, such as alumina, can mimic iron, but a Perls’ stain is usually discriminatory. Macrophage accumulation may be seen in other interstitial lung disorders, but these are usually characterized by a predominance of other histological features (e.g. organizing pneumonia, fibroblastic foci, marked fibrosis and honeycomb change, hyaline membranes, asbestos bodies, etc.) that are not seen in RB/RB-ILD. Where subpleural fibrosis is a prominent feature, the distinction from fNSIP occurring in a smoker may be difficult, especially in limited biopsy material. In NSIP the fibrosis more diffusely involves the lobules and there is usually more inflammation, but correlation with clinical and radiological features, in particular the presence of an underlying CTD, may help establish the correct diagnosis. Distinction from DIP is described below.

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Chapter 10: Interstitial lung diseases Table 10 Histological features of RB/RB-ILD

Key histological features Bronchiolocentric accumulation of alveolar macrophages Mild bronchiolar fibrosis and chronic inflammation may be seen Macrophages have fine brown cytoplasmic pigmentation (positive for iron stain) Pertinent negative findings No diffuse accumulation of macrophages No interstitial fibrosis or honeycomb change Source: modified from7.

Prognosis Improvement of symptoms is seen in many patients following cessation of smoking.202,204,205,220 In persisting or progressive cases, steroids and in some instances immunosuppressive agents are used, and the 5-year survival rate approaches 100%. Corresponding improvements in PFT and HRCT abnormalities are also described.221 Some patients fail to show significant improvement of either symptoms or lung function, and this may reflect a degree of residual fibrosis.196,204,220 Rare cases showing chronic symptomatic deterioration are described,202,207,222 and in a few patients, serial HRCT studies have shown an evolution of RB/RB-ILD to emphysema.218

Desquamative interstitial pneumonia Desquamative interstitial pneumonia is a form of IIP characterized by diffuse accumulation of pigmented macrophages within the airspaces. It was first described by Liebow et al. in 1965,223 who used the epithet “desquamative”, as they believed the cells represented desquamated alveolar epithelial cells. They are now recognized as alveolar macrophages, but the misnomer remains. Although initially thought to represent a cellular form of UIP, patients with DIP have a better prognosis than those with UIP and it is now regarded as a distinct pattern of interstitial pneumonia with characteristic clinicopathological features more closely related to RB/RB-ILD.224 Desquamative interstitial pneumonia is an uncommon form of interstitial pneumonia, and with few epidemiological studies in the literature, the exact incidence and prevalence of disease are unknown. Up to 90% of patients are smokers.220,224 However, in contrast to RB/RB-ILD, DIP may be seen in non-smokers in association with a variety of conditions, including pneumoconiosis,225 drug reaction226 and CTD.227 In rare cases, especially in children, there is no evidence of an underlying trigger (idiopathic DIP).138 A focal accumulation of macrophages resembling DIP can also be seen in a number of unrelated conditions, such as surrounding tumors, or any other process that stiffens the alveolar walls, and is termed “DIP-like reaction”. Only those cases lacking any known cause, other than smoking, are accepted as “idiopathic” DIP in the current ATS/ERS classification.

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Figure 28. HRCT scan of DIP showing patchy ground-glass opacity in a predominantly subpleural distribution; the minute cystic airspaces may represent a background of centrilobular emphysema or traction bronchiolectasis. (Image courtesy of Professor D. Hansell, London, UK.)

Clinical presentation Patients with DIP are typically middle-aged with a male predominance. Individuals usually present with gradual onset of dyspnea and/or dry cough, though rarely systemic symptoms such as fever may be present. Clinical examination reveals bibasal inspiratory crackles, while clubbing is reported in up to 50% of patients.205,220,224 Pulmonary function tests show a reduced DLCO and varying degrees of restrictive defect. Bronchoalveolar lavage specimens may show increased numbers of pigmented alveolar macrophages, sometimes with eosinophilia,169 but lymphocytosis and neutrophilia are rare. The disease is also rarely reported in children, where it may be familial and in some cases appears related to an inborn error of metabolism.228,229

Radiological findings Chest radiographs are normal in up to 22% of biopsy-proven cases,7 but may show patchy opacities. High-resolution computed tomography typically shows bilateral, ground glass opacification, more prominent in the lower lobes and subpleural zones (Figure 28). There may be a fine reticular pattern, associated with mild localized fibrosis that is usually limited to subpleural areas.230 Over time some cases may show an NSIP-like appearance on HRCT.138 Honeycombing is not usually seen.

Pathology Widespread accumulation within alveolar spaces of lightly pigmented alveolar macrophages (Figure 29) characterizes DIP histology. The macrophages are identical to those seen in RB, but in contrast to RB the whole lobule tends to be involved, without bronchocentric accentuation. As in RB, interstitial chronic inflammation, including eosinophils (Figure 30), interstitial fibrosis (Figure 31) and localized lymphoid follicles, may be present. However, their extent is

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Figure 29. Desquamative interstitial pneumonia pattern. (a) Diffuse intra-alveolar accumulation of alveolar macrophages is obvious. (b) Elastic van Gieson stain highlights mild interstitial fibrosis. (c) A CD68 stain stains the macrophages.

greater in DIP than in RB.138 Co-existent emphysematous changes can be seen, although honeycombing is nearly always absent. Indeed, the presence of honeycomb change should concern the pathologist that there might be incidental DIPlike changes in a case of UIP/IPF. The histological features are summarized in Table 11.

Differential diagnosis The differential diagnosis of hemosiderosis following alveolar hemorrhage has been discussed above. Interstitial fibrosis may be seen in patients with DIP, but the presence of fibroblastic foci and honeycomb changes are more indicative of UIP. The UIP pattern rarely features diffuse intra-alveolar macrophage accumulation. The distinction from fNSIP may be more

problematic, as there can be some overlap of histological features, particularly as the numbers of intra-alveolar macrophages vary across the biopsy. Macrophages may be washed out of alveolar spaces during overly intense formalin distension of surgical biopsies. This leaves mild interstitial fibrosis that resembles fNSIP. In these cases correlation with clinical and radiological features is necessary. Rarely eosinophils are so prominent in DIP that a diagnosis of eosinophilic pneumonia may be considered but, in contrast to the latter disease, eosinophilic microabscesses are not a feature of DIP. Correlation with clinical and imaging data resolves this problem. Langerhans cell histiocytosis is also a smoking-related disease and characterized by accumulation of Langerhans cells within the lung parenchyma. However, the Langerhans cells are usually interstitial and form nodular

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Figure 30. Alveolar macrophages with admixed eosinophils. Table 11 Histological features of DIP

Key histological features Uniform involvement of lung parenchyma Intra-alveolar accumulation of lightly pigmented alveolar macrophages Mild-moderate interstitial fibrosis may be seen Mild chronic inflammation Pertinent negative findings Inconspicuous or absent dense extensive fibrosis No honeycomb change Inconspicuous or absent fibroblastic foci and organizing pneumonia Rare or absent eosinophils Rare or absent smooth muscle proliferation Source: modified from7.

aggregates, often with cystic changes and foci of stellate fibrosis. Langerhans cells have quite typical cytological features with folded nuclei and pale “tissue paper”-like cytoplasm. In equivocal cases immunohistochemistry for S100 protein and CD1a decorate the Langerhans cells. Rarely Langerhans cell histiocytosis and DIP may coexist in the same case. A DIP-like reaction can also be seen with drug reactions, dust inhalation and in association with other interstitial lung diseases. Rare cases of non-small-cell lung cancer can also exhibit a predominantly or exclusively intra-alveolar dissociated pattern of growth.231 These can be readily distinguished by the presence of cytological atypia, though this may be minimal, and immunohistochemical staining for cytokeratins and macrophage markers.

Pathogenesis Since DIP is seen primarily in association with cigarette smoking, the underlying pathogenesis in these cases is likely

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Figure 31. DIP with diffuse interstitial fibrosis.

to be similar to RB-ILD. Other triggers of macrophage accumulation and activation are likely to play a role, as a DIP-like reaction pattern is also described with dust inhalation, drug reactions and inborn errors of metabolism, as well as in association with other ILDs and surrounding tumors. Rare idiopathic cases are seen in the absence of any known association.

Prognosis and natural history Without treatment many patients with DIP experience progressive worsening of symptoms. Patients usually improve with smoking cessation and steroid therapy, with or without the addition of immunosuppressive agents. The prognosis is good with 5-year and 10-year survival rates of 95 and 70%138,224 and is significantly better than seen in patients with UIP and fNSIP.114,115 Some patients are left with a degree of respiratory compromise, probably reflecting underlying interstitial fibrosis. A small number of patients show progressive disease with no response to steroid therapy230 and the role of cytotoxic and immunosuppressive agents in such cases is unclear. Recurrence of disease after lung transplantation has also been reported.232,233 In children the prognosis is worse, probably reflecting the difference in etiology, as pediatric cases appear to be due to surfactant protein gene mutations (see Chapter 3).234

Provisional and recently described entities Idiopathic pleuroparenchymal fibroelastosis is a rare idiopathic entity with unique pathological features that do not fit into the current classification of IIPs. First described by Frankel et al., it is characterized by pulmonary symptoms with marked apical pleural thickening and subpleural upper lobe fibrosis on CT. The pathological features are of prominent subpleural fibroelastosis with marked fibrous thickening of the overlying visceral pleura (Figure 32). The interface between the fibroelastosis and underlying lung parenchyma is abrupt

Chapter 10: Interstitial lung diseases

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(b)

Figure 32. Pleuropulmonary fibroelastosis. (a) Dense subpleural fibroelastosis with overlying fibrous pleural thickening typifies this entity. (b) An elastic van Gieson stain highlights the striking elastosis.

Figure 33. Rare fibroblastic focus at margin of pleural fibroelastotic tissue and normal lung parenchyma.

although rare fibroblastic foci can be seen (Figure 33). Some patients with this condition die of progressive respiratory failure.235,236 The histological features are similar to those seen in apical cap (see Chapters 2 and 36). However, apical cap is an asymptomatic focal lesion. In contrast the patients with pleuroparenchymal fibroelastosis present with clinically significant chest symptoms, including dyspnea and cough, and have extensive radiological abnormalities on imaging. Spontaneous pneumothoraces complicated by bronchopulmonary fistula have been reported in one case.236 Since the ATS/ERS classification of IIPs was published in 2002, a number of novel or provisional interstitial pneumonias have been described which show overlapping histological features with varying degrees of bronchiolocentric fibrosis, inflammation and peri-bronchiolar metaplasia (Figure 34). These entities have been given a variety of names, including

Figure 34. Peribronchial metaplasia associated with mild peribronchial interstitial fibrosis. This is a nonspecific pattern of lung injury, which may be seen in association with a variety of conditions and exposures.

airway centered interstitial fibrosis, peribronchiolar metaplasia and bronchiolitis interstitial pneumonia.237–239 The published series are small; however, female patients appear over-represented and the mean age at presentation is similar (47–57 yr). Symptoms include chronic cough and dyspnea, and radiological findings include diffuse reticular and reticulonodular changes and peribronchovascular fibrosis. Patients generally show a restrictive pattern of PFTs. The histological features described are relatively nonspecific and can be seen following bronchiolar injury due to a variety of causes, including aspirational/inhalational injury, chronic HP, and CTD. In view of the small numbers of patients, and limited and variable followup data, it is not possible as yet to clearly define an idiopathic form of disease.

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Pulmonary hypertension and interstitial lung disease Pulmonary hypertension (PH) is found in many patients with diffuse interstitial lung disease, although the reported incidence varies and it is most commonly seen in IPF, CTDassociated lung disease (particularly scleroderma), sarcoid and pulmonary Langerhans cell histiocytosis (see Chapters 13, 18, 21 and 34). In IPF, the prevalence of PH is reported as ranging between 31–85% of patients listed for lung transplantation,16,240–244 but it is probably lower in patients with early-stage disease. There is no correlation between the degree of PH and disease extent, as assessed by HRCT, PFTs or composite physiological index. Pulmonary hypertension may be seen in patients without hypoxemia and with relatively little interstitial fibrosis.241,243,245 This suggests the development is not solely secondary to chronic hypoxia or interstitial fibrosis, but may also represent an intrinsic part of the disease process (termed disproportionate PH by some groups). Gene expression analysis shows different gene profiles for IPF-associated PH compared to idiopathic pulmonary arterial hypertension, implying the pathophysiology is different in these conditions.246 The presence of severe PH is associated with a significant increase in mortality in patients with IPF241,242,247 even following transplantation.248 The effect of mild and moderate PH on prognosis remains unclear. A spectrum of histological changes is described in the pulmonary vasculature in IPF biopsies. In areas of established fibrosis arteries show nonspecific medial hypertrophy and intimal fibrosis. Fibroblastic foci usually have no microvessels, but microvessel density (MVD) is increased in the underlying fibrous tissue.249 In areas of preserved lung architecture, mild muscularization of pulmonary arterioles has been described,250 sometimes with associated alveolar capillary proliferation.249,250 The intimal fibrosis reported within venules and small veins in 65% of patients suggests to some more global vascular dysfunction, rather than a response to the interstitial fibrosis.250 The etiology of PH in IPF is probably multifactorial, and may be different at different stages of disease. In advanced fibrosis, chronic hypoxic vasoconstriction and fibrous obliteration of vessels are probably the main factors affecting the pulmonary vasculature. In early/active disease the pro-fibrogenic microenvironment of the fibroblastic focus probably also plays a role in local vascular remodeling, as many fibrogenic mediators, such as TGF-b, TNF-a, platelet-derived growth factor, fibroblast growth factor and endothelin-1 (ET-1), are also involved in angiogenesis and angiostasis.251 There is evidence that both occur in IPF, with increased microvessel density near fibroblastic foci, and vascular ablation with reduced MVD in areas of established fibrosis.249,252 In early disease, intermittent episodes of hypoxia during sleep or exercise may initiate local vascular remodeling, possibly through elevation of inflammatory mediators and ET-1.253

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Recent gene array analysis has detected abnormal vascular phenotypes in patients with IPF and pulmonary hypertension,254 with upregulation of a variety of factors in endothelial cells including endothelin-1.254,255 Endothelin-1 has a number of biological effects, including regulation of vasodilatation, recruitment of inflammatory cells and proliferation of fibroblasts/myofibroblasts.256 It can also trigger release of free radicals and fibrogenic cytokines from macrophages. It is possible that, once initiated, abnormal vascular remodeling may in turn stimulate the matrix remodeling that characterizes IPF. Vascular smooth muscle dysfunction is also likely to contribute to abnormal vascular remodeling, with abnormal migration and extracellular matrix production reported in vascular smooth muscle cells in IPF.257–259 Furthermore, vascular remodeling may result in generation of pulmonary systemic anastomoses and right to left shunting, exacerbating local hypoxemia. The pathogenesis of pulmonary hypertension in other interstitial lung diseases is less well characterized, but chronic hypoxia rather than vascular remodeling is likely to account for pulmonary hypertension associated with smoking-related interstitial lung disease. Vascular inflammation and thrombosis may be significant in cases associated with an underlying collagen vascular disease.260,261

Pulmonary calcification and ossification Pulmonary calcification and ossification includes a number of entities characterized by the deposition of calcium salts and/or bone within lung tissue.262 They frequently occur secondary to a variety of local and system diseases, though rare idiopathic forms are also recognized.

Pulmonary calcification Pulmonary calcification is usually grouped into two forms, dystrophic and metastatic. Dystrophic calcification is characterized by the deposition of calcium in areas of tissue scarring or necrosis, as well as being associated with foci of chronic granulomatous inflammation and infection. In the lung it is especially common following tuberculosis, histoplasma or viral pneumonia (particularly chickenpox pneumonia) as well as being associated with chronic parasitic infections. Calcium deposition, in association with hemosiderin or amyloid, can also occur. In all cases the calcified foci may undergo secondary ossification with formation of lamellar bone, sometimes with associated marrow elements. So called “metastatic” calcification is characterized by the deposition of calcium in normal tissues and usually occurs secondary to a disturbance in calcium or phosphorus metabolism. It frequently involves multiple organs, in particular the heart, stomach and kidney, as well as the lung. It is often classified into benign and malignant forms.262 The benign form is commonly seen with hypercalcemia associated with chronic renal failure,263 but is also reported in primary

Chapter 10: Interstitial lung diseases

Figure 35. Metastatic calcification with deposition of calcium within alveolar walls.

hyperparathyroidism,264 sarcoidosis,265 following liver transplantation266 and in association with systemic alkalosis, such as in chronic renal failure.267 The malignant form occurs with hypercalcemia secondary to malignancy such as with myeloma, bony metastases,268 or tumor-related paraneoplastic syndromes.269 Occasional idiopathic cases have been described in which no underlying abnormality of calcium metabolism is detected. The underlying etiology is unclear, but local factors such as pH and phosphate levels probably influence the degree of calcium deposition. Metastatic pulmonary calcification can occur at any age and is frequently asymptomatic, though some patients can develop dyspnea with restrictive PFTs and reduced diffusion capacity.270–272 Rare cases presenting with fulminant respiratory failure have also been reported.273 The calcification may be too fine to be detected on routine radiology but is usually apparent on CT and dual-energy digital radiology. The findings are varied and include centrilobular calcified nodules, calcification of vessels and bronchial walls and ground-glass opacification which can mimic pulmonary edema.262 Some cases show a zonal distribution with upper lobe predominance. Grossly the lungs are heavy with a pale gray cut surface and gritty consistency. On microscopy there are fine granular and linear deposits of calcium on the elastic fibers of the alveolar walls, vessels and bronchial mucosa (Figure 35). These may be associated with a foreign-body giant cell reaction or with a fibrinous alveolar exudate which can progress to intraalveolar fibrosis.271 Rarely the changes are confined to the airways.274 The features can be subtle and Von Kossa stain will highlight calcium deposition not obviously apparent on H&E sections. Treatment is aimed at correction of the serum levels of calcium and/or phosphate, often by treating the underlying cause. Pulmonary calcification may progress despite normal serum and phosphate levels.262 Death is usually due to cardiac involvement.275,276

Figure 36. Pulmonary ossification with lamellar bone expands and distorts the alveolar interstitium.

Pulmonary ossification Pulmonary ossification is characterized by the presence of lamellar bone within the lung parenchyma. Incidental localized deposition of bone is common and frequently follows dystrophic calcification. In contrast diffuse pulmonary ossification is rare and two histological patterns have been described, both preferentially involving the lower lobes. The most common is a nodular form, more often seen in men, which is usually associated with longstanding pulmonary congestion, particularly secondary to mitral stenosis, although it is also reported with aortic stenosis, left ventricular failure and pulmonary venous hypertension.277 Rarely no underlying association is found. The nodules of bone are circumscribed and tend to fill alveolar spaces, and marrow tends to be absent. The second form is characterized by a dendriform or branching pattern of ossification, which tends to involve the interstitium as well as alveolar spaces, with branching spicules of bone, often containing marrow (Figure 36).278–281 This pattern is commonly seen in cases of idiopathic pulmonary ossification (IPO) in which there is no underlying tissue damage.278,282–284 It is also often seen in fibrosing lung disease, cystic fibrosis, asbestosis281 and following chronic busulfan treatment,285 where it probably represents a more extensive form of dystrophic calcification. The incidence of pulmonary ossification tends to increase with age, but it is rarely symptomatic and diagnosis is usually made incidentally on imaging or at post mortem. Occasionally patients with extensive ossification present with dyspnea and restrictive PFTs, and experimental treatments directed at inhibiting bone metabolism have been attempted.262

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Figure 37. HRCT of pulmonary alveolar microlithiasis showing a nodular pattern with a high attenuation component (particularly around the mediastinal border) and minor peripheral paraseptal emphysema. (Image courtesy of Professor D. Hansell, Royal Brompton Hospital.)

Pulmonary alveolar microlithiasis Pulmonary alveolar microlithiasis (PAM) is a rare diffuse lung disease, in which diffuse micronodular calcification develops throughout the lungs in the absence of any underlying abnormality of calcium metabolism. Both sporadic and familial forms exist, the latter exhibiting an autosomal recessive pattern of inheritance.286 The sporadic form is more commonly seen in males, whereas the converse is found in familial cases. Pulmonary alveolar microlithiasis is found worldwide, but an increased incidence is reported in Turkey,287,288 Japan and Italy.289 Mutations of the SLC34A2 gene have recently been described in both familial and non-familial cases.290,291 This gene codes for a phosphate transporter protein that is expressed in alveolar type II pneumocytes and transports phosphorus ions from the alveolar space into the type II cell.290 Mutation in this gene prevents normal function, and it is likely that PAM results from the accumulation of phosphorus ion within the alveolar spaces, resulting in secondary precipitation of calcium phosphate, the major constituent of the microliths in this disease.292,293 Rare sporadic cases have been reported following the inhalation of sand particles.288,294 Most patients are diagnosed in their second or third decade, though rare cases are seen in children and the elderly.295 Diagnosis is often made incidentally on chest X-ray as patients are frequently asymptomatic, even with extensive disease,288,292,296 and the discrepancy between the degree of radiological changes and symptoms is a clue to this disorder. Symptoms usually present late in the disease with cough or spontaneous pneumothorax and patients show impaired lung function on spirometry.297 Involvement of other organs including pleura and kidney has been reported.298,299 Chest X-ray findings are of diffuse bilateral micronodular calcification, particularly in the lung bases and hilar regions.287,296 On HRCT an intra-alveolar pattern of calcification

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Figure 38. Microliths in a bronchoalveolar lavage specimen.

is seen, as well as calcification along bronchovascular bundles, interlobular septa and subpleurally (Figure 37).300 Bronchoalveolar lavage fluid may contain large numbers of calcified bodies (Figure 38),292 although this is not a specific finding, as occasional calcified bodies may be seen in patients with COPD. The macroscopic findings are of firm gritty lungs, which are often difficult to slice. On microscopy there are numerous intra-alveolar and interstitial laminated calcifications composed mainly of calcium and phosphorus (Figure 39).292,301 They are frequently concentrated in the subpleural space, interlobular septa and the bronchial mucosa, vary in size from a few mm to several millimetres and may show focal ossification. The calcifications must be distinguished from corpora amylacea and blue bodies (see Chapter 2). The disease has a variable clinical course though most patients are stable. In some the disease is slowly progressive with eventual death from respiratory insufficiency, albeit several decades after diagnosis.293,296,301 Rapid deterioration or spontaneous remission is very rare.302 Treatment with repeated BAL,293,296,303,304 inhibitors of bone metabolism (e.g., disodium etidronate),292,293,305 and lung transplantation306–308 have been attempted with limited success.

Pulmonary alveolar proteinosis Pulmonary alveolar proteinosis (PAP) is a rare diffuse lung disease characterized by the accumulation of granular lipoproteinaceous surfactant material within the alveolar spaces. Approximately 90% of cases are idiopathic but congenital and secondary (including dust-related) forms are recognized, accounting for approximately 2 and 5–10% of cases, respectively.309,310

Etiology The etiology is multifactorial but involves abnormalities of surfactant metabolism, in which alveolar macrophages play an

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Figure 39. Alveolar microlithiasis. (a) Wedge biopsy specimen demonstrates alveolar filling with microliths and significant ossification. (b) Laminated intra-alveolar microliths distort airspaces.

important role.311 Granulocyte-macrophage colony-stimulating factor (GM-CSF), which promotes terminal differentiation and maturation of alveolar macrophages, plays a key pathogenic role.312 Idiopathic PAP is thought to represent an autoimmune phenomenon, in which production of neutralizing antibodies to GM-CSF results in poor macrophage function and impaired surfactant clearance.313 It is more common in males and smokers and is usually seen in the third–fifth decades, though rare cases are reported in children and the elderly. Increased levels of anti-GM-CSF antibody are detectable within serum and BAL fluid. The levels of anti-GM-CSF antibody do not correlate with disease severity. Secondary alveolar proteinosis is thought to result from impaired macrophage activity and/or oversecretion of surfactant, resulting in poor clearance.313 It is associated with a variety of systemic conditions including malignancy (especially hematological malignancy), immunodeficiency (including HIV), viral infection,314 drug reaction315 and following lung transplantation.316,317 It is also seen following acute exposure to dusts, particularly crystalline silica (acute silicosis),318,319 although cases have also been reported following exposure to aluminum320 and kaolin.321 Cases presenting in infancy and childhood are almost always associated with an underlying disorder of surfactant metabolism, and over 30 surfactant or surfactant-associated protein mutations have been described, with both autosomal dominant and autosomal recessive patterns of inheritance.313 These are now collectively referred to as pulmonary surfactant metabolic dysfunction disorders, and are frequently

characterized by accumulation of surfactant in alveoli, often with abnormal function or composition. They show a spectrum of disease severity, with some mutations (surfactant protein B) incompatible with survival beyond birth, whilst others (surfactant protein C) are characterized by chronic lung disease in infancy and childhood. In less than 1% of cases, there is a mutation in the alpha chain of the GM-CSF receptor (CSF2RA), which shows an autosomal recessive pattern of inheritance. The mutation impairs GM-CSF binding and receptor signaling, resulting in a functional deficiency of GM-CSF. These patients often show elevated levels of serum GM-CSF. Mutations in the GM-CSF gene itself have not yet been detected. Pulmonary alveolar proteinosis is also reported in patients with the inherited disorder of lysinuric protein intolerance, caused by a mutation in an amino acid transporter.322 Many of these congenital cases also show features of interstitial lung disease such as cellular pneumonitis of infancy and NSIP, in which the proteinosis may be a minor component. Rare familial cases of PAP may represent a milder form of inherited surfactant deficiency manifest in adulthood.

Clinical features Patients commonly present with dyspnea and cough, and about 50% have a low-grade fever. Some complain of hemoptysis, fatigue and chest pain. About a third of patients are asymptomatic.323,324 Clinical examination may be normal, or may reveal mid-inspiratory crackles, but clubbing is rare.

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Pulmonary function tests show impaired DLco with a restrictive pattern in some patients.325 The disease is usually bilateral, affecting multiple lobes, but it may be patchy and rarely involves just one lung.324 Nonspecific serological findings include elevated levels of lactate dehydrogenase and surfactant proteins.

Radiological findings Radiological features include airspace consolidation with perihilar accentuation and on HRCT there may be thickening of the interlobular septa in areas of ground-glass attenuation, resulting in a characteristic “crazy paving” pattern (Figure 40).326 The radiological abnormalities are often

Figure 40. HRCT in a case of alveolar proteinosis showing typical widespread “crazy paving” pattern (thickened interlobular septa on a background of ground-glass opacification, separated from normal lung by sharp geographical border). (Image courtesy of Professor D. Hansell, London, UK.)

(a)

discordant with the degree of clinical symptoms, which are frequently mild. The diagnosis may be suspected at the time of BAL, as the lavage fluid has a characteristic white, “milky” appearance. Microscopy of the lavage fluid shows casts of granular PASpositive proteinaceous material and can be sufficient for diagnosis (Figure 41).324,327,328 The presence of large quantities of mineral dusts or eosinophils may be a clue to underlying mineral dust exposure or drug reaction in some cases.329

Pathology Macroscopic features in the lung are of patchy, firm yellow consolidation, sometimes with exudation of milky fluid from the cut surface. The histological changes can be patchy and may not be apparent on small biopsy material. There is intraalveolar accumulation of amorphous granular eosinophilic material, that is lipid rich and may contain cholesterol clefts and foamy macrophages (Figure 42). It shows strong PAS positivity, which is resistant to diastase pre-digestion, corresponding to large amounts of glycosylated surfactant apoprotein. There is often a small amount of admixed degenerate cellular material and some degree of type II pneumocyte hyperplasia may be seen (Figure 43). On immunohistochemistry the amorphous material stains for surfactant and on electron microscopy numerous lamellated bodies are seen in alveolar macrophages.330,331 These may have abnormal morphology in congenital forms of disease.332 In longstanding disease, secondary changes including interstitial fibrosis and chronic inflammation may be present, especially in patients with underlying immunosuppression.333,334 In congenital cases there may be an associated diffuse interstitial lung disease, such as NSIP or DIP, which may be the presenting feature. About 16–18% of patients develop an opportunistic infection,309 and this should be suspected where (b)

Figure 41. Pulmonary alveolar proteinosis. (a) Amorphous granular alveolar casts in a broncholalveolar lavage sample. (b) Strong PAS positivity of amorphous casts.

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(a)

(b)

Figure 42. Pulmonary alveolar proteinosis. (a) Intra-alveolar accumulation of granular eosinophilic material. (b) Cholesterol clefts.

reported as highly predictive for the disease.328 In idiopathic cases the presence of anti-GM-CSF antibodies in the serum or BAL fluid is specific for the disease and they can be detected quickly.339 In hereditary cases serum levels of GM-CSF may be elevated.340

Differential diagnosis

Figure 43. Mild type II pneumocyte hyperplasia associated with alveolar proteinosis.

there is a significant inflammatory infiltrate. Typical infectious agents include Nocardia and a variety of fungi, though mycobacterial infection is also seen.335–337 The growth of microorganisms appears to be enhanced by the lipoproteinaceous material and impaired macrophage function.338 Patients are also at increased risk of systemic infection, which may be related to a relative deficiency of GM-CSF.309 Histological diagnosis is regarded as the “gold standard” and may be made on transbronchial or wedge biopsy of lung. The presence of more than 18 proteinaceous casts in BAL fluid is

References 1. Flaherty KR, King TE Jr, Raghu G, et al. Idiopathic interstitial pneumonia: what is the effect of a multidisciplinary approach to diagnosis?

The main differential diagnosis is with pneumocystis infection, in which the exudate has a more bubbly appearance (see Chapter 7). Fungal stains should be performed, especially in immunosuppressed individuals, to exclude this possibility. Pulmonary edema fluid may superficially resemble proteinosis at scanning magnification, but this does not have the granular proteinaceous appearance or foamy macrophages seen at high power. Similar changes can be seen focally, secondary to bronchial obstruction,341 as well as with hypersensitivity pneumonitis or in rare cases of surfactant-secreting adenocarcinoma.342 The clinical course is variable and prior to treatment with BAL, approximately a third of patients show some improvement, a third stabilize and a third die of progressive disease.309,324 Spontaneous regression has been rarely reported.343 Current treatments include repeated whole lung lavage, which removes the proteinaceous material as well as the auto-antibody, and injections of GM-CSF.310,344–346 The 5-year survival rate is now reportedly > 90%. Secondary PAP may regress following treatment of the underlying disease; however, the prognosis of congenital cases is generally poor, despite surfactant replacement therapy and lung transplantation. Recurrence following lung transplantation has been reported.317,347

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344. Kavuru MS, Sullivan EJ, Piccin R, Thomassen MJ, Stoller JK. Exogenous granulocyte-macrophage colonystimulating factor administration for pulmonary alveolar proteinosis. Am J Respir Crit Care Med 2000;161:1143–8.

342. Vazquez M, Sidhu GS. Surfactant production by neoplastic type II pneumocytes. Ultrastruct Pathol 1988;12:605–12.

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345. Seymour JF, Presneill JJ, Schoch OD, et al. Therapeutic efficacy of granulocyte-macrophage

colony-stimulating factor in patients with idiopathic acquired alveolar proteinosis. Am J Respir Crit Care Med 2001;163:524–31. 346. Tazawa R, Hamano E, Arai T, et al. Granulocyte-macrophage colonystimulating factor and lung immunity in pulmonary alveolar proteinosis. Am J Respir Crit Care Med 2005;171:1142–9. 347. Parker LA, Novotny DB. Recurrent alveolar proteinosis following double lung transplantation. Chest 1997;111:1457–8.

Chapter

11

Metabolic and inherited connective tissue disorders involving the lung Gail Amir and Annick Raas-Rothschild

Introduction Lung involvement presenting in the course of metabolic disorders is usually overshadowed by features of the underlying disease. Pulmonary involvement is rarely the initial presenting feature. This constitutes a diagnostic challenge, particularly in adults, since the changes on biopsy are frequently nonspecific and a diagnosis of metabolic lung disease may not be considered. The pulmonary pathologist needs to be aware of the repertoire of lung pathology in these diseases to facilitate early diagnosis. This is ever more important, not only for genetic counseling but also for prompt therapy.

Lysosomal storage diseases Lysosomal storage diseases constitute a group of inherited disorders characterized by lack of a protein essential for normal lysosomal function. As a consequence, substrate accumulates in cells of various organs. Marked phenotypic heterogeneity characterizes many of these diseases with regard to age of onset, severity of symptoms and organs affected including the central nervous system (CNS). In many metabolic diseases the lungs are involved and in some cases the pulmonary involvement is significant. These usually present with interstitial infiltration, airways obstruction or pulmonary hypertension.

Gaucher disease Introduction

Gaucher disease (GD) is an autosomal recessive inborn error of glycosphingolipid metabolism caused by loss-of-function mutations in the gene encoding lysosomal glucocerebrosidase. This results in accumulation of glucosylceramide in tissue macrophages, systemic macrophage activation, and a complex systemic phenotype that includes three types based on the presence or absence of neurological manifestations and their rate of progression.1

Epidemiology Gaucher disease is the commonest sphingolipidosis. Over 90% of affected individuals have type 1 disease. The prevalence of

type 1 GD is 1/40 000 to 1/60 000 in the general population.2 Type 1 GD is the most frequent inherited lysosomal storage disease in the Ashkenazi Jewish population, among whom the carrier frequency reaches 1/14.3 Type 2 GD occurs in 1/500 000 live births and is panethnic. Type 3 GD occurs in less than 1/100 000 live births.3 The largest group of patients with type 3 GD has been reported from the province of Norrbotten in Sweden.4

Genetics Gaucher disease is inherited as an autosomal recessive disorder. The gene for glucocerebrosidase is located on chromosome 1q21. More than 250 different disease-causing mutations are recognized.5 Patients with GD have highly variable presentations and symptoms that, in many cases, do not correlate well with specific genotypes. The mutations are distributed throughout the gene. Most are missense mutations, but frame shift, splice site insertion, deletions and recombinant alleles carrying multiple mutations have been described. The mutations 1226G (N370S), 1448C (L444P), 84GG, and IVS2 (þ1) account for over 95% of the Jewish Ashkenazi alleles. In nonJewish patients, the commonest mutation is 1448G.6 Patients with the mutation N370S do not present with neurological symptoms. The L444P mutation is often associated with types 2 and 3 GD with severe manifestations, including perinatal lethal disease. L444P homozygotes appear to be at major risk for development of pulmonary disease at a young age.7

Clinical manifestations Three major clinical subtypes are recognized, based on the presence and age of onset of the neurological manifestations.1 Type 1 GD is the most frequent and is variable in symptoms and progression. Type 2 GD, the acute neuronopathic variant, and Type 3, the chronic neuronopathic disease, are progressive diseases. The main clinical features are summarized in Table 1. Nearly 70% of patients with type 1 GD have some degree of abnormal pulmonary function. This includes airways obstruction, reduced lung volume and alveolar-capillary diffusion abnormalities.8

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 11: Metabolic and inherited connective tissue disorders involving the lung Table 1 Epidemiological and clinical features of Gaucher disease

Characteristic

Type 1 GD

Type 2 GD

Type 3 GD

Age at onset

Childhood/adult

Infancy

Childhood

Ethnic group

Panethnic/Ashkenazi Jews

Panethnic

Panethnic/Norrbottnian (Northern Swedish)

Hepatosplenomegaly

Mild-severe

Mild

Mild-severe

Neurodegenerative

None

Severe

Mild-progressive

Course/Survival

Childhood/adult

< 2 years

Childhood/adult

Figure 2. Gaucher disease. Gaucher cell cytoplasm has a striated tissue-paper-like appearance. Figure 1. Gaucher disease. Five-year-old child with type 1 disease. A CT of the chest shows reticulonodular infiltrates, a ground-glass appearance and accentuation of the interlobular septa. (Courtesy of Dr Irith Hadas-Halpern, Jerusalem, Israel.)

Overt lung disease is seen in a minority of GD patients. Approximately 5% of patients with type 1 GD present with clinically or radiologically significant pulmonary involvement, usually in the form of interstitial lung disease.9 Pulmonary hypertension, the most serious pulmonary complication of GD, is detected in 7% of patients.10 Risk factors for progressive life-threatening pulmonary hypertension in Gaucher patients include family history, female gender, splenectomy, ACE 1 gene polymorphism, and non-N370S GD gene mutation.11 Pulmonary involvement is seen in two-thirds of infants with type 2 GD and is the most severe non-neurological feature of this subtype.12 It usually takes the form of bilateral interstitial lung disease and may lead to respiratory insufficiency and death.13 These infants also suffer from recurrent aspiration pneumonia, resulting from uncoordinated swallowing and episodes of laryngeal spasm that frequently constitute the immediate cause of death.12 Lung involvement may be asymptomatic early in the course of type 3 GD but it is also ultimately one of the major causes of death in patients affected with this subtype. Restrictive lung disease can develop secondary to thoracic skeletal abnormalities.14

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Pulmonary radiology HRCT demonstrates reticulonodular infiltrates in type I GD (Figure 1).15 Interlobular septal thickening and an irregular interface at the pleural surfaces correspond to the interstitial component of the disease. A ground-glass appearance reflects interstitial or intra-alveolar infiltration by Gaucher cells.16 Changes indicative of pulmonary hypertension may be seen.17 Chest radiographs of infants with type 2 GD frequently show an interstitial pattern and/or a ground-glass appearance, consistent with either Gaucher cell infiltration or peribronchiolar infiltrates, secondary to chronic aspiration.18 Interstitial lung disease is observed radiologically in most patients with type 3 GD. Findings vary from interstitial reticulonodular infiltrates to pulmonary fibrosis or a ground-glass appearance.

Pathology The morphological hallmark of GD is the Gaucher cell. It is a 20–100 µm in diameter round, polygonal or spindled cell of monocyte/macrophage origin with abundant pale cytoplasm and delicate linear striations, imparting a wrinkled tissue-paper appearance (Figure 2). The characteristic cytoplasmic striations are caused by lysosomes engorged with non-metabolized glycolipid. These striations are accentuated by staining with periodic

Chapter 11: Metabolic and connective tissue disorders involving the lung Figure 3. Gaucher disease. The intra-alveolar spaces of the lung are stuffed with Gaucher cells. There is preservation of the alveolar septa.

Figure 4. Gaucher disease. Nodular collections of Gaucher cells expand alveolar septa.

Gaucher cells are seen in alveolar capillaries of all patients with extensive GD and this feature is probably not confined to the lungs. There are only rare descriptions of the pathology of pulmonary hypertension in GD. These include capillary plugging by Gaucher cells, multiple bone marrow emboli and a primary pulmonary hypertension-like picture with angiomatoid lesions (see Chapter 18).19,20 Pulmonary involvement in infants with type 2 GD shows an intra-alveolar accumulation of Gaucher cells and/or aspiration pneumonia.21,22 At autopsy, type 3 GD patients with pulmonary involvement show mainly interstitial Gaucher cells infiltrate. Alveolar and capillary plugging may also be seen.17,23,24 Lung fibrosis is uncommon in this subtype. Figure 5. Gaucher disease. Cells expand an alveolar septal capillary. (arrow)

acid Schiff (PAS) and anti-CD68 immunohistochemical antibody. Gaucher cells stain pale blue to gray with the Romanovsky stain and also stain with nonspecific esterease. They contain iron micelles and tartrate-resistant acid phosphatase. Our knowledge of the pulmonary pathology in GD is gleaned primarily from autopsies of patients with advanced GD. Varying numbers of Gaucher cells are seen in the lungs of all patients dying of GD and significant lung pathology is seen in approximately one-third of these subjects. Pulmonary disease in GD usually accompanies severe systemic involvement. Four patterns of pulmonary involvement are recognized:16,17,19 (1) filling of alveolar spaces by Gaucher cells, giving rise to a lipid pneumonia-like pattern with preservation of the alveoli (Figure 3); (2) multifocal massive alveolar septal accumulation of Gaucher cells, causing septal thickening (Figure 4); (3) peri-lymphatic infiltration by Gaucher cells; (4) Gaucher cells in alveolar septal capillaries (Figure 5).

Cytology Gaucher cells can be recognized in bronchoalveolar lavage fluid by their distinctive cytoplasmatic striations.25

Electron microscopy The cytoplasm of Gaucher cells contains dilated, elongated, membrane-bound vesicles (0.6–4 µm) with ∼350 Å in diameter twisted tubular structures. About 90% of each tubule is composed of glucosylceramide (Figure 6).

Laboratory findings Definitive diagnosis of GD is via assay demonstrating reduced β-glucocerebrosidase activity in the peripheral blood in conjunction with DNA mutation analysis.26

Pathophysiology GD is an autosomal recessive inborn error of glycosphingolipid metabolism resulting from deficient activity of the lysosomal enzyme glucocerebrosidase. The substrate glucocerebroside accumulates in monocyte-macrophage cells, particularly in the liver, spleen, lymph nodes and bone marrow.

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(a)

(b)

The etiology and pathogenesis of pulmonary hypertension in type 1 GD are unclear and are probably multifactorial. Pulmonary capillary plugging by Gaucher cells, hypoxia due to alveolar septal involvement, intra-pulmonary arterio-venous shunts, thromboembolic disease due to hypercoagulability, and/or multiple bone marrow emboli are all possible causes. Substrate burden and macrophage activation probably contribute to the disease.27 Inflammatory markers, such as C-reactive protein, are predictors of pulmonary hypertension in GD patients but the mechanism is not known.27

Differential diagnosis Pulmonary involvement in patients with GD is usually associated with multiorgan disease and is rarely the presenting feature. Conditions characterized by accumulation of distended macrophages in the lung may mimic GD histologically but usually a correct diagnosis can be made if the clinical context and unique wrinkled appearance of the Gaucher cells are taken into consideration. Desquamative interstitial pneumonia is a smoking-related disease characterized by intraalveolar accumulation of macrophages (see Chapter 10). Exogenous lipid pneumonia due to mineral oil aspiration usually occurs in the lung bases and features large vacuoles within macrophages and multinucleated giant cells. These macrophages are smaller than Gaucher cells and contain intracytoplasmic fat. The macrophages in exogenous lipid pneumonia are also not usually PAS-positive. Endogenous lipid pneumonia develops in a segment of lung distal to bronchial obstruction. Pseudo-Gaucher cells may be seen in atypical mycobacterial infection (usually Mycobacterium avium-intracellulare), but massive numbers of acid-fast bacilli will be found in the macrophages (see Chapter 6).28 Intra-pulmonary storage cells are seen in some other hereditary diseases including Niemann-Pick disease, Hermansky-Pudlak syndrome, Krabbe disease, Farber disease, GM1 gangliosidosis and Wolman disease (see Table 2).29 The pulmonary manifestations are rarely the presenting event in these conditions. In most of the lysosomal storage diseases, other than GD, the macrophages have

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Figure 6. Electron photomicrographs of Gaucher cells. (a) Intracytoplasmic membrane-bound lysosomal vesicles are abundant. (Courtesy of M. Selig, Boston, MA, USA.) (b) Packed tubular structures are noted within the vesicles. (Courtesy of Z. Ne’eman, PhD, Jerusalem, Israel.)

vacuolated cytoplasm. The storage cells in some cases of GM1 gangliosidosis are similar morphologically to Gaucher cells on light microscopy but not ultrastructurally. Clinical features, biochemical assay and molecular studies are required for diagnosis confirmation. Other rare pulmonary entities that morphologically mimic GD include crystal-storing histiocytosis with or without an underlying lymphoproliferative disorder, Rosai-Dorfman disease, Erdheim Chester disease, malakoplakia and Whipple disease.30 Crystal-storing histiocytosis is a rare manifestation of plasma cell dyscrasias/B-cell lymphoproliferative diseases, characterized by an accumulation of histiocytes that have phagocytosed an abnormal crystalline immunoglobulin (see Chapter 34).30,31,32,33 The crystal-bearing histiocytes have eosinophilic cytoplasm with numerous non-birefringent needleshaped eosinophilic crystals. These cells differ morphologically from Gaucher cells (Figure 7). Furthermore the crystals stain with PTAH, are negative for PAS and their characteristic appearance can be demonstrated by electron microscopy. Rosai-Dorfman disease (sinus histiocytosis with massive lymphadenopathy) may occur in the lung. It is usually in the form of tumor masses affecting large airways and occasionally as diffuse parenchymal involvement. The histiocytes are large, contain intact lymphocytes in their cytoplasm (emperipolesis) and stain strongly and diffusely with S100 protein (see Chapter 34). In Erdheim Chester disease there are typical radiological findings in the long bones. Pulmonary involvement shows a sharply defined lymphangitic distribution and is composed of finely vacuolated histiocytes, inflammatory cells and fibrosis (see Chapter 34).34 Fibrosis is uncommon in GD. Pulmonary malakoplakia is usually due to Rhodococcus equi infection and usually occurs in HIV-positive patients. There is a solitary or multinodular infiltrate composed of finely vacuolated macrophages. Distinctive calcospherites, known as Michaelis-Gutman bodies, are seen in and around the macrophages. They stain with von Kossa and PAS (see Chapter 4).

Chapter 11: Metabolic and connective tissue disorders involving the lung Table 2 Features of storage cells in various lysosomal storage diseases

Gaucher Light microscopy : a 20–100 μm in diameter lipid-filled macrophage with striated “crumpled tissue-paper” cytoplasm Histochemistry : the cytoplasm is strongly PAS-positive and is pale blue to gray with the Romanovsky stain; it also stains with nonspecific esterease and contains iron micelles and tartrate resistant acid phosphatase Ultrastructure : the cytoplasm of Gaucher cells contains dilated membrane-bound vesicles (0.6–4 μm) containing twisted structures composed mainly of glycosylceramide Niemann-Pick Light microscopy : a 25–75 μm in diameter foamy macrophage with numerous uniform-sized lipid droplets. Droplets are birefringent under polarized light and greenish to brownish yellow under UV light Histochemistry : on frozen section the lipid droplets stain with Sudan black B and oil red O. The Schultz reaction for cholesterol is positive. N-P cells stain poorly with PAS. So-called “sea-blue histiocytes” may be seen on Giemsa staining of bone marrow cells Type C N-P cells stain variably with PAS, Luxol fast blue, Sudan black and strongly with Schultz reaction and acid phosphatase. Cultured type C N-P fibroblasts fluoresce with filipin Ultrastructure : the foam cells contain numerous 0.5–50 μm in diameter frequently multilaminated secondary lysosomes. They are variable in appearance but are generally smaller than those in Tay-Sachs disease

Krabbe Light microscopy : multinucleated “globoid” macrophages in the brain; large intra-alveolar macrophages, some multinucleated, with eosinophilic cytoplasm and large brown or amber colored intracytoplasmatic inclusions Histochemistry : PAS-positive Ultrastructure : large amorphous electron-dense membranebound inclusions in the cytoplasm of pulmonary macrophages Pompe Light microscopy : vacuoles in muscle fibers Histochemistry : PAS-positive diastase sensitive, acid phosphatase Ultrastructure : membrane-bound and free glycogen; autophagic vacuoles in the infantile type Farber Light microscopy : foam cells and/or macrophages Histochemistry : PAS-positive Ultrastructure : banana-shaped Farber bodies

Fabry Light microscopy : large vacuolated cells with Maltese cross birefringence under polarized light microscopy Histochemistry : modified PAS and Sudan black Ultrastructure : characteristic concentric or lamellar inclusions (zebra bodies) Hermansky-Pudlak Light microscopy : vacuolated type II pneumocytes and intraalveolar macrophages, autofluorescent ceroid in intra-alveolar and interstitial macrophage Histochemistry : phospholipid can be demonstrated by the acid hematin method on frozen sections; the ceroid can be demonstrated by PAS, acid-fast stains and the Schmorl method Ultrastructure : large or giant lamellar bodies in type II pneumocytes and ceroid-like material in macrophages Mucopolysaccharidoses Light microscopy : enlarged vacuolated balloon cells Histochemistry : colloidal iron positive and less frequently Alcian blue positive Ultrastructure : dilated lysosomes that are empty or contain finely granular GAG or circular circumscribed electron dense bodies (zebra bodies) GM1 gangliosidosis Light microscopy : cells with small pyknotic eccentric nuclei and abundant granular or vacuolated cytoplasm Histochemistry : intensely PAS-positive; also stains with Alcian blue and toluedine blue Ultrastructure : macrophages contain numerous lysosomes bearing lamellated membrane structures

Figure 7. Crystal-storing histiocytosis. There is a diffuse infiltrate of histiocytes stuffed with refractile eosinophilic crystals that mimic the linear striations seen in Gaucher cells.

Whipple disease is caused by infection with Tropheryma whipplei. Rare cases of clinically significant pulmonary Whipple disease occur, characterized by parenchymal nodules or endobronchial lesions. Infiltration by PAS-positive histiocytes is seen on lung biopsy (see Chapter 4).35

Prognosis and natural history Enzyme replacement therapy (ERT) with recombinant acid β-glucosidase is the standard treatment of symptomatic type I GD. It produces dramatic resolution of hepatosplenomegaly and hematological symptoms. A few treated patients experience clinical amelioration of pulmonary symptoms, although pulmonary function disturbances and disturbances of lung architecture are slow to respond.9

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Pulmonary infiltrates in subjects with types 2 and 3 GD do not respond to ERT.36 Alternative therapeutic approaches are currently being tested, including substrate inhibition therapy, which has recently been approved, gene therapy and enzyme enhancement therapy (chemical chaperones). The latter is based on the use of chaperone molecules that assist the folding of mutated enzymes and improve their stability and lysosomal trafficking.37 Hematopoietic stem cell transplantation offers GD patients the potential of permanent cure. There are no clinical trials comparing the safety and efficacy of this method with more conservative treatment modalities.38 There is a single report of bilateral pulmonary transplantation for end-stage lung disease due to severe interstitial fibrosis associated with type 1 GD, without evidence of recurrent disease in the transplanted lungs.39 An association between ERT and pulmonary hypertension is controversial. In some subjects pulmonary hypertension develops or is even exacerbated after commencement of ERT.10 This has been attributed to closure of arteriovenous shunts in the lung, following the reduction of hepatomegaly by the ERT or to possible contaminants in the ERT.10 Others report an improved outcome of the pulmonary hypertension in patients receiving ERT.11,12

Niemann-Pick disease, types A and B Introduction

Niemann-Pick disease (NPD) types A and B (NP-A, NP-B) are rare inherited lysosomal storage disorders caused by deficient activity of the enzyme acid sphingomyelinase (ASM). This enzyme hydrolyzes sphingomyelin to ceramide and phosphocholine. Niemann-Pick disease types A and B are characterized by storage of sphingomyelin in various tissues and organs, especially the liver and spleen. Niemann-Pick type A runs a rapidly neurodegenrative course, which causes death by 3 years of age. Niemann-Pick type B patients have a less severe form with mild or no neurological involvement.

Epidemiology The estimated prevalence of NP-A and NP-B together is 1/264 000. Ashkenazi Jews have a higher incidence of NP-A, with an estimated carrier frequency of about 1/90 and an estimated birth rate of 3/100 000.40 Niemann-Pick type B is panethnic but appears to be more frequent in individuals of Middle Eastern and North African descent.41

Genetics Patients with NP-A and NP-B have an inherited deficiency of ASM produced from a gene (SMPD1) located on chromosome 11. Over 100 mutations causing ASM deficiency are described and some phenotype-genotype correlations have been made.41 DNAbased carrier screening of Ashkenazi Jews is informative, since three mutations account for over 90% of NPD in this population.

Clinical manifestations Niemann-Pick disease type A presents in early infancy with failure to thrive, hepatosplenomegaly and neurological abnormalites. Neurodegeneration leads to death within 3 years.42

414

Figure 8. Niemann-Pick, type B disease. Niemann-Pick cells have distended finely vacuolated cytoplasm.

Niemann-Pick disease type B is phenotypically variable. It presents later, usually in childhood, with hepatosplenomegaly. Patients usually do not have neurological involvement and often survive into adulthood. Pulmonary parenchymal involvement is seen in both subtypes of NPD. Patients are asymptomatic or suffer from shortness of breath and a non-productive cough. Infiltration of the pulmonary interstitium by NP cells occurs in all NP-A patients and may be out of proportion to the clinical manifestations. These patients suffer recurrent respiratory infections. The usual immediate cause of death is respiratory failure with or without pneumonia.42 In NP-B pulmonary involvement is rarely a presenting feature but most patients develop abnormal pulmonary function and radiological evidence of lung disease in the course of the disease.43 More severely affected patients often suffer considerable morbidity and mortality from progressive pulmonary infiltrates and recurrent pulmonary infections.44 Disease with cor pulmonale is also described.45

Pulmonary radiology On chest radiographs, diffuse, bilateral, reticular/nodular interstitial infiltrates are seen in most patients with NP-A. On chest radiographs and computed tomography (CT) scans 90% and 98% of patients with NP-B have evidence of lung disease, respectively.46,47 The changes are often out of proportion to the clinical manifestations. On high-resolution CT (HRCT) a ground-glass pattern is seen in the upper zones of the lungs, corresponding to airspace consolidation. Thickening of the interlobular septa is seen in the lower lung zones, matching the mild interstitial fibrosis and interstitial expansion by NPD cells.48,49 50 Rarely lung cysts are seen.51

Pathology The lipid-laden foamy macrophage, known as the NP cell, is the pathological hallmark of NPD. These cells have a small dense nucleus and finely vacuolated foamy cytoplasm, composed of uniform lipid droplets (Figure 8). They range in size

Chapter 11: Metabolic and connective tissue disorders involving the lung

Figure 9. Niemann-Pick, type B disease. Lung biopsy features endogenous lipid pneumonia with an accumulation of foamy macrophages within the alveoli. Foamy macrophages are also seen in the interstitium. There is little loss of alveolar architecture. (Courtesy of Dr A. Nicholson, London, UK.)

Figure 10. Niemann-Pick, type B disease. Ciliated epithelial cells show cytoplasmic vacuoles similar to those seen in the macrophages. (Courtesy of Dr A. Nicholson, London, UK.)

from 25 to 75 µm in diameter, which is smaller than the average Gaucher cell. Many of the droplets are birefringent under polarized light and greenish-yellow or brownish-yellow under UV light. On frozen section the lipid droplets stain with Sudan black B and oil red O.52 The Schulz reaction for cholesterol is positive in most NPD cells and the cells stain poorly with PAS. These two reactions distinguish the cells of NPD from those of GD. The foamy NP cells are found in neurons, glial cells, splenic red pulp and the hepatic sinusoids. In bone marrow NP cells have been called sea-blue histiocytes, because of the bluish granulations seen on May-Grünwald-Giemsa staining. This finding is not pathognomonic of NP. A lipoid pneumonia with filling of the alveolar spaces by foamy macrophages is the commonest histological finding. Interstitial infiltration is also observed (Figure 9).48,53 Underlying lung architecture is preserved, although mild interstitial fibrosis may be seen.54 Foamy macrophages may also be seen in pulmonary arteries and lymphatics. In NP-B ciliated airway epithelial cells but not type II pneumocytes are vacuolated (Figure 10).48 This feature helps to distinguish between NP-B and NP-C2 (see below).

Cytology Foamy macrophages may be identified in bronchoalveolar lavage fluid from patients with NPD.55

Electron microscopy On electron microscopy, the cytoplasmatic vacuoles are enlarged secondary lysosomes. These often contain coarse osmiophilic membranous bodies with concentric lamellar stacks within a clear matrix (Figure 11).56

Laboratory findings The most consistent laboratory finding in NPD is a highly atherogenic lipid profile. The diagnosis of NP-A and NP-B is

Figure 11. Electron photomicrograph of an epithelial cell from an individual with adult form of Niemann-Pick disease. Numerous intracytoplasmic secondary lysosomes with coarse lamellated membrane structures are seen. (Courtesy of M. Selig, Boston, MA, USA.)

made by enzymatic determination of acid sphingomyelinase activity in leukocytes, fibroblasts, and/or tissue extracts. Heterozygote detection by enzyme assay is unreliable and molecular studies are required. Prenatal diagnosis can be performed by enzymatic or molecular analysis of DNA extracted from cultured amniocytes or chorionic villi.57 Preimplantation genetic diagnosis is possible when the mutation is known.

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Pathophysiology Lysosomal acid sphingomyelinase participates in membrane turnover and degradation. It plays a role in plasma membrane reorganization in response to stress and other factors leading to apoptosis or senescence of cells.58 Altered macrophage function and surfactant dysfunction may account for the extent of lung disease and recurrent infections in NPD. An ASM knock-out (ASMKO) mouse model of NPD with pulmonary involvement, similar to that of human NPD, demonstrates massive accumulation of storage macrophages in the lungs and inflammatory cells in the airways. In addition there is increased chemokine production, upregulation of genes involved in inflammation and/or immunity and abnormal surfactant function.59,60 The accumulation of NPD cells, together with fibrosis, is responsible for impaired gas exchange. Excess sphingomyelin interferes with surfactant function and catabolism, causing an alveolar proteinosis-like picture in this model. This may also enhance susceptibility to infection.61

Differential diagnosis In addition to the differential diagnosis for pulmonary involvement in GD discussed above, amiodarone toxicity must be excluded. Useful features are age, heart and drug history. Foamy macrophage infiltration in amiodarone is less dense than that seen in NP-B. Type B disease also features foamy cytoplasm in ciliated epithelial cells.48 Other mimics of NPD include exogenous and endogenous lipid pneumonia and diffuse panbronchiolitis. The latter occurs mainly in Japan, and is usually associated with chronic sinusitis. This rare process is characterized by an acute and chronic cellular bronchiolitis with peribronchiolar foam cells (see Chapter 17).

Prognosis and natural history There is no specific therapy for NPD. Whole lung lavage may cause symptomatic improvement61 although poor response to this modality has also been described.44 Good outcome has been reported in a few patients with NP-B after bone marrow transplantation, including resolution of pulmonary infiltrates.62 Another approach has been partial or total splenectomy.63 These procedures may enhance the rate of progression and severity of pulmonary disease.40 Enzyme replacement therapy in ASMKO mice demonstrates promising results for reticulo-endothelial mobilization of storage cells and delayed, but good pulmonary clearance of storage cells.64 The delay may be due to the relatively long life of the pulmonary foamy NPD cells.40 Direct pulmonary delivery results in good pulmonary clearance and may represent an alternative route of delivery.65 In view of these and other findings in NPD animal models, clinical trials evaluating the efficacy of ERT in patients with non-neurological ASM-deficient NPD have recently been approved. Gene therapy has also achieved marked visceral organ improvement in ASMKO mice, including reversal of pulmonary pathological changes.65,66

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Niemann-Pick disease, type C Introduction

Niemann-Pick disease type C (NP-C) is an autosomal recessive sphyngolipidosis with protean clinical manifestations.67 It arises from mutations in the NPC1 or NPC2 gene. Both genes probably play a role in intracellular cholesterol and glycolipid trafficking. Free cholesterol accumulates in the peripheral organs, while glycosphingolipids GM2 and GM3 gangliosides accumulate in the brain.67 Niemann-Pick disease type C is characterized by progressive neurodegeneration. The clinical manifestations of NP-C1 and NP-C2 are similar because the respective genes are both involved in the egress of lipids, particularly cholesterol, from late endosomes or lysosomes.68

Epidemiology The prevalence of NP-C is 1/150 000, making it a commoner phenotype than both NP-A and NP-B combined.63,65 The true prevalence of NP-C has been underestimated, due to evolving recognition of different phenotypes and testing methods. Niemann-Pick disease type C is panethnic although genetic isolates of NP-C have been described in Novia Scotia and southern Colorado.69,70

Genetics Approximately 95% of patients have mutations in the NPC1 gene and 5% in the NPC2 gene. The NPC1 gene is mapped at 18q11 and encodes a large membrane glycoprotein, primarily located in late endosomes. The NPC2 gene is mapped at 14q24.3.71 Molecular analysis of NP-C patients is challenging because of the size of the genes and the number of private mutations.

Clinical manifestations Niemann-Pick disease type C presents in childhood and patients die in the second or third decade. Most patients have progressive neurological disease. Other phenotypes include hepatic disease, fetal ascites, fatal neonatal liver disease, early infantile onset with hypotonia and delayed motor development, and adult variants with psychiatric illness and dementia.67 The lungs are usually spared in NPC1, but ultimately patients develop dysphagia, and death is often due to related pulmonary complications. In the minor subgroup, NP-C2 (minor complementation group), patients may develop early severe respiratory failure.55,72–74

Pulmonary radiology Chest radiographs and CT of patients with NPC2 with pulmonary involvement show diffuse reticulonodular infiltrates or ground-glass type consolidation.55,74 These findings have also been described in some patients with NPC1 and do not appear to correlate well with clinical symptoms.55

Pathology Both foamy macrophages (NP cells) and sea-blue histiocytes may be seen in visceral organs in NP-C. They stain variably with PAS, Luxol fast blue and Sudan black, and strongly with

Chapter 11: Metabolic and connective tissue disorders involving the lung

the Schultz reaction for cholesterol and acid phosphatase. Cultured type C NP fibroblasts fluoresce with filipin. The cardinal pathological features of NP-C are visceral infiltration by foamy macrophages, involving spleen, liver, lymph nodes, tonsil, lung, and neuronal storage. The literature on pulmonary pathology in NP-C is limited. Changes are similar to those observed in NP-B and include an endogenous lipid pneumonia-like picture and pulmonary fibrosis.48 While cytoplasmatic vacuolation of type II pneumocytes is seen in NP-C, unlike the case in NP-B, ciliated epithelial cells are not involved.48 An alveolar proteinosis-like picture with intra-alveolar foamy macrophages has been described in siblings with type NP-C2.75,76 In another patient with this subtype, pulmonary infiltration by storage cells was accompanied by irregular emphysema, probably due to accumulation of foam cells in bronchiolar lumina.73

Electron microscopy See ultrastructural features of NP-A and B.

Laboratory findings Diagnosis of NP-C is based on the demonstration of a lysosomal accumulation of unesterified cholesterol (Filippin test) and the esterification test on cultured fibroblasts.71. Molecular analysis can confirm the diagnosis and distinguish between NP-C1 and NP-C2.67

Pathophysiology NP-C1 and NP-C2 proteins are essential for proper cellular cholesterol and glycolipid trafficking but their precise functions and relationships are unknown.77 Visceral organ macrophages exhibit marked cytoplasmic vacuolation, due to accumulation of cholesterol, phospholipids and glycolipids. This causes deregulation of lysosomal calcium.78 It is not known why NPC2 is associated with rapidly progressive respiratory disease, although in a few cases storage has been demonstrated.

Treatment and prognosis Cholesterol-lowering drugs do not improve neurological symptoms.79 A child with NP-C had a good visceral response to bone marrow transplantation with reversal of pulmonary interstitial involvement, but neurological deterioration was not halted.80 Recently substrate reduction therapy with miglustat, an inhibitor of glucosylceramide synthase, has been used to treat progressive neurological disease in adult and pediatric patients with NP-C, but it is not expected to have an effect on systemic manifestations.81

Fabry disease Introduction

Fabry disease (FD) is an X-linked recessive storage disease caused by deficient α-galactosidase A activity. This results in abnormal accumulation of glycosphingolipids in lysosomes, primarily in the form of globotriaosylceramide (Gb3). Substrate deposition occurs mainly in body fluids and in vascular endothelial, smooth muscle and perithelial cell lysosomes of

affected hemizygous males, as well as female carriers and children.

Epidemiology Fabry disease is a panethnic disease with an estimated incidence of 1/40 000 to 1/117 000 live male births.82,83 Heterozygous women were thought to be healthy carriers of FD but it has been shown such women can be affected with varying degrees of severity. Affected males present the classic phenotype with symptoms beginning in childhood, while females are symptomatic in later life.

Genetics The gene coding for α-galactosidase A is located on X q21–22. More than 300 mutations have been identified in the GLA gene. Point mutations, missense or nonsense mutations are the most frequent, but small and large deletions or insertions are also seen. Mutations leading to complete gene product loss of function are associated with classic disease, whereas missense mutations might be associated with a milder phenotype. The clinical presentation is variable with respect to the age of onset, rate of progression and organ manifestations, even within one family.84

Clinical manifestations Fabry disease is a progressive multisystem disease presenting in childhood or adolescence. Clinical features include severe pain and parasthesiae of the extremities, angiokeratomas of the skin and mucosa, hypohidrosis and corneal and lenticular opacities. It may progress to cardiovascular, cerebrovascular and renal complications. Clinically significant respiratory impairment, not associated with smoking or cardiac disease, occurs in 61% of men and 25% of women with FD. The disease becomes progressively more severe with age in men.85 Pulmonary symptoms and abnormalities include cough, dyspnea, wheezing, hemoptysis, pneumothorax and recurrent respiratory infections. Exercise intolerance, due to hypohidrosis, pulmonary disease or cardiomyopathy, may begin early in childhood.86 A large proportion of patients show airway obstruction on spirometry. Airway obstruction initially involves small airways and progresses to upper lobe emphysema.85,87

Pulmonary radiology Chest radiographs are normal or demonstrate bullous emphysema in the upper lobes.87–89 There is a single report of severe bilateral, predominantly upper lobe, mosaic attenuation on chest CT reflecting ground-glass opacities and air trapping.90 The ground-glass opacities, but not the air trapping, improve on ERT, implying that the opacities represent alveolar-filling by glycosphingolipid.90

Pathology On light microscopy, large vacuolated cells infiltrate various organs. Sudan black and modified PAS histochemical stains

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Chapter 11: Metabolic and inherited connective tissue disorders involving the lung Figure 13. Fabry disease. Electron photomicrograph of the apical part of a ciliated bronchial epithelial cell showing numerous myelin-figure-like whorls in the lysosomes. (Courtesy of Dr M. Elleder, Prague, Czech Republic.)

Figure 12. Fabry disease. Transverse section of a muscular pulmonary artery. The glycosphingolipid-laden intimal myofibroblasts have proliferated and induced deposition of elastic fibers around individual cells. Elastic van Gieson. (Courtesy of Dr P. Hasleton, Manchester, UK.)

highlight vacuoles. The accumulated crystalline glycosphingolipids are birefringent and show a Maltese cross configuration under polarized light.86 There are few well-documented descriptions of the pulmonary pathology in FD. Lungs of patients with FD are heavier than normal. There is peribronchiolar smooth muscle hyperplasia and fibrosis.91 The muscular pulmonary arteries, arterioles, veins and venules are hypertrophied and show vacuolation of medial smooth muscle cells and intimal myofibroblasts (Figure 12).92,93 Centrilobular and panacinar emphysema have been described.

damage. Vascular endothelial cell storage leads to ischemic events in different sites by three possibly related mechanisms: luminal narrowing and occlusion, altered vascular reactivity, and a prothrombotic state.83,97 The airways obstruction in some FD patients probably results from fixed narrowing of the airways by glycosphingolipid accumulation in epithelial and/or smooth muscle cells.87 As the disease progresses, larger airways and other pulmonary structures become involved, particularly in males.

Cytology

Prognosis and natural history

Glycosphingolipid accumulation may be confirmed by ultrastructural examination of ciliated epithelial and goblet cells obtained by induced sputum, bronchoalveolar lavage or bronchial brushings in patients with pulmonary involvement.94,95

Genotypic and phenotypic correlations are noted but not well understood.98 Before the advent of ERT, life expectancy was approximately 50 years in men and 70 years in women. Obstructive airways disease, respiratory infection and cardiac complications are the usual causes of death.83 Enzyme replacement therapy in the form of agalsidase α and β slows disease progression and prolongs life.99 Dramatic responses, including weaning from supplemental oxygen, have been reported.90

Electron microscopy Intracellular pulmonary lipid deposits are seen in type II pneumocytes, goblet cells, ciliated epithelial cells, pulmonary capillary and arteriolar endothelial cells, arteriolar smooth muscle cells and bronchial smooth muscle cells but not in pulmonary macrophages.88,91–93 The lipid is stored in lysosomes in the form of characteristic myelin figure-like whorls or parallel arrays of membranes (“zebra bodies”) and differs morphologically from the concentric lamellar structures normally seen in type II pneumocytes (Figure 13).85,93

Laboratory findings Clinical diagnosis is confirmed by assay of α-galactosidase A activity in plasma and/or leukocytes, and gene-sequencing analysis. Genetic testing is also performed in females.96

Pathophysiology Lysosomal accumulation of Gb3 leads to lysosomal and cellular dysfunction. Secondary inflammation leads to irreversible cellular

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Hermansky-Pudlak syndrome Introduction

Hermansky-Pudlak syndrome (HPS) is a rare genetically heterogeneous autosomal recessive disorder caused by defects in lysosome-related organelles, including melanosomes and platelet-dense bodies.100,101 It is characterized by tyrosinasepositive oculocutaneous albinism, platelet storage pool deficiency, and an accumulation of lysosomal ceroid lipofuscin in various tissues, particularly the lung and gut.

Epidemiology Type 1 HPS (HPS-1) accounts for the preponderance of this disease in the world by virtue of a genetic isolate in northwest Puerto Rico, where it is thought to affect 1/1800 individuals with a carrier rate of 1/21.102 It is also relatively frequent in an

Chapter 11: Metabolic and connective tissue disorders involving the lung

Figure 14. Hermansky-Pudlak syndrome. High-resolution computed tomographic chest scan shows thickening of interlobular septa, reticular pattern of fibrosis with focal honeycomb change, ground-glass opacities and traction bronchiectasis. (Courtesy of Dr B. Gochuico, Medical Genetics Branch, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD, USA.)

isolated village in the Swiss Alps.103 The disease is rare in other parts of the world, with a prevalence of 1/500 000 to 1/1 000 000.104 Men and women are equally affected but lung involvement is twice as common in females.105 Pulmonary disease is seen mainly in patients with mutations in the HPS1 and HPS4 genes, with an estimated 80% of HPS1 subtypes afflicted.106 Lung disease is a major cause of morbidity and mortality except for subjects with the BLOC-2 mutation, who do not develop pulmonary fibrosis (see below).

Genetics There are eight known human HPS genes, each of which can lead to a specific clinical variant of the disease. There are at least 16 mouse models.100 Only five of the genes have known functions, and four have roles in regulating membrane/vesicle and protein trafficking. The eight gene products operate in distinct complexes, e.g., BLOC-2 contains HPS3, HPS5 and HPS6 subunits. Mutations in one of the genes coding for HPS3, HPS5 and HPS6 are associated with mild clinical involvement. Mutations in the genes coding for the subunits HPS1 and HPS4, which form the complex BLOC-3, cause more severe complications. Mutations in the subunit HPS2 from the adaptor protein (AP3) complex are associated with immune deficiency.107 The human HPS-1 gene is located on chromosome segment 10q23.1–q23.3. Twenty-three disease-causing mutations have been reported. The commonest mutation, found exclusively in the Puerto Rican population, is caused by a 16-bp frameshift duplication in exon 15.108

Clinical manifestations Patients exhibit varying degrees of hypopigmentation, bleeding diathesis, impaired visual acuity and ophthalmic involvement. A more severe phenotype is observed in type 1 and type

Figure. 15. Hermansky-Pudlak syndrome. Section through lungs at autopsy of a patient shows advanced fibrosis with honeycomb change. Cystic changes are more prominent in the upper lobes. (Courtesy of Dr N. Nakamura, Yokohama, Japan, and Dr Yukio Nakatani, Chiba, Japan.)

4 HPS. Restrictive lung disease and granulomatous colitis are major causes of morbidity and mortality in these two types and both are associated with storage of lysosomal ceroid lipofuscin. Pulmonary fibrosis usually manifests in the third and fourth decades of life and accounts for premature death in 50% of HPS patients, generally in their fifties.109 Patients typically present with an insidious onset of dyspnea and maintain normal oxygen saturation at rest, despite significant impairment of pulmonary function. In other HPD subtypes there is minimal or no pulmonary disease but definite conclusions are limited by small patient numbers.100

Pulmonary radiology Approximately half the patients with HPS1 have abnormal chest radiographs and over 80% have HRCT abnormalities.106 High-resolution CT is the imaging method of choice for evaluating the extent of pulmonary disease. The changes are diffuse with a predominantly peripheral distribution and include interlobular septal thickening, reticular disease, subpleural cysts, and ground-glass areas (Figure 14).106 With disease progression, subpleural cysts appear and the central lungs show increasing peribronchovascular thickening and traction bronchiectasis.110 The severity of the pulmonary fibrosis on HRCT correlates well with the forced vital capacity.110

Pathology The lungs of patients dying from respiratory involvement by HPS are firm and rubbery, with a bosselated, cirrhosis-like appearance. There is honeycomb change with cystic structures, frequently more prominent in the upper and middle lobes (Figure 15).

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Chapter 11: Metabolic and inherited connective tissue disorders involving the lung

(a)

(b)

(c)

Figure 16. Hermansky-Pudlak syndrome. (a) There is marked interstitial fibrosis with remodeling of the pulmonary architecture. (b) Interstitial fibrosis, patchy chronic inflammatory cell infiltrates and clusters of clear cells are seen. (c) On high-power examination the clear cells are type II pneumocytes. (Courtesy Dr Douglas B. Flieder, Philadelphia, PA, USA.)

The histological pattern is one of chronic fibrosing interstitial pneumonia superficially similar to usual interstitial pneumonia (UIP) (Figure 16a). Unlike UIP, the fibrotic process is more advanced around respiratory bronchioles than in the peripheral part of the lobule. Constrictive bronchiolitis and bronchiolectasis are often seen.111 Features indicative of HPS include patchy clusters of clear vacuolated type II pneumocytes and intra-alveolar macrophages, ceroidal material in intraalveolar and interstitial macrophages and marked chronic inflammatory cell infiltrates (Figure 16b,c).109,112 The vacuolated type II pneumocytes contain phospholipids, which can be demonstrated on frozen section by the acid hematin method (Figure 17). These pneumocytes are only weakly immunoreactive for surfactant, despite the presence of giant lamellar bodies. The ceroidal material is brown, finely granular and autofluorescent under ultraviolet light. The material can be highlighted with PAS, acid-fast stains, and the Schmorl

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Figure 17. Hermansky-Pudlak syndrome. Phospholipid in type II pneumocytes is demonstrated by the acid hematin staining method. (Courtesy of Dr Yukio Nakatani, Chiba, Japan.)

Chapter 11: Metabolic and connective tissue disorders involving the lung

Differential diagnosis The main differential diagnostic consideration is UIP. However, the distribution of the fibrosis differs from UIP. In HPS, fibrosis is more prominent in the middle and upper lobes and centered on the small airways, while UIP is usually subpleural with lower lobe predominance. Vacuolated type II cells are also particular to HPS.

Prognosis and natural history

Figure 18. Electron micrograph of pneumocytes in a patient with Hermansky-Pudlak syndrome: there are numerous giant lamellar bodies. (Courtesy of Dr Yukio Nakatani, Chiba University Hospital, Chiba, Japan and Dr Mutsue Mizushima, Gifu, Japan.)

method. Similar ceroid-filled macrophages are seen in patient bone marrow, spleen and liver samples.111

Cytology Ceroid–filled macrophages may be demonstrated in bronchoalveolar lavage fluid.113

Electron microscopy Numerous large and/or gigantic lamellar bodies are seen in type II pneumocytes compressing the nuclei and occasionally disrupting the cytoplasm (Figure 18).111 Ceroid-like material is seen in macrophages.114

Pathophysiology The pathogenesis of pulmonary fibrosis in HPS is unclear and may be related to ceroid storage. Ceroid is an autofluorescent lipid protein complex of unknown significance in many tissues and in the urine of affected individuals. Type II pneumocyte function is probably disrupted by intracellular ceroid. This may trigger an inflammatory cascade with cytokine production and ultimately fibroblast proliferation.115 However, the amount of ceroid pigment deposition does not correlate with the degree of inflammation and/or fibrosis. An alternative theory is based on the finding that degenerate giant lamellar bodies in type II pneumocytes accumulate surfactant or surfactant derivatives. The lamellar body is considered to be the fourth organelle affected by HPS, in addition to the lysosome, the melanosome and the plateletdense granule.111 It is postulated that impaired surfactant trafficking and secretion is the basic impairment, leading to apoptosis of type II pneumocytes, inflammation, macrophage dysfunction and fibrosis.116,117

Respiratory disease, followed shortly by death, usually strikes in the fourth decade of life but there is wide variation. This suggests that environmental factors and/or modifying genes may trigger or exacerbate the pulmonary pathology. Severe disease observed when in HRCT is predictive of imminent mortality.110 The aim of therapy is to preserve existing pulmonary function, especially in patients with the HSP-1 mutation. This includes avoidance of smoking and exposure to second-hand cigarette smoke and other lung toxins, prompt treatment of pulmonary infections, prophylactic immunization for influenza and pneumococcus, regular exercise and a healthy diet. Pirfenidone, a compound with anti-inflammatory, antioxidant and anti-fibrogenic effects, may slow the progression of the pulmonary fibrosis in patients who have sufficient residual pulmonary reserves.118 One successful bilateral lung transplant has been performed.112

Mucopolysaccharidoses Introduction

The mucopolysaccharidoses (MPS) are a group of lysosomal storage disorders caused by lack of specific enzymes involved in the degradation of mucopolysaccharides, more accurately known as glycosaminoglycans (GAG). Each of these disorders is characterized by progressive multisystem disease. The skeleton, joints, somatic tissues including airways, heart and, in some of the disorders, the CNS are involved. The various disease types show a spectrum of manifestations and clinical courses ranging from early onset with rapid progression leading to early death, to more attenuated forms, characterized by later onset and slower progression, often resulting in a normal lifespan.119,120

Epidemiology Hurler and Hunter syndromes are the commonest subtypes of MPS. Both are panethnic. The estimated incidence of the three phenotypes of Hurler syndrome is 1/100 000 births121 and that of Hunter syndrome 1/140 000 to 1/330 000 births.122

Genetics There are 11 identified enzyme deficiencies causing seven distinct MPS clinical presentations.120 All the MPS are autosomal recessive diseases, except MPS II (Hunter syndrome), which is X-linked.

Clinical manifestations All the MPS feature a progressive course, multisystem involvement, organomegaly, dysostosis multiplex and coarse facies. Other manifestations include disorders of hearing, vision,

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Chapter 11: Metabolic and inherited connective tissue disorders involving the lung Figure 19. Electron micrographs of a skin biopsy from a boy with mucopolysaccharidosis IIIA. (A) Skin fibroblasts and endothelial cells contain varying amounts of lysosomes. (B) Large fibroblasts feature membrane-bound lysosomes that contain fine fibrillary material. (Courtesy of Dr J. Alroy, Boston, MA, USA.)

cardiovascular function and joint mobility, and large airways involvement. The presentation and progression of signs and symptoms of MPS are variable. MPS I is the commonest form and is composed of three clinical syndromes in diminishing order of severity: Hurler, Hurler-Scheie, and Scheie syndromes. The differences in severity are due primarily to various mutations, some of which permit residual enzyme activity. Marked mental retardation is typical of MPS IH (Hurler syndrome), the severe form of MPS II (Hunter syndrome) and MPS III (Sanfilippo syndrome). Bony lesions of varying degrees may be seen in all the subtypes but are more characteristic of MPS IV (Morquio syndrome) and MPS VI. Most children with Hurler syndrome have recurrent upper respiratory tract and ear infections, noisy breathing and copious nasal discharges.120 Airways obstruction is common and is due to a combination of enlarged tongue, tonsils and adenoids, a large epiglottis, supraglottic edema, thickened vocal cords, narrowing of the trachea and thickening of the mainstem bronchi, as well as abnormal cervical vertebrae and a short neck.120 The obstruction may initially be intermittent, presenting as sleep apnea.123 Chronic hypoxemia follows, which can lead to pulmonary hypertension. Recurrent pneumonia is common. In addition there may be a restrictive component due to rib cage and vertebral column deformities and elevation of the diaphragm by an enlarged liver and spleen.122,124 Patients with MPS present anesthetic problems. Perioperative mortality approaches 20% owing to airways obstruction and coronary artery disease.125 Difficult or failed intubation occurs in MPS I and II, and patients with most subtypes, particularly MPS IV, are at risk of spinal cord compression from atlanto-axial subluxation, secondary to hypoplasia of the

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odontoid peg.126 Intraoperative cardiac arrest due to coronary artery narrowing is another risk.

Pulmonary radiology Tracheal narrowing is apparent on chest X-ray and CT. Ribs are oar-shaped and kyphosis is common.127 In MPS IV, the patients are short-trunked dwarfs with barrel-shaped, fixed chest walls, due to pectus carinatum deformity.128

Pathology The epiglottis, aryepiglottic folds and vocal cords are enlarged and the walls of the trachea and the mainstem bronchi are thickened with variable luminal narrowing. This is due to GAG deposition in extracellular connective tissue, mesenchymal and epithelial cells. Intracytoplasmic GAG accumulation gives rise to enlarged cells with vacuolated cytoplasm known as balloon cells, clear cells, Hurler cells or gargoyle cells.129,130 These cells are seen in many tissues and organs including liver, spleen, bone marrow, heart, arteries, cartilage and brain.131–133 The vacuolated cells stain with colloidal blue and less frequently with Alcian blue.129,134 Pulmonary deposition of GAG leading to chronic interstitial lung disease is rare and is usually progressive.135 Alveolar spaces may be filled with vacuolated macrophages and the alveolar lining cells and bronchial cartilage chondrocytes may be vacuolated.136 Pneumonia is a common terminal event.

Electron microscopy Vacuolated cells contain dilated lysosomes that appear empty or contain finely granular stored GAG. Some vacuoles contain well-circumscribed, electron-dense circular bodies or inclusion bodies, called zebra bodies (Figure 19a,b).137

Chapter 11: Metabolic and connective tissue disorders involving the lung

Laboratory findings Urine GAG-derived mucopolysaccharide level quantification is a sensitive and specific screening test for diagnosis of MPS but does not discriminate between subtypes. Specific enzyme analysis, as well as genetic testing, is required to confirm the diagnosis.138,139 These methods may be used for prenatal diagnosis.

Pathophysiology Accumulation of lysosomal GAG molecules results in cell, tissue and organ dysfunction and death in early childhood. GAG fragments generated by an alternative pathway are excreted in the urine. Progressive deposition of GAGs in the upper respiratory tract soft tissues probably contributes to airway dysfunction and can lead to airways collapse during sleep or anesthesia.140 Other contributing features are abnormally shaped stiff ribs and abdominal organomegaly. General anesthesia is a high-risk procedure in MPS because intubation is hampered by the short neck, immobile jaw and the pathological changes in the upper airways.140

Prognosis and outcome In severly affected patients, death usually occurs in the second decade of life due to respiratory obstruction, neurological involvement or cardiac failure. At the opposite end of the phenotypic spectrum, patients are spared cognitive involvement but experience all of the somatic signs and symptoms.140 In this attenuated phenotype, patients typically survive into adulthood.119,121,122,141 Treatment of MPS patients is in most cases symptomatic. Specific treatment for airways obstruction includes tonsillectomy, adenoidectomy, laser excision of tracheal lesions,142 and non-invasive mechanical ventilation and tracheostomy.143 Since 1980 over 500 patients with MPS I have undergone hematopoietic stem cell transplantation (HSCT), accounting for the highest number of patients with lysosomal storage disorders treated by this modality. It is associated with significantly improved life expectancy, due in part to prevention or remission of cardiorespiratory complications144 but there is also considerable graft failure, morbidity and mortality. Enzyme replacement therapy is available for patients with MPS I, MPS II and MPS VI. It produces various clinical benefits, including improved lung function and reduced GAG storage in patients with MPS I and VI.145,146 It is indicated for patients with milder forms of MPS without CNS involvement, as the freely circulating enzymes administered during ERT do not cross the blood–brain barrier.144,147,148 Gene therapy is under investigation and shows promising results in animal models.149

GM1 gangliosidosis

enzyme acid β-galactosidase, mapping at chromosome 3p21.33. The enzyme hydrolyzes the terminal β-galactosyl residues from GM1 ganglioside, glycoproteins and glycosaminoglycans.150 This deficiency results in lysosomal accumulation of GM1 ganglioside and related glycoconjugates, leading to cellular damage and organ dysfunction. GM1 gangliosidosis can be divided into three types: type 1, infantile; type 2, juvenile; type 3, adult. The severity of each type is related to the residual acid β-galactosidase enzyme activity.

Clinical manifestations Affected individuals rarely have clinically significant pulmonary involvement. However, the neurodegenerative processes indirectly involve the lungs. Young patients may suffer from aspiration and recurrent secondary respiratory infections. Bronchopneumonia is the usual cause of death.

Pathology Storage macrophages have small, pyknotic, eccentric nuclei and abundant granular or vacuolated cytoplasm. They are intensely PAS-positive and stain with Alcian blue and toluidine blue. These cells are seen in numerous organs including the lung. Gaucher-like cells have been described in GM1 gangliosidosis.151 At autopsy the lungs of patients with types 1 and 2 GM1-gangliosidosis are enlarged and foamy macrophages are seen in alveoli, alveolar septa and around blood vessels.152–154 A 14-month-old boy with GM1 gangliosidosis who died of respiratory failure had numerous intra-alveolar foam cells at autopsy.155

Electron microscopy Lamellated membrane structures are seen in affected cells, including macrophages (Figure 20).156

Prognosis and natural history At present only symptomatic and supportive measures are available to patients with GM1 gangliosidosis. Bone marrow transplantation does not influence the neurological outcome of the disease.157

Krabbe disease (globoid leukodystrophy) Introduction

Krabbe disease or globoid cell leukodystrophy (GLD) is an inherited autosomal recessive neurological disease. It is caused by mutations in the GALC gene, which encodes the lysosomal enzyme galactocerebrosidase galactosylceramide β-galactosidase. This enzyme is responsible for the degradation of galactosylceramide and psychosine (galactosylsphingosine), both important glycosphingolipids in myelin.

Introduction

Clinical manifestations

GM1 gangliosidosis is is a group of three inherited autosomal recessive lysosomal disorders caused by deficiency of the

The pathological consequences of the GALC deficiency are mostly confined to the white matter of the central

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Chapter 11: Metabolic and inherited connective tissue disorders involving the lung Figure 20. Electron micrographs of the cerebellum from a 5½-month-old boy with GM1-gangliosidosis. The low-magnification image (left) prominently features neurons and a capillary. Cytoplasmic lysosomes are filled with different storage materials. The neuron (right upper) contains lamellated membrane structures, i.e. “finger prints” which represent the storage of glycolipids. Endothelial cells (right middle) contain oligosaccharides, and the neuron (right lower) contains spheroids. (Courtesy of Dr J. Alroy, Boston, MA, USA.)

and peripheral nervous system. Respiratory involvement is not a feature of Krabbe disease, although recurrent aspiration secondary to neurological impairment might be anticipated. Rare cases of primary pulmonary involvement are reported.158

Pulmonary radiology Chest X-ray may demonstrate pronounced hyperinflation and diffuse bilateral interstitial markings.158

Pathology Pathological changes are practically limited to the nervous system. The histological hallmark is accumulation of multinucleated “globoid” macrophages, particularly in regions of active demyelination and around blood vessels (Figure 21). At necropsy the lungs are firm and finely granular. Involved alveolar ducts and sacs are distended by numerous large macrophages, some multinucleate, with densely eosinophilic cytoplasm and large brown or amber colored PAS-positive intracytoplasmic inclusions. Typical globoid macrophages may be seen.158

Electron microscopy Ultrastructurally the globoid cells, Schwann cells and perineural macrophages contain abnormal cytoplasmic inclusions with straight or curved hollow tubular profiles in longitudinal section that appear irregularly crystalloid in cross section (Figure 22).159 In the case described above, large amorphous electro-dense membrane-bound inclusions were scattered throughout the cytoplasm of the pulmonary macrophages.158

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Figure 21. Krabbe disease. Cerebral cortical clusters of globoid macrophages are usually perivascular. (Image courtesy of Dr D. Flieder, Philadelphia, PA, USA.)

Prognosis and treatment The early onset type of Krabbe disease is invariably fatal and patients do not usually survive beyond the age of 2–3 years. Hematopoietic stem cell transplantation (HSCT) is indicated

Chapter 11: Metabolic and connective tissue disorders involving the lung

Figure 22. Electron photomicrograph of a peripheral nerve from a patient with Krabbe disease. A Schwann cell has intracytoplasmic tubular inclusions. (Courtesy of Dr Dov Soffer, Hadassah-Hebrew University Medical Center, Jerusalem, Israel.)

for asymptomatic infants or older patients with only minimal neurological involvement.160,161

Pompe disease (glycogen storage disease type II) Introduction

Pompe disease (acid maltase deficiency or glycogen storage disease type II) is an autosomal recessive disorder in which deficient activity of the lysosomal hydrolase acid α-glucosidase causes intra-lysosomal accumulation of glycogen in skeletal muscle and other tissues.151 It is the only glycogen storage disease that is also a lysosomal storage disease.

Figure 23. Pompe disease. A chest radiograph of a 7-year-old boy shows a tracheostomy tube (long arrow) and bibasilar consolidations with volume loss due to aspiration (short arrows). (Courtesy of Dr Dorrith Shaham, Jerusalem, Israel.)

Pulmonary radiology

Genetics

In the infantile form, there is cardiomegaly, atelectasis and pneumonia due to hypoventilation (Figure 23). Atrophy and fat replacement of paraspinal and chest wall muscles may be seen.166

Glycogen storage disease type II is an autosomal recessive disorder caused by multiple mutations in the acid maltase gene.

Pathology

Clinical features Pompe disease has been classified into infantile and late-onset forms, reflecting differences in age of onset, severity of symptoms and rate of disease progression.162 Classic infantile Pompe disease presents in the first months of life with severe muscle weakness, cardiomyopathy and respiratory failure. Most infants die within 1 year of diagnosis from cardiorespiratory insufficiency.162,163 Late-onset Pompe disease (juvenile and adult types) is a milder subtype with a better prognosis. It presents predominantly as a slowly progressive proximal myopathy. Respiratory muscles are affected, usually late in the course of the disease, although some patients present with respiratory failure.164 Nocturnal hypoventilation, caused by diaphragm involvement, may be the initial symptom of respiratory decompensation, and is associated with pulmonary hypertension.165 Respiratory failure is a major cause of death.162

Light microscopy of muscle, irrespective of the site of the biopsy, reveals excessive PAS-positive, diastase-sensitive, glycogen vacuoles. The degree of vacuolation is greatest in the infantile variety and decreases in the late-onset types.167 Acid phosphatase staining indicates lysosomal dysfunction. A normal muscle biopsy does not exclude Pompe disease.168 Other sites of glycogen accumulation in the infantile type include the heart and liver. These sites are not usually affected in the late-onset forms of Pompe disease.

Electron microscopy Muscle fibers in addition to other cell types contain membrane-bound and free glycogen (Figure 24). Autophagic vacuoles are present, particularly in the infantile type.

Prognosis and treatment Patients with classic infantile Pompe disease rarely survive beyond 1 year of age.169 In adults the disease is slowly

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Chapter 11: Metabolic and inherited connective tissue disorders involving the lung

involvement.172 Additional manifestations include CNS and lower motor neuron involvement, hepatosplenomegaly, lymphadenopathy and cardiac involvement. There are seven clinical subtypes, based on age of onset, mean age of death and degree of neurological involvement. Type 1 (classic) is the commonest subtype. Some severely affected children develop aphonia, feeding and respiratory difficulties, such as difficulties with swallowing, vomiting and pulmonary consolidation associated with fever. Aspiration pneumonia is a common terminal event in Farber disease, secondary to neurological deterioration and/or laryngeal involvement.

Pathology Figure 24. Electron micrographs of a skin biopsy from a 3-month-old boy with Pompe disease. Eccrine gland ductal cells and a few fibroblasts contain numerous lysosomes. Inset: lysosomal glycogen is noted. (Courtesy of Dr J. Alroy, Boston, MA, USA.)

progressive. Nocturnal hypoventilation and the resulting pulmonary hypertension can be reversed by nightly positivepressure ventilation. Patients in more advanced stages of lateonset Pompe disease treated with non-invasive mechanical ventilation or tracheostomy may experience brief improvement of respiratory symptoms due to improved vital capacity volumes.170 Currently the only specific treatment available is ERT. A high dose is required, the response is delayed and lifelong treatment is needed. The best clinical response is obtained by patients who are still in the early stages of the disease.169,171 Gene therapy and chaperone-based treatments are being tested on animal models.169,171

Farber disease Farber disease (Farber’s lipogranulomatosis or acid ceramidase deficiency) is a rare autosomal recessive lipid storage disease. It is characterized by accumulation of ceramide in lysosomes in various tissues, due to deficiency of lysosomal acid ceramidase.172,173

Epidemiology Only 80 cases of Farber disease have been reported.174 Three cases in Indian children have been described but the disease is not limited to any ethnic group.174,175

Genetics The disease is caused by mutations in the N-acylsphingosine amihydrolase (ASAH1) gene, mapped at 8p22–8p21.3, which codes for the acid ceramidase enzyme. The clinical course cannot be predicted from the genotype.

Clinical features Farber disease usually presents in the first few months of life with a classic triad of painful joints with progressive deformity, subcutaneous nodules, particularly near joints and over pressure points, and progressive hoarseness, due to laryngeal

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In early lesions there is accumulation of foam cells and in established lesions granulomas with PAS-positive lipid-laden macrophages and fibroblasts are seen. The granulomatous infiltrates involve subcutaneous nodules, joints and other organs. The granulomas contain ceramide. Older lesions undergo fibrosis. The lungs are involved in severely affected patients. Peribronchial and peribronchiolar foamy storage cells or fibroblasts are identified. Alveolar septa and airspaces are infiltrated by massive numbers of macrophages. Granulomas may be seen on the visceral and parietal pleura.172,176,177

Electron microscopy Membrane-bound curvilinear tubular structures (“Farber bodies” or banana bodies) are observed in fibroblasts, macrophages and endothelial cells on ultrastructural examination. Neurons and endothelial cells may also contain zebra bodies.

Laboratory findings Diagnosis is confirmed by demonstrating deficient lysosomal ceramidase activity in white blood cells, cultured skin fibroblasts and amniocytes or by loading studies with labeled precursors in cultured cells.172 Other diagnostic methods include demonstration of high ceramide levels in cultured cells, biopsy samples or urine as well as ultrastructural identification of “Farber bodies”. Prenatal diagnosis is possible on cultured amniotic cells or chorionic villi.172

Pathophysiology Farber disease is extremely rare and the underlying pathophysiology is not described.178 Accumulation of storage cells and granulomas in the upper and lower airways and aspiration secondary to CNS involvement probably play roles.

Treatment and prognosis There is no effective therapy for Farber disease. Patients with type 1 Farber disease die due to neurological deterioration. Hematopoietic stem cell transplantation confers no benefit.179 Laryngeal and pulmonary involvements require close supervision and tracheostomy may be required.172 Patients without CNS involvement (types 2/3) usually die in the second decade as a result of respiratory insufficiency.172 Hematopoietic stem cell

Chapter 11: Metabolic and connective tissue disorders involving the lung Figure 25. Lysinuric protein intolerance. A chest X-ray of an asymptomatic 15-yearold girl features reticulonodular densities in both lungs. (Courtesy of Dr F. Santamaria, Naples, Italy.)

transplantation in a small group of such patients led to symptomatic improvement.180

Aminoaciduria Lysinuric protein intolerance Introduction

Lysinuric protein intolerance (LPI) is an autosomal recessive aminoaciduria caused by defective cationic amino acid (CAA) transport at the basolateral membrane of renal and intestinal epithelial cells. Metabolic derangement is characterized by reduced CAA absorption from the intestine, and increased renal excretion of CAA with excessive urinary excretion of orotic acid (orotic aciduria).181 Orotic acid is an intermediate in the pyrimidine biosynthetic pathway. It is markedly increased in many inborn errors of the urea cycle and in a number of other disorders involving arginine metabolism.

Epidemiology Lysinuric protein intolerance is most prevalent in Finland (1/76 000 births). Families are also clustered in southern Italy and Japan. Sporadic cases have been described worldwide.182

Genetics The gene for LPI is solute carrier family 7 (cationic amino acid transporter, yþ system), member 7 (SLC7A7). It contains 11 exons, while the translation initiation begins in exon 3. This gene encodes the yþLAT-1 protein, which belongs to the family of heterodimeric amino acid transporters. There is no genotype/phenotype correlation.

Clinical manifestations The clinical presentation of LPI can be extremely variable, even in the same family. Some infants affected by LPI present with gastrointestinal symptoms, such as feeding difficulties, vomiting and/or diarrhea, soon after weaning.181 Other major clinical manifestations include failure to thrive, aversion to dietary protein, hepatosplenomegaly, bone marrow abnormalities, osteoporosis, episodes of coma following protein absorption, mental retardation, lung involvement, altered immune response and chronic renal disease. Pulmonary manifestations range from radiological interstitial lung disease without clinical symptoms to life-threatening unexpected respiratory complications. The latter leads, in some cases and at variable ages, to multiorgan failure and death.183,184 Patients present with fatigue, dyspnea on exertion, cough and rarely hemoptysis. Interestingly, although most adult patients have radiological evidence of pulmonary disease, few have respiratory impairment. Children with LPI have a marked tendency to develop alveolar proteinosis and pulmonary hemorrhage.

Pulmonary radiology High-resolution CT and radioisotopic studies are the most sensitive methods for early diagnosis of lung disease. Disease is detected in most patients by these modalities, even those

Figure 26. Lysinuric protein intolerance. CT scan of the same patient shows thickening of bronchial walls with bronchiectasis, small ground-glass densities, moderate thickening of the interlobular septa and small subpleural blebs. (Courtesy of Dr F. Santamaria, Naples, Italy.)

without clinical impairment.183 In asymptomatic patients HRCT reveals irregularities of the bronchial tree, bronchiectasis, localized small acinar nodules, localized interstitial intralobular and/or interlobular thickening, and/or thin-walled subpleural cysts.183,184 Diffuse reticulonodular interstitial densities and alveolar densities are seen in patients with severe respiratory insufficiency (Figures 25 and 26). Radioisotope studies show an uneven distribution of perfusion and ventilation, and confirm the presence of segmental and/or diffuse pulmonary functional defects.183

Pathology Progressive interstitial lung changes are frequently detected in those patients without clinical symptoms183 At necropsy the

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lungs are heavy, airless and edematous. Pulmonary alveolar proteinosis is a consistent finding (see Chapter 10).185 Other pulmonary changes include cholesterol granulomas,186,187 organizing pneumonia186 and intrapulmonary hemorrhage.185 The latter may be seen in the course of the disease or may be a terminal event associated with multiorgan failure and coagulopathy.

Electron microscopy Alveolar macrophages of patients with LPI have an increased number of multilamellar structures and contain excess iron.186

Laboratory findings Impaired intestinal absorption and renal resorption of CAA cause a metabolic derangement characterized by increased CAA urinary excretion. Low plasma CAA concentrations and dysfunction of the urea cycle lead to post-prandial hyperammonemia and orotic aciduria.

Pathophysiology While it is uncertain how alveolar proteinosis develops, researchers suggest that low airway bioavailability of surfactant protein D (SP-D) may play a role.181 A 2-year-old patient with LPI was noted to have partially degraded SP-D entrapped in unusual surfactant lipid tubules with a circular lattice.188–190 Supplementing SP-D and GM-CSF increased the uptake of protein and dying cells by healthy LPI alveolar macrophages, ex vivo.188 GM-CSF treatment drastically increased the number of granulomas, whereas SP-D treatment counteracted the adverse effect of GM-CSF.

Treatment and prognosis Lysinuric protein intolerance was once considered relatively benign and treatable with a low-protein diet and oral supplementation of citrulline, a neutral amino acid that ameliorates urea cycle dysfunction by converting to arginine. However, this treatment does not prevent the major causes of morbidity and mortality, namely alveolar proteinosis and/ or renal disease.182 A childhood history of pulmonary densities is associated with a poor prognosis.186 Bronchoalveolar lavage has been effective in a few patients with respiratory exacerbations.191 Pulmonary disease may be ameliorated by high-dose steroids. Heart-lung transplantation performed on a child with LPI and alveolar proteinosis was followed by recurrent pulmonary disease.192

Inherited connective tissue disorders Marfan syndrome and Ehlers-Danlos syndrome are inherited multisystem disorders of connective tissue synthesis and/or structure. Emphysema and spontaneous pneumothorax are seen in both. Recent breakthroughs in the understanding of the pathophysiology of these diseases, particularly Marfan syndrome, may provide insights into the mechanism of nonsyndromic emphysema.193

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Marfan syndrome Marfan syndrome (MFS) is an autosomal dominant connective tissue disorder with multiple systemic manifestations, caused by heterozygous mutations in the gene FBNI. It gives rise to a variety of clinical manifestations in the cardiovascular, pulmonary, skeletal and ocular systems.

Epidemiology The estimated prevalence of MFS ranges between 1/5000 and 1/10 000 live births. There is no ethnic or gender bias.194

Genetics Marfan syndrome is an autosomal dominant disorder with high penetrance but variable expression. The main cause of MFS is mutation in the fibrillin-1 gene FBN1 on chromosome 15 that encodes for fibrillin-1, a complex connective tissue extracellular matrix glycoprotein. One of the functions of fibrillin-1 is to bind transforming growth factor β (TGFβ) in the extracellular matrix and keep it inactive.195 The gene mutation leads to enhanced proteolytic degradation and malfunction of fibrillin-1. Heterozygous mutations in TGFβ receptor 2 on chromosome 3 cause a second type of MFS.196

Clinical features The clinical expression of MFS is variable and diagnosis is based on the Ghent classification of 1996.197 Some of the commoner manifestations of MFS include skeletal abnormalities such as thoracic deformities, above average height, hypermobility and luxation of joints, ectopia lentis, dilatation or dissection of the aorta and mitral valve prolapse. The leading cause of morbidity and mortality is aortic root aneurysm and subsequent aortic dissection. Pulmonary abnormalities occur in approximately 10% of patients with MFS. Spontaneous pneumothorax and apical blebs on chest radiography constitute two minor criteria for diagnosis of MFS.197 Spontaneous pneumothorax occurs in 5% of patients, usually adolescents, and tends to be bilateral and recurrent (see Chapter 36).198–202 Emphysema develops in adults, especially smokers.203 Other intrinsic pulmonary parenchymal diseases include bronchogenic cysts, bronchiectasis, upper lobe fibrosis, “honeycomb lung”, recurrent respiratory infections and aspergillomas.135,203–205 The latter probably arises in preformed bullous or cystic cavities. Pulmonary dysfunction may be restrictive due to chest wall abnormalities, such as pectus excavatum or kyphoscoliosis.206 Patients with MFS also have a high prevalence of obstructive sleep apnea207–209 thought to be due to increased upper airways “collapsibility”.208,210 Neonatal MFS lies at the severe end of the MFS clinical spectrum and emphysema is common in this group.211,212

Pulmonary radiology Chest radiographs typically show bilateral bullae in the upper lungs, with or without emphysema (Figure 27).203 In addition

Chapter 11: Metabolic and connective tissue disorders involving the lung

(a)

Figure 27. A 28 year-old male with Marfan syndrome. CT scan of the chest with IV contrast injection. (a) This lung window demonstrates biapical bulla. (courtesy of Dr Klaus Irion, Liverpool, UK.) (b) This mediastinal window features a 3.8 cm aneurysm of the descending thoracic aorta (arrow). (Courtesy of Dr Dorrith Shaham, Jerusalem, Israel.)

(b)

Pathophysiology Fibrillin-rich microfibrils form peripheral components of normal elastic fibers and provide an architectural foundation and tissue elasticity.219 All tissues in MFS contain these microfibrils. Originally MFS was believed to result from the production of abnormal fibrillin-1, yet animal studies indicated that many MFS manifestations are related to impaired inactivation of TGFβ, due to deficient fibrillin-1.220 Fibrillin-1-deficient mice are born with impaired alveolar septation and later develop emphysema. The impaired septation is associated with upregulation of TGFβ and is prevented by TGFβ inhibition.221,222 Mitral valve and aortic wall pathology can also be reversed by administering TGFβ antagonists.221

Prognosis and treatment Figure 28. Marfan syndrome. Emphysema and subpleural blebs are seen.

chest wall and spinal abnormalities, such as pectus excavatum, pectus carinatum and scoliosis, may be seen.197

Pathology Architectural abnormalities of the lung have been reported at autopsy of young subjects, including a monolobed left lung, a vestigial right middle lobe, an abnormally large lingula and distorted pleural cavity, due to kyphoscoliosis.213 Older patients show subpleural bullous emphysema, mostly at the lung apices.214 Histologically there is dilatation of distal airspaces and bleb or bulla formation (Figure 28).215 There are conflicting reports on the elastic staining properties of the alveolar septa ranging from normal216,217 to an increased amount of elastic tissue209 to abnormally thick, irregular, tortuous and interrupted elastic fibers.215,218

Electron microscopy Some elastic fibers appear normal while others are frayed with increased osmophilia and mucopolysaccharide deposition. The background is clear with a meshwork of small spheres and threads.215

The primary cause of death in subjects with MFS is cardiovascular collapse due to aortic dissection, rupture and pericardial tamponade. The clinical outcome and prognosis for people with MFS has improved steadily over the last three decades, due to medical and surgical advances. The average age at death and the median cumulative probability of survival were 32 and 48 years, respectively, in 1972 and improved to 41 and 72 years, respectively, in 1995.223,224 Pulmonary abnormalities place patients at risk of spontaneous pneumothorax and increase the danger of tension pneumothorax, when associated with positive-pressure ventilation. Bullae and blebs require pleurodesis or surgical excision. Spontaneous pneumothorax in MFS should be treated with early surgery in most cases due to the high risk of recurrence.198,199 TGFβ antagonists are a potential treatment strategy for human subjects with MFS and clinical trials are currently underway.221

Ehlers-Danlos syndromes Ehlers-Danlos syndromes (EDS) represent a clinically and genetically heterogeneous group of heritable connective tissue disorders. Most involve defects in the primary structure or post-translational processing of fibrillar collagen.

Epidemiology The estimated prevalence of EDS is 1/10 000 to 1/25 000 live births,225 with no gender or ethnic predisposition.

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Chapter 11: Metabolic and inherited connective tissue disorders involving the lung Table 3 Classification and genetics of Ehlers-Danlos syndrome

Phenotype

Genotype/biochemistry

Inheritance

Classic EDS

COL5A1/COL5A2

AD

Classic-like

TNX-B

AR

Vascular

COL3A1/ biochemical analysis of type III collagen

AD

Vascular-like

COL1A1/biochemical analysis of type I collagen

AD

Cardiac valvular

COL1A2/biochemical analysis of type I collagen

AR

Kyphoscoliotic

PLOD1/lysyl hydroxylase-1 activity

AR

AD, autosomal dominant; AR, autosomal recessive.

Figure 30. Ehlers-Danlos syndrome. A chest CT demonstrates a right lower lobe thick-walled 1.5 cm cystic lesion with adjacent soft tissue nodule. (Courtesy Dr D. Flieder, Philadelphia, PA, USA.)

anastomoses, a predisposition to asthma,227 recurrent sinusitis228 and tracheobronchomegaly (Mounier-Kuhn syndrome) (see Chapter 3).228–230

Pulmonary radiology

Figure 29. Ehlers-Danlos syndrome. A chest CT shows subpleural bullae (arrow). (Courtesy of Dr Takashi Ishiguro, Dr Noboru Takayanagi and Dr Yoshinori Kawabata, Saitama, Japan.)

Genetics The molecular basis of EDS is extremely heterogeneous. Some of the major types are shown in Table 3.

Clinical features The main clinical characteristics of EDS are skin hyperextensibility, delayed wound healing with atrophic scarring, joint hypermobility, easy bruising and generalized fragility of the soft connective tissues.226 The vascular type of EDS (also known as vEDS or type IV EDS) is the most severe form. It accounts for 5–10% of all cases and is associated with severe fragility of connective tissues, as well as fragility of arterial walls and hollow viscera. Pulmonary involvement is rare and has been described mainly in vEDS. The commonest manifestation is pneumothorax. Hemoptysis may be secondary to pulmonary artery rupture or tears in lung parenchyma and is potentially fatal. Other pulmonary abnormalities include pulmonary arteriovenous

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Radiological findings include bullous lung disease, diffuse panacinar emphysema, pulmonary cysts, lower lobe saccular bronchiectasis, and thick-walled cavities, resulting from previous rupture of lung (Figures 29 and 30).231–235 Scattered parenchymal fibro-osseous nodules may be seen.236 Skeletal abnormalities affecting the chest wall include pectus excavatum, thin downward sloping ribs, scoliosis and straight back syndrome.228

Pathology The lung parenchyma in EDS is abnormally friable. Histological changes are divided into bona fide disease and iatrogenic insults.233,237 Iatrogenic tears often occur during lung biopsy or resection. Lung disease may manifest with acute hemorrhage and hematomas, and sometimes with peculiar fibrous nodules. Hematomas cavitate and may be associated with pleural fibrosis or adhesions. In addition, organizing pneumonia, as well as intraluminal and interstitial hemosiderosis, may be observed. Iron deposition in alveolar and/or vessel walls can elicit a foreign-body reaction, so-called endogenous pneumoconiosis. Partial vascular wall disruption with secondary fibrosis is also seen. All types of emphysema including bullous and paracicatrical patterns are also described.

Chapter 11: Metabolic and connective tissue disorders involving the lung

(a)

(b)

(d)

(e)

(c)

Figure 31. Ehlers-Danlos syndrome. (a) Striking arterial adventitial fibrosis appears to infiltrate surround alveolar lung. (b) Cellular nodules are scattered throughout lung parenchyma. (c) Recent lesions are hypercellular. Large oval and spindle cells are embedded in a fibromyxoid stroma. Hemosiderin is also noted. (d) Nodules become hyalinized. (e) Ossification is frequently observed. (Courtesy Dr D. Flieder, Philadelphia, PA, USA.)

Parenchymal tears causing hemorrhage, hematoma and vascular wall disruption are thought to lead to fibrous nodule formation. The nodules may be smooth and bulbous or stellate and pseudo-infiltrative. Histologically one sees hypercellular proliferations of enlarged spindle cells admixed with fibromyxoid connective tissue. Hyperchromasia, prominent nucleoli and scattered mitotic figures may be noted. Large and irregular-shaped nodules become hypocellular and hyalinized over time. Ossification with bone marrow formation is not uncommon (Figure 31).237,238 If the surgical pathologist is not familiar with this disease, he/she may consider the fibrous nodules a low-grade sarcoma.

Laboratory findings A clinical diagnosis of Ehlers-Danlos is confirmed by biochemistry of different collagens using fibroblasts and/or molecular testing. Mutational analysis of specific abnormal collagens can be performed or linkage analysis, if there is a family history.

Pathophysiology The commonest variant of EDS, vEDS, is caused by a deficit of type III collagen, a constituent of the walls of arteries and the digestive tract.239 Structural changes in the bullae of patients with EDS are also due to deficiency of type III collagen.239

Prognosis and treatment Vascular EDS is the most lethal variant and carries a high risk of vascular, intestinal and uterine rupture. Arterial rupture and intestinal perforation occur in 25% of EDS subjects before the age of 20 years and in 80% before the age of 40.240 The median age at death is 50 years.241 Intestinal perforations occur mainly in the sigmoid colon and are less likely to be fatal. Maternal mortality due to obstetric complications occurs in about 12% of cases of the vascular type EDS.241,242 In the absence of specific therapy, medical treatment is focused on prophylactic measures, symptomatic treatment and genetic counseling. Surgery should be avoided, particularly in the vascular type, unless required urgently to treat

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potentially fatal complications.241 Clinical trials are evaluating the value of long-term beta-blocker treatment to prevent vascular complications in vEDS.241 Other therapeutic proposals

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Chapter

12

Hypersensitivity pneumonitis John C. English

Introduction Hypersensitivity pneumonitis (HP), also known as extrinsic allergic alveolitis (EAA), is a pulmonary disease typically manifested in the sensitized host by cough and dyspnea. It results from repeated inhalational exposure to any of numerous identified environmental antigens.1 The exposure context may be occupational, recreational or domestic. The antigens are classically small organic substances derived from animal, avian and fungal proteins, although a number of low molecular weight chemical compounds are also implicated. Hypersensitivity pneumonitis, in its various subsets, is immunologically mediated. In susceptible hosts, it produces a hypersensitivity reaction in the lung involving inflammation, stereotypically granulomatous, of the small airways (bronchiolitis), the alveolar parenchyma and airspaces. Hypersensitivity pneumonitis is to be distinguished from other inhalatory pulmonary injuries, such as “organic dust toxic syndrome” (pulmonary mycotoxicosis, silo-unloader’s syndrome).2 Hypersensitivity pneumonitis has been characterized as a “multifaceted” disorder3 and its clinical and pathological expressions may overlap with other lung conditions, presenting as diffuse acute, subacute or chronic interstitial disease.

Clinical features Classification The heterogeneous modes of clinical presentation of HP are reflected by different classification systems currently in use. The traditional scheme includes acute, subacute and chronic phases. In an attempt to accommodate the more protean aspects of the disease, more recent interpretations suggest “acute progressive”, “acute intermittent non-progressive” and “recurrent non-acute disease” patterns (Table 1).1,4,5 It may be difficult to distinguish with precision the various clinical phases of HP and it is likely that clinical variability is more related to host factors and circumstances of exposure rather than individual antigen characteristics.6 It is also important to acknowledge that acute disease does not necessarily progress to chronic, despite the continued presence of an offending antigen.7,8 Subclinical disease is also recognized, wherein

immunological and inflammatory processes are present in the host, without symptomatology.9 A current report addresses the integration of pathophysiology and clinical symptomatology in formulating a relevant classification.10 In this study, 168 patients were divided into two clusters; both clusters contained some proportion of the traditional acute, subacute and chronic designations. Cluster 1 patients tended to have greater recurrent systemic symptomatology and normal chest radiographs; patients in cluster 2 demonstrated greater clubbing, hypoxemia, restrictive physiological deficits and fibrosis on high-resolution computed tomography (HRCT). The data led the authors to hypothesize that a two-category classification system would be the most relevant, reinforcing the notion that chronic HP is not necessarily the sequel of acute HP.10 There is no single clinical element, laboratory test or other “gold standard” that is diagnostic of HP.11 The diagnosis is ultimately made by a combination of features that incorporate patient history, clinical examination, pulmonary function evaluation, radiological imaging and laboratory tests. The latter can include immunological testing, as well as histopathological examination of biopsied lung tissue. Lacasse and colleagues in the HP study group sought to identify criteria that would allow an accurate prediction tool for the clinical diagnosis of HP (exclusive of inactive and chronic forms).6,11 Significant predictors of HP included: (1) exposure to known offending antigen, (2) positive precipitating antibodies to the offending antigen, (3) recurrent symptomatic episodes, (4) inspiratory crackles on auscultation, (5) occurrence of symptoms 4–8 hours post exposure, and (6) weight loss.11 In their cohort, exposure to a known offending antigen was the single strongest predictor. Clinical diagnostic criteria for HP have been proposed12–16 and are reviewed.6

Incidence and prevalence The incidence and prevalence of HP are notoriously difficult to estimate. Reasons cited for this include difficulties in defining what is an acknowledged complex clinical entity, recognized

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 12: Hypersensitivity pneumonitis Table 1 Clinical classification of hypersensitivity pneumonitis

Classification

Temporal features

Clinical features

(Clinical) Pulmonary function tests

Acute HP (most common presentation)

Exposures classically short-term and high-level Symptoms begin 2–9 h. post-exposure Peak at 6 and 24 h Duration hours to days Symptoms usually improve with antigen avoidance Symptoms may recur with subsequent antigen exposure

Influenza-like symptomatology: chills, chest tightness, fever, sweats, myalgias, headache and nausea Cough and dyspnea typical but not universally present Bibasilar crackles on auscultation may be present

Restrictive defect Reduced diffusion capacity Hypoxemia

Subacute HP

Temporal relationship to antigen exposure not always precise Appears gradually over several days to weeks Patients experience acute episodes Initial symptoms may be few, if any Course may be subacute or chronic punctuated by acute exacerbations

Cough and dyspnea, may be progressive to severe respiratory embarrassment, cyanosis and hospitalization Bibasilar crackles on auscultation

Restrictive defect Reduced diffusion capacity Hypoxemia

Chronic HP

Exposures classically low-level, persistent Exposures may be recurrent as with acute and subacute episodes that go unrecognized Insidious onset over months Patients do not experience acute symptoms Course often chronic, progressive, with irreversible pulmonary decline despite antigen avoidance

Few, if any, symptoms in early stages Progressive cough and dyspnea on exertion Fatigue and weight loss Right-sided heart failure Digital clubbing

Restrictive defect Obstructive defect with reduced expiratory flow rates may dominate Reduced diffusion capacity Hypoxemia

Acute progressive HP (analogous to subacute – Selman1,3)

Persistent symptomatology requiring antigen avoidance and treatment

Debilitating symptoms post exposure and progression of symptoms with each subsequent exposure Patient often recognizes nature of symptoms, association with antigen and may modify behavior

Acute intermittent HP (Acute intermittent non-progressive)

Acute reactions spontaneously dissipate but recur closely after subsequent exposure

Similar, but less intense classical symptomatology Paradoxical reduction in intensity of symptoms with subsequent exposures Disease may stabilize

Chronic, non-progressive HP (Recurrent non-acute disease)

May occur in patients following acute symptomatic episodes or with no clear-cut temporal relationship to antigen exposure Symptoms stabilize, improve or disappear spontaneously or following treatment

Chronic dyspnea, anorexia, weight loss, malaise, cough Permanent disability

Chronic progressive

Insidious onset Symptoms evolve into end-stage pulmonary disease with diffuse fibrosis

Table prepared from information presented in references 1, 3, 5, 6, 9, 24, 66.

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Impaired lung function

Chapter 12: Hypersensitivity pneumonitis

regional, seasonal and climatic variances in presentation, individual susceptibility to inciting antigens and the influence of other cofactors, such as smoking.17 Epidemiological assessment of incidence and prevalence has been hampered by lack of consistent and standardized forms of documentation and is likely also to be complicated by patient selection bias.5 In a general population-based United Kingdom cohort study, the incidence of HP in the general population was estimated at 0.9 cases/100 000.18 Many such estimates of the clinical burden of HP are limited since studies often focus on cohorts with increased exposure risk. Rates of conversion to clinical HP in exposed individuals are also variable. For farmer’s lung, a Finnish study recorded an incidence of five per 100 000 farmers19 and in Sweden the rate was estimated at two to three per 10 000 per year.20 Similarly, for pigeon breeders, the prevalence has been reported as > 10%.21 In general, there is no gender bias in HP, although this does appear in certain specific patient cohorts. For example, in considering occupational forms of HP, pigeon keepers and farmers are typically male. In a recent series from Spain, 75% of the patients were female, with social conditions predisposing them to increased domestic contact with pet birds.17 Females are also affected twice as often as males in Japanese summer-type HP.22 Peak seasonal variations in developing HP have been noted in farmer’s lung, in particular late winter, when stored hay stocks are dispersed for cattle feed.5 In bird fancier’s lung (BFL), the exposure to avian antigens is maximized during the sporting season.23 Climatic effects are exemplified by the finding that HP is common in years with increased rainfall during springtime, hay mowing and baling.1 Similarly, household mold contamination in hot, humid summers, following seasonal rains, characterizes Japanese summer-type HP.22 Although HP typically presents in adults,24 the pediatric population, from infancy to adolescence, may also be affected.25 The exposure context is usually domestic or related to hobbies and the offending antigens are typically avian, although molds and other antigens have been documented as causes.25,26 Up to 50% of individuals exposed to environmental antigens capable of inducing HP may develop a lymphocytic alveolitis but remain asymptomatic.27 Up to 15% of persons exposed to high levels of recognized antigen sources will manifest clinical disease.1,28 It is well-recognized that many cases of HP cannot be directly associated with an identifiable antigen. In a recent study, 25% of patients could not be linked to a specific antigen, either through historical association or serological testing, even though surgical lung biopsy histology supported the diagnosis of HP.29 A recent case of “consort hypersensitivity pneumonitis” (transmitted by partners) has been described and is now a factor to be considered in the clinical investigation.30 The proportion of interstitial lung disease (ILD) that is attributable to HP has been estimated at between 4 and 13% in European registries31 but less than 2% in a study originating from New Mexico.32

Acute exacerbations of chronic HP Rapid clinical deterioration in patients with interstitial lung disease has been termed acute exacerbation (AE) and has mainly been described in cases of idiopathic pulmonary fibrosis.33,34 Acute exacerbations may complicate other fibrotic interstitial lung diseases including chronic HP.35,36,37,38 A case definition of AE of fibrotic HP has been proposed by Olson et al.37 and includes: consensus diagnosis of fibrotic HP prior to onset of AE, unexplained worsening or development of dyspnea within two months, new bilateral radiographic opacities, absence of detectable clinical infection, and absence of identifiable etiology (other than HP) for the clinical and radiological features. The mortality rate is high (86% in the study by Miyazaki et al.38) but not uniformly fatal. Risk factors for developing AE of fibrotic HP appear to include initial usual interstitial pneumonia (UIP)like pattern of fibrosis, history of smoking, reduced lymphocytes and increased neutrophils in bronchoalveolar lavage (BAL) fluid, but not the duration of the underlying disease.38 Precipitating factors are unknown but are considered to include: accelerated or fulminant expressions of the underlying disease process, occult/unidentified infection, and unrecognized aspiration of gastric contents in the context of gastroesophageal reflux disease.37 The pathological expression of acute lung injury is typically acute/organizing diffuse alveolar damage.

Clinical testing

Pulmonary function testing and pathophysiology Although both alveolar/parenchymal and airway compartments are involved in HP, there is a diverse spectrum of disease expression and this is reflected in the variability of pulmonary function test (PFT) results observed in these patients, both at presentation, and as the disease evolves. Physiological alterations may vary with intensity and duration of exposure.39 Pulmonary function testing cannot specifically differentiate HP from other diffuse lung diseases11 although both share many of the same functional deficits.40 Early studies were able to identify differences in PFTs between acute, subacute and chronic forms of HP.39 In acute/subacute forms, there is typically good correlation of the functional deficit in relation to clinical and radiological features. This suggests there is a more uniform, or idiotypic, pathological expression at this stage. In chronic HP, however, pulmonary function tests may be at variance with clinical and radiological observations. This is in accord with our understanding of the different patterns of chronic lung injury (see below).41 The typical pattern, at least for earlier forms of HP, is that of a restrictive defect with reduced static lung volumes (reduced forced vital capacity (FVC) and total lung capacity (TLC)), accompanied by reduced compliance/increased elastic recoil.1,42 Resting hypoxemia, oxygen desaturation with

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exercise, and increased alveolar-arterial oxygen gradient (P[A-a] O2) are seen.3 A recent study of 85 HP patients demonstrated the following pulmonary function abnormalities: 53% restrictive, 15% obstructive; 12% nonspecific abnormality; 10% isolated reduction in diffusing capacity; 10% within normal limits.29 In a study of 86 patients with BFL, the acute and subacute phase patients showed restrictive deficits in 81% and 68%, respectively. In the same study, obstructive physiology was observed in 3% of acute HP and rose to 15% in subacute patients.17 Obstructive changes (manifest as decreased FEV1, FEV1/FVC ratio and FEF 25%–75%) can be related to both large and small airway inflammation although the small airway component may be the dominant element.17,42 Mixed patterns of obstructive and restrictive changes were not seen in any acute HP cases but were present in 9% of the subacute cohort.17 In the subacute phase of HP, the pathology is that of a variably dense mononuclear inflammatory infiltrate involving lobular bronchioles and the surrounding interstitium (see below). Airflow abnormalities are probably the product of the bronchiolitis43 with luminal obstruction by inflammatory cells and organized exudates. It can be difficult to determine the proportion of obstructive deficits in smokers with HP because of the potential for concurrent smoking-related small airways disease and chronic obstructive pulmonary disease (COPD) not due to HP.44 The HRCT correlate of the small airway pathology (see below) is that of centrilobular nodularity, which represents airway and peri-airway inflammation, and mosaic attenuation and lobular air trapping on expiratory phase scans.45,46 This latter finding reflects the increase or relative preservation of residual volume (RV) revealed by pulmonary function tests.3,46 Reduced diffusion capacity for carbon monoxide (DLCO, also known as the transfer factor for carbon monoxide, TLCO) is considered by some to be universally present,47 or at least the most common finding, in either acute or chronic HP.40 It may be normal in up to 22% of patients.48 In the acute and subacute phases of the disease, DLCO was reduced in 89% and 75%, respectively.17 Since DLCO is the product of alveolar volume (VA) and the rate of alveolar uptake of CO (KCO, carbon monoxide transfer coefficient),49 a low diffusion capacity may be the result of architectural effacement of the alveolarcapillary bed (emphysema, honeycomb fibrosis) and/or alterations in the alveolar-capillary membrane (cellular interstitial pneumonitis, alveolar type II cell hyperplasia, interstitial edema and fibrosis). In the acute and subacute clinical phases, it is likely that alveolitis and bronchiolitis rather than fibrosis contribute to this decline of DLCO values. Abnormalities of surfactant properties may promote alveolar collapse and loss of gas exchange units with attendant restrictive physiology.50 In chronic HP, patients were classified into an “interstitial” disease profile (39%), “obstructive” profile (29%), combined airway and interstitial patterns (15%), “nonspecific” abnormalities (12%) and normal (5%).41 Another study demonstrated restriction in 92% and obstructive changes in 13% of cases.17

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In chronic HP interstitial and airway fibrosis as well as emphysematous changes (either related to smoking or as a part of the HP disease process itself) can be expected with greater frequency. Emphysematous parenchymal destruction results in loss of fibro-elastic support for small conducting airway patency. Combined with bronchiolitis and scarring, this results in the obstructive profile present in some patients, particularly those with farmer’s lung.41,51 DLCO was reduced in 100% of chronic HP cases17 and the reduction was not statistically different in either recurrent or insidious-onset HP.44 Gas exchange is more likely to be reduced in cases of fibrotic rather than non-fibrotic HP.52 Increased restrictive-type physiology and gas exchange abnormalities were exaggerated when patients with chronic HP showing UIP features were compared with other patterns.52 In advanced cases of HP, the diminished DLCO probably results more from obliteration of the capillary bed and fibrous expansion of the alveolar membrane than from cellular infiltrates.

Immunological testing Detection of serum-precipitating IgG antibodies to putative antigens remains an important diagnostic test for HP. The enzyme-linked immunoabsorbent assay (ELISA) is the preferred and most widely employed method.3 There are a number of caveats related to this test. While the development of immune sensitization indicates exposure to a putative provoking antigen, it does not confer an absolute diagnosis. In addition it is not necessarily proportional to symptoms, since asymptomatic contacts may demonstrate an antibody response.53 Likewise, inability to identify specific antibodies, due to either test insensitivity or exclusion of certain antigens in the test panel, does not exclude the presence of HP.13 Of note, improvements in methodology suggest to some that seronegative symptomatic disease is a very rare occurrence.54 There may be cross reactivity between antigens of different avian sources.17 In patient cohorts with higher exposures (greater number of birds, for example) there is an increased detection rate of elevated IgG levels;17 this finding is replicated in clinical presentation groups, where higher rates of detection of specific IgG levels may be seen in those groups with recurrent (typically greater numbers of bird-exposures, as seen in pigeon breeding) versus insidious (fewer bird-exposures, as with indoor pets) modes of presentation.44 A significant association between the mean level of immunoglobulin and intensity of antigen exposure53 was documented. Antibody titer may decrease with continuing antigen exposure or smoking.54 Thus, this test is reasonably sensitive but not specific for the diagnosis of HP.

Bronchoalveolar lavage Bronchoalveolar lavage cellular profiles may provide corroborative evidence of HP but are not diagnostic. The types and proportions of leukocytes are variable and probably dependent

Chapter 12: Hypersensitivity pneumonitis

on antigen characteristics, mode of exposure, temporal relationship of BAL sampling to antigen exposure,55 individual host immune factors, and smoking status.1,56 In addition, significant BAL lymphocytosis may be seen in asymptomatic individuals following antigen exposure.9,27,47 Bronchoalveolar lavage neutrophilia is seen within 24 h of antigen exposure in acute HP or following challenge or repeated exposures.57 Plasma cells are also noted early in the development of HP and may indicate a more active alveolitis.58 In the subacute phase neutrophilia dissipates, to be supplanted by lymphocytosis that is initially CD8þ suppressor T-cell dominant.17 It shifts to a relative increase in CD4þ helper T-cells in cases following antigen withdrawal.59 Alveolar macrophage proportions may decline at the expense of increased lymphocytes but still remain elevated relative to normal.24 Chronic HP features BAL lymphocytosis,17,60 but usually not to the same levels seen in the acute and subacute phases.3 Although this simplified concept acknowledges a CD4þ T-cell dominance in the chronic phases, literature support is inconsistent. Increased prevalence of CD4þ T-cells has been associated with fibrotic forms of chronic HP,61 but this may reflect a more long-term, insidious type of exposure, as may be encountered in some forms of BFL. In chronic phases of HP investigators report increased neutrophils in BAL fluid17 or in tissues, which correlates with the degree of pulmonary fibrosis.62 In smokers with HP, the CD4þ/CD8þ lymphocyte ratio is said to be increased.56

Chest X-ray findings are generally not specific for HP.68 The value of CT scan in the diagnosis of HP is well-established11 and may reveal characteristic features in some patients whose chest roentgenogram is normal.11 Although it is accepted that up to 8% of patients with proven HP may have normal CT scans,11 most believe that a normal HRCT is an argument for a different diagnosis.68 A false-negative CT reading may be due to a combination of subtle disease expression, slice interval11 and the propensity to misdiagnose subtle opacification as dependent density.69 HRCT examination can also document resolution or changes in pattern expression with different phases of the disease.

Inhalational challenge

Acute HP

Provocation tests to elicit symptoms and signs are sometimes employed when other diagnostic modalities fail to yield a definitive diagnosis.1 The test may be performed in a clinical laboratory under controlled conditions or by way of re-exposure in an occupational or environmental setting. Some investigators have demonstrated that the test can be useful in the differential of chronic HP, which is often difficult to distinguish from other fibrosing interstitial pneumonias.63,64 Pyrexia, cough/dyspnea, peripheral leukocytosis and altered pulmonary function tests (decreased PaO2, SaO2%, increased P(A-a)O2, and decreased FVC)63,64 typically manifest within 4 h of challenge, and resolve spontaneously by 18–24 h. Concurrent BAL fluid analysis demonstrates a lymphocytic and neutrophilic alveolitis63 proportional to symptomatology.65 The role of the provocation test is still not certain in routine clinical practice. Use of this test is attended by certain caveats, namely that it be carried out by skilled personnel in specialized environments that can accommodate occasionally serious side-effects.17,64 Other limiting factors include: difficulties in objectively defining a positive test, lack of standardized antigen reagents, individual clinical responses, and clinical overlap with other non-allergic inflammatory responses such as organic toxic dust syndrome and inhalational fevers.5 Due to these limitations, this test remains one of the last in the

Chest radiographs demonstrate mid and lower lung zone changes similar to acute pulmonary edema.69,70 CT scans are usually not obtained in acute episodes of HP, a situation which reflects the typical rapid resolution of symptoms.70 When performed, the findings are those of transient patchy or confluent ground-glass attenuation or consolidation densities.45,71

diagnostic algorithm and is sometimes used when a surgical lung biopsy is contraindicated.1

Skin testing The utility of skin testing in the diagnosis of HP, although advocated by some,17 remains uncertain.24,66 Immediate hypersensitivity reactions have been attributed to an IgG subclass rather than IgE.67 This result is seen in a high proportion of BFL patients and also a significant number of asymptomatic exposed individuals.17 Approximately one-third of patients demonstrated depressed cellular immunity on delayed hypersensitivity cutaneous testing.17

Radiological findings Introduction

Subacute HP Chest radiography in the subacute phase demonstrates ground-glass opacification and poorly defined nodules which may be diffuse but are usually distributed in the lower lung zones.45,72 Sparing of the basal segments/costophrenic angles has been described.73 Diffuse consolidation is rarely present.45 Occasionally, disrupted parenchyma secondary to airway obstruction can produce pneumomediastinum, pneumothorax, and subcutaneous emphysema.74 Pleural effusions have rarely been reported in HP.75 The prototypic HRCT image of subacute HP consists of patchy or diffuse ground-glass opacities which are symmetric and bilateral (Figure 1), poorly defined, usually numerous small centrilobular nodules (Figure 2) and mosaic attenuation (inspiratory series) with lobular air trapping (expiratory series) (Figure 3).45,46,69,70 Although these findings may be sufficient to allow for a confident diagnosis of HP,70 variations in

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Figure 1. Five millimeter thickness CT image at the level of the right hemidiaphragm shows diffuse bilateral ground-glass opacities. The findings are consistent with subacute HP. (Courtesy of Dr Nestor L. Müller, Vancouver, Canada.)

Figure 3. High-resolution CT image at the level of the lower lung zones demonstrates bilateral ground-glass opacities and a few lobular areas of decreased attenuation and vascularity. The findings are characteristic of HP. (Courtesy of Dr Nestor L. Müller, Vancouver, Canada.)

distribution and intensity are responsible for a spectrum of radiological patterns. Ground-glass opacification (regions of hazy increased attenuation that do not obscure margins of underlying airways and vessels)76 is characteristically extensive.46 However, it may be patchy and/or asymmetric.69 Its pathological correlate is typically cellular interstitial pneumonitis; however, minor degrees of organizing airspace exudate may also combine to produce this image.69 Centrilobular nodules are 3–5 mm in diameter ground-glass opacities45 that spare the perilobular and paraseptal parenchyma.70 Their radiological attributes reflect the combination of cellular bronchiolitis, often with focal luminal obstruction, and peribronchial inflammation, of which small amounts of organizing

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Figure 2. High-resolution CT image at the level of the lower lung zones shows bilateral ground-glass opacities and poorly defined centrilobular nodules. Also noted are a few localized areas of decreased attenuation and vascularity in the right lung. The findings are characteristic of subacute HP. (Courtesy of Dr Nestor L. Müller, Vancouver, Canada.)

pneumonia may combine with cellular interstitial pneumonitis. Their margin is poorly defined but, when a sharper interface is present, the characteristics of the nodules may mimic a miliary pattern on HRCT.70 Decreased attenuation and vascularity associated with expiratory lobular air trapping (one of the causes of so-called mosaic attenuation)76 is often seen as a polygonal focus that may approximate the size of a secondary lobule. It may be located centrally or peripherally within the parenchyma77 and is assumed to be secondary to bronchiolar obstruction. This pattern has been cited as the most common CT abnormality in some studies,46 although it is usually not widely manifest. A number of other features may coexist and are of less discriminating value in the diagnosis. Reticulation may be superimposed on ground-glass opacification and may thus suggest a picture of fibrotic nonspecific interstitial pneumonia (NSIP).78 When reticulation is expressed in a bilateral, subpleural fashion with honeycomb remodeling, the pattern of usual interstitial pneumonia/idiopathic pulmonary fibrosis comes to mind.69 Consolidation density (homogeneous increase in parenchymal attenuation that obscures vascular and airway margins)76 reflects organizing airspace exudates and is usually focal in HP. If present in significant amounts, it probably signifies underlying infection or acute exacerbation of HP.69 Randomly distributed, thin-walled 3–25 mm parenchymal cysts are noted in 13% of cases79 and appear similar to those described in lymphoid interstitial pneumonia (see Chapter 34).80 They probably represent changes secondary to bronchiolar obstruction.

Chronic HP Chest roentgenograms and CT scans demonstrate reticular opacities often attenuated by honeycomb remodeling and

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(a)

(b)

Figure 4. (a) Inspiratory high-resolution CT image at the level of the right middle lobe bronchus shows mild bilateral ground-glass opacities, subtle localized areas of decreased attenuation and vascularity, and mild peripheral reticulation. (b) Expiratory high-resolution CT image demonstrates bilateral lobular areas of decreased attenuation and vascularity consistent with air trapping. The findings are characteristic of a combination of subacute and chronic HP. (Courtesy of Dr Nestor L. Müller, Vancouver, Canada.)

Figure 5. High-resolution CT image at the level of the bronchus intermedius shows bilateral ground-glass opacities and peripheral reticulation. The findings are consistent with chronic HP. (Courtesy of Dr Nestor L. Müller, Vancouver, Canada.)

volume loss (Figures 4, 5 and 6).45 The ability of HRCT to discriminate chronic HP from other chronic interstitial diseases is the subject of recent study. The pathological expressions of chronic HP overlap with a number of fibrosing interstitial pneumonias, most particularly usual interstitial pneumonia/idiopathic pulmonary fibrosis (UIP/IPF) and NSIP. Because of its relative lack of fibrosis, subacute HP is generally easier than chronic HP to distinguish from IPF81 radiologically. Three radiological features tend to distinguish chronic HP from UIP/IPF and NSIP: lobular areas with decreased attenuation (80% of HP), centrilobular nodules (56% of HP) and absence of lower zone predominance (31% of HP).81 The centrilobular nodules corresponded to histological features of cellular bronchiolitis, non-necrotizing granulomas and bronchiolocentric interstitial pneumonitis,

typical changes of subacute-type injury. Interestingly, the prevalence of honeycomb remodeling was not significantly different between UIP/IPF and chronic HP cases, although the honeycomb change was more likely to be basal predominant in UIP/IPF. The diagnostic accuracy for HP was 85% with HP as a first-choice diagnosis, 88% with HP as a second-choice diagnosis and 70% with HP as a probable diagnosis.81 In a radiological study examining patients with CT findings consisting primarily of centrilobular nodules, all 15 patients with HP contained ill-defined ground-glass nodules, and 60% of cases demonstrated upper lung zone predominance.82 The nodules corresponded to pathological features of interstitial lymphocytic infiltrates and poorly defined granulomas distributed around bronchioles. There was no bronchial wall thickening or luminal dilatation.82 Just as HRCT has been employed for the diagnosis and prognostic evaluation of other interstitial lung diseases, some investigators have attempted to quantify fibrosis and to relate radiological imaging patterns to survival (see Chapter 10). One study of 69 patients with chronic HP, 75% of which had histological confirmation of the diagnosis, divided patients into fibrotic (38%) and non-fibrotic (63%) cohorts. CT findings of parenchymal fibrosis were associated with adverse outcome in chronic HP and the extent of fibrosis was positively related to mortality.83 In this study, there was no proportional difference between fibrotic and non-fibrotic cohorts with respect to ground-glass opacification, centrilobular nodules or mosaic attenuation. Honeycomb remodeling also had no predictive value, possibly due to the small numbers of cases (7%) demonstrating this feature.83 An earlier study examined CT findings in a group of 26 patients with chronic HP (58% fibrotic, 42% non-fibrotic, by pathological assessment)52 and found no difference in survival between groups radiologically designated as UIP/IPF, HP or mixed patterns. This was despite showing a significant decreased survival in

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(a)

(b)

Figure 6. (a) High-resolution CT image at the level of the lower lung zones shows extensive bilateral ground-glass opacities, reticulation, traction bronchiectasis and bronchiolectasis. The findings are characteristic of fibrotic NSIP yet represent chronic HP. (b) Transverse section of the right lung from the same patient. The chronic HP lung is diffusely fibrotic in a pattern of fibrosing NSIP (see Figure 22 for greater detail). Although subpleural honeycomb changes are minimal, parenchymal cysts representing traction bronchiectasis and bronchiolectasis are noted (arrows). (Bouin’s-fixed lung explant.) (Radiology courtesy of Dr Nestor L. Müller, Vancouver, Canada.)

the group with histologically evident fibrosis. Lack of a significant association may be the result of small patient numbers, time between pathological diagnosis and CT examination, and differences in levels and intervals of antigen exposure.

Etiology and pathogenesis Inciting agents Numerous substances have been reported to cause HP, and antigenic sources include those derived from bacteria, fungi, animal and plant proteins and low molecular weight bioactive chemical compounds (see Table 2). Most reported forms of HP are related to thermophylic actinomyces, fungi and avian protein exposures.1,84 Many of these sources are associated with disease syndromes, often fancifully named, and for which the concept of hypersensitivity pneumonitis is often better recognized (e.g. farmer’s lung, BFL). The site of exposure may be domestic, recreational or occupational. While initial reports of HP in the modern literature were described in the occupational setting (farmer’s lung,85,86 bagassosis87 and bird breeder’s disease88), it is now apparent that many cases are now identified in a domestic context.3,29,66,89 The relative decrease in farmer’s lung is probably secondary to recognition and better industrial hygiene, leaving BFL as the most common form of HP in many clinical practices.9 Antigen-related factors that influence the clinical expression of HP include antigen concentration, solubility, size of respirable particulates, duration and periodicity of exposure, and factors related to workplace hygiene.5 The particulates are categorized as being between 1 and 3 µm, a size that allows for alveolar deposition.5,90 It is recognized that a particular “dust” can harbor numerous antigens. For example, 46 antigens were recovered from Saccharopolyspora and multiple antigens have been identified in a spectrum of avian components.54,91 Interestingly, the wide variety of implicated antigens produces

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similar lung disease. This finding suggests that pulmonary manifestations are a common pathophysiological response.54 The prevailing opinion is that acute forms of HP arise secondary to intense, periodic exposure, while subacute HP follows less intensive exposure, although probably on a more continuous basis. Chronic HP can be the product of both acute and subacute phases but also as a result of much lower exposures over a prolonged period of time.5 The concept of “bird-years” in calculating exposure risk for BFL has been advanced.92

Immunological background The pathogenesis and exact molecular mechanisms that give rise to HP are unknown. Laboratory investigations often use soluble and cellular material fractions from BAL fluid, which have been considered representative of inflammatory events within the lung in HP.54 Surgical biopsies also yield valuable information, derived not only from histological interpretation, but also from other studies including gene expression analysis.93 In addition to human studies, a number of animal models and in vitro methods explore putative immunological pathways of HP development. These models employ (1) nasal or intratracheal instillation of antigens (most notably S. rectivirgula, SR) on strains of mice sensitive to SR,94–96 (2) initial sensitization using intraperitoneal or intravenous administration of antigen followed by intratracheal challenge97 and (3) adoptive transfer of previously sensitized immune cells into naive animals followed by intratracheal antigen challenge.98 Gene analysis offers insight into the factors that might influence individual susceptibility to HP. Polymorphisms involving the major histocompatibility complex, tumor necrosis factor-α (TNFα),99 transporter associated with antigen processing (TAP) 1100 and tissue inhibitor of metalloproteinase 3101 are associated with development or resistance to HP. Additional

Chapter 12: Hypersensitivity pneumonitis Table 2 Selected etiological agents in hypersensitivity pneumonitis

Disease/syndrome

Exposure source

Antigen/substance

Diseases due to bacteria and fungi Farmer’s lung

Moldy hay

Saccharopolyspora rectivirgula (Micropolyspora faeni) Thermoactinomyces vulgaris

Composter’s lung

Compost

Thermoactinomyces vulgaris

Mushroom worker’s lung

Mushroom compost

Thermoactinomyces sacchari Agaricus hortensis spores

Bagassosis

Moldy sugarcane/residues (bagasse)

Thermoactinomyces sacchari Thermoactinomyces vulgaris

Humidifier / air conditioner lung

Water reservoirs

Thermoactinomyces candidus Thermoactinomyces sacchari Thermoactinomyces vulgaris

Suberosis

Cork dust

Aspergillus fumigatus Penicillium frequentans

Stipatosis

Esparto grass fibers (manufacture of ropes, mats, stucco)

Aspergillus fumigatus Thermophillic actinomyces

Malt worker’s lung

Moldy grain/barley

Aspergillus clavatus Aspergillus fumigatus

Potato-riddler’s lung

Moldy hay around potatoes

Thermophilic actinomycetes

Woodworker’s lung

Oak/cedar/mahogany dust; pine/spruce pulp

Penicillium chrysogenum Alternaria spp. Wood dust

Wood trimmer’s lung

Moldy wood trimmings

Rhizopus spp. Mucor spp.

Dry rot lung

Rotten wood

Merulius lacrymans

Thatched roof lung

Dead grasses/leaves

Saccharomonospora viridis

Tobacco-worker’s lung

Tobacco mold

Aspergillus spp.

Peat moss plant worker’s hypersensitivity pneumonitis

Peat moss processing environment

Monocillium spp. Penicillium citreonigrum

Cheese washer’s lung

Cheese mold

Penicillium casei

Winegrower’s lung

Grape mold

Botrytis cinerea

Sewage-worker’s lung

Sewage

Cephalosporium

Detergent worker’s lung

Detergent enzymes

Bacillus subtilis

Machine-operator’s lung

Contaminated aerosolized metalworking fluids

Pseudomonas fluorescens Mycobacterium immunogenum

Familial hypersensitivity pneumonitis

Contaminated wood dust in walls

Bacillus subtilis

Sequoiosis

Moldy sequoia (saw)dust

Aureobasidium pullulans

Maple-bark stripper’s lung

Cryptostroma corticale

Paprika slicer’s lung

Paprika pods

Mucor stolonifer

Hot tub lung

Contaminated hot tub water (mist); ceiling mold

Mycobacteria avium intracellulare Cladosporium spp.

Sauna-taker’s disease

Contaminated sauna water

Aureobasidium spp.

Basement shower hypersensitivity pneumonitis

Shower mold

Epicoccum nigrum

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Chapter 12: Hypersensitivity pneumonitis Table 2 (cont.)

Disease/syndrome

Exposure source

Antigen/substance

Summer-type hypersensitivity pneumonitis

Household mold, damp wood and mats (Japan)

Trichosporon asahii Trichosporon mucoides

Sax lung

Saxophone mouthpiece

Candida albicans

Dog house lung

Animal bedding

Aspergillus versicolor

Contaminated water

Naegleria gruberi Acanthamoeba polyphaga Acanthamoeba castellani

Bird-fancier’s lung (BFL) Bird-breeder’s lung Pigeon-breeder’s lung

Birds (pigeon, dove cockatiel, budgerigar, turkey, duck); feather comforters, pillows, feathers used in hobbies, decorations etc.

Avian proteins contained in excreta (pigeon intestinal mucin and pigeon IgG/IgA), urine, serum (γ-globulins, albumin), feathers (bloom – a waxy substance coating feathers; pigeon IgG/IgA)

Animal handler’s lung Laboratory worker’s lung

Rats, gerbils

Urine, serum, pelt proteins (rodent dander)

Furrier’s lung

Animal pelts

Animal fur dust

Pituitary snuff-taker’s lung

Pituitary snuff

Bovine/porcine pituitary proteins

Fishmeal worker’s lung

Fish

Fishmeal dust

Bat lung

Bat excreta

Bat serum protein

Miller’s lung

Flour, infected grain

Wheat weevil (Sitophilus granarius)

Sericulturist’s lung

Silkworm larvae

Silkworm larval proteins

Mollusc worker’s lung

Sea-snail shells (manufacture of nacre buttons)

Sea snails (Tectus niloticus)

Soybean worker’s lung

Soybeans

Soybean hulls

Coffee worker’s lung

Coffee beans

Coffee bean dust

Lycoperdonosis

Lycoperdon spp. (puffballs)

Puffball spores

Pauli’s reagent alveolitis

Laboratory reagent (chromatography)

Sodium diazobenzene sulphate

Painter’s lung

Paints, plastics, paint hardener

Isocyanates

Chemical worker’s lung (Plastics worker’s lung)

Paints, plastics, elastomers, polyurethane foams, special glues

Isocyanates, trimellitic anyhydride

Epoxy worker’s lung

Heated epoxy resins

Phthalic anhydride

Pyrethrum pneumonitis (insecticide lung)

Insecticide

Pyrethrum

Vineyard sprayer’s lung

Vineyard fungicide

Bordeaux mixture (calcium hydroxide/copper sulphate)

Diseases due to amebae Humidifier lung

Diseases due to animals

Diseases due to plants

Diseases due to chemicals

Unknown Bible printer’s lung

Moldy typesetting water

Coptic lung (mummy handler’s lung)

Cloth mummy wrappings

Grain measurer’s lung

Cereal grain

Tap water lung

Contaminated tap water

Table compiled from information presented in references 1, 3, 9, 66, 84.

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linkages between HLA types and HP disease have been identified in farmer’s lung (HLA B8) and Japanese summer-type hypersensitivity (HLA-DQw3).102 Microchimerism predisposes individuals to HP.103 Patients with HP demonstrate an increased frequency of male microchimerism. These cells can be of several lineages, including macrophages, CD4þ or CD8þ T lymphocytes. Furthermore, in keeping with a view that microchimerism may be involved in the pathogenesis of some autoimmune diseases, the presence of microchimeric cells might increase the severity of HP.103 The initial events leading to pulmonary inflammation in HP are not clear. The antigenic particulates that cause HP are approximately 1mm in diameter.9 The initial cellular response appears to be neutrophilic with polymorphonuclear leukocytes appearing in the BAL fluid between 24 and 48 hours postexposure.55,96,104 In a murine model of SR-induced HP, raised BAL fluid levels of C-C chemokines, monocyte chemoattractant protein-1 (MCP-1) and macrophage inflammatory protein-1α preceded the intense neutrophilic exudate and subsequent increase in lymphocytes and macrophages. This led the investigators to suggest local chemokine production by alveolar macrophages may be an initial event that influences inflammatory cell exocytosis into the alveolar compartment.96 Barrera et al.105 provide evidence that the BAL fluid inflammatory cell immunological (immunophenotypic) profile differs between the subacute and chronic inflammatory states. In chronic HP compared to subacute HP, for example, the CD4þ:CD8þ ratio is increased and there is an increase in terminally differentiated memory CD4þ and CD8þ cells. Lower IFN-γ production is noted and CD8þ T lymphocytes exhibit decreased cytotoxic activity. This profile suggests that the antigen specificity of the memory T-cell lineage becomes exhausted. In addition, the authors also provide further evidence for a skewing of T-cells towards a Th2 phenotype relative to Th1 in chronic HP.105 In a microarray study, using tissue derived from lung biopsies, Selman and colleagues identified gene expression patterns that could differentiate IPF from HP.93 This analysis also reveals upregulation of a number of genes related to differentiation of the Th17 cell lineage, a recently described distinct T-cell subset identified in chronic inflammatory and autoimmune diseases.106,107 Recent experimental mouse models of HP demonstrate an increased Th17-polarized Tlymphocyte response with increased concentrations of interleukin-17.108,109 IL-17 may have a role in the development of pulmonary fibrosis and is a potentially attractive target for pharmacotherapy.

Effect of smoking on HP

Hypersensitivity pneumonitis is more likely (80–95%)24 to occur in non-smokers as compared to current or former smokers, given equivalent exposures.1,28,110,111 Cigarette smoke contains over 4000 chemicals112 and nicotine is recognized as having

immunosuppressive effects.113 Other active ingredients in cigarette smoke are probably responsible for immune cell dysfunction.114 Cigarette smoke may blunt or alter the expression of HP, since specific IgG antibody levels are reportedly lower in smokers than in non-smokers.21,115 In a murine model of chronic smoke exposure, dendritic cells produce less IL-12p70, a key polarizing cytokine linked with activation of ERK-dependent pathways.116 Cigarette smoke extract also imparts inhibitory effects on dendritic cell function related to T-cell priming and skews the immune cell population towards a Th2 profile.114 Putative protective effects of smoking may also be exerted through decreased levels of B7 family co-stimulatory molecules on alveolar macrophages.113,117 This suggests either an active suppression of macrophage activity by smoking or the selective ability of tobacco smoke to only recruit alveolar macrophages with low constituent B7 expression.117 It is possible cigarette smoking suppresses the key immunological components necessary for acute forms of HP but allows for a more insidious disease course in which established pulmonary fibrosis is already manifest at the time of diagnosis. This hypothesis would explain, in part, the recognized poorer outcome of the smoking cohort.111 In addition, smokers are apparently more prone to develop acute exacerbations of HP.38

Pathological findings Role of the surgical lung biopsy in HP Indications for a surgical lung biopsy in the diagnosis of HP may vary between centers, depending on the degree of institutional experience and confidence in the clinical diagnosis. Thus, in many instances, biopsies may be submitted from only the most challenging cases. Typical indications for obtaining a lung biopsy include: (1) to address diagnostic uncertainty when clinical and radiological features are not definitive, including cases where no offending antigen is identified,118 or where environmental antigenic sources are ubiquitous and exposure is indiscriminate;1 (2) to verify the initial clinical impression, when the course or response to treatment deviates from the expected; (3) to add prognostic information, particularly in assessing the presence and degree of fibrosis; and (4) to exclude infectious processes.6,24 Although many reviews acknowledge the transbronchial biopsy as an option, these are of limited utility.119 Wedge biopsies, obtained through either video-assisted thoracoscopic surgical (VATS) or open thoracotomy procedures, are preferred. As with other interstitial lung disease, biopsy of more than one lobe is recommended. The pathology of HP is considered to be more or less uniform, although a few cases may yield discordant lobar biopsy findings.118,120 Several studies document the utility of surgical lung biopsy in the diagnosis of HP. A recent publication estimated interobserver variation in the pathological diagnosis of diffuse

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parenchymal lung disease in surgically obtained lung biopsies. The final weighted kappa (k) coefficient of agreement for HP was 0.47 (moderate/satisfactory). This value was less than that for other interstitial diseases (sarcoidosis k ¼ 0.75; organizing pneumonia k ¼ 0.70; UIP k ¼ 0.59) although greater than for NSIP (k ¼ 0.40).120 The lower kappa values generated for HP by the 10-member expert pathology panel may reflect the relative multiplicity of histopathological elements required for the diagnosis.120 It is salutary that a companion interobserver study121 by radiologists had a kappa value in this disease of 0.6. It should be noted, however, that radiologists have the ability to scroll slices in real time and to view coronal and sagittal reformatted images in addition to the standard axial views. Although some biopsy findings may present very compelling features, there is no universally established diagnostic “gold standard”. The pathological diagnosis of HP, as with all diffuse parenchymal lung diseases, is likely to be more accurate with clinical and radiological correlation.122 A study examining the multidisciplinary approach to the diagnosis of idiopathic interstitial pneumonias found histopathological data (and pathologist input) had the single greatest impact on refining the final diagnosis. This was pronounced in cases where the initial clinical diagnosis was other than idiopathic pulmonary fibrosis.123

Acute HP Histopathological descriptions of acute HP are scarce, and most of the small series of cases that are repeatedly referenced in standard textbooks originate in the 1960s.124–127 Some of these reports are compromised to the extent that there does not appear to be a consistent (or any) case definition of “acute” HP at that time. In fact, one series of cases had biopsies taken 3, 5 and 9 weeks and 7 months post onset and all patients were treated with corticosteroids in the intervening period.125 Nevertheless, these reports tend to describe a bronchiolocentric cellular interstitial pneumonitis dominated by lymphocytes. Several also described obstructive small airway disease, seemingly representing obliterative bronchiolitis. Histiocytic giant cells and non-necrotizing granulomas are common. This aspect of the pathology appears more consistent with what has subsequently become accepted as the classic subacute stage (see below). In addition, some cases also showed airspace edema, fibrinous exudates, erythrocytes and neutrophils, now generally referred to as “organizing acute lung injury”. Two reports identified a neutrophilic capillaritis as a component of the injury pattern.125,126 However, whether this represented a neutrophilic leukostasis, as part of a generalized inflammatory reaction, or a specific immune-type vasculitis is not certain.

Subacute HP The classic histopathological disease pattern in this stage features the triad of (1) cellular bronchiolitis, (2) cellular

450

Figure 7. Early/subacute HP. Low-power micrograph demonstrating the classic centrilobular mononuclear interstitial infiltrate that becomes attenuated towards the lobular periphery. Wedge biopsy, inflated with formalin. Asterisk, membranous bronchiole; S, secondary lobular septum.

(essentially lymphocytic) interstitial pneumonitis and (3) small, poorly formed (“vague”) non-necrotizing histiocytic granulomas and/or single multinucleated histiocytic giant cells. These primary features are often attended by other inflammatory changes, including proliferative bronchiolitis obliterans, organizing pneumonia and variable amounts of fibrosis. Despite finding these classical histological features, some investigators only identify a specific exposure in approximately one-third of cases.128

Bronchiolitis Although bronchiolitis is a significant feature, classic subacute HP is usually not considered a primary bronchiolitis but rather classified within the group of “interstitial lung diseases with prominent bronchiolar involvement”. The airflow obstructive component of HP is predicated upon this involvement.129 A cellular infiltrate of variable density, composed primarily of lymphocytes and lesser numbers of plasma cells, is present within bronchiolar walls and extramurally, with possible extension into alveolar duct walls (Figures 7 and 8).128 The bronchiolar epithelium may130 or may not128 demonstrate squamous metaplasia. There may be associated smooth muscle hyperplasia and fibrosis.130 Segments of the mucosa may ulcerate in the presence of intraluminal buds of organizing connective tissue (proliferative bronchiolitis obliterans).127 Inflammation, fibrosis and smooth muscle hyperplasia can combine to produce compressive luminal encroachment, similar to constrictive bronchiolitis obliterans.130 Small peribronchial lymphoid aggregates are commonly noted and occasionally manifest as follicles with germinal centers (Figure 9),130 suggestive of inducible bronchus-associated lymphoid tissue (BALT).131

Chapter 12: Hypersensitivity pneumonitis

Figure 8. Early/subacute HP. In this example, which is fairly typical of HP, the bronchiolitis is mild and seen as scattered lymphocytes percolating within the epithelial layer of a terminal bronchiole (asterisk) as well as extending into the peribronchial interstitium. The arrows indicate airspace aggregates of foamy histiocytes which are often detected adjacent to inflamed or obstructed airways.

Figure 9. Subacute HP. A longitudinally sectioned bronchiole is seen along the top of the photomicrograph. A lymphoid follicle with germinal center is noted in the peribronchiolar interstitium (arrow). The mononuclear inflammatory infiltrate within the adjacent alveolar interstitium demonstrates the density that is characteristic of HP. The alveolar walls are expanded, indicating the beginning of fibrosis.

Figure 10. Subacute HP. The nodular quality of the interstitial and airspace inflammation can be appreciated here. In this example the wedge-shaped, or segmental, lobular distribution of the inflammatory process is apparent and could be responsible for the focal atelectasis and fibrosis identified in more advanced cases (see Figure 25).

Figure 11. Subacute/chronic HP demonstrating a cellular/fibrosing NSIP pattern. The characteristic lymphohistiocytic interstitial infiltrate is more or less equally distributed throughout the entire lobule. Note that the process is much less dense in the adjoining lobules. Arrows, interlobular septa.

Interstitial pneumonitis The interstitial pneumonitis is invariably mononuclear with lymphocytes predominating but they are combined with plasma cells and histiocytes.127,132 Small numbers of neutrophils and eosinophils may be present, with eosinophils being the more common of the two.132 The infiltrate can be patchy and of variable density, presumably related to exposure sequence and dose.133 The greatest density is in the centrilobular or peribronchial compartment, with progressive tapering towards the lobular periphery (Figures 9 and 10).24 This focality is

the pathological correlate of the small ground-glass nodules observed on CT scans (Figure 2). Pneumonitis in HP may also manifest as a pattern of NSIP, as either a cellular, fibrotic or mixed variant,134 in which case the inflammation is more diffuse (Figure 11).

Granulomas Granulomatous inflammation, indicative of the underlying cell-mediated immune reaction, is one of the histological hallmarks of HP. The granulomas are non-necrotizing, composed of epithelioid histiocytes with or without multinucleated

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(a)

(b)

Figure 12. Case of subacute/chronic HP showing inflammatory changes within the peribronchiolar interstitium. (a) At medium magnification, there is mild to moderate expansion of the alveolar walls by a predominantly lymphocytic infiltrate. A cluster of airspace macrophages is identified (arrow). The peribronchiolar connective tissue contains a poorly formed granuloma (arrow). (b) Higher magnification of the peribronchiolar granuloma. The large multinucleated histiocytic giant cells are obvious; however, the associated mononuclear histiocytes (adjacent to the giant cell) blend in with lymphocytes dispersed in the stroma. This lack of a defined boundary is characteristic of granulomas in HP.

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Figure 13. Subacute HP. The alveolar interstitium contains a modest lymphoplasmacytic infiltrate causing slight expansion of the alveolar walls. An aggregate of two multinucleated histiocytic giant cells is noted within.

Figure 14. Subacute HP. Histiocyte accumulation in a case of subacute HP. The arrows indicate a small interstitial granuloma in which multinucleated giant cells contain acicular cytoplasmic cholesterol clefts and an aggregate of moderately cohesive histiocytes in a nearby airspace.

histiocytic giant cells, and are typically loosely formed.128 This latter quality is responsible for the term “vague”, as applied to the histiocytic aggregate. The size is of an order smaller than sarcoid granulomas135 and there tends not to be the perigranulomatous fibrosis that is often seen in sarcoid (see Chapter 13). The granulomas are classically situated in the bronchiolar walls or peribronchiolar interstitium (Figure 12) but may extend, with the pneumonitis, into the alveolar duct region. A chronic inflammatory infiltrate typically invests or surrounds the

granulomas. Multinucleated giant cells, of Langhans or foreign body type,127 are noted but not invariably present.127 They may be prominent in some cases.136 Sometimes, only solitary giant cells within a sparse cellular interstitial pneumonitis are observed (Figure 13). Histiocytic aggregates and giant cells may also be seen within the airspaces. The histiocyte cytoplasm can contain acicular, clear, cholesterol clefts (Figure 14) or other inclusions, such as Schaumann bodies, asteroid bodies, or oxalate crystals (see Chapters 2 and 13).24

Chapter 12: Hypersensitivity pneumonitis

Figure 15. Maple bark stripper’s disease. Two conidia (spores) of Cryptostroma corticale are identified within the cytoplasm of a multinucleated histiocytic giant cell (arrowhead). The organisms typically display a brownish coloration and may range in size from 4.0–6.5  3.5–4.0 micrometers in dimension. (Image courtesy of Dr DB Flieder, Philadelphia, PA, USA.)

Figure 16. Small foci of organizing airspace/airway tissue (Masson body) are typical of subacute HP. They are often seen in conjunction with aggregates of foamy alveolar histiocytes (arrows), reflecting focal airway obstruction. Such features, often identified within regions of collapsed and inflamed lung, may be clues to the diagnosis of HP.

Granulomas are not always present, possibly secondary to limited tissue sampling, antigen-related factors and/or disease stage. Within the literature, granulomas have been quoted as present in 66%132 and 70%127 of cases, with lower rates in more chronic cases (see below). The appearance of the granulomas may vary with the cause. For example, those associated with BFL have been described as smaller and more poorly differentiated than those of farmer’s lung.130,137 By contrast, in “hot tub lung” (see below), granulomas are better formed and frequently involve airway lumina.138 As with any granulomatous pneumonitis, special stains for microorganisms are mandatory but are unlikely to reveal a specific pathogen. Rare exceptions are atypical mycobacteria, actinomyces and the distinctly pigmented spores of Cryptostroma corticale (maple bark stripper’s lung) (Figure 15).

Airspace organization (intraluminal fibrosis) Organizing airspace and airway luminal exudates are frequently noted in HP,125,127 and are present in over half of the cases in some series.128,132,139 Lesions may be more prevalent in the early course of the disease.125 These can be classified as proliferative bronchiolitis obliterans (see above), when the organizing exudate involves lumina of the bronchioles, and organizing pneumonia (OP) when distal airspaces are affected.128 A descriptive morphological classification relevant to a number of interstitial lung diseases has been described in HP.139 Intraluminal buds correspond to the “Masson bodies”, and are typically identified in alveoli and alveolar ducts (Figure 16). Lesions are composed of loose masses of inflammatory and connective tissue cells within a collagenous and proteoglycan matrix. These probably develop from acute fibrinous exudates, secondary to alveolar capillary leakage.

Figure 17. Mural incorporation of organizing airspace exudates. The arrows indicate foci of pale staining fibroblastic tissue adherent to underlying alveolar walls and lined, superficially, with hyperplastic type II pneumocytes. The arrowhead indicates a cluster of foamy alveolar histiocytes.

Intraluminal buds are epithelialized and vascularized prior to resorption.132 Obliterative changes, where organizing connective tissue fills and obliterates entire lumina of either airspaces or conducting airways, signify a more severe injury pattern and more likely progress to permanent remodeling.139 Mural incorporation is characterized by aggregates of organizing connective tissue, similar to the above. There is, however, evidence of incorporation or blending into airway or alveolar septal walls, often with surface epithelialization (Figure 17). In diffuse pulmonary

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Figure 18. Mucostasis. Two pools of inspissated mucin, containing degenerated inflammatory cell debris, are noted within a respiratory bronchiole and alveolar duct (asterisk). The surrounding interstitium contains the characteristic mononuclear infiltrate with “vague” granulomas (arrowheads).

diseases this can be associated with significant parenchymal remodeling, such as airway and alveolar wall thickening, although in HP this is more of a focal process.139 Patchy foci of obstructive pneumonia are often seen adjacent or distal to organizing luminal exudates. These foci may be inconspicuous and are typically limited to the peribronchiolar region24 and feature alveolar histiocytes, including multinucleated histiocytes, with vacuolated cytoplasm (Figures 8, 16 and 17).127 A desquamative interstitial pneumonia-like (DIP-like) pattern has also been described.140 Mucostasis can also be identified but represents a more specific marker of small airway obstruction (Figure 18).

Chronic HP Chronic HP has been the topic of many recent publications, especially as it relates to other chronic fibrosing interstitial pneumonias in general, and UIP in particular. The spectrum of pathological changes in chronic HP may recapitulate a number of patterns of interstitial lung disease, as embraced by the 2002 American Thoracic Society/European Respiratory Society consensus classification of idiopathic interstitial pneumonias.60,78 It is, perhaps, self-evident that pulmonary fibrosis caused by antigen-induced hypersensitivity can be categorized as “chronic HP”. However, within this spectrum the co-expression of other pathological features more typical of HP is variable. Cases included in studies addressing the histopathological features of chronic HP appear to be drawn from a variety of clinical presentations, antigen sources and exposure contexts. Tissue analysis in these studies originates from both surgical biopsy and autopsy material.

Granulomas There is a general understanding that granulomatous inflammation tends to dissipate with chronicity.125,135,141 Chronic

454

Figure 19. Finding isolated multinucleated histiocytic giant cells (arrow) within a region of honeycomb or otherwise fibrotic lung warrants a consideration of chronic HP.

HP studies that include histopathological evaluation indicate a wide variation of cases (0–100%) in which the characteristic histiocytic inflammation (individual multinucleated giant cells and/or epithelioid histiocytic granulomas) is present. For example, in six cases of chronic HP, Seal et al.125 found “scattered giant cells” in all cases but no granulomas. Suda et al.131 found granulomas in two of five cases. In 26 cases of chronic BFL, Ohtani and colleagues60 identified granulomas in approximately 20% of patients. Churg and associates142 found either isolated interstitial giant cells, poorly formed granulomas or both in all of their 13 cases. Trahan et al.118 described 15 patients with chronic HP and found granulomas and isolated multinucleated giant cells in 58% and 71%, respectively, of their cases. Hayakawa et al.143 failed to find any granulomas in their series of five patients. Of note, granulomas can be identified in some patients with acute-onchronic HP.143 The point has been made that incidental giant cells and granulomas can be identified in a wide variety of lung diseases and, by themselves, do not confer an unimpeachable diagnosis of HP.144 Conversely, reliance on the identification of granulomas to confirm the diagnosis in the chronic setting may also pose some difficulty since these structures are only expected in 60–70% of subacute cases on average (0% in some series134). In the opinion of some,144 a confident diagnosis of chronic HP, in the context of UIP-like fibrosis, should only be made when coexisting areas of more typical HP are present (bronchiolocentric cellular interstitial pneumonitis with giant cells or granulomas). Regardless of the ability to impart a precise diagnosis on an individual case, any features suggesting HP, even a solitary granuloma (Figure 19), are worthy of note since they may suggest a different clinical focus and, ultimately, prognosis (see below).

Chapter 12: Hypersensitivity pneumonitis

Figure 20. A case of chronic HP showing a UIP pattern. There is a subpleuralpredominant interstitial fibrosis with alveolar septal thickening and (microscopic) honeycomb remodeling. The honeycomb spaces contain inspissated mucin. Centrilobular bronchioles are indicated by an asterisk. P, pleura.

Figure 21. Chronic HP showing fibroblastic foci composed of immature, myxoid collagenous tissue plaques lined with hyperplastic type II alveolar pneumocytes (black arrow). The remnants of a fractured, calcified Schaumann body are noted in collapsed, fibrotic parenchyma (white arrow); this finding is considered a footprint of granulomatous inflammation and in a lung biopsy should raise the possibility of HP.

Bronchiolitis, interstitial pneumonitis Active inflammatory/remodeling processes such as bronchiolitis, alveolitis and intraluminal fibrosis, including proliferative bronchiolitis obliterans and organizing pneumonia patterns, may be seen in chronic HP. Bronchiolitis was reported in 20%,143 33%,125 54%,60 96%,118 100%131 and 100%130 of cases of chronic HP. Alveolitis, as a separate feature, was noted in 83%,118 100%143 and 100%125 in some of the same studies. Intraluminal fibrosis, categorized as “bronchiolitis obliterans organizing pneumonia” (BOOP), was identified in 8%,60 40%131 and 42%118 of cases. Takemura et al.135 reported cellular NSIP/organizing pneumonia in 30% of chronic HP and Churg et al. found “areas of typical subacute HP” in seven of their thirteen cases.142 Some of these changes may be accompanied by fibroblastic foci (see below).

Fibrosis A number of investigators have attempted to define and quantify more precisely certain manifestations of fibrosis in lungs with chronic HP. Some newer usages of older terms and introduction of novel descriptors have imparted a degree of complexity to such an analysis. In general, the various forms of fibrosis potentially present in chronic HP include UIP with associated honeycomb remodeling, fibroblastic foci, fibrotic NSIP, “bridging fibrosis” and degrees of lobular atelectasis. In addition a number of terms that describe what is implied to be fibrotic conversion of interstitial (and possibly airspace) inflammation in the central region of the secondary pulmonary lobule are used. This latter concept and its terms overlap with those entities contained under the rubric of “centrilobular/bronchiolocentric interstitial

pneumonias” (see below). In this chapter the term “centrilobular fibrosis (CLF)” will be used as an all-encompassing term to describe fibrotic change, with or without coexistent inflammation that affects both the bronchiole and the radial peribronchiolar parenchyma. The most commonly described pattern of fibrosis in protracted cases of chronic HP is UIP. In several series, UIP, or UIP-like areas (Figure 20), were present in 40%,143 42%,60 69%,142 72%145 and 100%146 of cases. When fibroblastic foci (Figure 21) are specifically described, they are typically present in the setting of honeycomb and UIP-like changes.142,146 Trahan and colleagues, however, describe biopsies in which fibroblastic foci are not associated with either regions of honeycomb remodeling or organizing pneumonia.118 Fibrosing NSIP (Figures 22 and 23) as a stand-alone pattern of fibrosis was identified in 16%,147 16%,145 30%142 and 30%60 in cohorts of chronic HP but may also coexist in cases with more typical UIP-like pathology.146 Akashi et al. use the term “atelectatic fibrosis” to describe the effects of continuous organization and inflammation that may be an extension of the process of “bridging fibrosis” to involve the entire lobule.146 Sections of chronic HP may also demonstrate sublobular or segmental areas of collapse, presumably the combined effect of inflammation and obstruction involving only one or several respiratory bronchioles and the subtended alveolar parenchyma (Figures 24 and 25). Centrilobular fibrosis is the product of the classic bronchiolitis and centrilobular-accentuated interstitial pneumonitis that is the hallmark of acute/subacute HP. There is intrinsic value in its recognition, particularly in the diagnosis of chronic

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(b)

(a)

(c)

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(d)

Figure 22. Chronic HP with a fibrosing NSIP pattern, corresponding to the previously illustrated case in Figure 6. (a) Gross photograph of the cut surface of a transverse lung slice. The arrowheads indicate the section edge with pleural surface visible in the left half of the photograph. The parenchyma is diffusely involved by alveolar septal fibrosis with a spongework of residual ectatic airspaces barely visible. Dilated airways (traction bronchiectasis/bronchiolectasis) can be seen in the right side of the photograph (Bouin’s fixation). (b) Low-magnification photomicrograph from an area similar to (a) showing two lobules with diffuse alveolar septal thickening in a pattern most consistent with fibrotic NSIP. Microscopic honeycomb remodeling is also noted. (c) Gross photograph of another region of lung from the same case. In this example there is some residual normal or near-normal lung (dark areas) with interspersed fibrotic patches (light regions, arrows) and areas of more conglomerate fibrosis (asterisk) (Bouin’s-fixed specimen). (d) Microscopic image from a similar region demonstrating paucicellular interstitial fibrosis with a slight centrilobular predominance. The appearance of the fibrosis is similar to NSIP although much of the lobule is relatively spared.

Figure 23. Fibrosing NSIP pattern in chronic hypersensitivity pneumonitis. A delicate lattice-like alveolar septal fibrosis involves the majority of two lobules. The arrows indicate two regions of a more enhanced fibrosis around the centrilobular structures. P, pleural surface.

Figure 24. Subpleural atelectasis in a case of chronic HP. A broad band of collapsed and fibrotic lung is enclosed by the two arrows and demonstrates a fairly abrupt interface with the underlying parenchyma. Two ectatic, caliberpersistent, membranous bronchioles (*) lead into the collapsed region.

fibrosing interstitial pneumonias, since it should lead at least to a consideration of HP in the differential diagnosis. Centrilobular fibrosis may contribute to extrinsic narrowing of small airways and has been recognized to develop in concert with

more distal parenchymal fibrosis. Thus, the obstructive physiological component, secondary to remodeling of the conductive airways, may be overwhelmed by the dominant restrictive defect imposed by alveolar fibrosis.130

Chapter 12: Hypersensitivity pneumonitis

Figure 25. Segmental lobular fibrosis in a case of chronic HP. A wedge-shaped area of fibrosis extends peripherally from the centrilobular region (asterisk) to the pleura (P, showing fatty metaplasia). This probably represents a region of post-inflammatory scarring and atelectasis with subtotal involvement of the lobule. The interlobular septa, defining the periphery of the lobule, are indicated by the arrowheads. There is widening of the septal lymphatics in the lower example due to overdistension with formalin.

Figure 27. Fibrotic patterns in chronic HP. Portions of three secondary pulmonary lobules are contained within this biopsy. Two lobules (upper and left-hand side) demonstrate paucicellular scarring with a nodular characteristic that is predominately centrilobular. The lobule in the lower portion of the micrograph demonstrates a more diffuse fibrosis that encroaches onto the interlobular septum.

In recounting their observations of CLF in HP cases, many authors do not specify whether the fibrosis is accompanied by the characteristic lymphohistiocytic infiltrates (Figure 26) or is relatively paucicellular (Figure 27). Some form of CLF was identified in 23%142, 42%60, 50%143, 72%145 and 100% (in 25% of which the foci were “prominent”)146 of cases of chronic

Figure 26. An example of the mixed inflammatory and fibrotic pericentral lobular parenchyma in a case of chronic HP. The arrow indicates residual interstitial histiocytic giant cells. The widened interstitium is lined with metaplastic cuboidal/ciliated epithelium. A, pulmonary artery; B, bronchiole.

HP. In a study of 31 biopsies from 15 patients, Trahan et al. described centrilobular changes in 54% of biopsies; in all but one case, the fibrosis was accompanied by chronic inflammation.118 In a cohort of 76 patients with chronic HP (defined as having fibrosis on the basis of HRCT findings), 35% had centrilobular pathology, 93% of which harbored some degree of CLF.147 Centrilobular fibrosis is typically identified in the context of UIP/honeycomb changes. All instances of CLF in the series by Akashi et al.,146 Churg et al.142 and Ohtani et al.60 were associated with UIP-like fibrosis. In a recent study, 83% of cases of chronic HP showing a UIP-like fibrotic pattern also harbored areas of CLF. In the same study, three of the 25 chronic cases manifested CLF as the sole fibrotic change.145 Forty-six percent of cases containing CLF in the Lima study also displayed honeycomb remodeling.147 Of two of five chronic HP patients with CLF, one had coexistent UIP.143 There is a sense that, with progression, dense fibrosis and honeycomb remodeling will efface enough of the lobular parenchyma to obliterate histopathological evidence of previous focal centrilobular injury. This has been documented in occasional cases which compared pathology at different stages of the disease148 and is reflected in the greater degree of CLF relative to UIP or honeycomb fibrosis in subacute/acute-on-chronic versus chronic cases.143 In concert with the general observation that granulomatous inflammation dissipates with time, granulomas and/or multinucleated histiocytic giant cells may not be found in chronic HP cases with well-developed CLF.143,146,147

Vasculopathy Pulmonary arterial vasculopathy, including autopsy evidence of cor pulmonale, has been noted since the first reports of chronic HP.125 The characteristic changes are those of intimal

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Figure 28. Vasculopathy in chronic HP. The pulmonary artery features circumferential fibrointimal stenosis leaving a residual slit-lit lumen. The image also depicts established collagenous fibrosis and small fibroblastic foci that impart a UIP-like pattern of remodeling.

and medial hyperplasia (Figure 28), alterations which are almost ubiquitous in any chronic fibrosing interstitial process (see Chapter 10).

Chronic HP with acute exacerbation In patients with chronic HP and acute clinical deterioration, surgical lung biopsies or autopsy findings typically demonstrate acute and/or organizing diffuse alveolar damage (DAD). Less common acute lung injury patterns include organizing pneumonia and diffuse alveolar hemorrhage.36–38 In one report, the typical background fibrotic pattern is described as UIP-like.38

Special forms of HP Hot tub lung Hot tub lung (HTL) is established as one of the prototypical forms of lung disease due to non-tuberculous mycobacteria (NTM). It is specifically associated with Mycobacterium avium-intracellulare complex (MAC).149,150 Whether it represents infection or is best characterized as a hypersensitivity reaction remains a matter of debate.13,151–154 Indoor hot tubs, jacuzzi spas, whirlpools or showers,152 high water temperatures, poor cleaning/maintenance and the ineffectiveness of many disinfectants all predispose to MAI growth.150 Water jets stimulate aerosolization of contaminated water and facilitate inhalation of the microorganisms. Hot tub lung patients are typically immunocompetent young to middle-aged individuals (average age 43.1 years; range 9–69 years) with a slight female predominance (52.7%).155 The median duration of exposure to hot tubs is 5 months (range 1–54 months)154 but only a minority of patients may be able to temporally associate hot tub use with onset of symptoms.

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Figure 29. Hot tub lung. The granuloma pictured here is larger than those usually seen in otherwise typical HP. Protrusion of the granuloma into the bronchiolar lumen is a characteristic feature. (Case courtesy of Dr H.D. Tazelaar, Scottsdale, AZ, USA.)

Typical presentation is that of an acute flu-like illness with cough and dyspnea while constitutional symptoms are less prominent.154 Respiratory function abnormalities demonstrate an obstructive-dominant pattern in most cases, in contradistinction to the restrictive pattern that characterizes HP.154 Restrictive or mixed physiological deficits may also be present in HTL.155 A review of published cases indicates positive cultures are obtained from sputum, BAL fluid, lung biopsy and hot tub (or other source) in 74.1%, 62.5%, 85.7% and 94.7% of cases, respectively.155 Chest radiography demonstrates diffuse nodular or interstitial infiltrates, but may be normal in 22%.154 Computed tomography reveals diffuse, poorly defined low-attenuation centrilobular micronodules (65%), diffuse ground-glass opacification (65%) and lobular air trapping (100%).154,156 The majority of reports emphasize lower lobe predominance.149 The features are very similar to subacute HP. However a peripheral “tree-and-bud” pattern, reflecting exudative or cellular bronchiolitis, seen in HTL, is a potential distinguishing feature.156 Histopathological features of HTL are those of prominent, often described as exuberant, non-necrotizing granulomas composed of epithelioid histiocytes with occasional multinucleated giant cells. The granulomas tend to be well-defined, with a cuff of lymphocytes and a minimal tendency to coalesce.138 Although the granulomas can be randomly distributed within airspaces and interstitium, a frequent bronchiolocentric location is recognized, with granulomas situated within bronchiolar walls or airway lumina (Figure 29)138,154 and occasionally causing erosion of the airway mucosa. Pleural and septal distribution, as seen in sarcoidosis, is not a feature. Necrotizing granulomas are noted in 7.3% of cases and, although considered a rare occurrence by some authors,

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literature review indicates that 25.9% demonstrate positivestaining for acid-fast bacilli (reviewed in155). The granulomas are attended by a cellular interstitial pneumonia with alveolar septal thickening, which is typically patchy and of variable severity, and by cellular bronchiolitis. Organizing pneumonia, characterized by airspace plugs of young collagenous tissue, is often present138 in up to half of cases.136 A point of emphasis is that in HTL the granulomatous disease dominates the interstitial pneumonitis. In HP the interstitial component is usually the most prominent feature and the small, loosely aggregated, “vague” granulomas may be fairly subtle or even absent. Treatment recommendations for HTL are uncertain since the medical field is not certain whether it is a primary infection or a hypersensitivity process. Hot tub avoidance alone is effective in facilitating recovery although some patients may require corticosteroids and, ultimately, specific antimicrobial pharmacotherapy.150

Japanese summer-type hypersensitivity pneumonitis (SHP) A distinctive summer HP is the most prevalent form in Japan,22,157,158 accounting for approximately three-quarters of all cases of HP. The northern portion of the country appears to be spared. The causative antigens are derived from the fungi Trichosporon asahii proposed (as T. cutaneum) as a candidate source in 1984159 and Trichosporon mucoides. These organisms colonize damp and decaying wood, as may be present in older domiciles with poor ventilation and decreased sunlight, in straw tatami floor mats and bedclothes.160 Females are affected twice as frequently as males, probably reflecting increased domestic exposure. The age range of affected patients is 2–86 years, the peak incidence being in the forties.22,158 Initial symptoms occur in the hot and humid summer to mid-autumn months that follow the rainy season.22 Recurrent seasonal symptoms are noted as are precipitous recurrences following re-introduction of the patient into contaminated home environments. In subacute cases, the histopathology is like other forms of HP with a near-universal alveolitis, typically with centrilobular accentuation, attended by varying degrees of bronchiolitis, organizing airspace exudates and granuloma formation.22,157 While SHP contributes to the bulk of acute/subacute presentations in Japan, this etiology represents a smaller number of cases of chronic HP. Yoshizawa et al. identified 36 cases of chronic HP of which ten were due to SHP.161 The remaining etiologies in this series included BFL, isocyanate-induced HP, farmer’s lung and home-related HP. The mean age of the patients in this series of mixed etiology was 57.7 years with an 8:2 female:male predominance. Only one of ten chronic SHP patients had an insidious onset. An additional two cases of insidious onset have been reported; in both instances the original diagnosis was that of UIP/IPF and the point is made that many cases of chronic HP may be misdiagnosed as an idiopathic interstitial pneumonia.162 Pathological descriptions of chronic SHP include the following features: UIP-like lesions in

association with “airway-centered fibrosis”,162 honeycombing, fibroblastic foci, “centrilobular fibrosis” and “bridging fibrosis”.163 In nine chronic SHP patients with surgical/transbronchial biopsy specimens, Yoshizawa et al. observed alveolitis in 9/9, Masson bodies in 2/9 and granulomas in 4/9 cases.161

Isocyanate-related HP Isocyanates are compounds used extensively in industry as cross-linking agents in the polyurethane industry, including paints. Spray painters are at particular risk of acquiring pulmonary disease when exposed to these compounds.164,165 The most common agents are toluene diisocyanate (TDI), diphenylmethane diisocyanate (MDI) and hexamethylene diisocyanate (HDI).164 The reactive NCO group on the molecules can bind to carrier proteins, an attribute that may be important in disease initiation. The majority of industrial health-related lung disease cases related to these compounds have occupational asthma; however, a small proportion of affected patients evince clinical features of hypersensitivity pneumonitis.165–170 Unfortunately, pathological descriptions of likely cases are few and often derived only from transbronchial biopsy samples. These reports indicate characteristic changes found in classic subacute HP, such as lymphohistiocytic alveolitis, organizing airspace exudates (Masson bodies), some degree of bronchiolocentricity and, on occasion, epithelioid histiocytic granulomas.166,170 Eosinophilic alveolar exudates and acute inflammation have been described.169

HP-like syndrome in occupational exposure to metal-working fluids

First described in 1995171 under the rubric of “machine operator’s lung”, occupational exposure to metal-working fluids (or metal removal fluids) used in metal forging, machining and stamping172 may produce an HP-like illness. The reported cases and outbreaks are associated with the automotive industry. It is agreed the water fraction of water-based fluids (emulsions, semisynthetic or synthetic compounds) harbors the offending microorganisms. These products become aerosolized, and therefore prone to inhalation during the machining process. This condition is often associated with occupational asthma.173 The original cases identified Pseudomonas fluorescens as the most common microbial isolate in culture and associated with serum precipitating antibodies; however, a variety of other organisms are also identified.171 Of late, Mycobacterium immunogenum is implicated as the likely causative agent in some single cases and outbreaks of this disease.150,174,175

Differential diagnosis of HP The clinical differential diagnosis is often broad in many cases and includes the various expressions of HP and also other diffuse parenchymal lung diseases, including idiopathic interstitial pneumonias, infections and other secondary causes (Table 3).1,11,24,78,135,176–178 The histopathological differential

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Table 3 Differential diagnosis of hypersensitivity pneumonitis

Hypersensitivity pneumonitis

Sarcoidosis

Lymphocytic interstitial pneumonitis

Nonspecific interstitial pneumonitis

Usual interstitial pneumonitis

Aspiration / foreign-body bronchiolitis

Granulomas in infections

Drug-related granulomatous pneumonitis

Frequency

Up to 2/3 of cases (subacute); transitory lifespan – dissipate with chronicity

All cases, more numerous generally than in HP; persistent

20–50% of cases; persistent

Absent

Absent

Common

Variable; more common in miliary forms

Present, by definition, although may be scant

Appearance

Non-caseating; variable sized, typically small; poorly formed or “vague’; ill-defined borders; blends in with surrounding interstitial infiltrates; variable mixtures of epithelioid histiocytes, multinucleated histiocytic giant cells; often a high concentration of intermixed lymphocytes; alveolar histiocytic aggregates coexist within airspaces

Well-defined, discrete granulomas; epithelioid histiocytes predominate, mixed with multinucleated giant cells; granulomas may become confluent; cellular early stage with progressive characteristic lamellar sclerosing collagenous fibrous replacement; typically nonnecrotizing with subpopulation showing bland, central “fibrinoid degeneration”

Usually poorly formed; some may be well delineated; noncaseating

–

–

Variable size; necrosis uncommon

Usually necrotizing (caseating or infarct-like necrosis); size variable – small, ill-defined forms may be seen in biopsies; microorganism may be demonstrable in granuloma

Variable forms: (a) poorly formed histiocytic granulomas with intermixed lymphoplasmacytic infiltrate; (b) wellformed sarcoid-like (may represent reactivation disease in IFNγ therapy); rare necrotizing granulomas have been described

Distribution

Usually random within lobule but some cases show a prominent peribronchiolar location; often noted within airway walls

Peribronchovascular / perilymphatic distribution; often noted for vascular wall invasion (typically pulmonary veins); common in larger airway walls

Random

–

–

Variable – bronchiolar or peribrochiolar parenchyma in aspiration (vs. intraor peri-capillary in IV drug abuse)

Random

Variable: (a) poorly formed granulomas may be randomly distributed in airspace and interstitium; (b) sarcoid-like granulomas may localize in characteristic sites as sarcoid

Feature

Granulomas

Within granulomas or isolated within interstitium; cytoplasmic inclusions: cholesterol clefts, Schaumann bodies, calcium oxalate crystals

Langhans-type giant cells; usually seen only within confines of granuloma; inclusions: calcium oxalate crystals common

Occasionally described

Absent

Absent (some authors allow rare giant cells in UIP but not typically considered part of the disease)

Foreign-body type; inclusions clue to diagnosis (plant residue, mineral dusts (talc, barium), cholesterol clefts)

Langhans-type

Occasional

Bronchiolitis

Cellular (lymphocytic) bronchiolitis frequent

Absent to minimal

Minimal

Minimal

Rare, typically minimal if present

With aspiration, yes; in other etiologies (IV drug abuse), no

Variable

Variable

Interstitial pneumonia

Mild to moderate density; virtually all subacute cases; chronic cases may contain only sparse interstitial pneumonitis

Modest if present at all

Characteristically prominent, expansile

Moderate density

Modest density

Variable intensity

Variable (uncommon in TB)

Variable expression and density

Composition

Lymphocytes (CD8þ T-cells dominate), plasma cells, histiocytes; eosinophils occasionally but not prominent; neutrophils in acute stage

Lymphocytes, CD4þ T-cell predominate

Interstitial cells small lymphocytes (predominately T-lymphocytes), plasma cells, histiocytes; lymphoid follicles common (B-lymphocytes)

Lymphocytes, plasma cells, histiocytes

Lymphocytes and plasma cells

Mostly lymphocytes and plasma cells

Lymphocytes predominant; CD4þ T-cells typically, around granulomas

Typically lymphocytes, plasma cells and histiocytes; some histiocytes may display foamy cytoplasm; eosinophils may be prominent but are not invariably present.

Distribution

Classically patchy peribronchiolar interstitium, attenuating towards distal lobule; may show diffuse lobular interstitial infiltrate

Inflammation predominates around granulomas

Diffuse; often involves pleura

Diffuse

Patchy, expression variable

Focal

Focal

Variable patterns: nodular, mimicking HP, diffuse or NSIPlike, bronchiolocentric

Giant cells

Table 3 (cont.)

Feature

Hypersensitivity pneumonitis

Sarcoidosis

Lymphocytic interstitial pneumonitis

Nonspecific interstitial pneumonitis

Usual interstitial pneumonitis

Aspiration / foreign-body bronchiolitis

Granulomas in infections

Drug-related granulomatous pneumonitis

Organizing airspace exudates (intraluminal fibrosis)

Variable degree, present in up to two-thirds of cases (mostly subacute)

Rare; minimal if present at all

Absent or very rare

Moderate

Absent or very rare

Very common in some studies; acute bronchopneumonia may be present

Occasional

Relatively common

Interstitial fibrosis

Typical of advanced cases, may be very dense

May be very dense in advanced cases

Slight, if present at all; dense fibrosis very unusual

Frequent

Present by definition; patchy

Possible in chronic or recurrent cases

Nodular characteristic

Multiple illcharacterized fibrotic reactions attributed to drugs; UIP, NSIP and other patterns possible

Fibroblastic foci

Occasional

Absent

Absent

Occasional

Frequent

Not described

–

Possible

Centrilobular fibrosis

Frequent, perhaps typical, in advanced cases

Occasional

Absent

Minimal

Minimal

Not well described; presumably a manifestation of aspiration in chronic cases

–

Not described

Honeycomb remodeling

Frequent in advanced cases

Possible in advanced cases

Microscopic honeycomb remodeling possible but unusual

Occasional, in fibrosing subtype

Frequent

Possible

–

Possible

Table developed, with modifications, from information presented in references 1, 24, 78, 135, 176, 177, 178.

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diagnosis may be equally difficult and the approach may well depend on the dominance (or mixture) of certain features, e.g. cellular interstitial pneumonitis, bronchiolitis, granulomas, and several patterns of fibrosing pneumonias.

Usual interstitial pneumonia/idiopathic pulmonary fibrosis In cases of chronic HP the combination of interstitial inflammation, fibrosis, honeycomb remodeling, and even fibroblastic foci may resemble UIP60,142–144,146 on biopsy. A crucial diagnostic dilemma also relates to the significance of a rare or otherwise inconspicuous granuloma. In the opinion of some142 the presence of multinucleated giant cells or granulomata in the setting of a UIP pattern of fibrosis confers a diagnosis of chronic HP. Others144 require the additional finding of areas of typical HP, specifically a bronchiolocentric cellular interstitial pneumonia with giant cells or poorly formed granulomas. In cases that closely resemble UIP, subtle differences, such as the lack of perilobular and presence of centrilobular fibrosis, may favour HP.142 In areas of UIP showing less fibrosis, the interstitial inflammatory infiltrate is typically less dense than that of HP.179

Nonspecific interstitial pneumonia The histological pattern of NSIP, including cellular and fibrosing types, has been identified in HP.60,146 This is the sole histopathological pattern in some cases, even in the absence of giant cells or granulomas.134 In retrospect several of the initially reported cases of NSIP180 were probably HP. A diffuse interstitial pneumonitis and/or fibrosis that demonstrates relative preservation of the underlying lobular architecture should prompt consideration of a diagnosis of NSIP. The presence of even a rare granuloma in this context should strongly raise consideration of HP or other diffuse interstitial lung processes that may commonly contain granulomas. These include lymphoid interstitial pneumonia (LIP), infections, connective tissue disease or certain drug reactions.

Lymphoid interstitial pneumonia Lymphoid interstitial pneumonia is well recognized by its diffuse alveolar interstitial polymorphous infiltrate of small lymphocytes, immunoblasts, plasma cells and histiocytes (see Chapter 34).181 The infiltrate is characteristically dense, with expansion of alveolar septa and some prominence around the lymphatic routes (bronchovascular bundles, interlobular septa and extension under the pleura). Follicles with germinal center development are common. While the interstitial infiltrate is composed mainly of T-lymphocytes with some polytypic plasma cells, the peribronchial germinal centers are composed of polyclonal B-lymphocytes.181,182 Small, non-necrotizing granulomas may be observed in 20–50% of cases (Figure 30),182,183 a feature that would suggest HP in the differential diagnosis. The essential difference between the two is that the diffuse

Figure 30. Interstitial granulomas in a case of lymphocytic interstitial pneumonia (LIP). Multinucleated histiocytic giant cells with acicular cholesterol clefts are identified by the arrow. The characteristic density of the interstitial infiltrate is much greater than is typical for HP.

lobular involvement with expanded, sometimes distorted, alveolar septa in LIP contrasts with the more bronchiolocentric but less dense lymphoid infiltrate with attenuation of the interstitial infiltrate towards the distal lobule in HP. Another helpful feature, if present, is the finding of foci of bronchiolitis obliterans in HP.179 Lymphoid interstitial pneumonia is renowned for its association with some connective tissue diseases, particularly Sjögren syndrome, and other conditions of immune dysregulation, therefore clinical history can be of great benefit.181–184

Organizing pneumonia and cryptogenic organizing pneumonia Organizing pneumonia, in either its idiopathic or secondary forms,78,185,186 is the product of alveolar injury. This results in a fibrinoid inflammatory exudate that becomes progressively organized, resulting in the formation of characteristic arborizing intraluminal buds (Masson bodies) (see Chapters 10 and 17). Although extensive areas of confluent pneumonia may be present, the injury may be more discretely nodular/ peribronchovascular187 and this could be confused with the small foci of centrilobular organization common in HP.179 In organizing pneumonias, including cryptogenic, the intraluminal pathology dominates the picture, with less prominent interstitial pneumonitis. The reverse is usually the case with HP.

Centrilobular/bronchiolocentric interstitial pneumonias A number of recent studies claim to have identified cohorts of patients with idiopathic interstitial pneumonias in which the pathology consists of combinations of fibrosis, chronic inflammation, intraluminal organization and epithelial (ciliary, cuboidal) metaplasia in and around the centrilobular region. Because of their recent advent in the literature, not all of these patterns have achieved complete acceptance and their relationship to each other is a matter of conjecture. Nevertheless,

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injury and reparative changes in this compartment naturally raise the possibility of HP (and other primary airway-related disease).188 Idiopathic bronchiolocentric interstitial pneumonia (“BrIP”)189 was published as a series of ten patients, with a female predilection, with restrictive lung disease in whom no inciting or otherwise environmental inhalatory exposures could be documented. Pathological features included centrilobular/ bronchiolocentric mononuclear inflammation with extension into the adjacent interstitium and tapering towards the peripheral lobule. Most cases also showed peribronchiolar fibrosis and cuboidal metaplasia. Rare interstitial foci of “fibromyxoid tissue” and airspace granulation tissue polyps were present. Granulomas were absent. The authors conceded this may represent residua of other disease processes, including chronic HP. Peribronchiolar metaplasia-interstitial lung disease (“PBM-ILD”)141 was described in 15 (13 females) who demonstrated heterogeneous combinations of restrictive, obstructive, or mixed, physiological deficits. Virtually the only pathology was a distinctive centrilobular/peribronchiolar distribution of cuboidal/ciliated cell metaplasia. There was no inflammation or fibrosis extending into the surrounding alveolar septa. Three cases contained small granulomas. Several patients had smoked or had serological markers for autoimmune disease and one kept pigeons. The authors indicated that no cases demonstrated the classical inflammatory patterns of HP but recognized that chronic HP may be difficult to exclude. Peribronchiolar metaplasia may be identified in other conditions, such as UIP (59%), NSIP (50%), HP (50%), respiratory bronchiolitis-interstitial lung disease (RB-ILD) (11%) and DIP (50%).141 Airway-centered interstitial fibrosis (“ACIF”)190 was a pattern described in 12 patients (eight females) with “airwaycentered” pathological changes that were manifested clinically as interstitial lung disease with a predominately restrictive lung function. The group contained a significant number of patients with inhalational exposure histories. CT scans showed fibrosis around larger airways not sampled for histology. The spectrum of pathological changes included: interstitial fibrosis centered on membranous and respiratory bronchioles, conglomerate fibrous masses juxtaposed to airways, fibrosis extending to the pleura, often in a linear pattern, peribronchiolar metaplasia and mild chronic interstitial inflammation. Granulomas were not identified. The authors state the changes are not like HP pathologically, radiologically or clinically but acknowledge some similarity to the cases of BrIP. 189 Bronchiolitis interstitial pneumonitis (“BIP”)191 was a series of 31 patients (male:female approximately equal), again with mostly restrictive but often combined or occasionally purely obstructive lung physiological deficits. No occupational or environmental exposures suggesting HP were uncovered. Histopathological findings were summarized as arborizing intraluminal tufts of immature fibroblastic tissue (“bronchiolitis obliterans”), often in combination with organizing alveolar pneumonia but less dominant than the

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interstitial component; interstitial fibrosis distant from the bronchiolar disease; architectural “simplification” with some degree of honeycomb change; chronic interstitial inflammation; and occasional peribronchiolar metaplasia. No granulomas were identified. The authors allowed that their cases might be variants of other interstitial pneumonias with prominent centrilobular injury and included HP in the differential diagnosis.

Granulomatous disorders to be distinguished from HP Sarcoidosis

Most cases of pulmonary sarcoid can be easily differentiated from HP, although potential for confusion does occur. The chief distinguishing characteristics between the two reside in the number and appearance of the granulomas and their distribution, as well as differences in associated interstitial pneumonitis and airspace cellularity (see Chapter 13). The typical sarcoid granuloma consists of a fairly discrete, well-circumscribed nodule of epithelioid histiocytes with multinucleated histiocytic giant cells. There is a small cuff of lymphocytes (typically CD4þ T-cells) at the margins, prompting the term “naked granuloma”. There is a tendency for granulomas to coalesce. Giant cells may be large and very prominent and often contain intracytoplasmic inclusions such as Schaumann bodies, asteroid bodies and oxalate crystals. Schaumann bodies are claimed to be more common in sarcoid than any other granulomatous process192 although they may be readily apparent in HP.193 The margin of sarcoid granulomas typically, with time, acquire a characteristic mantle of lamellar, hyalinized fibrosis. This scar tissue often progressively replaces the granuloma, possibly leaving behind a solitary giant cell (or even an embedded Schaumann body). Areas of central necrosis may be identified in sarcoid granulomas but it is typically minimal and likened more to fibrinoid “degeneration” than outright caseation.194 Earlier reports195 describe a focal mononuclear interstitial pneumonitis, without predilection for perivascular or bronchovascular localization. This was considered the earliest manifestation of the process that generates the granulomas. This inflammation is transient and dissipates with the advent of well-formed granulomas and fibrosis. It is conceivable that nascent granulomas of sarcoid may resemble those of HP, particularly on small biopsies. In contrast, in HP, at least in the classic subacute stage, the interstitial pneumonitis overshadows the granulomas. Granulomas tend to be more numerous in sarcoid than in HP and demonstrate a predilection for the lymphatic pathways: pleura, peribronchovascular sheath and secondary lobular septum. In HP, the granulomas are usually smaller, loosely formed (less discrete), and are concentrated in the peribronchial region. In addition, small foci of organizing pneumonia and foamy alveolar histiocytes are present and these may provide valuable secondary clues to the nature of the inflammation.

Chapter 12: Hypersensitivity pneumonitis

Drug-related interstitial lung disease Many forms of drug-related pneumonitis have been described (see Chapter 16).196–198 An HP-like reaction is one of the rarer forms of injury198,199 demonstrating combinations of a cellular interstitial pneumonitis and granulomas. In most cases, the granulomas are described as loose/poorly formed and nonnecrotizing and may be seen in the context of a cellular interstitial pneumonitis (Figure 31). The alveolar spaces may contain, and sometimes be filled by, a loose aggregate of

epithelioid histiocytes, admixed with lymphocytes. Single multinucleated histiocytic giant cells may sometimes be identified within this infiltrate. The agents most often cited as producing an HP or granulomatous interstitial pneumonia include: methotrexate, nitrofurantoin, BCG, fluoxetine, procarbazine, acebutalol, sulfasalazine, statins, interferons, sirolimus, cromolyn sodium and cocaine.196–198 Clinical history is required to arrive at the correct diagnosis. Some of the agents that can produce this reaction may be administered in the context of an immunosuppressed state, so the coexistence of infectious agents should always be considered.

Cholesterol granuloma Pulmonary cholesterol granulomas may be identified in patients with endogenous lipoid pneumonia, pulmonary alveolar proteinosis or lysinuric protein intolerance, as an incidental finding at autopsy. These granulomas are associated with gastroesophageal reflux disease and aspiration,200 and in up to 25% of patients with severe pulmonary hypertension.201,202 In one series the granulomas are described as airspace and interstitial collections of multinucleated histiocytic giant cells with acicular, clear, cholesterol clefts in their cytoplasm (Figure 32). The same cholesterol clefts may be seen free within this infiltrate; lymphocytes are also usually noted.202 These lesions tended to be situated in centrilobular stellate fibrous foci, which, by virtue of this particular location, suggests a diagnosis of HP. Granuloma morphology and clinical history should allow easy distinction. Figure 31. Drug-related granulomatous pneumonitis. This biopsy from a 64-year-old male treated with Docetaxel for prostatic adenocarcinoma shows two small granulomas (arrows) within airspaces. The background injury pattern was a diffuse organizing subacute lung injury; microbiological stains were negative.

(a)

Pneumocystis pneumonia Pneumocystis jirovecii can manifest as a granulomatous pneumonia in the context of immunosuppression related to human (b)

Figure 32. Cholesterol granuloma. (a) Low-magnification micrograph demonstrating numerous airspace cholesterol granulomas in a woman with thrombotic pulmonary arteriopathy. The arrow indicates the largest granuloma; the arrowhead points to a pulmonary artery with thrombotic occlusion. (b) Higher magnification of two cholesterol granulomas. The mono- and multinucleated histiocytes demonstrate a cohesive quality and have ample eosinophilic cytoplasm containing acicular cholesterol clefts.

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immunodeficiency virus/acquired immune deficiency syndrome,203 malignancy204 and transplantation205 (see Chapter 7). Within the spectrum of this uncommon histopathological reaction, well-formed and focally necrotizing granulomas (sometimes with fibrosis and calcification) are unlikely to cause difficulty in the differential diagnosis of HP. The pneumonic form, however, with poorly defined granulomas composed of loosely arranged histiocytes and occasional giant cells (Figure 33) may be problematic, particularly since the

Figure 33. Granulomatous pneumonitis in Pneumocystis jirovecii. A prominent multinucleated histiocyte is seen within a vague airspace granuloma (arrowhead). The background inflammatory pattern features organizing acute and subacute injury with cellular airspace aggregates of mixed lymphocytes and histiocytes. Rare Pneumocystis organisms were identified (not shown).

(a)

characteristic foamy eosinophilic alveolar exudate of Pneumocystis pneumonia is often absent. Histiocytes may also be identified within the interstitium. The organisms of Pneumocystis are easily demonstrated with the Gomori methenamine silver (GMS) stain and are identified within the confines of the granuloma. The density of organisms in this setting may be less than that typically associated with the classic foamy alveolar exudates.

Aspiration bronchiolitis/pneumonia Occult micro-aspiration and gastroesophageal reflux disease (GERD) are associated with a variety of pulmonary disorders including granulomatous pneumonitis and organizing pneumonia. These diseases are putative etiological factors in some of the fibrosing interstitial pneumonias, particularly IPF and scleroderma-related interstitial lung disease.206 A histological pattern of centrilobular or bronchiolocentric pneumonitis with granulomas/multinucleated histiocytic giant cells and some degree of organizing airspace exudates superficially describes HP. Thus aspiration bronchiolitis should be included in the pathological differential diagnosis of HP. Finding vegetable or meat fragments, pharmaceutical tablet compounds or other amorphous material (Figure 34) in the context of a foreign-body granulomatous response, particularly with some degree of suppuration, facilitates the diagnosis of aspiration disease (see Chapter 17). A number of cases, however, may present with an atypical radiological distribution not specific for the characteristic dependent regions of the lung, and without clinically recognized aspiration events. Younger patients without obvious neurological impairment may not initially be considered as having chronic aspiration. In some reports, HP was an initial consideration for the diagnosis, on either clinical (b)

Figure 34. Aspiration bronchiolitis. (a) An interstitial pneumonitis and airspace granulomatous process is seen in the peribronchiolar parenchyma. There is variable widening of alveolar septa and focal organization of airspace exudates seen in conjunction with multinucleated histiocytic giant cells. (b) Higher magnification showing foreign material engulfed by a giant cell. Although characteristic features of vegetable matter and meat particles are often illustrated, the observer should recognize amorphous eosinophilic material as potentially foreign.

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grounds207 or in pathological consultation material.208 In a recent review of aspiration-associated pulmonary pathology, the most common findings were bronchiolar/peribronchiolar organizing airspace exudates and multinucleated giant cells. All of these cases contained demonstrable foreign material, thus allowing a confident pathological diagnosis. In cases where the possibility of aspiration is overlooked, the combination of a bronchiolocentric interstitial and airspace reaction with histiocytic giant cells may erroneously focus the diagnosis towards HP. Although incompletely documented, long-term aspirationinduced bronchiolocentric fibrosis is conceivably a sequel to occult aspiration. With the group of so-called bronchiolocentric interstitial pneumonias (see above), this form could also overlap with certain manifestations of chronic HP. In the series of Mukhopadhyay and Katzenstein, four cases showed peribronchiolar fibrosis and chronic inflammation; these particular cases did not demonstrate organizing pneumonia or giant cells.208 De Souza et al. have recently described a distinctive bronchiolocentric lesion, “centrilobular fibrosis (CLF)”, that was often an isolated lesion or associated with NSIP in systemic sclerosis patients. Giant cells and foreign material were sometimes observed. The authors postulate a link between this injury and esophageal dysmotility, and suggest a relationship to the more diffuse interstitial parenchymal pathology.209 A recent experimental rodent study utilized periodic instillation of small volumes of fractionated gastric contents. In this study, the food particle fractions, isolated or combined with other components, were required for a granulomatous response. This was independent of the pH of the solution as hydrochloric acid alone (or bile alone) had no effect different than controls.210

Treatment, prognosis and survivial The variegated nature of HP is reflected in the lack of uniform prognostic data. Reported mortality rates for HP demonstrate a range from 1 to 29%.137,211 A recent UK study determined that the 5-year survival for their study group of HP patients was 82%.18 Mortality rates of HP in the United States were analyzed for the period 1980–1998, using ICD-9 subclassification. Hypersensitivity pneumonitis deaths attributed to “unspecified allergic alveolitis and pneumonitis” accounted for 55.5% of the total, farmer’s lung 37.3% and BFL 4.4%.212 This study also determined that, in the same period, ageadjusted mortality rates increased significantly from 0.09 to 0.29 per million population.212 In general, the prognosis of acute BFL is excellent, even despite continued antigen exposure in some cohorts,213 and avian HP-related fatalities are rare.214 In chronic forms of the same disease, the mortality is increased, up to 25% within 5 years of initial diagnosis.137 The presence of fibrosis at the time of biopsy portends a poor outcome. Median survival times in this situation have been reported as 4 years (UIP-like or fibrotic NSIP-like patterns),60

2.8 years (UIP-like fibrosis) and 2.1 years (NSIP-like fibrosis).145 In the latter study, 68% of patients with chronic HP were current or former smokers and the influence of smoking on survival may contribute to the poor prognosis.145 For any form of HP, the prime treatment directives are early diagnosis and avoidance of (or reduction of exposure to) the offending antigen(s). In acute and subacute HP, antigen avoidance should produce clinical resolution.215 An allergenfree environment may be difficult to achieve since the source of antigen may not be apparent, even after diligent searching. Antigen avoidance may not be possible if it entails rigorous employment or lifestyle changes that are unacceptable to the patient. Indeed, some patients may actively obfuscate details of their situation if they perceive impending loss of income or a beloved pet. Progressive changes in workplace environmental hygiene have mitigated some of these risks. Reduction in farmer’s lung may be anticipated through conversion to silage for animal fodder and technical improvements in ventilation and drying of hay to reduce mold accumulation.216 Other strategies include the use of propionic acid (an antiseptic and dehumidifying agent) treatment of hay to reduce microorganism concentration,217 and the use of appropriate facemasks to reduce the burden of respirable antigenic particulates.218 Masks, however, may still transmit particles less than 1 mm and their effectiveness depends on proper fitting. Bird-related HP poses slightly different problems. In less developed countries birds, e.g. pigeons, may cohabitate closely with humans48 and high levels of antigens may persist in the environment despite eradication efforts that include removal of the birds and environmental cleansing.219 Pigeon fanciers are encouraged to reduce the time spent in activities associated with the highest exposure4 and to increase loft ventilation.5 In the industrial sector, recognition of the hazards associated with metal-working fluids has generated recommendations for adopting new management and technical processes and worker education.220 Corticosteroids represent the only standard pharmacological therapeutic option, although, as in many studies, individual case details of antigen dose and type, smoking history and individual host susceptibility may impose a non-uniform response. Dosing schedules typically parallel those used for other non-infectious chronic diffuse interstitial lung diseases.1 In an often-quoted double-blinded placebo-controlled study involving acute HP, prednisolone treatment was effective in improving lung function in the short term. It had no significant effect in long-term follow-up.221 Short-term clinical benefit may be achieved even though functional testing parameters and radiological changes may not demonstrate significant improvement.222 Corticosteroid therapy may predispose patients to recurrence in the face of sustained antigen exposure.221 The role of steroid therapy in chronic HP is not yet defined although it may be indicated in cases of chronic progressive disease.

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148. Ohtani Y, Inase N, Miyake S, Yoshizawa Y, Saiki S. Fatal outcome in chronic bird fancier’s lung. Am J Med 2002;112:588–90. 149. Waller EA, Roy A, Brumble L, et al. The expanding spectrum of Mycobacterium avium complex-associated pulmonary disease. Chest 2006;130:1234–41. 150. Griffith DE, Aksamit T, Brown-Elliott BA, et al. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial disease. Am J Respir Crit Care Med 2007;175:367–416. 151. Embil J, Warren P, Yakrus M, et al. Pulmonary illness associated with exposure to Mycobacterium-avium complex in hot tub water. Hypersensitivity pneumonitis or infection? Chest 1997;111:813–6. 152. Marras TK, Wallace RJ Jr, Koth LL, et al. Hypersensitivity pneumonitis reaction to Mycobacterium avium in household water. Chest 2005; 127:664–71. 153. Rickman OB, Ryu JH, Fidler ME, Kalra S. Hypersensitivity pneumonitis associated with Mycobacterium avium complex and hot tub use. Mayo Clin Proc 2002;77:1233–7. 154. Hanak V, Kalra S, Aksamit TR, et al. Hot tub lung: presenting features and clinical course of 21 patients. Respir Med 2006;100:610–5. 155. Sood A, Sreedhar R, Kulkarni P, Nawoor AR. Hypersensitivity pneumonitis-like granulomatous lung disease with nontuberculous mycobacteria from exposure to hot water aerosols. Environ Health Perspect 2007;115:262–6. 156. Hansell D. Nontuberculous (atypical) mycobacterial infection. In Müller NL, Silva CI S, eds. Imaging of the Chest. Philadelphia: Saunders Elsevier, 2008. pp. 342–55. 157. Kawai T, Tamura M, Murao M. Summer-type hypersensitivity pneumonitis. A unique disease in Japan. Chest 1984;85:311–7. 158. Ando M, Arima K, Yoneda R, Tamura M. Japanese summer-type hypersensitivity pneumonitis. Geographic distribution, home environment, and clinical characteristics of 621 cases. Am Rev Respir Dis 1991;144:765–9.

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159. Shimazu K, Ando M, Sakata T, Yoshida K, Araki S. Hypersensitivity pneumonitis induced by Trichosporon cutaneum. Am Rev Respir Dis 1984;130:407–11. 160. Yoshida K, Ando M, Sakata T, Araki S. Environmental mycological studies on the causative agent of summer-type hypersensitivity pneumonitis. J Allergy Clin Immunol 1988;81:475–83. 161. Yoshizawa Y, Ohtani Y, Hayakawa H, et al. Chronic hypersensitivity pneumonitis in Japan: a nationwide epidemiologic survey. J Allergy Clin Immunol 1999;103:315–20. 162. Ohtani Y, Ochi J, Mitaka K, et al. Chronic summer-type hypersensitivity pneumonitis initially misdiagnosed as idiopathic interstitial pneumonia. Intern Med 2008;47:857–62. 163. Inase N, Ohtani Y, Usui Y, et al. Chronic summer-type hypersensitivity pneumonitis: clinical similarities to idiopathic pulmonary fibrosis. Sarcoidosis Vasc Diffuse Lung Dis 2007;24:141–7. 164. Pronk A, Preller L, Raulf-Heimsoth M, et al. Respiratory symptoms, sensitization, and exposure-response relationships in spray painters exposed to isocyanates. Am J Respir Crit Care Med 2007;176:1090–7. 165. Raulf-Heimsoth M, Baur X. Pathomechanisms and pathophysiology of isocyanate-induced diseases – summary of present knowledge. Am J Ind Med 1998; 34:137–43. 166. Baur X. Hypersensitivity pneumonitis (extrinsic allergic alveolitis) induced by isocyanates. J Allergy Clin Immunol 1995;95:1004–10. 167. Vandenplas O, Malo JL, Saetta M, Mapp CE, Fabbri LM. Occupational asthma and extrinsic alveolitis due to isocyanates: current status and perspectives. Br J Ind Med 1993; 50:213–28. 168. Vandenplas O, Malo JL, Dugas M, et al. Hypersensitivity pneumonitis-like reaction among workers exposed to diphenylmethane [correction to piphenylmethane] diisocyanate (MDI). Am Rev Respir Dis 1993;147:338–46. 169. Charles J, Bernstein A, Jones B, et al. Hypersensitivity pneumonitis after exposure to isocyanates. Thorax 1976;31:127–36.

170. Nakashima K, Takeshita T, Morimoto K. Occupational hypersensitivity pneumonitis due to isocyanates: mechanisms of action and case reports in Japan. Ind Health 2001;39:269–79. 171. Bernstein DI, Lummus ZL, Santilli G, Siskosky J, Bernstein IL. Machine operator’s lung. A hypersensitivity pneumonitis disorder associated with exposure to metalworking fluid aerosols. Chest 1995;108:636–41. 172. Rosenman KD. Asthma, hypersensitivity pneumonitis and other respiratory diseases caused by metalworking fluids. Curr Opin Allergy Clin Immunol 2009;9:97–102. 173. Robertson W, Robertson AS, Burge CBSG, et al. Clinical investigation of an outbreak of alveolitis and asthma in a car engine manufacturing plant. Thorax 2007;62:981–90. 174. Shelton BG, Flanders WD, Morris GK. Mycobacterium sp. as a possible cause of hypersensitivity pneumonitis in machine workers. Emerg Infect Dis 1999;5:270–3. 175. Beckett W, Kallay M, Sood A, Zuo Z, Milton D. Hypersensitivity pneumonitis associated with environmental mycobacteria. Environ Health Perspect 2005; 113:767–70. 176. Leslie KO. Pathology of interstitial lung disease. Clin Chest Med 2004;25:657–703. 177. Barrios R. Hypersensitivity pneumonitis (extrinsic allergic alveolitis). In Tomashefski Jnr JF, ed. Dail and Hammar’s Pulmonary Pathology, 3rd ed. New York: Springer, 2008. pp. 650–67. 178. Corrin B, Nicholson AG. Diffuse parenchymal disease of the lungs. In Pathology of the Lungs, 2nd ed. Philadelphia: Churchill Livingstone Elsevier, 2006. pp. 267–306. 179. Katzenstein A-LA. Immunologic lung disease. In Katzenstein A-L A, ed. Katzenstein and Askin’s Surgical Pathology of Non-Neoplastic Lung Disease, 4th ed. Philadelphia: Saunders Elsevier, 2006. 180. Katzenstein A-L, Fiorelli R. Nonspecific interstitial pneumonia/fibrosis: histologic features and clinical significance. Am J Surg Pathol 1994;18:136–47.

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181. Swigris J, Berry G, Raffin T, Kuschner W. Lymphoid interstitial pneumonia: a narrative review. Chest 2002;122:2150–64. 182. Nicholson AG, Wotherspoon AC, Diss TC, et al. Reactive pulmonary lymphoid disorders. Histopathology 1995;26:405–12. 183. Kradin RL, Mark EJ. Benign lymphoid disorders of the lung, with a theory regarding their development. Hum Pathol 1983;14:857–67. 184. Cha S-I, Fessler MB, Cool CD, Schwarz MI, Brown KK. Lymphoid interstitial pneumonia: clinical features, associations and prognosis. Eur Respir J 2006;28:364–9. 185. Cordier JF. Cryptogenic organising pneumonia. Eur Respir J 2006; 28:422–46. 186. Epler GR, Colby TV, McLoud TC, Carrington CB, Gaensler EA. Bronchiolitis obliterans organizing pneumonia. N Engl J Med 1985;312:152–8. 187. Lee KS, Kullnig P, Hartman TE, Müller NL. Cryptogenic organizing pneumonia: CT findings in 43 patients. Am J Roentgenol 1994;162:543–6. 188. Cordier J-F. Challenges in pulmonary fibrosis. Bronchiolocentric fibrosis. Thorax 2007;62:638–49. 189. Yousem SA, Dacic S. Idiopathic bronchiolocentric interstitial pneumonia. Mod Pathol 2002; 15:1148–53. 190. Churg A, Myers J, Suarez T, et al. Airway-centered interstitial fibrosis: a distinct form of aggressive diffuse lung disease. Am J Surg Pathol 2004;28:62–8. 191. Mark EJ, Ruangchira-urai R. Bronchiolitis interstitial pneumonitis: a pathologic study of 31 lung biopsies with features intermediate between bronchiolitis obliterans organizing pneumonia and usual interstitial pneumonitis, with clinical correlation. Ann Diagn Pathol 2008;12:171–80. 192. Hsu RM, Connors AF, Tomashefski JF. Histologic, microbiologic, and clinical correlates of the diagnosis of sarcoidosis by transbronchial biopsy. Arch Pathol Lab Med 1996;120:364–8.

a review with particular emphasis on unusual and underrecognized features. Int J Surg Pathol 2009; 17:219–30. 195. Rosen Y, Athanassiades TJ, Moon S, Lyons HA. Nongranulomatous interstitial pneumonitis in sarcoidosis: relationship to development of epithelioid granulomas. Chest 1978;74:122–5. 196. Myers JL, El-Zammar O. Pathology of drug-induced lung disease. In Katzenstein A-L A, ed. Katzenstein and Askin’s Surgical Pathology of Non-Neoplastic Lung Disease, 4th ed. Philadelphia: Saunders Elsevier, 2006. pp. 85–125. 197. Flieder DB, Travis WD. Pathologic characteristics of drug-induced lung disease. Clin Chest Med 2004;25:37–45. 198. Myers JL, Limper AH, Swensen SJ. Drug-induced lung disease: a pragmatic classification incorporating HRCT appearances. Semin Respir Crit Care Med 2003;24:445–53. 199. Myers JL. Other diffuse lung diseases. In Churg AM, Myers JL, Tazelaar HD, Wright JL, eds. Thurlbeck’s Pathology of the Lung, 3rd ed. New York: Thieme, 2005. pp. 601–73. 200. Fisher M, Roggli V, Merten D, Mulvihill D, Spock A. Coexisting endogenous lipoid pneumonia, cholesterol granulomas, and pulmonary alveolar proteinosis in a pediatric population: a clinical, radiographic, and pathological correlation. Pediatr Pathol 1992;12:365–83. 201. Caslin AW, Heath D, Madden B, et al. The histopathology of 36 cases of plexogenic pulmonary arteriopathy. Histopathology 1990;16:9–19. 202. Nolan RL, McAdams HP, Sporn TA, et al. Pulmonary cholesterol granulomas in patients with pulmonary artery hypertension: chest radiographic and CT findings. AJR 1999;172:1317–9. 203. Bleiweiss IJ, Jagirdar JS, Klein MJ, et al. Granulomatous Pneumocystis carinii pneumonia in three patients with the acquired immune deficiency syndrome. Chest 1988;94:580–3.

193. Rosen Y. Pathology of sarcoidosis. Semin Respir Crit Care Med 2007;28:36–52.

204. Bondoc AYP, White DA. Granulomatous Pneumocystis carinii pneumonia in patients with malignancy. Thorax 2002;57:435–7.

194. Cavazza A, Harari S, Caminati A, et al. The histology of pulmonary sarcoidosis:

205. Gal AA, Plummer AL, Langston AA, Mansour KA. Granulomatous

Pneumocystis carinii pneumonia complicating hematopoietic cell transplantation. Pathol Res Pract 2002;198:553–8. 206. Morehead RS. Gastro-oesophageal reflux disease and non-asthma lung disease. Eur Respir Rev 2009;18:233–43. 207. Barnes TW, Vassallo R, Tazelaar HD, Hartman TE, Ryu JH. Diffuse bronchiolar disease due to chronic occult aspiration. Mayo Clin Proc 2006;81:172–6. 208. Mukhopadhyay S, Katzenstein A-LA. Pulmonary disease due to aspiration of food and other particulate matter: a clinicopathologic study of 59 cases diagnosed on biopsy or resection specimens. Am J Surg Pathol 2007;31:752–9. 209. de Souza RBC, Borges CTL, Capelozzi VL, et al. Centilobular fibrosis: an underrecognized pattern in systemic sclerosis. Respiration 2009;77:389–97. 210. Downing TE, Sporn TA, Bollinger RR, et al. Pulmonary histopathology in an experimental model of chronic aspiration is independent of acidity. Exp Biol Med 2008;233:1202–12. 211. Kokkarinen J, Tukiainen H, Terho EO. Mortality due to farmer’s lung in Finland. Chest 1994;106:509–12. 212. Bang KM, Weissman DN, Pinheiro GA, et al. Twenty-three years of hypersensitivity pneumonitis mortality surveillance in the United States. Am J Ind Med 2006;49:997–1004. 213. Bourke SJ, Banham SW, Carter R, Lynch P, Boyd G. Longitudinal course of extrinsic allergic alveolitis in pigeon breeders. Thorax 1989;44:415–8. 214. Zacharisen MC, Schlueter DP, Kurup VP, Fink JN. The long-term outcome in acute, subacute, and chronic forms of pigeon breeder’s disease hypersensitivity pneumonitis. Ann Allergy Asthma Immunol 2002;88:175–82. 215. Wells AU, Hirani N, et al. Interstitial lung disease guideline: the British Thoracic Society in collaboration with the Thoracic Society of Australia and New Zealand and the Irish Thoracic Society. Thorax 2008; 63(Suppl V):v1-v58. 216. Dalphin JC, Pernet D, Reboux G, et al. Influence of mode of storage and drying of fodder on thermophilic actinomycete aerocontamination in

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dairy farms of the Doubs region of France. Thorax 1991;46:619–23. 217. Reboux G, Dalphin JC, Polio JC, et al. Influence of buffered propionic acid on the development of micro-organisms in hay. Mycoses 2002;45:184–7. 218. Hendrick DJ, Marshall R, Faux JA, Krall JM. Protective value of dust respirators in extrinsic allergic alveolitis: clinical assessment using inhalation provocation tests. Thorax 1981;36:917–21.

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219. Craig TJ, Hershey J, Engler RJ, et al. Bird antigen persistence in the home environment after removal of the bird. Ann Allergy 1992;69:510–2. 220. Bracker A, Storey E, Yang C, Hodgson MJ. An outbreak of hypersensitivity pneumonitis at a metalworking plant: a longitudinal assessment of intervention effectiveness. Appl Occup Environ Hyg 2003;18:96–108.

221. Kokkarinen JI, Tukiainen HO, Terho EO. The effect of corticosteroid treatment on the recovery of pulmonary function in farmer’s lung. Am Rev Respir Dis 1992;145:3–5. 222. Cormier Y, Israel-Assayag E, Desmeules M, Lesur O. Effect of contact avoidance or treatment with oral prednisolone on bronchoalveolar lavage surfactant protein A levels in subjects with farmer’s lung. Thorax 1996;51:1210–5.

Chapter

13

Sarcoidosis Douglas B. Flieder, Abraham Sanders and Michael N. Koss

Introduction Sarcoidosis is a relatively uncommon multi-systemic immunological disorder of unknown cause(s) with variable clinical manifestations and outcomes. Many of the earliest investigators are associated with sarcoidosis in eponymous perpetuity.1 Jonathan Hutchinson, a British dermatologist, first reported and illustrated a case of sarcoidosis in 1877 under the title of “Case of livid papillary psoriasis”. In 1895 he presented the case of a 64-year-old woman with facial and upper extremity skin lesions, which were neither tuberculosis nor lupus and named it Mortimer’s malady.2,3 Yet Mrs Mortimer was not willing to undergo a skin biopsy and only in 1897 did Caesar Boeck manage to biopsy a 34-year-old policeman with cutaneous lesions.4 The term “sarcoid” derives from Boeck’s initial histological description of the tumoral non-necrotizing granulomas as sarkoid (Greek for flesh- or sarcoma-like). Dr Arthur Conan Doyle, a London contemporary of Dr Hutchinson, was probably the first to describe familial sarcoid and made cutaneous sarcoid an integral part of the plot of The Adventures of the Blanched Soldier.5,6 Visceral involvement, including the first description of lung disease, was reported during the First World War.7 Swedish physicians Jorgen Schaumann and Sven Löfgren applied chest radiography to sarcoid patients and defined hilar and mediastinal nodal disease in the first two decades of the twentieth century; years before Löfgren described the syndrome of erythema nodosum, polyarthritis, hilar adenopathy and fever that now bears his name.8,9 Morton Ansgar Kveim noted that intradermal inoculation of sarcoid lymph node tissue elicited a skin reaction in virtually all sarcoid patients. Louis Siltzbach modified this test using splenic tissue from affected individuals and organized an international study:10,11 hence the Kveim-Siltzbach test. Although Hutchinson was a well-known physician and many European doctors were aware of his and other descriptions of sarcoidosis, the disease was not of great interest in the United States until the second half of the twentieth century. All editions of William Osler’s Textbook of Medicine are mute on the condition, even though Osler appears to have described the

disease in a 14-year-old African-American girl in 1898.12,13 The disease was first formally presented to American physicians in a 1936 Johns Hopkins Hospital review.14 While once figuratively buried under the cloak of tuberculosis, sarcoidosis has spawned its own journal, Sarcoidosis, its own association, the World Association of Sarcoidosis and Other Granulomatous Disorders (WASOG), and regular World Congresses. The present day descriptive definition encompasses clinical, radiographic and histological findings. The American Thoracic Society, European Respiratory Society and WASOG agree that: Sarcoidosis commonly affects young and middle-aged adults and frequently presents with bilateral hilar adenopathy, pulmonary infiltration and ocular and skin lesions. The liver, spleen, lymph nodes, salivary glands, heart, nervous system, muscles, bones and other organs may also be involved. The diagnosis is established when clinicoradiographic findings are supported by histological evidence of non-caseating epithelioid cell granulomas. Granulomas of unknown origin and local sarcoid reactions must be excluded. Frequent observed immunological features are depression of cutaneous delayed-type hypersensitivity and a heightened Th-1 immune response at sites of disease. Circulating immune complexes along with signs of B-cell hyperactivity also may be found.15

This chapter strives to present a current discourse on classic sarcoidosis and its variants touching on all relevant clinical, radiographic and pathological aspects of this enigmatic disease.

Epidemiology Epidemiological generalizations about sarcoidosis are difficult owing to its rarity, lack of precise case definition, varying methods of case ascertainment, variations in presentation, including possible absence of clinical manifestations, and the lack of definitive diagnostic tests.16,17 Many sarcoidosis patients are probably admixed with tuberculosis cases. As a historical example, Robert Louis Stevenson spent time at Edward Livingston Trudeau’s Saranac Lake Hospital/Adirondack Cottage Sanatorium in 1887–1889 for presumed

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 13: Sarcoidosis Table 1 Variations in sarcoidosis incidence across ethnic groups

Ethnic group

Incidence per 100 000

Peak decade of incidence

Percent increased risk in females

EuropeanAmericans

3–10

4th–5th

10–20

AfricanAmericans

35–80

3rd–4th

30

Northern Europeans

15–20

3rd

30

Southern Europeans

1–5

4th–5th

33

Japanese

1–2

3rd

10–20

Data from Rybicki, et al.

39

tuberculosis, yet it is likely he suffered from sarcoidosis and probably died of cardiac sarcoidosis.13,18 The first epidemiological study on sarcoidosis appeared in 1950, based on 350 cases detected in American World War II veterans.14,19 Many studies have since been undertaken and much is known about the disease. Sarcoidosis occurs around the globe and affects men and women of all races and ages. Geographic, ethnic and sex differences figure prominently. The disease usually manifests before 50 years of age, with general incidence peaks in the third decade.20 Sarcoidosis is less common in children than in adults.21 Ethnic differences are huge. For example, the age-adjusted annual incidence rate for African-Americans is 35.5, but only 10.9 for Caucasian-Americans.20,22–24 Age-specific incidence peaks are also different in diverse ethnic groups (Table 1). The peak incidence in African-Americans is in the fourth decade of life, while in both Scandinavia and Japan there is a second peak incidence in women older than 50 years of age.20,25,26 Geography also plays a role. The highest incidence rates are reported in Northern Europe among Swedes and Danes and in the United States (up to 80 cases per 100 000 people). In Japan the annual incidence is only 1 to 2 per 100 000 people.27,28 The disease is relatively infrequent at latitudes close to the equator.29,30 Seasonal clustering in the spring has been noted, in both Europe and New Zealand.31–34 Across all ethnic groups, slightly higher disease rates are noted in women. In the United States, incidence rates are 6.3 and 5.9 per 100 000 for women and men, respectively.23 Sarcoidosis is infrequently reported in Spain, Portugal, South America and India. This is due to the high incidence and prevalence of infectious granulomatous diseases, such as tuberculosis and leprosy, and the absence of mass screening programs.35,36 Clinical presentations, organ involvement and severity of disease, including morbidity and mortality rates, also follow ethnic and/or racial lines (see below).

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Genetic associations and etiological considerations Elucidating the etiology of this multisystem granulomatous disease is a challenge, since any proposed cause(s) must not only produce the established histological findings but also account for the varied clinical manifestations across different geographic and ethnic groups. The lack of a known etiological agent is certainly not due to lack of medical interest. Research over the past decade, including the recently completed National Institutes of Health-funded A Case Control Etiological Study of Sarcoidosis (ACCESS) study, provides new insights into possible etiologies.37–39 Of significant note, the ACCESS study failed to identify a single cause of sarcoidosis and confirms the belief that the disease develops in genetically susceptible hosts in response to specific environmental agents. Amidst the thousands of publications and pronouncements regarding etiological agents, experts only agree that sarcoidosis is not an autoimmune disorder. Genetic susceptibility is not surprising given the markedly different incidence rates according to race and ethnicity. Familial sarcoidosis was first reported in two German sisters and a recent registry-based study indicates an 80-fold increased risk in monozygotic twins, compared to an increased risk in dizygotic twins of only 7-fold.40,41 Furthermore, ACCESS data reveal that patients with sarcoidosis have either siblings or parents with the disease five times as often as controls.42 Curiously, North American and British Caucasians have a markedly higher familial relative risk, compared with African-Americans.42,43 Associations between sarcoidosis and class I and class II HLA antigens are beyond doubt and they probably play complementary roles in the disease (Table 2).44–47 Class I HLA-B8 is associated with acute disease. Class II antigens, encoded by HLA-DRB1 and DQB1 alleles, are thought to confer a predisposition to disease phenotype rather than to susceptibility.46,48,49 HLA-DQB1*0201 and HLA-DRB1*0301 are strongly associated with acute disease and a good prognosis, while HLA-DQB1*0602 is associated with severe pulmonary disease.49,50 Genome-wide scans to date yield inconsistent findings, which suggests genetic heterogeneity. Strong linkage signals at chromosomes 3p and 6p are seen in German Caucasians, while 5p and 5q strong signals in African-Americans are reported.51,52 For example, the butyrophilin-like 2 gene (BTNL2), a probable co-stimulatory molecule in the T cell activation pathway, found in the MHC region on chromosome 6p, might be a risk susceptibility factor.52–55 Other studies suggest 3p and 5q11.2 as sarcoidosis susceptibility genes in African-Americans, while protective genes inhabit the 5p15.2 region.56 Associations with genes encoding for tumor necrosis factor-a (TNFa), interferon-g (INF-g), chemokine receptors and vascular endothelial growth factor and its receptors seem logical candidates but associations have not been confirmed.57–61 Fas promoter genetic variants may be related to disease risk in African-Americans.62

Chapter 13: Sarcoidosis Table 2 Summary of HLA association studies of sarcoidosis

Table 3 Suggested etiological agents in sarcoidosis

HLA

Risk alleles

Finding

HLA-A

A*1

Susceptibility

HLA-B

B*8

Susceptibility in several populations

HLADQB1

*0201

Protection, Löfgren syndrome, mild disease in several populations

Infectious Viruses Herpes simplex Human herpes virus-8 Epstein-Barr Coxsackie B virus Cytomegalovirus

*0602

Susceptibility/disease progression in several groups

Non-tuberculous mycobacteria

*0301

Acute onset/good prognosis in several groups

Propionibacterium acnes

*04

Protection in several populations

Chlamydia

*1101

Susceptibility in whites and AfricanAmericans; stage II/III chest X-ray

Mycoplasma

*1501

Associated with Löfgren syndrome

*0101

Susceptibility/disease progression in whites

HLADRB1

HLADRB3

Modified from Iannuzzi, et al.57

If one considers this disease a genetic-based dysregulated hyperimmune response, then it follows that susceptible individuals develop disease when exposed to particular environmental agents.63 Given the frequent involvement of the lungs, skin and eyes, airborne antigens are likely triggers and infectious agents and non-infectious organic and inorganic substances top most etiological lists (Table 3).15,63 Historic and recent epidemiological studies, some lacking well-defined, matched control groups, associate sarcoidosis with rural or coastal residences, fireplaces, wood stoves, home mold, central air conditioning and exposure to pine pollen and pica. In addition, occupations such as pesticide-using industries, the lumber industry, the military and firefighting and rescue workers involved in the 2001 World Trade Center attacks, middle and high-school teachers and healthcare workers have been associated with the disease.22,63–76 A recent publication even suggests photocopier toner dust may be a candidate antigen.77 Interestingly, tobacco smoking, despite the thousands of particles and agents contained in each and every drag, is negatively associated with sarcoidosis.63,68,78 The degree of alveolitis in smokers with sarcoidosis is less intense than that of non-smokers with an equivalent amount of disease.79 Tobacco probably modulates the intrapulmonary immune response, but smoking does not improve the clinical course or prognosis of the disease.80–82 For over 100 years investigators have been trying to prove that sarcoidosis has an infectious etiology and most research has targeted mycobacteria.83,84 Epidemiological data, including familial and seasonal clustering, suggest the

Mycobacterium tuberculosis Borrelia burgdorferi Rickettsia

Inorganic Aluminum Zirconium Talc Photocopier toner dust Organic Pine tree pollen Clay

unknown antigen(s) is/are transmissible, although the lung transplant literature discounts this notion.85–88 Recent molecular investigations reveal mycobacterial DNA, RNA and cell wall components in sarcoidal tissue.89–93 Many additional microbes, including but not limited to Propionibacterium sp., rickettsia, borrelia, chlamydia, Epstein-Barr virus, herpes simplex virus and human herpes virus-8, have also been touted or suggested as etiological agents or a co-agents.90,94–103 While multiplexed liquid chromatography, non-highperformance liquid chromatography coupled to electrospray or matrix-assisted laser desorption/ionization mass spectrometry findings are thought-provoking, over 100 years of morphological investigations should not be relegated to the historical dust heap when sifting through these twentieth and twenty-first century high-tech reports.57,104–106 To quote the twentieth-century New York Yankee baseball icon Yogi Berra, “you can observe a lot by watching”, and neither morphologists nor microbiologists have yet to link any infectious agent with sarcoidosis histopathology.106–110 Thus, Koch’s postulates remain unfulfilled and the etiology remains an enigma. However, the possibility that host immune responses to mycobacterial protein remnants, such as mycobacterial catalase-peroxidase (mKatG), play a role in a subset of sarcoidosis patients cannot be entirely discounted.75,87,104,111–113

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Clinical manifestations As the granulomatous inflammation of sarcoidosis involves different organs, patients come to the attention of different clinical specialists. Patients may present with symptoms from one or several organs simultaneously. The most commonly involved organs are the lungs and upper airways (90%), lymph nodes (80–90%), eyes (25–80%), skin (25–35%), heart (5–25%), nervous system (10–25%), liver and spleen (10–20%), bones and joints (14–38%). All other organs may also be involved. The percentage of patients with sarcoidosis harboring lesions in each of the above organs varies greatly depending on age, geographic, racial and ethnic groupings (Table 1).39 For example, those diagnosed at less than 40 years of age are more likely to have extrathoracic lymphadenopathy, compared to older individuals. Women are more likely to have ocular and neurological involvement, as well as erythema nodosum. Men more frequently experience abnormal calcium metabolism, due to the disease. Interestingly, African-American women rarely present with erythema nodosum but endure lupus pernio more often than Caucasians. In addition, children may have pulmonary disease, rather than constitutional symptoms, peripheral adenopathy, ocular symptoms and skin involvement.21,114–116 Statistics also differ depending on whether one is interested in clinical disease or disease noted at autopsy.117 For example, only 8% of surveyed Germans with sarcoidosis reported cardiac disease, while Japanese studies report rates as high as 78%.118–120 In the United States, clinical manifestations, such as conduction defects, mitral insufficiency, ventricular aneurysms and congestive heart failure, are apparent in only 5% of all patients but myocardial or conduction system granulomas are identified in approximately 25% of patients at autopsy.121 Only 50% of afflicted individuals have symptoms.122,123 Patients usually complain of constitutional symptoms, such as fever, fatigue, weight loss and depression with or without obvious organ involvement.124 Respiratory symptoms are most common, affecting one-third of patients, and complaints are of dry cough, dyspnea on exertion and chest discomfort. Hemoptysis is rare.125 Patients may present to ophthalmologists with anterior and posterior uveitis and to dermatologists with rashes, macules, papules, plaques, erythema nodosum or lupus pernio. Cardiologists encounter patients with arrythmias, heart failure, pulmonary hypertension and rarely sudden death. Rheumatologists see those with chronic arthralgia, bone lesions and muscle weakness. Neurologists treat cranial nerve palsies, headaches, ataxia, cognitive dysfunction, weakness and seizures. Nephrologists evaluate renal calculi, hypercalcemia and rarely renal failure. Internists are often the first physicians to investigate peripheral lymphadenopathy. Many patients are asymptomatic and the disease is discovered when chest radiographs are obtained. Physical findings are usually unremarkable. Crackles are heard in less than

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20% of patients, even when radiographic infiltrates are extensive, and clubbing is almost non-existent.126 A notable exception is Löfgren syndrome (erythema nodosum, polyarthritis, hilar adenopathy and fever). The erythema nodosum component is more common in women, while ankle joint disease without erythema nodosum is more common in men.127 These men and women represent 20 to 50% of those with acute disease and up to 34% of all sarcoidosis patients.128 Lung function studies are often abnormal and may not correlate with radiographic or morphological findings.129–132 Twenty percent of patients without radiographic evidence of lung disease have abnormal pulmonary function tests. Up to 80% of most patients with lung involvement show restrictive impairment with reductions in lung volumes and carbon dioxide diffusing capacity.27,133,134 The diffusing capacity reduction is less pronounced than in idiopathic pulmonary fibrosis (see Chapter 10).135 These changes reflect the fibrosing nature of the pulmonary parenchymal involvement. Expiratory airflow obstruction is also seen in over 30% of patients. Causes include airway granulomas and/or fibrosis, bronchial hyperreactivity, peribronchiolar fibrosis, extrinsic compression by enlarged mediastinal lymph nodes, or pulmonary fibrosis.136–141 Pulmonary function tests at the time of diagnosis do not predict long-term outcome in patients with sarcoidosis but sequential studies allow one to follow disease progression and assess response to therapy.142,143 Measured physiological improvements following therapy are usually evident within 6 to 12 weeks.142

Radiographic findings Chest radiographic features of sarcoidosis are variable and encompass the entire spectrum of intrathoracic radiological findings from hilar adenopathy to cavitary parenchymal lesions. Remarkably, routine chest radiographs usually suffice in the diagnosis and management of patients with sarcoidosis. Although computed tomography (CT) studies offer far more detail than conventional chest radiographs, suggested uses are limited to atypical clinical and/or chest radiographic findings, a normal chest radiograph in patients with a clinical suspicion of disease, and the detection of pulmonary complications.15 In this subset, pulmonary high-resolution CT (HRCT) may discriminate between inflammation and fibrosis and correlate with functional impairments. This diagnostic tool can influence therapeutic decisions in a small minority of patients.144–148 More than 80% of patients with sarcoidosis have intrathoracic adenopathy at presentation (Figure 1).149 While only hilar, aortic pulmonary window and right paratracheal lymph nodes are easily identified on posteroanterior and lateral chest radiographs, CT examination highlights many more lymph node stations.150 Surprisingly, nodal calcification, not unlike that associated with infectious and environmental diseases, is noted in approximately 50% of patients.151,152 Pulmonary parenchymal disease has a strong predilection for the upper lung fields.153 A central distribution, unrelated to

Chapter 13: Sarcoidosis

Figure 1. Chest radiograph of stage I sarcoidosis. Bilateral relatively symmetrical hilar adenopathy is apparent.

Figure 2. Chest radiograph of stage II sarcoidosis. Bilateral interstitial markings in the absence of striking hilar adenopathy are not specific for sarcoidosis but represent one piece of evidence in the diagnostic evaluation.

hilar lymph node involvement, is more common than diffuse or peripheral findings. Reticular, reticulonodular or focal alveolar opacities are the most characteristic chest radiographic features while consolidation, well-circumscribed nodules and ground-glass opacities are less common (Figure 2).154–159 Nodular lesions are usually bilateral and rarely solitary (see Sarcoidosis variants below). Computed tomography and especially HRCT scans demonstrate the interstitial nature, i.e. the vascular, airway, and/or lymphatic distribution of the disease.160,161 The most common parenchymal abnormalities are lymphangitic 0.2 to 1.0 cm sharply defined irregular-edged nodules along the small and large bronchovascular bundles and subpleural parenchyma.162 Opacities may coalesce into nodules (so-called nodular sarcoidosis; see below), areas of consolidation or ground-glass opacities. Air-trapping is another common CT feature of pulmonary sarcoidosis, reflecting small airway narrowing secondary to granulomas.163 End-stage disease involves the central and upper lung fields and manifests as masses, septal bands, hilar retraction, upper lobe volume loss and honeycomb change with large bullae on chest radiographs and CT scans (Figure 3). Paracicatricial emphysema is rare. Mycetomas, most often aspergillomas, may be seen in upper lobe fibrotic lung and bullae. Advanced sarcoidosis rarely features HRCT findings identical to those expected in idiopathic pulmonary fibrosis (IPF), especially since IPF is most often lower lobe predominant (see Chapter 10).164 A chest radiographic classification proposed almost 50 years ago remains clinically relevant.165 The schema groups the most common pulmonary radiographic patterns and is associated with prognosis (Table 4). The incidence of radiographic stages varies depending on geographic, racial and ethnic considerations. Scandinavian studies note a predominance of radiographic stages I and II disease while British and

American studies often feature a majority of radiographic stages III and IV disease.166–169 Prognosis is best with radiographic stage I and worsens through to stage IV. Pulmonary function abnormalities also correlate with this staging system but not with functional impairments.15 Of note, some patients have radiographs that do not fit into the classification and CT is not consistently superior to chest radiography in this regard.148,170 Other imaging modalities including magnetic resonance imaging (MRI), gadolinium-enhanced MRI, gallium-67 scintigraphy scanning, lung clearance of inhaled technetium99m-labeled diethylenetriaminepentaacetic acid scanning (99mTc-DTPA) and proton-emission tomography with 18 F-fluorodeoxyglucose (PET-FDG) may be valuable in detecting unsuspected sites of disease or monitoring disease in other locations. These include the central nervous system or musculoskeletal system, but are not routinely used in the evaluation of thoracic disease.

Macroscopic pathology In the early stages of pulmonary sarcoidosis the lungs usually show no gross abnormalities. The first findings are yellowwhite 0.1 to 0.2 cm gritty nodules along subpleural lung, interlobular septa and around bronchovascular bundles (Figure 4). Gray leathery fibrosis encircles pulmonary lobules (Figure 5). Solitary or multiple usually bilateral up to 8.1 cm nodules are seen in less than 5% of cases (see Sarcoidosis variants below).171–174 Bronchial and bronchiolar disease can manifest as submucosal nodularities, which compromise lumina and may lead to post-obstructive pneumonia (Figures 6 and 7). Bronchioloectasis and bronchiectasis develop in progressive disease and may result in cyst formation (Figure 8). Bullous emphysema may rarely be seen.175 These changes are

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(a)

(b)

Figure 3. Chest radiograph and computed tomogram of stage IV sarcoidosis. (a) The CXR demonstrates bilateral symmetrical reticulonodular infiltrates and lower lobe hyperinflation. (b) The corresponding CT shows interstitial markings and striking cystic change. Table 4 Chest radiograph staging system of sarcoidosis

Radiographic stage

Radiographic finding

Percentage of patients at presentation

0

Normal chest X-ray

0–15

I

Bilateral hilar lymphadenopathy (BHL)

35–40

II

BHL þ parenchymal infiltrates

35–40

III

Parenchymal infiltrates

10–15

IV

Fibrosis and bullae formation

5

BHL, bilateral hilar lymphadenopathy.

more likely to involve the upper lobes and lobar collapse may ensue. Longstanding cavities may contain saprophytic fungal disease (mycetomas) (Figure 9). Interestingly, honeycomb change is unusual.176 Raised, white pearly visceral pleural plaques are not uncommon, but thick diffuse rinds rarely form (see below) (Figure 10). Bilateral lymph node involvement frequently accompanies parenchymal disease. Involved intrathoracic lymph nodes, including intrapulmonary lymph nodes, are enlarged, firm, gray-tan and often adherent to or poorly demarcated from

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Figure 4. Early pulmonary sarcoidosis. This ill-defined multilobulated firm gray nodule centered on a bronchovascular bundle is the most obvious finding in this field. Interlobular septa are slightly more prominent than usual (arrows).

adjacent soft tissue or lung. Black carbon specks may litter the lymph node cut surface (Figure 7). Partial lymph node involvement is not unheard of.

Chapter 13: Sarcoidosis Figure 5. Pulmonary sarcoidosis. The lymphangiitic distribution is apparent in this autopsy specimen. Lobules are outlined by firm gray infiltrates. Exquisite bronchovascular disease with minimal acinar involvement is seen.

Figure 6. Pulmonary sarcoidosis. Extensive lymphangitic disease compromises the bronchial lumen. (Courtesy of L. Litzky, MD, Philadelphia, Pennsylvania, USA.) Figure 8. Pulmonary sarcoidosis with lobar collapse and cystic change. Severe disease has a predilection for the upper lobes. In this case the upper lobe is almost entirely replaced with cysts while parenchymal disease including bronchiectasis affects remaining lung. Parietal pleural and diaphragmatic adhesions indicate involvement of these organs as well.

Figure 7. Bronchial and lymph nodal sarcoidosis. This lobar bronchus features a thickened wall with endobronchial nodularity. Involved peribronchial lymph nodes are enlarged and replaced with firm white tissue. Pulmonary ventilation and perfusion can be greatly affected by this pathology. (Courtesy of L. Litzky, MD. Philadelphia, Pennsylvania, USA.)

Histopathology Sarcoidosis, whether involving the lungs, lymph nodes or other organs, is characterized by non-necrotizing granulomas. Yet the earliest stage of the disease features a mild interstitial or alveolar lymphocytic infiltrate with few plasma cells but no granulomas (Figure 11).129,177,178 These findings are neither diagnostic nor even suggestive of sarcoidosis. Sarcoidal granulomas are well-circumscribed collections of epithelioid macrophages with light eosinophilic cytoplasm and reniform to oval vesicular nuclei, surrounded by a rim of

lymphocytes and fibroblasts (Figure 12). Multinucleated Langhans-type giant cells, probably resulting from the fusion of epithelioid mononuclear cells, are often but not always noted.179 Scattered lymphocytes, and rarely plasma cells, neutrophils and even eosinophils, can be part of the granulomas. Apoptotic bodies may also be seen. As lung disease progresses, morphology also changes. Granulomas are cellular and discrete in the early stages of the disease, and either resolve or become fibrotic in longstanding cases.180 Fibrosis appears to progress from the outer rim inward, so that concentric collagen entombs the central “active” portion of the granuloma before entirely obliterating the cellular constituents (Figure 13).181,182 Longstanding disease usually mixes cellular, partially fibrotic and obliterated granulomas.

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Figure 10. Pleural sarcoidosis. Either visceral pleural or subpleural pulmonary disease manifests as raised pleural nodules. Nodules may coalesce into patches but pleural fibrosis is distinctly uncommon. (Courtesy of J. English, MD, Vancouver, British Columbia, Canada.) Figure 9. Aspergilloma arising in sarcoidosis. Upper lobe cysts may be colonized with fungi. Underlying bronchiectasis and parenchymal sarcoidal changes are noted adjacent to this aspergilloma. (Courtesy of Y. Rosen, MD, North Bellmore, New York, USA.)

Figure 12. Sarcoidal granuloma. This typical non-necrotizing granuloma features many epithelioid histocytes with a few admixed multinucleated giant cells and lymphocytes. Surrounding lymphoplasmacytic infiltrates expand edematous connective tissue. Figure 11. Early pulmonary sarcoidosis. A lymphangiitic lymphoplasmacytic infiltrate is the first microscopic finding in pulmonary sarcoidosis. The almost nodular area suggests the development of a granuloma.

While most of the granulomas are non-necrotizing, up to 40% contain small foci of central fibrinoid, granular or eosinophilic necrosis (Figure 14). This material is not unlike that observed in infectious processes.181,183,184 Extensive necrosis usually indicates an infectious process but necrotizing sarcoid granulomatosis is a differential consideration (see below).

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The lymphangitic distribution characterizing pulmonary sarcoidosis encompasses bronchovascular bundles, interlobular septa and the visceral pleura (Figure 15). At first one finds scattered compact granulomas merely expanding the connective tissue at these locations but over time fibrosis leads to coalescence and nodular destruction of underlying lung (Figure 16). When only nodules are seen, one refers to the process as nodular sarcoidosis (see Sarcoidosis variants below). The lymphangitic pattern is always appreciated in less involved

Chapter 13: Sarcoidosis

Figure 13. Longstanding sarcoidal granuloma. Fibrous obliteration of granulomas proceeds from the periphery of the lesion toward the center. Collagen appears to strangle the cellular constituents, eventually leading to nodular scars.

Figure 14. Sarcoidal granuloma with punctate necrosis. While most granulomas are non-necrotizing, central necrosis is not uncommon. Since sarcoidosis is always a diagnosis of exclusion, special stains must be performed and tissue findings correlated with the clinical scenario.

Figure 15. Pulmonary sarcoidosis. The striking lymphangitic distribution is obvious in this example. Airspaces are unaffected at this early stage of disease but bronchiolar constriction and distortion are seen.

Figure 16. Pulmonary sarcoidosis. Alveolar parenchyma is obliterated by coalescing granulomas. While individual granulomas are not fibrotic, large areas of infarct-like fibrosis and elastosis are probably secondary to vascular insufficiency. Interestingly, desquamative interstitial pneumonia-like reactions are rarely seen in sarcoidosis.

areas of tissue and may be the only clue to the diagnosis in advanced disease. Granulomas always involve and distort bronchi and bronchioles (Figure 17). While alveolar spaces are preserved in

early disease, in progressive disease significant fibrosis or bullous change replace lung tissue (Figures 18 and 19).175 While small airways may be destroyed, bronchiolitis obliterans is uncommon.185

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Figure 18. Significant pulmonary sarcoidosis. This whole mount demonstrates cyst formation in the presence of lymphangitic granulomas. The bronchus is only minimally affected but normal parenchymal architecture is lost.

Figure 17. Bronchiolar sarcoidosis. Granulomas even adjacent to but not directly involving airways distort ventilatory function. In this example the bronchiole is compressed and partially filled with macrophages and mucus.

Figure 20. Bronchovascular granulomas. While the bronchiole is distorted, the bulk of disease surrounds the accompanying artery. Intimal thickening is apparent.

Figure 19. End-stage pulmonary sarcoidosis. Twisted bronchioles are the only evidence that this tissue is lung parenchyma. Remaining alveoli are rounded and overrun by longstanding fibrotic granulomas. (Glass slide courtesy of J. English, MD, Vancouver, British Columbia, Canada.)

Angiocentricity is common and granulomas can be angioinvasive.186 Arterial and venous vessels, including bronchial arteries of all sizes, are affected. Blood vessel involvement often overshadows other distribution patterns (Figure 20).

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Granulomas usually infiltrate the vessels from the adventitial layer inward (Figure 21). Medial involvement is frequent but intimal is less common. The elastic laminae are disrupted, but vessel wall necrosis is not seen (see Necrotizing sarcoid granulomatosis below) (Figure 22). Lumenal compression along with secondary intimal and medial thickening of medium to large caliber arteries may lead to pulmonary hypertension and even mimic pulmonary veno-occlusive disease, but thrombosis, aneurysm formation or infarction has not been reported (Figure 23).187,188

Chapter 13: Sarcoidosis

Figure 22. Sarcoidal vascular involvement. Perivascular and adventitial granulomas infiltrate this pulmonary vein. The internal elastic lamina is shredded and an intimal proliferation is apparent. (Courtesy of Y. Rosen, MD, North Bellmore, New York, USA.)

Figure 21. Sarcoidal vascular involvement. The artery is virtually entombed in fibrosis and granulomas (arrows). Only the vessel outline is preserved while the lumen is compressed by granulomas.

Figure 23. Sarcoidal vascular involvement. Compact non-necrotizing granulomas markedly compress this arterial lumen. Hemodynamic alterations may develop.

Figure 24. Asteroid body. This multinucleated giant cell contains a classic asteroid body. The central core, rays and vacuoles are apparent.

Inclusions Several morphological curiosities are frequently identified in sarcoidal granulomas. Asteroid bodies, Schaumann (conchoidal) bodies and calcium oxalate crystals are cytoplasmic inclusions found within macrophages. These curiosities, as well as Hamazaki-Wesenberg bodies, found in sarcoidal lymph nodes, are nonspecific findings of no diagnostic utility.183,189,190 From 2% to 9% of sarcoidal giant cells contain asteroid bodies.183,184 These complex, star-shaped, spiculated, intracytoplasmic inclusions range from 5 to 30 mm in diameter and may feature over 30 gently curved threads or rays emanating from a central 1 mm. core. The rays are surrounded by vacuoles (Figure 24).

Asteroid bodies should not be confused with infectious organisms even though they are also found in tuberculosis, leprosy, histoplasmosis and schistosomiasis as well as in foreign-body granulomas.184 Ultrastructurally, myelinoid membranes envelop the filamentous vimentin-positive arms; the presence of calcium and/or phosphorus is disputed.191–193 These structures form from either cytoskeletal structures or extraneous phospholipid components after histiocyte fusion.191,192,194–196 Schaumann (conchoidal) bodies, first described and named by Jorgen Schaumann in 1941, are 25 to 200 mm. Intracytoplasmic, lamellar calcifications are found in up to 88% of sarcoidal cases

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Chapter 13: Sarcoidosis Figure 26. Schaumann (conchoidal) bodies. Transbronchial biopsy samples may on occasion contain these structures. Structures aggregate and represent tombstones of long dissolved giant cells. (Glass slide courtesy of L. Litzky, MD, Philadelphia, Pennsylvania, USA.)

Figure 25. Schaumann (conchoidal) bodies. Lamellar cytoplasmic calcifications are often seen in sarcoidal giant cells but also in many other diseases. (Courtesy of Y. Rosen, MD, North Bellmore, New York, USA.)

Figure 28. Hamazaki-Wesenberg bodies. One to 15 mm round to oval brown structures noted in the subcapsular sinuses of lymph nodes look like and stain similarly to a variety of yeast forms. In the setting of granulomatous lung disease, an erroneous interpretation of infection is possible. (Glass slide courtesy of K. Cooper, MD, Burlington, Vermont, USA.) Figure 27. Calcium oxalate crystals. Block-like inclusions noted in giant cells do not represent foreign material but rather intrinsic cellular debris. (Courtesy of Y. Rosen, MD, North Bellmore, New York, USA.)

(Figure 25).197 Also seen in chronic berylliosis, hypersensitivity pneumonitis, Crohn disease and tuberculosis, the basophilic calcifications are composed of calcium oxalate, aluminum, phosphorus and iron.193,197 Nodules may also contain lipoproteins, acidic and non-acidic mucoproteins.194 These masses often burst from giant cells and aggregate with other calcium salts (Figure 26). One may find them in cytological preparations, including bronchoalveolar lavage (BAL) samples.198–200 Schaumann bodies probably originate as calcium oxalate crystals deposited on myelinoid membranous inclusions (Figure 27).193 The calcium oxalate serves as a nidus for further deposition of macrophage lysozyme products in the formation of Schaumann bodies. These crystals occur both

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within and independently of Schaumann bodies.189,201,202 The poorly stained, lucent, block-like or irregular but never needlelike 1 to 2 mm brilliantly birefringent crystals are noted within giant cells and macrophages of not only sarcoidal, but also tuberculous, and histoplasmosis granulomas as well as in lymph nodes draining carcinomas.189 Hamazaki-Wesenberg bodies, also known as yellow-brown bodies, yellow bodies, spindle bodies and chromogenic bodies, were first described by Yukio Hamazaki in 1938 and revisited by W. Wesenberg in 1966.203 While historically associated with systemic sarcoidosis, this incidental finding is probably no more prevalent in this disease than in other conditions, including appendicitis, hepatic cirrhosis and colon cancer.204 The 1 to 15 mm round to oval, yellow-brown structures are most often found free or within giant cells in lymph node subcapsular sinuses throughout the body (Figure 28).

Chapter 13: Sarcoidosis Figure 29. Fine-needle aspiration sample, consistent with sarcoidosis. Aggregates of epithelioid histiocytes and occasional lymphocytes against a clean background are commonly seen. Epithelioid cells have elongated nuclei and abundant amphophilic cytoplasm.

Figure 30. Fine-needle aspiration sample, consistent with sarcoidosis. Samples rarely feature granulomas with giant cells but rather scattered solitary giant cells. The location of the nuclei is of no help in differentiating sarcoidosis from other pathological processes. Small round to oval nuclei with occasional nucleoli are the norm but reactive nuclear changes may be seen.

Pulmonary collections have not been reported. Ultrastructural studies suggest the bodies represent large lysosomes and residual bodies, rather than lipofuscin.194,205–207 The yeastlike morphology and histochemical staining characteristics, namely Grocott-methenamine silver (GMS), periodic acidSchiff and Ziehl-Neelsen (ZN) positivity, encourage erroneous diagnoses of fungal disease. Hamazaki-Wesenberg bodies stain with Fontana-Masson silver stain, while fungi, apart from Cryptococci, are non-reactive.207

walls, as well as alveolar capillary endothelial cell basal lamina layering, swelling, and bleb formation.178 These findings probably represent endothelial cell degeneration. Uninucleate epithelioid cells are secretory cells, while multinucleated giant cells have both secretory and phagocytic abilities.212–215 Both cell types contain cytoplasmic well-developed Golgi apparatuses, rough endoplasmic reticulum and numerous mitochondria. Epithelial cells also have membrane-bound cytoplasmic vesicles, with electron-lucent material (lysozyme), while multinucleated cells also contain phagolysosomes.212,216 Interestingly, the alveolar macrophages in sarcoidosis are larger due to more numerous and bigger lysosomes and complex pseudopodia than control subject macrophages.217 Lymphocytes may also be larger, with more lysosomes.217 These findings support the cytomorphological description of “cellular reactive changes”.198 Extensive cytoplasmic membrane folding and intimate associations between macrophages and lymphocytes suggest an information-sharing system. Subplasmalemmal linear densities link the macrophages, while small tongues of organelle-free, lymphocyte cytoplasm invaginate into macrophage cytoplasm.217

Cytology Cytology samples are never diagnostic of sarcoidosis but retrospective correlative studies delineate typical morphological findings in sputum, lavage and fine-needle aspirate samples.198,200,208 First and foremost, classic sarcoidosis cytology features a clean background, devoid of acute inflammation.198,209,210 While sputum samples are rarely informative, one may see multinucleated giant cells, epithelioid cells and lymphocytes.200 Bronchoalveolar lavage specimens with alveolar macrophages, epithelioid cells and more than occasional multinucleated giant cells suggest a diagnosis of sarcoidosis.210,211 Interestingly, macrophages, epithelioid cells and giant cells are more likely to be individually scattered on the slide, rather than clustered into granulomas.198 Fine-needle aspirate samples of the lung demonstrate similar findings. Cytomorphologically, multinucleated giant cells usually have a horseshoe distribution of nuclei, but randomly scattered nuclei are also seen. Some nuclei might be enlarged with open chromatin and small but prominent acidophilic nucleoli. Rare cytoplasmic inclusions may be noted. The uninuclear alveolar macrophages are cytomorphologically similar to the giant cells. The epithelioid cells, the hallmark of granulomatous disease, feature central oval to elongated nuclei and significant amounts of amphophilic cytoplasm without carbon particles (Figures 29 and 30). Bronchoalveolar lavage studies are discussed below.

Electron microscopy Ultrastructural features of alveolitis in sarcoidosis note lymphocytes, monocytes and macrophages in the alveolar

Diagnosis of sarcoidosis The diagnosis of sarcoidosis requires a clinical picture consistent with the disease, a biopsy consistent with sarcoidosis, and the exclusion of other diseases with similar clinical presentations. Most commonly patients present with symptoms in one organ, while multiple organ dysfunction is less common. When the diagnosis is suspected, evaluation of all organ systems should be undertaken to look for involvement, even if not obvious. All patients should receive a thorough history and physical examination including an ophthalmological exam, chest imaging and echocardiogram, blood count and liver function studies, serum calcium, urinalysis and 24-h collection for calcium excretion, tuberculin skin test and pulmonary function studies. Since 95% of patients have clinical evidence of pulmonary involvement and more than 40% have skin, peripheral lymph node or eye disease, informative samples can be procured from

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most of these sites. Liver sampling is discouraged, since hepatic granulomas are nonspecific.27 The guiding principle should be to biopsy a site with the least morbidity. In the absence of skin lesions, thoracic disease is usually sampled. Conjunctival biopsies are also reasonable in individuals with conjunctival follicles or ocular abnormalities consistent with sarcoidosis.218 Few clinical pulmonary presentations may be assumed to be sarcoidosis without tissue confirmation. Even in such clinical Table 5 Rarely sampled clinical presentations of sarcoidosis

Löfgren syndrome Bilateral hilar lymphadenopathy on chest X-ray Erythema nodosum skin lesions Fever (frequent) Arthralgias/arthritis of the ankle (frequent) Herfort syndrome Uveitis Parotiditis Fever (frequent) Bilateral hilar lymphadenopathy on chest X-ray without symptoms Panda sign Parotid and lacrimal gland uptake on gallium-67 scan Lambda sign Bilateral hilar and right paratracheal lymph node uptake on gallium-67 scan

scenarios infectious etiologies, e.g. coccidiodomycosis mimicking Löfgren syndrome, must be excluded (Table 5).219,220 Given the relatively recent appearance of acquired immunodeficiency syndrome (AIDS), therapeutic use of TNF antagonists and the ability to sample mediastinal lymph nodes through minimally invasive biopsy procedures rather than mediastinoscopy, one could persuasively argue that all patients suspected of having sarcoidosis should have tissue sampling. In addition to morphological findings, the procured tissue can be examined for infectious agents. The latter consideration is not trivial, since over 10% of histochemical stain-negative transbronchial lung biopsy (TBLB) cases of suspected sarcoidosis have positive microbiological cultures.190 As sarcoidosis features an exquisite lymphangitic distribution, flexible fiberoptic bronchoscopy with transbronchial and/or endobronchial biopsy can easily sample affected bronchovascular bundles. Historical studies demonstrate transbronchial lung biopsy (TBLB) sensitivity rates from 50 to 90%.183,221–223 The higher diagnostic rates are reported in patients with radiographic stage II or III disease, yet virtual bronchoscopy-guided TBLB may improve the diagnostic yield in all stages of disease.222,224 Four to six tissue samples usually suffice but up to ten may be required to make a diagnosis of non-necrotizing granulomas in radiographic Stage I disease (Figure 31).222,225 Since endobronchial involvement is common in the disease, endobronchial biopsies performed alongside TBLB can increase the diagnostic yield (Figure 32).226

Modified from Judson.261

Figure 31. Transbronchial biopsy, consistent with sarcoidosis. The lymphangitic distribution of the disease lends itself to transbronchial sampling. While up to six tissue samples may be needed to find a single granuloma, sometimes a single biopsy contains many non-necrotizing granulomas.

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Figure 32. Endobronchial biopsy, consistent with sarcoidosis. Endobronchial biopsies can complement transbronchial samples owing to the wealth of bronchial wall lymphatic channels. This sample features submucosal granulomas surfaced with metaplastic respiratory epithelium. Such microscopic nodularity corresponds to gross findings (see Figures 6 and 7).

Chapter 13: Sarcoidosis

Until recently, sampling hilar and mediastinal lymphadenopathy required mediastinoscopy. Although this undertaking yields “gold standard” tissue, newer procedures greatly lessen the need for a surgical intervention. When mediastinal or hilar lymphadenopathy is present on chest CT, transbronchial needle aspiration (TBNA) biopsies with Wang 18, 19 or 22 gauge cytology needles demonstrate findings consistent with sarcoidosis in 63 to 90% of patients.227–233 Furthermore, combining TBLB and TBNA increases the yield in radiographic stage I and II patients from 66 to 83% and 76 to 86%, respectively.229,234–236 Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a recently touted adjuvant and even alternative to CT-guided TBNA and TBLB in individuals with a high clinical suspicion of having sarcoidosis.209,237–239 The technology visualizes paratracheal, subcarinal and hilar lymph nodes and allows for minimally invasive and safe lymph node sampling. Although diagnostic rates greater than 90% are reported, results from all radiographic stages of sarcoidosis are not known and published lower diagnostic yields highlight the procedure’s dependence on the bronchoscopist’s skills and need for on-site cytology screening.209 Combining EBUS-TBNA with TBLB or endobronchial biopsies probably obviates mediastinoscopy but any talk of replacing TBLB with EBUS-TBNA is premature.209,235–237,240–244 Although BAL is performed at most bronchoscopy procedures, and the general cytomorphological features for sarcoidosis have been described, this test is not diagnostically accurate. While a lymphocytic alveolitis is noted in 90% of sarcoidosis patients at the time of diagnosis, BAL lymphocyte counts and elevated CD4/CD8 ratios do not independently distinguish this disease from other processes, most notably chronic hypersensitivity pneumonitis.245 However, combining suggestive BAL with TBLB and TBNA findings, diagnostic sensitivity can reach 100%.246 Lavage samples also provide material for microbiology studies.

Laboratory findings Laboratory studies are nonspecific in patients with sarcoidosis. Anemia, leukopenia, lymphocytopenia, hypergammaglobulinemia, elevation of liver enzymes, hypercalcemia and hypercalcuria can be seen. Hypercalcemia, for example, is reported in less than 10% of sarcoidal patients and hypercalcuria occurs in about 30%.247 The former is probably due to macrophage overproduction of the active form of vitamin D3, leading to increased calcium resorption.248 Angiotensin-converting enzyme (ACE) is normally produced by pulmonary capillary endothelial cells, but activated macrophages in sarcoidal granulomas also produce the enzyme. Serum levels (SACE) are elevated in 30 to 90% of patients.249–251 Elevated levels are thought to be a marker of total granuloma burden and positive and negative predicted values of only 84% and 74%, respectively, have been reported.252 Levels are influenced by ACE gene

polymorphisms and elevations are seen in other diseases, including but not limited to mycobacterial and fungal infections, berylliosis and Hodgkin lymphoma. Therefore the test should not be used for diagnosis and probably not as a surrogate for disease activity.253–257 The Kveim-Siltzbach test is no longer a standard diagnostic test but retains historical and investigation interest. In 1941 Morten Ansgar Kveim reported the test, using lymph node tissue from sarcoidosis patients, but Louis Siltzbach popularized a modified form using splenic tissue.10,11 Intradermal inoculation with sarcoidal splenic tissue produces a skin nodule composed of non-necrotizing granulomas in 4–6 weeks in approximately 50% of patients with sarcoidosis.11,258 Though highly specific, this insensitive test is dependent on the particular splenic suspension.259,260 Since the antigen is unknown, a commercially available form does not exist for this human extract. As the test is not approved by the United States Food and Drug Administration, this study is not clinically useful.261 However, preparations may yield clues regarding the inciting antigen.262–264

Immunopathogenesis and clinical associations Clinical symptoms and organ dysfunction in sarcoidosis are attributable to granuloma formation and fibrosis. The immunobiological development of the sarcoidal granuloma is fairly well elucidated and does not reflect an autoimmune phenomenon. Alveolar macrophages and lymphocytes are the main players. The local T-cell immune response is peculiar, in that it features an oligoclonal pattern, biased in expression of genes for the a- and b-chain variable region of the T cell receptor.265,266 The process begins with the digestion and processing of the unknown insoluble or poorly soluble antigen(s) by alveolar macrophages. The antigen is then presented to CD4þ T-cells, through the classic MHC-antigen complex.267 An increased density of class II MHC molecules and other molecules on the alveolar macrophages has been noted and may indicate a genetic predisposition to granuloma formation.268–271 Many cytokines, especially IFN-g and interleukin-2 (IL-2), are secreted by the macrophages and T-cells, and constitute the “T-helper 1 immune response (Th1)”.267,272–274 Cytokines, chemokines and other substances, such as serum amyloid A, stimulate the macrophages to differentiate into epithelioid cells. This endows them with secretory functions, at the expense of phagocytic functions, and perhaps fosters the ability to fuse with other epithelioid cells and form giant cells.275–278 These factors form and maintain the granulomas and also modulate inflammatory responses through autocrine and paracrine loops. For example, IL-12, secreted by macrophages, upregulates the development of Th1 cells and amplifies IFN-g secretion.279 Interleukin-18 acts synergistically with IL-12 to enhance T-cell cytotoxicity. IL-18 is postulated as a central factor for macrophage activation, interferon-g production and also granuloma formation.279,280

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Factors responsible for downregulation of the lymphocytic alveolitis and granulomas and/or development of parenchymal fibrosis are not well understood but T-helper 2 immune response (Th2) may facilitate fibrosis.281 High levels of Th2 cytokines IL-4 and IL-13, known to contribute to extracellular matrix deposition, and fibroblast stimulating cytokine CCL18 may be central actors.282,283 Increases in IL-8 have also been associated with transforming-growth-factor-b, a cytokine known to promote parenchymal fibrosis,284 while activated lymphocytes, macrophages, neutrophils, eosinophils and mast cells can all contribute to fibrosis. Additional cytokines including IFN-g, IL-1, IL-6, fibronectin, alveolar macrophagederived growth factor and platelet-derived growth factor, as well as oxygen radicals and matrix metalloproteinases, probably also contribute to pulmonary injury and fibrosis.285–287 Additional abnormalities, some paradoxical, in cell-mediated and humoral immunity are noted in sarcoidosis patients.288 Cutaneous anergy is common despite the brisk Th1 immune response. Possible explanations include peripheral T-cell lymphopenia and/or expansion of CD25bright cells. This subset of regulatory CD4þ cells inhibits IL-2 production and T-cell proliferation.289,290 Peripheral blood studies reveal a reverse pattern of T-cell subtypes, namely increased numbers of CD8þ suppressor-cytotoxic T-cells and decreased numbers of CD4þ helper-inducer T-cells.267,291 Blood dendritic cell function is also diminished.292,293 B-cell hyperactivity, including an increase in polyclonal immunoglobulins (hypergammaglobulinemia) and circulating immune complexes, is most likely an epiphenomenon secondary to B-cell activation, but an unknown pathogenetic role has not been entirely excluded. Peripheral blood monocytes inhibit INF-g after exposure to provocative stimuli.294,295 Remarkably these immunopathogenic findings centered on enhanced Th1 immunity ring true in clinical subsets. Pregnancy appears to lessen disease activity, while clinical exacerbations follow several months after delivery.296,297 This behavior is thought to be at least partially explained by the cytokine profile shift away from Th1 reactivity of sarcoidosis to Th2 reactivity of pregnancy.298,299 Sarcoid-like granulomatous response is a rare complication of human immunodeficiency virus (HIV) infection.300–302 Most cases appear 1–2 months after treatment with highly active antiretroviral therapy (HAART) and are an uncommon manifestation of the immune reconstitution inflammatory syndrome (IRIS).303 The recovery of CD4þ cell function directed toward ubiquitous antigens is probably responsible. Chest radiographs and tissue morphology are similar to non-HIV sarcoidosis and the process probably reflects immune reconstitution with an influx of naïve and IL-2 receptor-positive CD4þ cells.304,305 Treatment of IRIS sarcoid-like reaction with corticosteroids is controversial.300 In other patients with HIV/AIDS, treatment with exogenous IL-2 in hopes of increasing CD4þ T cells may also precipitate sarcoid-like lesions.306,307 Symptoms resolve following IL-2 therapy cessation.

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Sarcoidosis-like reactions are also a rare complication of immunomodulating therapies. Type 1 interferons (IFN-a and IFN-b) are used to treat viral hepatitis, autoimmune diseases, including multiple sclerosis, and malignancies. These include chronic myelogenous leukemia, non-Hodgkin lymphoma, multiple myeloma, Kaposi sarcoma, malignant melanoma and renal cell carcinoma.308–311 Recombinant type I interferons (rIFN) increase IFN-g and IL-2 levels and evoke a Th1-driven process.312 The best-studied cases involve hepatitis C and smaller numbers of hepatitis B virus-infected patients treated with rIFN-a.313,314 Incidence rates range from < 0.5% to 5% and constitutional symptoms, as well as thoracic and dermatological complaints, usually develop within 6 months of antiviral therapy.315 Pulmonary morphology is similar to that observed in de novo sarcoidosis.316 Most cases resolve upon rIFN-a dose reduction or cessation. Corticosteroid use may be required but such therapy increases viral loads.315 Anti-TNFa therapy for rheumatological diseases has also been reported to produce pulmonary sarcoidosis-like processes. Given the role of this cytokine in granuloma formation, it is paradoxical that the very agents used in cases of refractory sarcoidosis (infliximab and etanercept) can also mimic sarcoidosis (see below).317–320 Sarcoidosis-like disease is also associated with common variable immunodeficiency (CVID).321,322 Although CVID is a heterogeneous syndrome, defined by impaired immunoglobulin production, peripheral T-cell dysfunction is noted in a substantial proportion of patients.323,324 Sarcoidosis-like disease develops in up to 10% of patients with CVID and rarely, especially in adults, CVID may follow a diagnosis of sarcoidosis.325 Since childhood sarcoidosis is so rare, CVID should be excluded before settling on a diagnosis of sarcoidosis. Presumed sarcoidosis patients with recurrent sinopulmonary infections and hypogammaglobulinemia, rather than hypergammaglobulinemia, might also have CVID.325 Malignancies and autoimmune processes can also induce sarcoidosis-like granulomatous reactions in the lung. Primary lung, breast, gastric, kidney, uterine, ovarian and testicular cancers, as well as lymphomas, may elicit limited reactions in the lung or regional draining lymph nodes (Figure 33).326–330 Whether sarcoidosis predisposes to malignancy and/or vice versa remains a contentious issue. Associations between multi-organ sarcoidosis and skin cancer, lung cancer, colon cancer, kidney cancer, testicular cancer, uterine cancer, Hodgkin and non-Hodgkin lymphoma and acute myelobastic leukemia have been postulated. However, the coexistence of these processes could be merely coincidental or represent the abovementioned sarcoidosis-like granulomatous reaction, secondary to malignancy or chemotherapy.331–341 Additional associations between sarcoidosis and autoimmune entities, including but not limited to systemic lupus erythematosus, Sjögren syndrome, Hashimoto thyroiditis, celiac disease, primary biliary cirrhosis, inflammatory bowel disease, membranous glomerulonephritis, vitiligo and amyloidosis, are noted.342–350 Diagnosing these processes in the presence of known sarcoidosis can be challenging.

Chapter 13: Sarcoidosis

Figure 33. Sarcoidosis-like granulomas in lung malignancies. Lungs riddled with metastatic carcinoma often have small lymphangitic sarcoidal granulomas. Many cases presumed to reflect the presence of both sarcoidosis and malignancy may simply represent a host immune response to tumor.

Differential diagnosis Sarcoidosis is well recognized as a diagnosis of exclusion at clinical, radiographic and morphological levels. The surgical pathologist is responsible for identifying and characterizing the granulomas and excluding other causes, while the clinician must collect and synthesize all the procured data. In many instances, the morphological differential diagnosis may not include legitimate clinical considerations. The gross and light microscopic differential diagnosis in the lung includes first and foremost infections, then pneumoconiosis, hypersensitivity pneumonitis (HP), foreign-body granulomas, interstitial lung diseases and drug reactions. While comprehensive descriptions and discussions of these topics are found elsewhere in this text, focused comments are offered. Bear in mind that although many differential considerations are easy to discriminate from sarcoidosis on large tissue samples, small biopsies can be very difficult if not impossible to interpret. Granulomatous inflammation in lung, lymph node and pleural samples should be considered infectious until proven otherwise. Mycobacterial and fungal infections often have a lymphangitic distribution. Whether epithelioid granulomas are necrotizing or non-necrotizing is also of little importance since mycobacterial, fungal and even non-mycobacterial infections can manifest with either finding.190,351 Infection with Pneumocystis carinii (jiroveci) can produce a necrotizing granulomatous response, while sometimes a non-necrotizing appearance predominates.352 Granulomatous bacterial infections, including Burkholderia pseudomallei and Brucella suis,

are usually suppurative and diagnosis depends on cultures. Several morphological features should further raise one’s suspicion of infection. Over one-third of infectious granulomas are solitary, while sarcoidal granulomas are rarely so. Neutrophils and/or eosinophils within the necrotic foci or adjacent to the granulomas also favors infectious etiologies over sarcoidosis. In immunocompetent patients, M. avium intracellulare may present as a predominantly non-necrotizing granulomatous pneumonitis; however, unlike in sarcoidosis, many of these granulomas are found in airspaces rather than in the interstitium and organizing pneumonia is obvious.353 Ziehl-Neelsen and GMS histochemical stains and perhaps fluorochrome stains, such as auramine-rhodamine, should be performed on at least two tissue blocks and correlated with microbiological studies.190,354 Unfortunately, non-necrotizing and/or hyalinized infectious granulomas usually fail to demonstrate organisms on special stains, especially in small biopsies. As mentioned above, slightly more than 10% of special stain-negative transbronchial biopsies demonstrating epithelioid granulomas had either mycobacteria or fungi isolated from cultures.190 Fungi, including Coccidioides sp., Blastomyces sp. and Histoplasma sp., are easier to identify on either hematoxylin and eosin-stained or GMS-stained tissue sections, than acid-fast organisms on any type of stained tissue section. Inhalation of inorganic dusts may be impossible to discriminate from sarcoidosis. Chronic berylliosis is the most common of these rarities and is almost always caused by repeated or longstanding environmental exposure to beryllium (see Chapter 14).355–357 Lung and extrapulmonary lesions are identical to those in sarcoidosis, although berylliosis may have less necrosis and/or a denser interstitial infiltrate.129,358–361 The correct diagnosis relies upon an occupational history, tissue analysis and/or a beryllium lymphocyte transformation test.357,362 Other pneumoconioses histologically indistinguishable from sarcoidosis are caused by zirconium, aluminum, titanium, barium, gold, copper, rare earth metals (lanthanides) and glass fibers, such as fiberglass and rock wool.363–367 In some instances HP may be difficult to separate from sarcoidosis (see Chapter 12). Although the classic triad of chronic interstitial inflammation, patchy bronchiolitis and scattered ill-defined non-necrotizing granulomas is unmistakable, histological overlap can be seen.368 Hypersensitivity pneumonitis granulomas are small, ill-defined and usually composed of several macrophages and occasional giant cells. Sarcoidal granulomas tend to be compact and well-formed with many epithelioid cells. Granulomas in HP are found adjacent to bronchi and bronchioles, as well as in the interstitium, but lack the complete lymphangitic distribution of those in sarcoidosis. Interstitial lymphoplasmacytic inflammation is also brisker in HP and is usually accompanied by organizing pneumonia. Bronchiolitis obliterans is common in HP yet granulomatous bronchiolitis is prominent in sarcoidosis. Longstanding sarcoidosis also leads to more interstitial fibrosis and granulomatous end-stage change than one usually sees in chronic HP.

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Foreign-body granulomas may involve the lung via intravenous injection or aspiration. Intravenous drug abusers injecting talc along with inert fillers, such as cornstarch, crospovidone and microcrystalline cellulose, develop giant cell reactions to birefringent yellow-pale green material in the walls of muscular pulmonary arteries, perivascular tissue and alveolar septa (see Chapter 16).369–371 In distinction to sarcoidosis, talc and filler granulomas do not demonstrate a diffuse lymphangitic pattern, and are neither compact nor contain epithelioid cells. The plate-like frontal and thin in cross-section talc inclusions are unlike the block-like calcium oxalate crystals of sarcoidosis. Aspiration pneumonia enters the clinical differential diagnosis for sarcoidosis but is not likely to be mistaken for sarcoidosis by a surgical pathologist. The process manifests as a necrotizing bronchocentric granulomatous process with spillage into surrounding airspaces. Food and chemical aspiration pneumonias may be unilateral in up to one-half of cases, lack the lymphangitic pattern of sarcoidosis while intense acute inflammation and fibrin often obliterate underlying lung architecture. The granulomas are composed of loose aggregates of multinucleated cells and epithelioid cells surrounding foreign material. Aspirated vegetable matter, skeletal muscle and fillers from oral medications may be mildly birefringent, due to cellulose.372 Chronic fibrosing interstitial pneumonias may also cause confusion. The clinico-radiographic features of many of these entities, including IPF, allow for distinction but patients presenting with atypical radiographic disease or longstanding disease may be problematic.373 For example, the lower lobe preference of usual interstitial pneumonia pattern may not be apparent and biopsies may demonstrate end-stage lung without typical sarcoidal granulomas. Similar comments pertain to other fibrosing lung diseases. Drug reactions can morphologically mimic sarcoidosis but this scenario rarely causes clinical confusion. As discussed above, sarcoidosis-like reactions occur in patients treated with INF-a and INF-b while smaller granulomas can be seen with etanercept, leflunomide, mesalamine, sirolimus and methotrexate (see Chapter 16).316,374,375 These instances serve as a reminder that sarcoidosis is not solely a morphological diagnosis.

Natural history, pathophysiology, prognosis and treatment The natural history of sarcoidosis is variable and patients often experience remissions and relapses. Spontaneous remission is common, occurring in nearly two-thirds, but a chronic or progressive course is seen in up to 30% of patients. Almost 20% of patients may have permanent dysfunction. Only 2–8% of patients with spontaneous remissions relapse at a later date.166,169,376 Tapering or cessation of therapy is often followed by relapse. The overall prognosis for those with sarcoidosis is usually good, with mortality rates of 5% or lower,

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Table 6 Sarcoidosis remission rates predicted by chest radiograph

Radiographic stage

Radiographic finding

Predicted remission rate (%)

0

Normal chest X-ray

N/A

I

Bilateral hilar lymphadenopathy (BHL)

55–90

II

BHL þ parenchymal infiltrates

40–70

III

Parenchymal infiltrates

10–20

IV

Fibrosis and bullae formation

0

Data from ATS.15

but is dependent on genetic and epidemiological factors.15 Most notably, African-American patients have higher rates of progressive disease, relapses and worse long-term prognosis.15 In the United States respiratory failure is the most common cause of death, while in Japan cardiac disease heads the list.117 Clinical presentation is associated with prognosis. Patients with Löfgren syndrome usually resolve spontaneously without treatment. Presentations associated with worse outcomes include African-American race, age of onset after 40, lupus pernio, chronic uveitis, chronic hypercalcemia, nephrocalcinosis, splenomegaly, nasal mucosal involvement, cystic bone lesions, neurosarcoidosis, myocardial involvement, progressive or prolonged symptoms for more than 6 months, including chronic respiratory dysfunction, or involvement of more than three organ systems.15,377–379 The radiographic staging of thoracic disease is prognostic with reproducible remission rates (Table 6). A recent study suggests an association between genetic polymorphisms and specific disease phenotypes. Particular polymorphisms in the caspase recruitment domain (CARD) 15 and the C-C chemokine receptor (CCR) predict radiographic stage IV disease.380 Adverse pulmonary findings aside from progressive fibrosis include pulmonary hypertension, bronchostenosis, mycetomas and pleural disease. Although pulmonary vascular involvement is noted in almost all cases of sarcoidosis, pulmonary hypertension (mean pulmonary arterial pressure > 25 mmHg at rest or 30 mmHg with exercise) probably afflicts no more than 5% of patients. Most have advanced fibrosis and three-quarters of sarcoidosis patients listed for transplants suffer pulmonary hypertension.124,381,382 Arterial luminal compression by granulomas is the most likely etiology but destruction of the alveolar capillary bed, venous obstruction and extrinsic compression of pulmonary arteries by hilar adenopathy also contribute.188,383–386 Pulmonary arterial hypertension is a poor prognostic finding, even when accounting for concomitant fibrosis. Pulmonary hypertension is the strongest predictor of mortality in patients awaiting transplantation and right ventricular failure is noted in up to 30% of sarcoidosis deaths.124,378,387

Chapter 13: Sarcoidosis

Airway involvement can also be debilitating. Airway involvement is practically a sine qua non of sarcoidosis and single, multifocal or diffuse narrowing of airways may develop.388 In addition to primary airways involvement, countless patients also have enlarged hilar lymph nodes compressing lumina or parenchymal fibrosis with large airway distortion. These anatomic lesions are responsible for cough, wheezing and stridor.388,389 Atelectasis and right middle lobe syndrome may also develop but clinically significant bronchial stenosis is seen in no more than 26% of patients and is rarely severe.389–393 Corticosteroids may be effective and mechanical dilatations can improve ventilation.388 Bronchiectasis and mycetomas (usually aspergillomas) may develop.394,395 Mortality rates from mycetomas exceeding 50% are probably due to underlying sarcoidosis but invasion into pulmonary vessels can cause fatal hemorrhage.391,396 Thus, surgical treatment is preferred to topical, intracavitary or systemic antifungal therapies or even vascular embolization.394,397–399 High-resolution CT studies identify uni- or bilateral pleural/subpleural nodules and upper lobe pleural thickening in up to 76% and 11% of patients, respectively, and 35% of lung wedge biopsies feature visceral pleural granulomas. Yet clinically significant pleural manifestations of sarcoidosis affect no more than 5% of patients. Since pleural disease is most often seen in patients with advanced disease, it is difficult to wholly ascribe restrictive physiology to pleural fibrosis alone.400–404 Pleural effusions are not uncommon in patients with advanced disease but may rarely be the initial presentation.183,194,401,405 Effusions may be transudative or exudative, and lymphocytosis is noted in two-thirds of cases with a predominance of CD4þ lymphocytes.401,406–409 Effusions are more probably due to increased capillary permeability, rather than pleural space involvement. Massive pleural effusions, effusions causing lung entrapment and chylothorax are quite rare.410–414 Effusions usually resolve spontaneously within 3 months.415 Pneumothorax secondary to bulla(e) rupture or subpleural lung collapse is also seen in advanced disease but may also be a presenting manifestation.416–418 Bilaterality is exceedingly rare.419 Hemothorax has also been reported, even as a presenting finding.420–422 Systemic or rarely topical corticosteroids are the mainstays of sarcoidosis treatment.126 Remarkably, the appropriate dose and duration of corticosteroid therapy has never been evaluated in controlled randomized trials. Indications for systemic corticosteroids are not universal but general guidelines suggest initiation for radiographic stage I patients only with extrapulmonary symptoms and treatment of symptomatic radiographic stage II and III disease after a 6 to 12 month observation period. However, severe symptoms or pulmonary dysfunction require immediate treatment. Patients with radiographic stage IV disease are treated but rarely improve. Treatment continues for at least 1 year with tapering and discontinuation. Up to one-third of patients may relapse within 2 years and require treatment again.15,169 Anecdotal success with second-line cytotoxic drugs, such as methotrexate and azathioprine, antimicrobial agents

including chloroquine, and immunomodulatory therapies, such as TNFa inhibitors and thalidomide, has been reported.423–426 In small series, prostacyclin analogs, endothelin receptor antagonists and phosphodiesterase-5 inhibitors improve hemodynamics, functional status and outcomes in patients with pulmonary hypertension.427 Single or bilateral lung transplantation is a reasonable option for those stricken with fibrotic, late-stage, treatmentrefractory sarcoidosis. Almost 3% of lung transplants are performed for this group of patients and survival rates are similar to those transplanted for other lung diseases.85,378,428,429 However, perioperative mortality for patients with this multisystem disease is higher than that noted for all others.430 Interestingly, sarcoidosis recurs in 47 to 67% of pulmonary allografts and is probably of recipient rather than donor origin.86,431,432 Nevertheless, recurrent disease does not compromise graft function or patient survival.429,433

Sarcoidosis variants Despite many clinical, radiographic and morphological similarities, pathologists and the thoracic literature historically separate nodular sarcoidosis and necrotizing sarcoid granulomatosis (NSG) from classic sarcoidosis. However, these two so-called entities probably represent rare radiographic and morphological variants of sarcoidosis.

Nodular sarcoidosis Nodular sarcoidosis, as alluded to in the radiology and pathology sections above, is probably nothing more than an uncommon presentation of pulmonary sarcoidosis. First reported as a radiographic mimic of metastatic cancer, more than 50 examples have since been published.158,159,171,391,434–443 Nodular sarcoidosis comprises approximately 4% of all sarcoidosis cases and clinical features are not unlike those of classic sarcoidosis. The majority of patients are women and in urban American study populations almost all are African-Americans in their third to fourth decades of life. Interestingly, the majority of patients studied were either active or former smokers.171,174 Almost half of patients also have extrapulmonary disease. Restrictive ventilatory defects are noted.171,174,443 Radiographically, almost all cases of nodular sarcoidosis feature numerous bilateral poorly defined hazy and fluffy lesions, while less than 20% of cases are solitary (Figure 34).391,440,442 An upper lobe predilection is reported and a minority of cases feature cavitation.171 Perhaps these cases represent so-called “cavitary sarcoidosis”.444,445 One cm to 8.1 cm, white to tan, firm, solid lesions tend to be peripheral/pleural-based.171,438 Hilar and/or mediastinal lymphadenopathy is usual while pleural thickening and/or effusions may accompany the lung disease in up to 33% of patients.171 Resections demonstrate well-demarcated, poorly circumscribed, round to oval, firm, gray-white nodules with irregular edges (Figure 35). Histologically the lung parenchyma is replaced by irregular, partially hyalinized, fibrous tissue

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Chapter 13: Sarcoidosis

Figure 34. Nodular sarcoidosis. This chest computed tomogram demonstrates multiple pulmonary masses without necrosis. (Courtesy of A. Soubani, MD, Detroit, Michigan, USA.)

Figure 36. Nodular sarcoidosis. The solitary subpleural poorly circumscribed hyalinized mass lesion has prominent peripheral tentacles. (Glass slide courtesy of J. English, MD, Vancouver, British Columbia, Canada.)

nodules replete with compact non-necrotizing granulomas (Figures 36–38).174 Although the periphery of nodules may have lymphangitic tentacles, typical bronchovascular and lymphangitic granulomas are not seen. Computed tomogram-guided FNAB and core biopsies, as well as TBLB, demonstrate compact “sarcoidal” granulomas and provide material for microbiology studies.437 Excisional biopsies remain the gold standard and the presence of either necrosis or vasculitis should lead one to seriously consider a diagnosis of NSG. Prognosis is usually favorable. Spontaneous or therapyinduced, i.e. corticosteroid-induced, remission is more likely than progression of disease; however, restrictive physiology may persist.126,171,174

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Figure 35. Nodular sarcoidosis. Typical gross pathology demonstrates a subpleural poorly circumscribed mass along with an enlarged peribronchial lymph node. Evidence of classic sarcoidosis is not apparent. (Courtesy of J. English, MD, Vancouver, British Columbia, Canada.)

Figure 37. Nodular sarcoidosis. Lesions feature peripheral non-necrotizing granulomas and central fibrosis. Vessels are often obliterated (arrow).

Necrotizing sarcoid granulomatosis Necrotizing sarcoid granulomatosis (NSG) is an extremely rare entity that shares features with both nodular and classic sarcoidosis. It was initially recognized and characterized as a primary angiitis and granulomatosis.446 Given the descriptive histopathological definition of the process, namely sarcoidosislike granulomas with areas of necrosis and vasculitis, most cases are likely to represent sarcoidosis. Yet the morphological findings alone leave open the possibility that the entities are not so closely related.

Chapter 13: Sarcoidosis

Figure 39. Necrotizing sarcoid granulomatosis. This HRCT window demonstrates bilateral thick-walled cavities along with small scattered nodules and enlarged hilar and mediastinal lymph nodes. (Courtesy of A. Nicholson, MD, London, UK.)

Figure 38. Nodular sarcoidosis. Granulomas are seen along the edges of the nodules and retain their typical compact configurations. Lymphoplasmacytic infiltrates percolate through the hyalinized fibrous tissue.

Not unlike classic sarcoidosis, NSG affects both children and adults.447 Most patients are 40 to 50 years old, while children as young as 8 years of age with the disease have been reported.448–450 Women outnumber men by more than 2:1. The disease has also been noted in a family with several cases of classic sarcoidosis.451 Associations with inflammatory bowel disease, Sjögren syndrome and idiopathic thrombocytopenia suggest an underlying immunological process.447,452,453 Necrotizing sarcoid granulomatosis is usually limited to the thorax and the most common symptoms are cough, dyspnea, chest pain and constitutional complaints.446,454–457 Up to 25% of patients are asymptomatic and extra-pulmonary disease affects less than 15% of patients in most studies. Visual disturbances and blindness due to iritis, uveitis and retinal involvement, primary central nervous system manifestations including headaches, transient hemiplegia and complaints related to diabetes insipidus, as well as skin rashes, are most common.448,450,452,458–464 Cardiac, hepatic, splenic and sinus involvements are rare.465,466 A variety of radiographic patterns have been described. Diffuse bilateral nodules, solitary nodules, nodular infiltrates and irregular infiltrates can be seen (Figure 39).446,454 A miliary pattern may precede the formation of nodules and these infiltrates have a peri-bronchovascular and/or subpleural distribution. Nodules measure from 0.5 to 5.0 cm, while solitary nodules may be the only radiographic manifestation in up to 33% of patients. On HRCT the nodules also have a peribronchoarterial and subpleural distribution.467 Observed nodules may resolve, increase in size or cavitate. Cavitation is

seen in up to 25% of cases and may also represent “cavitary sarcoidosis” in the clinical literature.444,445 Pleural effusions or thickening are not infrequent.468 Bilateral hilar and/or mediastinal lymphadenopathy is not nearly as common as in classic sarcoidosis, with only 7% to 65% of patients demonstrating this radiographic finding.446,454–456,467–469 Rare macroscopic descriptions of lesions reveal white-tan, semi-firm nodules with irregular outlines (Figure 40).467 These nodules are usually larger than those seen in classic sarcoidosis. Smaller satellite lesions may be seen. Morphologically there are three defining criteria for NSG. Granulomatous pneumonitis composed of sarcoidosis-like granulomas, variable amounts of necrosis, and granulomatous vasculitis must be identified. These discrete but loose clusters of epithelioid histiocytes, giant cells and lymphocytes form round and irregular nodules (Figure 41). The granulomas have a lymphangitic distribution and not unlike classic sarcoidosis coalesce (Figure 42). Submucosal airway involvement and an intra-alveolar component may be seen, yet visceral pleural involvement is rare.450,454,466,468 Hyalinized granulomas may also be seen. If these were the only findings, then one would consider a diagnosis of nodular sarcoidosis. However, parenchymal necrosis distinguishes NSG from nodular sarcoidosis. Small foci of fibrinoid-type necrosis within the center of the compact granulomas suffice but most of the time one finds impressive geographic and infarct-like zones of necrosis (Figures 43 and 44). The vasculitis involves muscular pulmonary arteries and veins. Three different types of vasculitis may be seen (in order of frequency); necrotizing granulomatous, mononuclear cell, or giant cell arteritis-like (Figures 45–48). The mononuclear cell type features lymphocytes, plasma cells and macrophages, while giant cells may be arranged circumferentially within large artery walls. The vasculitis is usually confined to vessel walls but endovascular involvement, so-called endarteritic

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Chapter 13: Sarcoidosis

Figure 40. Necrotizing sarcoid granulomatosis. A subpleural ill-defined mass with central necrosis and hemorrhage has a few satellite lesions. (Courtesy of J. English, MD, Vancouver, British Columbia, Canada.)

Figure 41. Necrotizing sarcoid granulomatosis. Microscopic lesions represent more of a granulomatous pneumonitis, rather than a lung with granulomas as one sees in both classic and nodular sarcoidosis.

Figure 42. Necrotizing sarcoid granulomatosis. The granulomas may coalesce into a lymphangitic distribution. Bronchovascular disease is more often seen at the periphery of the lesion.

pattern, may be seen (Figures 49 and 50). Of note, systemic vasculitis does not occur. Bronchiolitis obliterans, often granulomatous in nature, along with post-obstructive pneumonia are common secondary findings observed in lung samples with NSG. Enlarged mediastinal lymph nodes feature parenchymal replacement with non-necrotizing granulomas.455,469 Extrapulmonary lesions feature granulomas but may not feature necrosis or vasculitis.464 Suffice it to say, NSG should only be diagnosed on excision specimens. Although TBNA and TBLB may be useful in

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Figure 43. Necrotizing sarcoid granulomatosis. Necrosis may be limited to the center of the lesion.

documenting granulomas and collecting tissue for microbiology studies, the diagnosis of NSG relies on a constellation of morphological findings not seen in small tissue samples. Laboratory findings are not well studied. Individual cases of elevated erythrocyte sedimentation rate, elevated SACE levels

Chapter 13: Sarcoidosis

Figure 45. Necrotizing sarcoid granulomatosis vasculitis. Necrotizing granulomatous vasculitis is the most common type of vasculitis and features destruction of vessel walls by epithelioid histiocytes and giant cells. Necrosis often distorts the vessel wall and luminal involvement may occur.

Figure 44. Necrotizing sarcoid granulomatosis. Necrosis is usually not subtle and may be geographic, as in this example, or even infarct-like. (Glass slide courtesy of J. English, MD, Vancouver, British Columbia, Canada.)

Figure 47. Necrotizing sarcoid granulomatosis vasculitis. Large muscular arteries can be attacked by giant cells and histiocytes.

Figure 46. Necrotizing sarcoid granulomatosis vasculitis. Lymphocytes and plasma cells can infiltrate arteries and veins. The lumen of this vein is severely narrowed by the inflammatory infiltrate.

and hypergammaglobulinemia have been reported.452,455,470,471 Pulmonary function test findings are varied with regard to ventilatory defects and carbon dioxide diffusion capacities.452,468,470 Bronchoalveolar lavage studies from one series

indicated an elevated number of recovered cells but variable and nonspecific findings. In two cases with increased lymphocyte percentages, the CD4þ/CD8þ ratios were 0.9 and 1.4.452 While the pathogenesis of NSG is unknown, the current most commonly held opinion is that it represents a variant of sarcoidosis. This interpretation is based on the overlap in clinical presentations, clinical course and radiographic, morphological and laboratory findings.454,466,469 Characteristic necrosis is thus probably due to vasculitis-induced parenchymal ischemia. Infectious and hypersensitivity processes have also been suggested but evidence is currently lacking.455,470

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Figure 48. Necrotizing sarcoid granulomatosis vasculitis. While confined to the artery wall, this giant cell arteritis type vasculitis will certainly destroy the vessel.

Figure 50. Necrotizing sarcoid granulomatosis vasculitis. The inflammatory infiltrate is confined to the vessel wall but bulges into the lumen. (Courtesy of Y. Rosen, MD, North Bellmore, New York, USA.)

The differential diagnosis of NSG, not unlike classic sarcoidosis, includes first and foremost infectious processes. Negative GMS and ZN histochemical stains, as well as microbiology cultures for fungi and acid-fast organisms, are

498

Figure 49. Necrotizing sarcoid granulomatosis vasculitis. This vein is overrun by granulomatous inflammation. The luminal diameter is markedly compressed.

absolutely necessary before diagnosing this rare entity.472 This point is stressed in one of the larger NSG series, where cases with “textbook” morphology were excluded on the basis of culture results.454 Recall that secondary vasculitis from infectious processes is the commonest cause of pulmonary vasculitis. Wegener granulomatosis, one of the original pulmonary angiitis and granulomatosis processes, can be distinguished from NSG on many grounds. Wegener granulomatosis is almost always a systemic disease with upper respiratory tract and renal manifestations and patients almost always have elevated antineutrophil cytoplasmic antibodies (ANCA) (see Chapter 19). While the two processes demonstrate vasculitis and necrosis, there are morphological differences. Wegener granulomatosis but not NSG features neutrophilic microabscesses and neutrophilic capillaritis, while compact non-necrotizing granulomas are seen in NSG but not in WG. The natural history of NSG appears to be indolent in most cases.446,450,452,454–456,468 Death attributable to pulmonary disease has not been reported. Lesions may disappear spontaneously, may be resected, or treated with corticosteroids. Up to 25% of patients relapse and require either additional corticosteroids or second-line therapies.446,455 Rarely disease relapses at extrapulmonary sites.452

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14

Occupational lung disease Thomas Sporn and Victor L. Roggli

Introduction Occupational lung disease is the most significant form of work-related illness in the United States in terms of its severity, frequency and cost to society.1 The US Department of Labor reported the occurrence of some 4.1 million workplace injuries and illnesses in 2006, including 17 700 respiratory ailments in private industry alone and an incidence of non-fatal occupational respiratory illness of 1.9 cases per 10 000 full-time workers.2 Occupational lung diseases result in one of the most significant causes of lost work productivity, with the highest rate of days away from work due to respiratory illness sustained by the mining industry.3 Occupational lung diseases are the third most prevalent (246 per 100 000 population) in the European Union, also with the highest proportion found in the mining industry. Technological advances in construction have led to new groups of at-risk workers in addition to the traditional occupations in mining and quarry work.4 Global estimates of disability and disease resultant from occupational exposure to airborne particulates also include 386 000 deaths from pneumoconiosis, asthma and other chronic obstructive lung diseases.5 The toll that occupational lung diseases exact upon society is reflected in estimated direct and indirect costs that number in the billions of dollars.6,7 Occupational lung diseases cause significant morbidity, which usually lacks curative medical intervention at the time of presentation, apart from jobsite or occupation modification. Stringent oversight of workplace conditions and permissible exposures on the part of governments and regulatory agencies, along with the retention of occupational health physicians on the part of larger firms, will hopefully mitigate the development of severe disease in the future. Occupational lung diseases are generally referable to a specific occupation or exposure to hazardous workplace materials, such as asbestos, coal, silica and other mineral and metal dusts, as well as gases and fumes. These workplace exposures may exacerbate underlying respiratory illnesses, such as asthma and other chronic obstructive lung diseases.

In addition they may directly cause a spectrum of neoplastic and non-neoplastic lung diseases. The pathological response to any given agent results from the intensity and duration of exposure, latency period following exposure, the quantity, size and physicochemical properties of the inhaled substance, the route and efficiency of any such clearance from the respiratory tract, and host responses and interaction between other environmental pollutants, such as cigarette smoke (Table 1). Varying pathological processes may occur in the lung as a response to inhalation of fibers, dusts and fumes, ranging from the minimal (e.g., macules, responses to nuisance dusts) to extensive late-stage fibrotic changes of the lung parenchyma (e.g., progressive massive fibrosis (PMF)), as well as diffuse acute lung injury, airways inflammation and carcinogenesis. This chapter will present a classification of occupational diseases, based on the particular etiological agent and occupational means of exposure. Malignant pleural mesothelioma, significantly associated with occupational (as well as paraoccupational) exposure to asbestos, and hypersensitivity pneumonia (extrinsic allergic alveolitis), which may result from the inhalation of organic antigens in the occupational setting, are discussed elsewhere in this text (see Chapters 12 and 36).

Asbestos Introduction and occupational risk

Derived from the Greek adjective “inextinguishable”, asbestos is a commercial rather than a mineralogical term for a group of naturally occurring fibrous hydrated silicate minerals. The physicochemical properties of high heat resistance, tensile strength, flexibility and resistance to chemical degradation shared by these minerals have allowed for much utility in the industrial setting, dating to antiquity. Asbestos is a regulatory term for a group of fibrous minerals belonging to the amphibole (actinolite, amosite, anthophyllite, crocidolite, tremolite) and serpentine (chrysotile) groups (Table 2a). This separation is based on physicochemical characteristics, which in turn are of significant pathophysiological importance.

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 14: Occupational lung disease Table 1 Pathological reactions in typical metal and mineral dust pneumoconiosis

Compartment of lung involved

Agent(s)

Examples of reactions

Airways

Aluminum, metal oxides

Asthma, bronchiolitis, metal fume fever

Parenchyma – interstitial and peribronchial

Silica, silicates, coal, asbestos, hematite

Micro/ macronodules, interstitial fibrosis, PMF

Interstitial – diffuse

Beryllium, talc, hard metal, nylon flock, welding fumes, silicates

Interstitial pneumonitis, granulomatous inflammation, interstitial dust accumulation

Alveoli

Aluminum, silica, beryllium, mercury, cadmium

Alveolar proteinosis, diffuse alveolar damage, desquamative interstitial pneumonia

Parenchyma – focal

Asbestos, cadmium, nickel, chromium

Carcinoma

Pleura

Asbestos, asbestiform silicates (erionite, zeolites, Libby amphibole)

Pleural effusions, plaque and diffuse pleural fibrosis, mesothelioma

PMF, progressive massive fibrosis.

Table 2a Types of asbestos

once ingested, prone to chemical degradation. Among the amphiboles, only crocidolite (“blue asbestos”) and amosite (“brown asbestos”) have been exploited commercially, although tremolite has been mined in open pits in western India.8 The non-commercial forms (actinolite, anthophyllite and tremolite) are important only with regard to their appearance as contaminants of other forms of asbestos, such as chrysotile ore, or non-asbestos minerals, such as talc and vermiculite.9 Despite the ubiquity of asbestos and its numerous applications in industrial settings, most clinically significant exposures to the mineral occur in relatively select groups and occupations. Genetic polymorphisms in oxidative metabolism and DNA repair processes suggest genetic susceptibilities for asbestos-related diseases, including neoplasms.10 Occupations exposed to asbestos with a risk of asbestosis are summarized in Table 3. Most notably, before the health risk of asbestos was well established, asbestos was a common component of commercial insulation materials in both the civilian and naval shipbuilding setting. Occupations provided exposure to asbestos, not only through direct handling of insulation materials, but also to bystander workers in trades, such as electricians, and later to those involved in maintenance of buildings containing asbestos insulation or in the removal of the mineral. In the United States, a minority of those sustaining occupational exposure were engaged in the mining or milling of asbestos, or in the manufacture of asbestos products.11 Potential for exposure to asbestos as sustained by garage mechanics through the course of performing vehicular brake and clutch repair has been closely scrutinized (see Chapter 36). Non-occupational exposures to asbestos may also cause significant lung disease. At-risk cohorts have included the household contacts of asbestos workers and those with environmental exposure to crocidolite mines and mine tailings.12

Background levels

Non-occupational risk and reference population levels

Unlike chrysotile, the amphiboles are chain silicates complexed with cations, including magnesium, calcium, sodium and iron (Table 2b). These minerals may have non-fibrous counterparts of identical chemical formulae, which are nonpathogenic. Chryostile is a fragile and curled magnesium sheet silicate, readily fragmented and cleared by macrophages, and

As measured by sensitive analytical techniques, some level of asbestos can be identified in virtually everyone’s lungs.13,14 The determination of asbestos content by different laboratories examining samples from the same patient can differ considerably, since there are important differences in methodology between laboratories.15 Epidemiological studies show asbestos-related diseases may develop in non-occupational settings.12,16,17 For all these reasons, it is of critical importance that laboratories performing such analyses of lung asbestos content determine a range of values from background exposures in an appropriate reference population. In this context, background refers to tissue asbestos levels resulting from living and working in an industrialized society in the absence of significant occupational, paraoccupational or environmental exposures. The background level of asbestos found in Western populations, where the mineral has been banned, is falling year

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Chapter 14: Occupational lung disease Table 2b Chemical components of different asbestos types

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Chapter 14: Occupational lung disease Table 3 Industries and occupations with potential for exposure to asbestos and asbestos-containing materials

Mining and milling of asbestos Asbestos textiles industry Ship building Insulators Pipefitters Welders Oil and chemical refinery workers Power plant workers Automotive/friction products Paper mill workers Sheet metal workers Construction workers Railroad workers Molten metal workers Service in the navy/merchant marine Machinist Boiler workers Para-occupational (household) exposure Environmental (dwelling near asbestos factories/dumps)

by year. Ideally pathologists should take control samples at post-mortem from patients with good occupational histories. This is not possible in most cases and is also often against the Human Tissue Act. Many pathologists in the UK avoid taking tissue at post-mortem, often at the behest of the Coroner. Asbestos exposures derived from environmental sources occur either from weathering of naturally occurring asbestos deposits,18 from tailings resulting from processing of asbestos containing ores,19 from living or working in a building with asbestos-containing materials (building occupant),20 or living in the vicinity of an asbestos mine or asbestos manufacturing plant.12,16,17 Such contacts are typically low-level exposures, and the greatest risk is the development of pleural plaques and even mesothelioma. Some cases of asbestosis may result from such exposures, although these are exceptional.18,19 Pan et al. reported an increased risk of mesothelioma in association with residential proximity to naturally occurring asbestos in California.21 Kurumatani and Kumagai mapped cases of mesothelioma in the vicinity of a large asbestos cement pipe plant in Japan and found an elevated standard mortality ratio up to 2.2 km from the plant and skewed in the direction of prevailing winds.22 To date, lung fiber burden studies have not been reported in any of these cases. In this regard, in a study of 1445 mesotheliomas, Roggli et al. found no cases from the United

States attributable to neighborhood exposures and three cases believed to be due to exposures as a building occupant.23 However, Howel et al. report high concentrations of asbestos fibers and in particular chrysotile, in mesothelioma patients in the Yorkshire area of the UK, where their only known exposure was living near an industrial source.24 Paraoccupational exposures can occur as a result of living in the household of an individual who is occupationally exposed to asbestos. Such exposures, also referred to as domestic exposures, are secondary to asbestos brought home on the worker’s clothing.25,26 They can result in asbestos-related diseases, particularly mesothelioma but also asbestosis and asbestos-related lung cancer in some cases.20,27–30 The reported cases occurred primarily in women, who traditionally dusted down and laundered the clothes of the exposed worker. In a study of 62 cases of mesothelioma in women, more than half were associated with exposures as a household contact.31 The lung fiber burdens in these cases are often equivalent to those observed among workers with light or moderate occupational exposures.20,23,29 The main fiber type in the studies by Roggli et al. was amosite,23,32 whereas Gibbs et al. reported elevated levels of amosite and/or crocidolite.29 In all three studies, some cases claiming exposure as a household contact had lung asbestos fiber counts indistinguishable from a reference or background population. One measure of asbestos content is the concentration of asbestos bodies per gram of wet or dry lung tissue. Asbestos bodies are readily detected with bright field light microscopy, and the agreement among different laboratories analyzing the same lung tissue samples is quite good.15 Asbestos bodies are found in about 90% of lung samples from members of the general population (i.e., non-occupationally exposed individuals).32 Smith and Naylor found fewer than 100 asbestos bodies per gram of wet lung tissue among members of the general population in the Chicago area.13 Roggli and colleagues found 0–20 asbestos bodies per gram of wet lung from non-exposed individuals in the Houston area and in Durham, North Carolina, USA.14,33 Similar ranges were reported by Dodson in eastern Texas and by Hammar in the Pacific Northwest.34 Hence there appears to be reasonable agreement among several laboratories and from several different regions in the United States, regarding the background or reference level for asbestos bodies. These asbestos bodies are formed predominantly on amphibole fibers (amosite in men and anthophyllite, actinolite or tremolite in women).35 Several investigators have attempted to define the range of lung fiber content present in a control or reference population (Table 4).27,30,36–39 The quantitative results vary from laboratory to laboratory, commensurate with differing methodology, including sample preparation, counting rules, and the detection system used.15 These observations underscore the necessity for such laboratories to determine their own reference ranges using their particular methodology. Our control cases were selected on the basis of having macroscopically normal lungs at autopsy and an asbestos body count within our

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Chapter 14: Occupational lung disease Table 4 Asbestos content of lung tissue in reference or control populations

Source

No. of cases

Method

Uncoated fibers/g dried lung

Whitwell et al.27

100

PCLM

0.007 (0–0.521)

Mowe et al.36

28

SEM

0.25 (0–4.8)

Gaudichet et al.37

20

TEM

11.2

Churg and Warnock35

20

TEM

1.29a (0.260–7.55)

23

TEM

0.62

20

SEM

0.031a (0.004–0.169)

Case et al.39 30

Roggli et al.

Values reported are the median counts for millions (106) of uncoated fibers per gram of dried lung tissue, with ranges indicated in parentheses, except for the study by Gaudichet et al.,37 where only the mean value for total fibers per g dried lung could be obtained from the data presented. a Values multiplied by a factor of 10 (approximate ratio of wet to dry lung weight) for purposes of comparison. PCLM, phase contrast light microscopy; SEM, scanning electron microscopy; TEM, transmission electron microscopy. Modified from reference 18.

previously determined normal range.40 There is at present no scientific evidence that background levels of asbestos exposure cause disease.

Regulatory activity and exposure standards Increased recognition of the health hazards posed by both occupational and non-occupational exposure to asbestos has spawned significant regulation of its usage. This has been followed by its diminution or elimination in industrial and consumer products. Outright bans on the importation and use of asbestos exist within the European Union, Japan, Australia and New Zealand. In the United States, the Environmental Protection Agency’s (EPA) recommendation to phase out and ban its usage was ultimately overturned. The Occupational Safety and Health Administration (OSHA) currently regulates workplace exposure to asbestos, and sets a permissible exposure level (PEL), an 8-hour time-weighted average for a 40-hour week work shift, based on counting fibers greater than 5 mm using phase microscopy.7 As concerns over the hazards posed by asbestos exposure and improvements in technology have only increased over time, there has been a concomitant reduction in the PEL. The first permanent standards, set in 1972, permitted 5 fibers/cm3. Successive reductions have led to the current PEL of 0.1 fiber/cm3. In the USA, all fiber types of asbestos are regulated to this level. By contrast, in 2005 the PEL, set by regulatory authorities in India, was 1 fiber/cm3 for asbestos. However, in some mills in that country, chrysotile and tremolite levels have been reported to be as high as 200–400 fiber/cm3 and 20–25 fiber/cm3, respectively.8 In some mill workers in Andra Pradesh, an asbestosis prevalence of 11.5% has been reported.8

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Deposition and clearance of asbestos fibers Inhaled particles deposit in the respiratory tract as a function of individual particle size, shape and a physical characteristic known as the aerodynamic equivalent diameter. The respiratory system is well-guarded against the deposition of inhaled particulate matter (see Chapters 1 and 4). The vibrissae in the nasal cavity filter particles of aerodynamic equivalent diameter, whereas particles that have the greatest probability of deposition and retention in the gas-exchanging compartments of the lung tend to measure between 1 and 5 mm. Fibrous dusts, such as asbestos, differ from compact or spherical particulates. This is because fiber diameter, rather than length, is the main determinant of penetrability into the alveoli.41–44 Accordingly, most fibers deposited in human lungs are 1 mm or less in diameter, but may exceed 200 mm in length. The difference in relative fiber characteristics confers significant differences in the fates of aerosols containing amphibole asbestos and chrysotile. The curled morphology of the long fiber chrysotile causes its deposition in the upper respiratory tract and subsequent mucociliary clearance. Long amphibole fibers tend to be straight, and therefore are more likely to penetrate into the gas-exchanging areas of the lung. Similarly, the physicochemical differences between the amphiboles and chrysotile confer differences in fiber retention. Chrysotile fibers that are deposited into the deeper lung are more likely to undergo fragmentation and subsequent macrophage clearance, or chemical degradation. The long, straight fibers of the amphiboles do not tend to fragment, nor do they readily undergo chemical degradation. It is estimated the effective clearance half-time of chrysotile is measured in months, in contrast to a clearance half-time for amphibole asbestos measured in years to decades.45 Animal models indicate that long fiber asbestos is significantly more potent, both oncogenically and fibrogenically, than short fibers.46–47 These animal studies, the marked differences in fiber characteristics and how these influence deposition, clearance and induction of disease have been proposed as the main pathological basis for the difference between chrysotile and the amphiboles in terms of the relative potential for inducing fibrotic and neoplastic disease in humans. Asbestos fibers may reach the parietal pleura and lymph nodes, via the lymphatics.48,49

Asbestos bodies and non-asbestos ferruginous bodies In cytology preparations or within tissue sections, the identification of the characteristically golden-brown, beaded or segmented asbestos body alerts the pathologist that the patient has sustained significant exposure to the mineral (Figures 1 and 2). The asbestos body is the sine qua non for the diagnosis of asbestosis. Originally believed to be pigmented crystals of uncertain significance, these structures were first termed “asbestosis bodies” in 1929.50 As it was later known that these

Chapter 14: Occupational lung disease

Figure 1. Asbestos bodies in sputum. Papanicolau stain.

Figure 2. Asbestos bodies partially engulfed by alveolar macrophages in the pulmonary interstitium.

Figure 3. A Perls’ stain highlights the interstitial asbestos body. Barbell-like clumps of iron are typical.

bodies were also found in lungs of workers who did not have asbestosis, the term asbestos body became preferred.51,52 Asbestos bodies form following fiber inhalation and deposition in peripheral lung. Macrophages phagocytose the fibers and coat them with iron and mucoglycoprotein. This coating process neutralizes cytotoxicity, fibrogenicity and diminishes the core fiber’s participation in the generation of reactive oxygen species.53–55 The coating imparts the segmented, beaded or dumbbell appearance of the asbestos body and the iron component allows strongly positive histochemical staining using Perls’ stain (Figure 3). Asbestos bodies generally measure 20 to 50 mm in length, but may be much longer, up to 500 mm. Asbestos body width is usually of the order of 2–5 mm.55 Only a small percentage of asbestos fibers in the lung at any given time are coated and there are several factors that determine whether fiber coating will occur. In addition to the macrophages of any individual patient’s intrinsic ability to coat fibers, fiber dimensions are important determinants in this regard.57 Fibers less than 20 mm in length rarely become coated, while almost all fibers greater than 80 mm are coated. Thicker fibers and those with surface irregularities are

Figure 4. A tangle of chrysotile asbestos bodies (scanning electron microscopic image).

also more likely to become coated.58,59 The proportion of coated fibers greater than 5 mm rises as a function of overall increased tissue fiber burden. The presence of other dusts in the lung probably also influences the coating process.32 The type of asbestos fiber itself also plays a role with regard to coating. Most asbestos bodies in human lung tissue contain an amphibole core. In men from the population at large and asbestos workers, this is likely to be amosite or crocidolite. In women from the general population, the fibers are more likely to have a non-commercial form (tremolite or anthophyllite). The presence of tremolite possibly reflects the contamination of talc used in cosmetics.60 Chrysotile asbestos bodies are distinctly uncommon in tissues, which is at odds with the commercial usage, where it predominates over the amphiboles (Figure 4). Chrysotile asbestos bodies are likely to be found in those exposed to long fiber chrysotile, such as miners and millers of chrysotile, or in asbestos textile workers. The rarity of chrysotile asbestos bodies probably stems from the physicochemical characteristics of the mineral

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Chapter 14: Occupational lung disease

Figure 5. Asbestos bodies (asterisk) within a lymph node. Note the histiocytic response.

described above, with its tendency for fragmentation into smaller fibrils that generally do not undergo the coating process. Accordingly, asbestos bodies are not indicative of overall tissue chrysotile burden, but correlate well with the burden of fibers 5 mm or greater in the asbestos-exposed population.60,61 Such fibers are also probably commercial amphiboles.60 Despite the presence of asbestos bodies in the lungs of the general population, their detection in tissue sections is indicative of significant exposure to asbestos. Churg has observed that approximately 500 asbestos bodies per gram of wet lung tissue are necessary, before any are detected in tissue sections.62 Asbestos bodies may also be identified in pulmonary lymph nodes (Figure 5), a finding predictive of a high nodal and lung asbestos body burden.32 Regarding the significance of the detection of asbestos bodies within tissue sections, there have been multiple studies addressing the asbestos body and fiber content of the population at large. Publications document the varying types and manners of exposure, which may be correlated with various asbestos-associated diseases. It is apparent that diseases are induced at fiber burdens greater than those observed in control populations. However, different diseases are induced at different mean fiber burdens, which also vary in accordance with fiber type. For example, the lowest burden of amphibole asbestos is seen in the general population, with increasing burdens observed in those with benign pleural disease (plaque), mesothelioma and asbestosis. For those exposed to pure chrysotile, mesothelioma is induced at the extreme level of tissue content, similar to those observed in cases of asbestosis, supporting the fact that chrysotile is a much weaker inducer of mesothelioma than the amphiboles.63 Fibrous dusts, other than asbestos, may undergo iron coating and resemble asbestos bodies. These include sheet silicates, carbon fibers, metal oxides, man-made mineral fibers and asbestiform minerals, such as erionite. In some cases, the optical characteristics of the core fiber, as observed at the light microscopic level, can help identify these as non-asbestos ferruginous bodies. For example, the carbon fiber cores observed in the lungs of coal miners are black and may be broad and

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Figure 6. Ferruginized refractory ceramic fiber, scanning electron microscopic image. Inset: energy dispersive analytic spectrum.

plate-like64 and the broad, yellow plates of talc or mica may also be identified. In other cases, ferruginous bodies forming around other silicates, refractory ceramic fibers and fibrous glass may be indistinguishable from true asbestos bodies at the light microscopic level, requiring additional analytic studies for confirmation of chemical composition (Figure 6) (see below).32

Asbestosis Asbestosis is defined as diffuse, bilateral, pulmonary interstitial fibrosis caused by inhalation of asbestos. The incorporation of asbestos-associated pleural processes described above, into term asbestosis, should be avoided. This is because it unnecessarily groups together disorders with different epidemiologies, pathophysiologies, clinical features and outcomes.

Epidemiology Asbestosis generally occurs in individuals sustaining heavy exposure to asbestos over long periods of time. Fiber burden analyses of patients with asbestosis indicate tissue fiber burdens higher than in any other form of asbestos-associated lung disease. Asbestosis may result from exposure to any of the commercial forms of asbestos, as well as the non-commercial amphibole, anthophyllite. The incidence appears to be higher in cigarette smokers than in non-smokers with similar degrees of exposure.65–67 Antao et al. describe the clear association between asbestos consumption and deaths from asbestosis. Per capita consumption of asbestosis in the USA peaked in 1951, but the persistence of the disease illustrates its latency period.68 Between 1970 and 2004, Bang et al. documented 25 413 deaths from asbestosis in the United States, with a maximum age-adjusted death rate of 6.9 per million population. The death rate was nearly 35-fold greater for men than for women.69

Chapter 14: Occupational lung disease

The areas of the country with the highest death rates were the coastal regions, with the shipbuilding industry sustaining an expected proportionate mortality ratio. In this series, insulators and boilermakers had the highest asbestosis mortality rates. This is in keeping with the historical observation that occupations leading to heavy exposures caused asbestosis. These occupations typically included spray insulators, asbestos miners and millers, asbestos textile workers and pipefitters. Brief but very intense exposures may suffice to cause asbestosis, as has been reported in insulators and one individual with a 9-month reported exposure to crocidolite in a dusty environment where cigarette filters were manufactured.65 There is believed to be a threshold of asbestos exposure, between 25 and 100 fibers per cm3 years, below which asbestosis is not observed. In this spectrum of exposures, commercial amphibole-induced asbestosis appears at lower dosages, whereas chrysotile-induced asbestosis seems to require the higher exposures.70 This difference probably results from the lesser capacity of chrysotile to induce fibrogenesis in comparison with the amphiboles on a per fiber basis. This is probably a function of the physicochemical differences in the fiber types, discussed above.

Mechanisms and pathogenesis of asbestos-related fibrosis The mechanisms and pathogenesis of asbestos-related fibrosis are well studied. Progression to pulmonary fibrosis following asbestos fiber inhalation entails the development of a protracted cycle of inflammatory cell recruitment, the generation of reactive oxygen species and release of proteases, epithelial injury, apoptosis and fibroblast proliferation.71–75 Upregulation of oncogenes, and macrophage expression of cytokines and growth factors are also implicated. The suggested role for cigarette smoke in the causation and progression of asbestosis probably results from increased fiber retention. This follows enhanced fiber penetration in smokers, reduced pulmonary fiber clearance and changes in alveolar inflammatory cell populations, resulting from smoking, as reported in bronchoalveolar lavage fluid studies.65 Genetic factors are believed to play a significant role in the development of asbestosis.10,76–80 Specifically, polymorphisms in the genes coding for glutathione-S-transferases (GST) have been implicated in the increased risk. Glutathione–Stransferases are critical enzymes, involved in the detoxification and inactivation of reactive oxygen and nitrogen species. These are involved in the inflammatory cascade following the phagocytosis of asbestos fibers by macrophages.81 The GST family contains a number of cytosolic isoenzymes, and polymorphisms in GTSP1, M1 and T1 isoenzymes are associated with an increased risk of the development of asbestosis. Such polymorphisms are relatively common, with approximately 50% of the Caucasian population affected with the null polymorphism of the GSTM1 and GSTT1 genes.78

Figure 7. Chest X-ray of asbestosis demonstrating characteristic lower lung zone reticulonodular opacities.

Clinical features The clinical features of asbestosis are not distinctive. There is dyspnea, dry cough, bibasilar rales, digital clubbing in advanced disease, and restrictive ventilatory defects with reduced diffusion capacity in asbestosis. These features may be also observed in any of the diffuse pulmonary fibrosing pneumonitides.

Radiology The radiological findings in asbestosis are those of lower lung zone reticulonodular infiltrates and small irregular opacities, detectable on plain films (Figure 7). The identification of bilateral pleural thickening and/or plaques heightens suspicion for asbestosis, as these findings are not typically observed in other forms of diffuse pulmonary fibrosis. Pleural changes may be observed in some 80% of cases on plain chest radiographs, which rises to 100% using high-resolution computed tomography (CT) imaging.82–84 Predominantly mid or upper lung zone distribution of infiltrates argues against the diagnosis of asbestosis. The principal radiographic differential diagnosis for asbestosis is usual interstitial pneumonia (idiopathic pulmonary fibrosis, UIP), as both entities feature predilection for the lower lung zones, with subpleural accentuation and progression to honeycomb changes at late stage (see Chapter 10). Computed tomography scanning is more sensitive at detecting the parenchymal changes of asbestosis at an early stage. Such findings may not be evident on plain radiographs, and in the presence or absence of supportive clinical data.82 Other findings on the chest radiographs of patients with asbestosis include “shaggy” cardiac silhouettes and indistinct diaphragmatic contours.82 The radiological findings are

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Chapter 14: Occupational lung disease

crucial to the establishment of an acceptable clinical diagnosis of asbestosis. The International Labor Office (ILO) developed a classification scheme for the radiographic assessment of pneumoconioses based on the size and profusion of radiographically detectable opacities (Table 5). The American Thoracic Society (ATS) proposed, in the appropriate clinical setting, that the ILO category of 1/1 reticulonodular opacities, accompanied by reductions in predicted forced vital capacity and diffusion capacity less than the lower limits of normal, was sufficient for the diagnosis.84 Inexplicably, the more recent ATS document concerning the diagnosis of non-malignant asbestos-related disease reduced the radiological criteria for the diagnosis of asbestosis, thus decreasing specificity at a time when the disease is becoming scarcer.85 Surgical lung biopsy is reserved only for the evaluation of pulmonary fibrosis where an asbestos exposure history is not compelling, and uncertainty exists on a clinical and/or radiographic basis.

Macroscopic pathology The optimal demonstration of fibrosis and coexistent pathological processes requires a well-distended, adequately fixed surgical or autopsy specimen. It is important not to misinterpret visceral pleural fibrosis as asbestosis. The diffuse parenchymal changes requisite for the diagnosis begin as gray streaks of fibrotic tissue at the lung bases and periphery, with sparing of the central lung zones (Figure 8). In autopsy cases, the fibrosis is bilateral with symmetric involvement. One mm to 15 mm honeycomb changes may be grossly apparent in advanced cases.65

Histopathology Acceptable histopathological definitions of asbestosis have been provided by the College of American PathologistsNational Institute for Occupational Safety and Health (CAPNIOSH)87 and by an expert group (in the so-called “Helsinki criteria”)88 whose recommendations are echoed by the ATS (Table 6).86 These definitions were recently updated by the Asbestosis Committee of the College of American Pathologists and the Pulmonary Pathology Society.89 In addition to an acceptable pattern of alveolar septal fibrosis, the histological diagnosis of asbestosis requires the identification of asbestos bodies. These may be found in alveolar spaces, embedded in a fibrotic interstitium or within giant cells. Iron stains should be routinely employed if asbestosis is suspected and asbestos bodies are not observed on routine-stained sections. The examination of multiple sections is recommended, if possible, as asbestos bodies may have an uneven distribution in lung tissue. Churg stated that a single asbestos body in the presence of interstitial fibrosis is a sufficient criterion to establish the diagnosis.70 He noted the improbability of such a finding in the lung tissue of an individual with background exposure. The

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likelihood of detecting an asbestos body in tissue sections in populations with background exposure is of the order of 1%, based on Roggli and Pratt’s calculations.70,90 Such findings are generally demonstrable only in surgical lung or autopsy specimens. However, the CAP and Pulmonary Pathology Society proposed that at least two asbestos bodies should be identified per cm2 of lung tissue examined, in the appropriate light microscopic setting.89 Transbronchial lung biopsy in the diagnosis of asbestosis shares the same limitations as it does in any other form of diffuse pulmonary fibrosis. Asbestos bodies may be found in the bronchoalveolar lavage fluid of 95% of studied populations with asbestosis (Figure 9).91,92 However, the presence of asbestos bodies in such cytological preparations is best considered a marker of asbestos exposure, and, while associated with high tissue levels, is not diagnostic of asbestosis. Similarly, the detection of an asbestos body(s) in the absence of interstitial fibrosis does not fulfill diagnostic criteria.89 The demonstration of fibrosis also requires a well-distended, adequately fixed surgical or autopsy specimen. To repeat, care should be taken not to misinterpret visceral pleural fibrosis as asbestosis. Because of the prevalence of cigarette smoking among asbestos workers, there may be significant parenchymal changes related to exposure to tobacco smoke, typically emphysema. The earliest pathological finding observed microscopically is the presence of increased collagen in the walls of respiratory bronchioles. It is recommended that fibrosis restricted to the bronchiolar walls be termed “asbestos-airways disease” rather than asbestosis.89 With disease progression, fibrosis extends from the respiratory bronchiole proximally into the terminal bronchioles as well as distally into the alveolar ducts and then to involve more of the lung acinus. Ultimately the fibrosis extends into surrounding alveolar septa. With late-stage disease, there is confluence of the separate fibrotic foci, resulting in diffuse fibrosis. Finally the fibrotic changes become confluent with the development of honeycomb change (Figure 10). The most extensive fibrosis is typically observed in the subpleural tiers of alveoli, and in those alveoli in closest proximity to the bronchioles. These small airways feature low cuboidal and columnar epithelium, and contain mucus and inflammatory debris. In early disease, Masson’s trichrome stain may help assess the extent and distribution of interstitial fibrosis. Additional and curious histological findings can be seen in asbestosis. Hyperplastic type II pneumocytes may contain deposits of waxy, deeply eosinophilic material with tinctorial and ultrastructural features typical of the Mallory hyaline seen in hepatitis (Figure 11). Not unique to asbestosis, as once believed, in such a setting this probably represents a nonspecific form of alveolar epithelial injury (see Chapter 2). Other nonspecific histopathological findings include dendriform pulmonary ossification, where branching spicules of bone can contain hematopoietic elements (see Chapter 10).

Chapter 14: Occupational lung disease Table 5 ILO international classification of radiographs of pneumoconioses

I. Parenchymal abnormalities Small and large opacities: Descriptors include profusion, affected zones of lung, shape and size. Major

Minor/subcategory

Small opacities Profusion:

Category Category Category Category

Shape/size:

Radiological small, round opacities: p ¼ diameter up to 1.5 mm q ¼ diameters up to 1.5–3 mm r ¼ diameters 3 mm – 10 mm

Shape/size:

Radiological small, irregular opacities: s ¼ opacities widths up to 1.5 mm t ¼ opacities widths 1.5–3 mm u ¼ opacities widths 3–10 mm

Large opacities

0: Normal 1: Mild 2: Moderate 3: Severe

0/
, 0/0, 0/1 1/0, 1/1, 1/2 2/1, 2/2, 2/3 3/2, 3/3, 3þ

Defined as having greatest dimension exceeding 10 mm:

Category A:

One large opacity having longest dimension up to approximately 50 mm, or several large opacities with the sum of their longest dimension not exceeding about 50 mm.

Category B:

One large opacity having longest dimension exceeding 50 mm, but not exceeding equivalent area of right upper zone, or several large opacities with sum of longest dimensions exceeding 50 mm but not exceeding equivalent area of right upper zone.

Category C:

One large opacity which exceeds the equivalent area of the right upper zone, or several large opacities which when combined exceed the equivalent area of the right upper zone.

II. Pleural abnormalities Pleural abnormalities are divided into plaques, costophrenic angle obliteration and diffuse pleural thickening. 1. Plaques (localized pleural thickening): Presence or absence of calcification, site and laterality are recorded, as is extent (recorded only for chest wall plaques). Extent 1: total length up to ¼ of lateral chest wall Extent 2: total length up to ¼ to ½ of chest wall Extent 3: total length exceeding ½ of chest wall 2. Costophrenic angle obliterations are recorded as present or absent, with designation as to side involved. III. Additional descriptors/symbols Radiographic features of importance, which may be relevant to dust exposure, are also given. Examples include: es (eggshell calcification; hilar or mediastinal nodes) em (emphysema) cp (cor pulmonale) ho (honeycomb lung) ca (cancer, thoracic malignancies excluding mesothelioma) Modified from: International Labour Office, International Classification of Radiographs of Pneumoconiosis, rev ed. Occupational Safety and Health Series, No. 22 Rev 2000, Geneva.

Differential diagnosis The principal differential diagnostic considerations include UIP and the fibrosing variant of nonspecific interstitial

pneumonia (NSIP). Similar to UIP, asbestosis features lower lung zone and subpleural accentuation, but is that of a temporally uniform and collagenous fibrosis, without the prominent fibroblast foci typical of UIP (Figure 12).

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Chapter 14: Occupational lung disease Table 6 Published criteria for acceptable histopathological definitions of asbestosis

I. College of American Pathologists – National Institute for Occupational Safety and Health – (CAP-NIOSH)87: “Demonstration of discrete foci of fibrosis in the walls of respiratory bronchioles associated with accumulations of asbestos bodies”. II. “Helsinki Criteria”88: “Diffuse interstitial fibrosis in well-inflated lung tissue remote from a lung cancer or mass lesion, plus the presence of either two or more asbestos bodies in tissue with a section area of 1 cm2, or a count of uncoated asbestos fibers recorded by the same laboratory for asbestosis”. III. Asbestosis Committee of the College of American Pathologists and the Pulmonary Pathology Society89: Acceptable pattern of alveolar septal (not bronchiolar) fibrosis and an average rate of asbestos bodies of at least 2/cm2. Cases with diffuse interstitial fibrosis and an asbestos fiber burden, determined by an experienced laboratory using electron microscopic techniques, within the range of values observed for bona fide cases of asbestosis are also likely examples of asbestosis. Figure 8. Asbestosis with late-stage interstitial fibrosis and lower lobe honeycomb changes.

Figure 9. Asbestos body, bronchoalveolar lavage fluid, Wright’s stain.

Honeycomb change is generally less marked in cases of asbestosis. The temporal and spatial uniformity of the interstitial fibrosis in asbestosis is similar to that of NSIP, but its distribution in the acinus of the lung is different. In contrast to asbestosis, NSIP features a more spatially uniform pattern of fibrosis, and may also feature a variable degree of cellular interstitial inflammation (Figure 13).89

Grading of asbestosis It is generally sufficient for pathologists to incorporate gross and microscopic features in order to estimate the extent of any fibrosing or destructive lung processes, and to classify such involvement descriptively as mild, moderate or severe. This is usually sufficient for routine practice, with reference to asbestosis. It is possible, using histological grading schemata, to assess the extent of asbestosis in a semi-quantitative manner and augment those qualitative descriptors. For example, the CAP-NIOSH grading system includes scores for both severity and extent of disease (Table 7a). This scheme allows 12 possible grades for each slide, but in more practical terms a simpler grading scheme has been proposed by us (Table 7b).65

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A common problem in referral clinical practice arises when asbestosis is suspected on the basis of exposure history or other clinical grounds. However, asbestos bodies are not identified in fibrotic lung tissue, even following iron stains. To this end, specialty laboratories are often contacted to perform analytic testing of lung tissue to measure and quantify asbestos fiber burdens. Roggli has shown that, in patients lacking asbestos bodies in histological sections, an uncoated asbestos fiber burden well below that observed in cases with bona fide asbestosis is to be expected.93 Gaensler et al. also showed that when asbestos bodies are identified in tissue sections in patients with interstitial lung disease and a history of asbestos exposure, high uncoated fiber burdens will be found on tissue analytic studies. In those cases where no asbestos bodies are identified, uncoated fibers are not elevated beyond those observed in control populations.94 The authors also further evaluated cases of diffuse pulmonary interstitial fibrosis with asbestos exposure but whose biopsies did not meet established criteria for asbestosis, and compared their respective fiber burdens with those of confirmed asbestosis cases. Of 86 cases, seven had asbestos body counts within the 95% predicted interval for asbestosis, but none of these had commercial amphibole fiber levels within the 95% prediction interval. This would indicate that in the cases of diffuse pulmonary fibrosis studied, most cases did not contain asbestos fibers within the range typically observed for asbestosis. In addition, a history of asbestos

Chapter 14: Occupational lung disease

(a)

(d)

(b)

(c)

(e)

Figure 10. Grades of asbestosis. (a) Asbestos airways disease. Fibrosis is restricted to the bronchiolar wall. (b) Grade 1 asbestosis. Fibrosis involves the bronchiolar wall with extension into the first adjacent tiers of alveoli. An asbestos body is noted in the peribronchiolar interstitium (arrow). (c) Grade 2 asbestosis. Peribronchiolar fibrosis and involvement of alveolar ducts in addition to more than two tiers of alveoli. Inset: A regional asbestos body is noted on a Perls’ iron stain. (d) Grade 3 asbestosis. Fibrosis involves alveolar septa between at least two adjacent bronchioles. A regional asbestos body is noted on a Perls iron stain. (e) Grade 4 asbestosis. The lung features late-stage fibrosis with progression to honeycomb change. A regional asbestos body is noted (arrow).

Figure 11. Asbestosis with intracytoplasmic accumulations of eosinophilic material similar to Mallory’s hyaline.

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(a)

(b)

Figure 12. Usual interstitial pneumonia (UIP). (a) Interstitial fibrosis and honeycomb changes predominate in lower lung zones, with subpleural accentuation. (b) Temporally and spatially heterogeneous interstitial fibrosis with areas of relatively normal lung.

Table 7a CAP – NIOSH asbestosis grading scheme

Figure 13. Nonspecific interstitial pneumonia (NSIP). Temporally and spatially uniform interstitial fibrosis are seen.

exposure alone is not sufficient for a diagnosis of asbestosis in this setting.95 Accordingly, in cases of pulmonary fibrosis where asbestosis is suspected on a historical basis, but no asbestos bodies are demonstrated despite careful histological examination of an adequate specimen, electron microscopy for asbestos fiber

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Grade 0:

No fibrosis associated with bronchioles.

Grade 1:

Fibrosis involves wall of at least one respiratory bronchiole with or without extension into septa of first adjacent tiers of alveoli.

Grade 2:

Fibrosis in Grade 1 lesions, plus involvement of alveolar ducts and two or more tiers of alveoli adjacent to the bronchiole with some spared alveoli between adjacent bronchioles.

Grade 3:

Fibrosis as in Grade 2 lesions, plus fibrotic thickening of the septa of all alveoli between at least two adjacent bronchioles.

Grade 4:

Fibrosis as in Grade 3 lesions, plus the formation of honeycomb changes (formation of cyst-like spaces larger than an alveolus which may be epithelialized).

Grading the extent of disease is classified according to the percentage of bronchioles showing excessive peribronchiolar fibrosis. Grade A:

Only occasional bronchioles involved.

Grade B:

More than occasional involvement, but less than half.

Grade C:

More than half of all bronchioles involved by fibrosing process.

Modified from reference 87.

Chapter 14: Occupational lung disease Table 7b Suggested modification of CAP–NIOSH histological grading schema for asbestosis.

Grade 0:

No appreciable peribronchiolar fibrosis or fibrosis confined to bronchiolar walls.

Grade 1:

Fibrosis confined to the walls of respiratory bronchioles and the first tier of adjacent alveoli.

Grade 2:

Extension of fibrosis to involve alveolar ducts and/or  2 tiers of alveoli adjacent to the respiratory bronchiole with sparing of at least some alveoli between adjacent bronchioles.

Grade 3:

Fibrotic thickening of the walls of all alveoli between  2 adjacent respiratory bronchioles.

Grade 4:

Honeycomb change.

Data from references 65 and 89.

analysis is not recommended and is unnecessary.95 In such instances, the diagnosis of a non-asbestos associated form of pulmonary fibrosis is quite permissible. However, there are extremely rare documented cases where large numbers of uncoated asbestos fibers have been demonstrated in instances where pulmonary fibrosis is observed, in the absence of asbestos bodies on H&E or Perls’ stained sections.86 In such instances, fiber analysis may serve as an adjunctive diagnostic aid, provided the assay is performed by an experienced laboratory. The result must be correlated with the range of values observed in bona fide cases of asbestosis.89 In the authors’ opinion, such instances are distinctly uncommon, and the approach described in the foregoing paragraph is amply justified. In everyday practice, fiber burden analysis cannot substitute for or overrule the histological diagnosis of asbestosis.89

Prognosis Similar to other forms of diffuse pulmonary interstitial fibrosis, asbestosis results in significant morbidity and mortality. The immunosuppressive therapy which constitutes the mainstay of medical intervention for other forms of pulmonary interstitial fibrosis is ineffective. Historically, deaths result from intractable respiratory failure, with the expected association of decreased life expectancy in proportion to disease severity. Other causes of death include cor pulmonale, carcinoma of the lung and mesothelioma. Studies from the United Kingdom and Finland document considerable and excessive mortality from carcinoma of the lung in workers with asbestosis.96,97 A review of 525 asbestosis autopsies from Japan from the years 1958–1996 noted an increased incidence of malignancy in 60% of cases, represented chiefly by carcinoma of the lung and mesothelioma.98 Selikoff et al. showed that the risk of lung cancer among insulators was increased five-fold, relative to a population unexposed to asbestos with similar smoking histories.99 Most of these individuals had asbestosis radiographically or pathologically.100 McDonald and McDonald reported a relative risk of mesothelioma of 46 among insulators.101

Hopefully, the recognition of the serious hazards posed by exposure to asbestos, the reduction in asbestos consumption, the improvements in regulation, occupational and industrial hygiene, and the scientific understanding of asbestos-associated illness down to the molecular level will result in the removal of this scourge. Statistical modeling indicates that asbestosis deaths are not due to decrease sharply over the next 10–15 years. The disease will claim the lives in excess of 29 000 individuals between the years 2005 and 2027.68

Asbestos and lung cancer The association between asbestos exposure and an increased risk of bronchogenic carcinoma is indisputable. Cases reporting this association were first published in 1935,102,103 and the relationship was confirmed in epidemiological studies in 1955.104,105 All types of commercial asbestos fibers have been implicated and a dose-response relationship has been established.106,107 Asbestos has been recognized as a Class A carcinogen by the International Association for Research on Cancer (IARC). Experimental animal studies provided additional support for the association between asbestos and lung cancer.108 There are several additional considerations with regard to the relationship between asbestos exposure and lung cancer. These include the interaction between asbestos and cigarette smoking in increasing lung cancer risk, differences in fiber types with respect to potency in the production of lung cancer, and the role of asbestos dose versus asbestosis as the underlying etiological factor. Tumor location and histological type are additional considerations. Finally, one must consider the role of fiber analysis of lung tissue in determining the cause of lung cancer in an individual case. Selikoff et al. first reported the synergistic effect between asbestos and cigarette smoking in the causation of lung cancer in a cohort of insulation workers.109 Subsequently, a number of studies have confirmed the synergistic relationship, although a few have reported additive or supra-additive (i.e., greater than additive but less than synergistic) effects.106 The precise mechanism involved in synergism is unknown. Cigarette smoking inhibits clearance mechanisms in the lower respiratory tract and thus can increase the effective dose of asbestos. Furthermore, carcinogens from cigarette smoke, adherent to the surface of asbestos fibers, may be more readily delivered to the respiratory epithelium.63,110 Although the difference in potency of the various asbestos fiber types with respect to mesothelioma has been recognized for some time, potency effects were less apparent for lung cancer until the analysis by Hodgson and Darnton.111 These investigators reported the amphiboles (amosite and crocidolite) were between 10 and 50 times more potent than chrysotile in the production of lung cancer on a fiber per fiber basis (the values for mesothelioma were from 100 to 500 times more potent) (Table 8). Berman and Crump reached a similar conclusion in their extensive literature review.112,113 However, the steep gradient for lung cancer risk in the South Carolina

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Chapter 14: Occupational lung disease Table 8 Exposure-specific risk of mesothelioma and lung cancer for three principal commercial asbestos types

I. Risk ratio for mesothelioma for chrysotile, amosite, crocidolite: 1:100:500 II. Risk differential for lung cancer for chrysotile alone versus amphibole fibers appears to be between 1:10 and 1:50 Modified from reference 111.

chrysotile textile plant is an outlier and remains unexplained.114 The differences in fiber potency are probably related to the greater biopersistence of amphiboles in comparison to chrysotile. One of the most controversial areas with respect to asbestos and lung cancer is whether asbestosis is an obligatory precursor of asbestos-related lung cancer (fibrosis/cancer hypothesis), or whether asbestosis and lung cancer are two independent endpoints, each related to the dose of asbestos (fiber burden hypothesis). Proponents of the fibrosis/cancer hypothesis note the oncogenic potential for severe interstitial lung fibrosis, and the tendency for carcinomas to develop in animal models only in cases of asbestosis. Proponents of the fiber burden hypothesis consider asbestos both an initiator and promoter of carcinogenesis, and regard fibrogenesis and carcinogenesis as distinct effects of asbestos exposure. The pro and con arguments in this regard have been presented in detail elsewhere, and have included the fundamental disagreements between experts on the criteria for the diagnosis of asbestosis, laboratory-specific variance in fiber burden analyses and criticisms of the imprecision of epidemiological studies.106,115–118 One of the strongest arguments for the fiber burden hypothesis is the distribution of the two diseases. Asbestosis is primarily a disease of the lung periphery with greatest severity typically in the lower lobes, whereas asbestos-related bronchogenic carcinoma is a more central process and commoner in the upper lobes. Some have argued that epidemiological studies favor asbestosis as the precursor to asbestos-induced lung cancer.115,119 However, studies from our laboratory indicated that epidemiological studies are unlikely to have the statistical power to distinguish between asbestosis versus a fiber burden equivalent to that seen in asbestosis, as the likely precursor.107 Cigarette smoking is an important confounding factor in the determination of an asbestos etiology for an individual lung cancer case. This is because 85 to 95% of lung cancers are related to cigarette smoking, whereas asbestos is a causative factor in fewer than 5% of lung cancer cases.107 Tumor location and histological type are of little assistance in making this determination. Although some studies have reported that asbestosrelated lung cancers are commoner in the lower lobes, others have found that they are more frequent in the upper lobes (similar to cigarette-smoking related lung cancers in the absence of asbestos exposure).107 Furthermore, there is no significant difference in the distribution of histological types between asbestos-related and non-asbestos-related lung

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cancers.33,107,110 All of the major histological types of lung cancer (adenocarcinoma, squamous cell carcinoma, small cell carcinoma, and large cell carcinoma) can be caused by asbestos. Analysis of lung tissue fiber content plays an increasing role in determining the causation of lung cancer in an individual case. In 1994, Karjalainen et al.119 reported that fiber burden was an independent predictor of lung cancer risk. This observation was supported by the consensus of experts that met in Helsinki, Finland, in 199788 but is disputed by others.117,118 Karjalainen et al. found an increased odds ratio for lung cancer among patients with more than one million amphibole fibers per gram of dry lung tissue as determined by analytical electron microscopy. Our studies show one asbestos body per cm2 of an iron-stained lung tissue section is equivalent to approximately 400 asbestos bodies per gram of wet lung tissue.90 We have also demonstrated a significant association between asbestos bodies per gram of lung tissue as demonstrated by light and scanning electron microscopy.32 We are not aware of any studies that have compared the findings of asbestos bodies or fibers per gram of wet lung as determined by either scanning electron microscopy (SEM) or transmission electron microscopy (TEM). In this regard, there is considerable variation in results analyzing the same sample by different laboratories, since different laboratories follow different protocols.15,30 With respect to the relationship between fiber counts by SEM and TEM, Karjalainen has shown that when samples from the same laboratory are analyzed by SEM and TEM at the same magnification, approximately three times as many fibers are found by TEM as by SEM. This difference is almost entirely due to the detection of chrysotile (Karjalainen, personal communication, academic dissertation, University of Helsinki 1994). Karjalainen et al. found an increased odds ratio for lung cancer among patients with more than one million amphibole fibers per gram of dry lung tissue as determined by analytical electron microscopy.119 This level has also been associated with the development of asbestosis.29 In a study of 234 lung cancer cases with varying degrees of asbestos exposure, Roggli and Sanders found a marked difference in the asbestos content of lung tissue among cases with histologically confirmed asbestosis compared to those without asbestosis (including cases with parietal pleural plaques but without asbestosis). Specifically, when comparing groups of patients with asbestosis, parietal plaques but not asbestosis, and groups with neither, the asbestos body count in the asbestosis cohort was more than 35 times greater than the cohort with plaque only, and more than 300 times greater than the group with neither plaques nor asbestosis. The total asbestos fiber count for the asbestosis cohort was nearly 20 times greater than the cohort with pleural plaque, and more than 50 times that of the cohort with neither. The difference was due almost entirely to commercial amphiboles.120 Hence pleural plaques are an insufficient marker to determine asbestos causation in a given case of lung cancer.88,120 To establish the diagnosis of lung cancer and determine the histological type, the pathologist should identify other

Chapter 14: Occupational lung disease

morphological features in the lung that determine the etiology of a given case.121–123 For example, smoking-related changes, such as emphysema, small airways disease or respiratory bronchiolitis-associated interstitial lung disease, may point to tobacco smoking as the etiology. The finding of asbestosis may point to this mineral as the etiological agent. These are of course not mutually exclusive, as cigarette smoking is a cofactor in most asbestos-related lung cancers. The finding of asbestos bodies in the absence of asbestosis is not sufficient to determine causation. It may be an indication for fiber analysis to determine whether the fiber burden is within the range of values observed for patients with asbestosis (see above). Despite decades of research and the development of considerable knowledge in the mechanisms of asbestos-related disease and the means for its diagnosis, the topic remains fraught with controversy, even amongst those considered as experts within the field. This is reflected in a so-called Delphi study of an empaneled group of authorities tendered as experts on the basis of asbestos-related disease publications. In this study, consensus on statements regarding asbestos-related disease was not attained in 9/32 examples. This included statements concerning the diagnosis of pleural and parenchymal lung disease, and the role of asbestos exposure in the causation of lung cancer.124

Methods of tissue analysis A variety of techniques have been developed for the identification of asbestos in tissue samples. Quantification requires removal of the organic matrix in which the fibers are embedded, typically accomplished by wet chemical digestion techniques or low-temperature plasma ashing. The residue can be collected on a membrane filter, which is analyzed by various techniques. The brightfield light microscope can be used for counting asbestos bodies, with fairly good interlaboratory agreement.15 Alternatively, an aliquot of the digestate can be placed in a counting chamber and the number of fibers determined by phase contrast light microscopy.125,126 These light microscopic techniques have several limitations. Asbestos bodies account for a fraction of the fiber content and there is a variable relationship between the numbers of asbestos bodies and uncoated fibers from case to case (although asbestos bodies are a fairly good marker for long amphibole fibers).31,126 Phase contrast is limited to fibers with a diameter of 0.2 µm or greater, so thinner fibers are missed with this technique.6 Furthermore, phase contrast does not permit distinction between asbestos and non-asbestos mineral fibers. The accurate detection of mineral fibers in lung tissue and distinction of asbestos from other non-asbestos mineral fibers generally requires the use of analytical electron microscopy, which consists of a transmission or scanning electron microscope equipped with an energy-dispersive spectrometer (EDS).6,126,127,128 These techniques require a high degree of technical expertise and are expensive and time-consuming. Preparation techniques have the potential for loss or addition of fibers

to the sample. Counting rules may differ from one laboratory to another. Furthermore, there is no standardized methodology for the analysis of human tissue samples. Consequently, examination of the same specimen by different laboratories using similar techniques may result in values that differ widely.15 Transmission and scanning electron microscopy have their own particular advantages and limitations that must be considered in the evaluation of results obtained with these techniques. Transmission electron microscopy is generally considered to be the most sensitive method for detecting fibers in tissues, especially for the detection of the finest/smallest fibrils. Because of the small sample size with TEM, the specimen has to be placed on a 3 mm diameter electron microscope grid. Therefore, a portion of the filter must be selected, which is assumed to be representative of the entire filter. The filter material must be removed, which is generally accomplished by means of cold finger reflux technique.129 Then a number of grid openings, assumed to be representative of the entire grid, are examined. The number of fibers of a given type per grid is extrapolated to the entire filter, which is in turn extrapolated to a given weight of lung tissue. Transmission electron microscopy can visualize the central capillary (core) of chrysotile fibrils, a useful identifying feature. Furthermore, selected area electron diffraction can be performed with the TEM, permitting the determination of the crystalline structure of an individual fiber. Finally, the addition of an energy-dispersive spectrometer (EDS) allows for the determination of the elemental composition of an individual fiber by means of energy-dispersive X-ray analysis (EDXA).29 Scanning electron microscopy allows for the use of a larger sample size, so an entire filter can be mounted on a substrate and examined. The filter is coated with a conducting material, such as gold or platinum, which reduces charging artifacts that may interfere with fiber detection. The number of fibers of a given type per field is again extrapolated to the entire filter, which is in turn extrapolated to a given weight of lung tissue.129 Energy-dispersive X-ray analysis provides information regarding the elemental composition of individual fibers (Table 2b). These data combined with fiber morphology permit accurate identification of most fibers. Asbestos body and fiber concentrations, as determined by SEM, correlate well with light microscopy asbestos body counts from the same specimen.29 For the quantification of asbestos fibers in lung tissue samples, some investigators have preferred analytical TEM130–132 whereas others have used the analytical SEM.35,133,134 Both methods have their limitations and biases. For example, although TEM has been touted as superior for the detection of individual fibrils, there is poor interobserver reproducibility for the detection of fibers less than 1 µm in length.134 Practically, only fibers > 5 mm in length merit detection, since shorter fibers are not implicated in the causation of fibrotic lung disease or neoplasia.135 Investigators who have used both methodologies note TEM is biased towards the detection of chrysotile, whereas SEM is biased towards the finding of amosite.136

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These biases and limitations are somewhat moot, since investigators using TEM have come to similar conclusions as those using SEM. These are that an elevated asbestos content is present in just under 90% of lung tissue samples from US mesothelioma cases and the predominant fiber type is amosite.23,35 TEM studies showed that in populations exposed to mixtures of asbestos fiber types (chrysotile and amphiboles), disease patterns correlate with amphibole fibers, but not with chrysotile.66,133,134,136–138

Silicotic lung disease Introduction Silicosis comprises a group of related lung diseases that result from the inhalation of crystalline silica. With the descriptions of silicosis in ancient Egyptian mummies, this may be the oldest-known pneumoconiosis.139,140 Incidents such as the Hawks Nest Tunnel disaster in West Virginia in the 1930s, where hundreds of exposed workers died of acute silicosis, underscore the durability of this disease through the passage of time.141

Mineralogy Silica, the mineralogical term for the oxide (SiO2) of the element silicon, is an extremely common component of the earth’s crust, and occurs in both crystalline and amorphous forms. Crystalline polymorphs of silica include quartz, tridymite, cristobalite, coesite and stishovite.142 Most occupational exposures are to quartz, the commonest of these crystalline polymorphs. Diatomaceous earth is a familiar example of amorphous (non-crystalline) silica. Silicates are higher oxidized forms of the element silicon (SiO4) combined with various cations, and the pneumoconioses associated with silicate exposure will be discussed below.

Occupations causing exposure to silica and pathogenesis An exposure to crystalline silica may occur in any occupation that disrupts rock in the earth, or processes rock and stone, owing to the abundance of silica in the earth’s crust. In addition, common industrial minerals, such as granite, sandstone and shale, contain considerable amounts of quartz. Occupations involving exposure to silica typically include construction, tunneling, blasting, mining and quarry work, as well as trades using silica-containing abrasives. In some countries with lax health and safety regulations, it is possible to see men surrounded by dust sanding the stone of new buildings. Silica also occurs in a biogenic form (phytoliths) in some plants143 and has been identified in urban air samples and tobacco smoke. Silica is often a component of coal dust (see below). The biological activity of silica particles is complex and depends on a number of particle and host factors. Crystalline

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forms of silica are much more toxic than amorphous forms, which have not been implicated in human disease.144,145 Particle size is important. Quartz particles in the size range 0.5–2.0 µm are the most fibrogenic. Impurities such as iron or aluminum in the crystal exert a protective effect and reduce the toxic bioactivity of silica.146 Quartz particles often contain a soluble amorphous surface layer, the Beilby layer, which reduces their biological activity. The toxicity of quartz particles is enhanced when this layer is removed by acid washing or when freshly cleaved crystal surfaces are exposed, as occurs in sandblasting. Tridymite, which is the most fibrogenic form of crystalline silica, lacks a Beilby layer.142 Current concepts of the mechanism of tissue injury by crystalline silica have focused on surface activity of the crystal. SiOH groups on the hydrated surface of the crystal are thought to form hydrogen bonds with cellular macromolecules, including phospholipids and proteins. This bonding can lead to protein denaturation and lipid membrane damage. Free radical generation from freshly fractured silica particles may also play a role in silica-induced cytotoxicity.144–147 The alveolar macrophage is the principal effector cell for silica-related lung injury. It is capable of mediating inflammation, epithelial proliferation and fibrogenesis, through the elaboration of growth factors, chemokines, cytokines and oncogenes, as well as facilitating the creation of reactive oxygen species and free radicals.75,147 Phagocytized silica particles interact with lysosomal and/or plasmalemmal membranes of the macrophage, leading to cell injury and death. Injured or dying macrophages release a soluble protein factor that stimulates fibroblast proliferation and collagen synthesis.148,149 Direct interaction between silica and fibroblasts may also be important.150,151 Immunological factors resulting from exposure of antigenic sites of denatured proteins have been suggested to play a role in the pathogenesis of silicarelated tissue injury.143 Latency periods for the development of silicosis typically measure 20–40 years, following initial exposure. The disease may develop after only 10–15 years and occasionally occurs more acutely in individuals with particularly heavy exposures.

Clinical features Clinically the patient may be asymptomatic, or there may be restrictive changes on pulmonary function tests and hypoxia causing cough and dyspnea. Pulmonary hypertension and cor pulmonale may supervene. Complicated silicosis has an increased risk of tuberculosis, which in the past has been reported in as many as 40–60% of cases.152 Cavitary lesions in these patients should always raise the possibility of mycobacterial infection. Other complications of silicosis are discussed below.

Radiological features Patients with simple silicosis are often asymptomatic, and may even have normal chest radiographs.153 The radiological

Chapter 14: Occupational lung disease

Figure 14. Simple silicosis. (a) Radiographic features of multiple small upper lung zone nodular opacities. (b) Peribronchial silicotic nodule with characteristic fibrotic gray discoloration.

diagnostic of silicosis, especially when combined with the presence of small rounded opacities in the upper lung zones. Complicated silicosis is often associated with coalescence of nodules and the radiographic demonstration of large and bilateral opacities, corresponding to the development of progressive massive fibrosis (PMF) and varying degrees of perifocal emphysema, a form of paracicatricial emphysema adjacent to dust nodules.153,154

Pathological features

Figure 15. Pulmonary silicotic nodule. Lung parenchyma is completely obliterated by whorled bundles of hyalinized paucicellular collagen. Peripheral carbon pigment is also seen.

features of simple silicosis are those of multiple well-defined nodular opacities that predominate in the upper and posterior lung zones (Figure 14a).154 “Eggshell” calcification of hilar lymph nodes may be present, resulting in a characteristic radiographic appearance. This radiographic finding may be

Nodular silicosis is characterized by hyalinized nodules, ranging from a few millimeters to more than a centimeter in diameter. Lesions are firm, spherical, and slate-gray to black, depending on the presence of other dusts inhaled along with the silica (e.g. coal dust) (Figure 14b). Silicotic nodules may be detected anywhere in the lung, although they tend to be more numerous in the upper lobes. The upper lobe predominance is thought to be related to less dense lymphatic drainage of these lung fields, in comparison with the lower and mid lung fields. Histologically one sees concentric, acellular, whorled bundles of dense hyalinized collagen fibers (Figure 15). Calcification may be present, and variable amounts of black pigment (carbon) are present centrally or at the periphery (Figure 16).152 The nodules often occur in perivascular or peribronchiolar locations but may be found anywhere within the lung parenchyma (Figure 17). These nodules may coalesce to form gray-black, irregular firm masses 2 cm or more in maximum dimension, usually in the upper lobes and often bilaterally (Figure 18). Such coalescent lesions are also referred to as conglomerate silicosis. These masses show more extensive

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Chapter 14: Occupational lung disease

Figure 16. Pulmonary silicotic nodule. Osseous metaplasia and central carbon pigment are occasionally seen.

Figure 18. Bilateral coalescent nodules of conglomerate silicosis in central lung zones. Note smaller nodules in upper lobe of left lung.

fibrosis and cavitation centrally, as a result of ischemia. When this occurs, superinfection chiefly caused by Mycobacterium tuberculosis should be suspected. Perifocal emphysema may be identified adjacent to fibrotic lesions. Nodules often extend to the pleura, and there may be extensive pleural adhesions.154 Hilar lymph nodes are almost always involved in silicosis. Abnormalities in the hilar nodes

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Figure 17. Pulmonary silicotic nodules. The lymphangitic distribution of disease is apparent. Small nodules often coalesce to form masses.

may be found in the absence of parenchymal nodules, especially in early or mild disease. The lymph nodes are typically black, enlarged, and have an extremely firm and rubbery consistency. The histological appearance of silicotic nodules within lymph nodes is identical to those observed within the lung parenchyma. However, a diagnosis of silicosis may not be made solely on the presence of such nodules within hilar lymph nodes. Extrathoracic lesions may also be found, especially in cases with heavy and prolonged exposures.155 The silica particles producing these lesions are transported from the lung to such sites hematogenously, or through lymphatics, where they are then phagocytized by histiocytes in the spleen, liver, bone marrow or lymph nodes. The most common sites for extrathoracic silicotic nodules are spleen, liver, bone marrow and abdominal lymph nodes. Peritoneal silicotic nodules have also been described.156 Silicotic nodules are typically bilateral and multiple and may be further distinguished microscopically from old healed tuberculous or fungal granulomas. The latter are unilateral, often solitary and usually have residual amounts of central necrosis. Examination of sections with polarized light is of limited use in this regard, because silica particles are weakly birefringent and small particles in tissue sections may be difficult to visualize.157 The brightly birefringent, platy or needleshaped particles sometimes seen in the periphery of silicotic nodules are not silica but silicates, which are inhaled along with the silica dust.157,158 Alveolar lipoproteinosis, sometimes termed acute silicoproteinosis (ASP), may result as an acute reaction to inhalation

Chapter 14: Occupational lung disease

Figure 20. Rheumatoid pneumoconiosis (Caplan’s lesion). This subpleural nodule is tan yellow and fibrous with obvious carbon deposition. Most lesions are larger than this example. (Image courtesy of Dr K. Kerr, Aberdeen, UK.) Figure 19. Silicoproteinosis. Intra-alveolar amorphous eosinophilic material surrounds a silicotic nodule.

of silica dust. Acute silicosis occurs in some individuals within a few years of intense exposures to very fine silica particles, and many of the reported cases have been in sandblasters.159 This form of silicotic lung disease may have a short latency period, reflecting such intense degrees of exposure, and lethal outcomes are frequent.75,159,160 It is postulated the dust injures macrophages and alveolar epithelium, altering surfactant production, its degradation, or both. Clinically, ASP is fulminant and accompanied by the radiographic findings of bilateral airspace consolidation and perihilar ground-glass opacities. Calcification has been reported in excess of 80% of such cases, which helps distinguish ASP from other causes of diffuse airspace consolidation.161 Pathologically, the lungs become consolidated with proteinaceous material that biochemically resembles surfactant but lacks surfacetension-reducing properties. Morphologically one finds alveolar spaces filled with granular eosinophilic material identical to that observed in idiopathic pulmonary alveolar proteinosis (see Chapter 10) (Figure 19). The eosinophilic material stains positively with periodic acid Schiff (PAS) but negatively with Alcian blue. A similar lesion has been reported in an individual exposed to high levels of fine aluminum dust (see below).162 A diagnosis of alveolar proteinosis should prompt an investigation into the patient’s occupational history.

Associated immune dysfunction Exposure to silica can result in systemic immune dysfunction.163 Patients with silicosis have impaired resistance to both tuberculous and non-tuberculous mycobacteria. Up to 5.0% of cases are complicated by the development of tuberculosis.164

Defects in immune and macrophage function probably account for the impaired resistance. This is due to silicainduced injury to alveolar macrophages, as well as a reduction in cell-mediated immunity, caused by silica exposure.165 Large numbers of patients with silicosis have various serological abnormalities, including circulating rheumatoid factor, antinuclear antibodies, hypergammaglobulinemia and immune complexes.166 Immune dysfunction induced by silica exposure might contribute to the development of scleroderma and renal disease in this population. In addition, coal miners with rheumatoid arthritis may develop rheumatoid pneumoconiosis (RP), historically termed Caplan’s lesion or Caplan syndrome, following its initial report in Welsh coalminers.167 This lesion has also been described in patients who are seropositive for rheumatoid factor in the absence of clinical arthritis, as well as in those exposed to silica and non-fibrous silicates.167,168 Rheumatoid pneumoconiosis lesions are a peculiar combination of silicotic nodules and rheumatoid nodules. Grossly, silicotic nodules with smooth borders and concentric internal laminations are seen (Figure 20). Microscopic features include peripheral, histiocytic palisading and focal areas of necrobiosis, similar to rheumatoid nodules in other sites (see Chapter 21). A peripheral and perivascular infiltrate of plasma cells may be present. Whether mineral dust inhalation-induced pulmonary fibrosis causes bronchogenic carcinoma is controversial. The IARC has classified silica as a definite human carcinogen169 leading some to suggest a putative association between silica dust exposure and lung cancer in humans.75,170 Conflicting data exist in animal models.171,172 Such an association has been suggested in a number of epidemiological studies. Some of these epidemiological studies lack proper control for

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(a)

(b)

Figure 21. Mixed dust pneumoconiosis. (a) Irregularly shaped fibrous nodules are centered on airways. (b) Crystalline material with optical features typical of silicates is visible under polarized light.

confounding factors, such as cigarette smoking or radon exposure in underground mining operations. Others observed an association between silica and lung cancer but it fell short of implicating this mineral in the induction of carcinoma of the lung.173–176 No such association was observed by Yu et al. in a large cohort of silicotic workers in Hong Kong.177 The authors of this chapter believe that current evidence is inconclusive with regard to a causal association between silica exposure and bronchogenic carcinoma in humans.

Future trends Bang et al. reported deaths in excess of 6000 from silicosis in the USA, for the period from 1981 to 2004. There was a decline in the overall age-adjusted mortality rates per million from 2.4 to 0.7, with approximately three deaths from silicosis per year since 1995. Such data indicate efforts to limit workplace exposures have been successful, and should be continued to eradicate this preventable disease.178

Mixed-dust pneumoconiosis and mixed pneumoconiosis Mixed-dust pneumoconiosis (MDP) has historically been the term for pulmonary fibrosis referable to the concomitant inhalation of silica and less fibrogenic dusts.179–180 The current consensus restricts the term to those lesions featuring dust macules or mixed dust nodules in the presence or absence of silicotic nodules in those patients with a history of mixed dust exposure.181 Other established pneumoconioses must be excluded.182 Typical occupations at risk of mixed dust pneumoconiosis include pottery and ceramic workers, foundry workers, metal miners and masons.182 The fibrotic response may result in part from the silica (quartz) content with modification of the response due to the silicate component. The role

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of the more numerous silicate particles in the fibrotic process is unclear. Diagnosis of mixed-dust pneumoconiosis requires a detailed occupational history and the finding of irregular opacities on chest radiographs. The pathological features of mixed dust pneumoconiosis include dust macules, nodules and PMF lesions with or without silicotic nodules. Mixed dust fibrotic (MDF) lesions are generally stellate areas of interstitial collagenization with numerous dust-laden macrophages and conspicuous birefringent particles, when viewed using polarized microscopy (Figure 21). These differ from the round opacities of silicosis. By definition the mixed dust fibrotic lesions should outnumber the silicotic nodules, otherwise silicosis is the preferred diagnosis.182 Mixed pneumoconiosis is a form of lung disease that results from exposure to two or more inorganic dusts, with the demonstration of pathological features characteristic of each component. Examples include siderosilicosis in hematite miners, in which diffuse interstitial deposits of iron oxides may be seen in combination with typical silicotic nodules. Another example is asbestosilicosis, in which peribronchiolar interstitial fibrosis and asbestos bodies may be seen along with typical silicotic nodules. This is a fairly common finding in shipyard workers, where sandblasting, boiler-scaling and insulating activities occur simultaneously in a confined space. An extreme case of mixed pneumoconiosis featuring silicosis, asbestosis, talcosis and berylliosis was reported.183

Respiratory diseases resulting from coal and coalmine dust Coal workers’ pneumoconiosis (CWP), chronic obstructive lung disease and pulmonary fibrosis number among the lung pathologies that may result from the inhalation of coal dust

Chapter 14: Occupational lung disease

during mining. The development of pulmonary disease depends on factors including the quantity and quality of inhaled dust, occupation within the mine, duration of exposure, interval since last exposure, and other considerations, such as cigarette smoking. Coal, derived from compressed organic material, principally of plant origin, is the earth’s most abundant fossil fuel, and is mined globally. In the United States, coal production and the number of coal miners is on the increase: 122 000 individuals in over 2000 mines in 26 states engaged in the production of more than one billion tons of coal in 2007.184 Improvements in mechanization have allowed increased productivity, but the amount of respirable dust generated remains considerable. Workers continue to develop respiratory disease, including advanced pneumoconioses, despite implementation of dust control measures and reduction in the PEL of respirable coal mine dust. In the US, the PEL is set at 2 mg/m3 after successive reductions pursuant to the Coal Mine Health and Safety Act of 1969.185 New cases continue to develop, even among miners who have worked exclusively under such limits.185–188 The last 30 years has seen a reduction in the prevalence of CWP in the USA from > 10% to less than 2% following the passage of such legislation.184 The risk of the development and progression of CWP reflects the total dust burden within the lung, as well as the rank of coal being mined. Coal is ranked to reflect its amount of fixed carbon, volatiles and heating value. Higher ranks (hard coal, anthracite) feature low moisture contents and higher amounts of fixed carbon than the lower-ranked soft coals (lignite, bituminous). Coal dust consists mainly of amorphous non-crystalline carbon together with varying amounts of crystalline silica (quartz), kaolin, mica and other silicates. The quartz content of coal dust is an important determinant of the pathological response. Such content varies geographically and with coal rank. Anthracite usually contains a higher percentage of quartz than bituminous or lignite coal. The intensity of quartz exposure also varies for different jobs, within any particular coal mine. Roof bolters drilling into the ceiling of a shaft or constructing communicating shafts between adjacent coal seams are exposed to higher levels of crystalline silica than individuals working at the coalface, or loading coal for transport. The geographic clustering of rapidly progressive CWP in young miners in the eastern USA, typically in smaller work sites mining high rank coal, has been reported.189

Pathogenesis Respiratory diseases resulting from coal develop from tissue injury, remodeling and modification of the extracellular matrix due to the elaboration of cytokines, growth factors, eicosanoids and fibrogenic factors. Chronic inhalation of coal dust results in increased numbers of pulmonary inflammatory effector cells, including neutrophils and macrophages. Coal dust serves as a significant source of both cellular and

non-cellular reactive oxygen species formation.190 Although quartz is highly fibrogenic, amorphous carbon is innocuous and is classified as a nuisance dust. Intratracheal instillation of amorphous carbon in experimental animals results in a substantial influx of macrophages into alveolar spaces, but no appreciable fibrosis.191

Coal workers’ pneumoconiosis (CWP)

The popular term “black lung disease” is a legal and occupational medicine term. It encompasses a broad variety of respiratory maladies suffered by coal miners in their occupational setting, and is not a synonym for CWP. With a prevalence in the USA among exposed populations as great as 10% during the 1970s, reductions in the PEL for coalmine dust have been met with concomitant declines in the prevalence of CWP to less than 2%, as reported by NIOSH in the Coal Workers Xray Surveillance Program.187 Similar trends have been reported in Europe and South Africa.188

Clinical features Patients with simple CWP may be asymptomatic. Owing to the progression of bulky parenchymal fibrosis in advanced disease, miners with complicated CWP and PMF often suffer significant pulmonary impairment with cough productive of black sputum, breathlessness and hypoxia. Restrictive and obstructive defects are noted. Pulmonary hypertension and cor pulmonale may follow. Coal workers also have an increased risk for tuberculosis, especially those with PMF. Historically, up to 40% of patients with PMF suffered from superimposed tuberculosis.158 The current incidence of complicating tuberculosis is thought to be lower.

Radiology The radiological classification of CWP separates the disease into simple and complicated forms. The simple form comprises macular and nodular lesions, with the complicated form representing PMF. Simple CWP features 1–5 mm round nodular opacities on chest X-ray, most profuse in the upper lung zones. Calcification may be observed in 10–20% of such cases, but in a finely nodular pattern that helps distinguish it from the “eggshell pattern” typical of calcification in silicosis.153 Progressive massive fibrosis lesions may mimic carcinoma of the lung radiographically. Positron emission tomography scans may be ambiguous in view of the intensive 18F-fluorodeoxyglucose (FDG) uptake by benign fibrotic masses. Magnetic resonance scanning has been reported to be of use in such instances, prior to a biopsy diagnosis.153

Pathology The defining lesion of CWP is the coal dust macule. These non-palpable lesions appear as 1–4 mm in diameter black areas distributed diffusely throughout the lung. An upper lobe preponderance is noted, owing to the relative reduction in density of lymphovasculature and particulate clearance in these areas.

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Figure 22. Simple coal workers’ pneumoconiosis. Coal dust macules expand alveolar septa.

Figure 23. Coal dust nodules. Note hyalinized collagen within nodule centers.

Together with a diffuse increase in background pigmentation, lesions impart a black appearance to the lung tissue. Macular lesions are typically more numerous in the upper lobes, and blackening also occurs along lymphatics within the secondary lobular septa and beneath the visceral pleura. Histologically, the macular lesion consists of focal collections of coal dust-laden macrophages at the division of respiratory bronchioles (Figure 22). These cells often extend into and fill adjacent alveolar spaces, as well as involving the peribronchiolar interstitium.158 Such collections of macrophages may be associated with a few reticulin fibers but mature collagen is inconspicuous. The macrophages may form a mantle around respiratory bronchioles and accompanying small pulmonary arteries. Nodular lesions, both micro- and macronodules, contain a collagenous center that may be hyalinized (Figure 23). Pigmented macrophages are found in the center of the nodules, as well as in the stellate mantle surrounding the lesion. The similar histology of these nodules to silicotic nodules demonstrates the presence of silica in coal dust. Emphysematous changes often occur in the zones adjacent to the coal dust macule, so-called focal emphysema. It is considered by some to represent an integral part of simple CWP.188 This lesion is limited in its extent to a small zone in the proximal part of the pulmonary acinus. It closely resembles centrilobular emphysema in its gross and histological features, differing only in its limited extent and invariable association with the dust macule. This process was shown by Heppleston to involve all three orders of respiratory bronchioles, whereas centrilobular emphysema only involves the distal respiratory bronchiole, with a proximal bronchiolitis (see Chapter 17).192 Perifocal or paracicatricial emphysema may also occur, especially adjacent to areas of PMF. Focal emphysema may develop in coal miners, who have never smoked cigarettes.

This process is probably mediated by antiproteases secreted by coal-dust-activated macrophages.193 A form of bronchitis, known as industrial bronchitis, is thought to occur in individuals working in a dusty environment, but this entity has not been well studied pathologically.

Complicated CWP and PMF The development of palpable nodular lesions in lung tissue indicates a change in the tissue pathological response. It is most likely related to the silica content of the inhaled dust. This process is sometimes referred to as anthracosilicosis. The spherical nodules are similar in appearance and distribution to those observed in silicosis, except they are more darkly pigmented. The nodules of CWP are classified into micronodules, which measure up to 0.7 cm, macronodules, which range from 0.7 to 2 cm, and PMF, which is more asymmetrical in distribution and irregular in shape, with lesions by definition at least 1 cm in one or more dimensions.158 Progression to complicated CWP, defined as the development of PMF, typically occurs on a background of simple CWP. Factors thought to be important in the evolution of simple coal workers’ pneumoconiosis into PMF include the silica/quartz content of the mine dust, infection with M. tuberculosis or other infectious pathogens, and/or other immunological factors. Cigarette smoking does not appear to play a role in the development of PMF.75 The cumulative exposure to respirable dust represents the most important factor in the development and clinicopathological progression of PMF.75,152 Although the relative contribution of each of these factors to PMF is at present controversial,158 the silica content in areas of massive fibrosis is greater than in adjacent nonfibrotic lung.194 Despite cessation of coal dust exposure, complicated CWP may progress, and some 4% of coal miners die of causes directly related to the disease.152

Chapter 14: Occupational lung disease Figure 24. Coal workers’ pneumoconiosis. This destructive black lesion demonstrates PMF with central cavitation. Note the smaller nodules in upper lobe, and enlarged black lymph node at hilum.

Figure 25. Coal workers’ pneumoconiosis. This PMF lesion obliterates bronchovascular structures.

Figure 27. PMF. Note presence of birefringent silicate particles adjacent to haphazard array of collagen bundles. Figure 26. Lymph node involvement with PMF.

Pathology The gross distribution of PMF lesions favors the upper and posterior lung zones, owing to reduced lymphatic flow and particulate clearance in these areas, and may be bilateral in advanced cases. Lesions may extend across fissures to involve adjacent lobes. Lesions are locally destructive, black and rubbery, and may undergo cavitation (Figure 24). The presence of cavitary lesions suggests M. tuberculosis infection, although ischemic necrosis also occurs. Liquified material in these cavities has the appearance of India ink. Microscopically, PMF contains collagen fibers, characteristically arranged in a haphazard fashion and separated by large amounts of coal dust. Bronchovascular structures are often obliterated, leaving only remnants of cartilage and elastin

fibers (Figure 25). Emphysematous change around PMF lesions is marked. Variable amounts of necrosis with cholesterol clefts may be associated with black lipid debris. Necrosis and giant cells suggest tuberculosis or fungal infection. However, the typical histological picture of tuberculosis may be lacking, even in cases where mycobacteria are demonstrable. The regional lymph nodes are jet-black, often enlarged and may contain silicotic nodules (Figure 26). Examination of sections with polarized light may demonstrate fairly numerous and brightly birefringent, platy silicate particles (Figure 27), but the optical characteristics of silica itself make it difficult to visualize with this technique.157

Differential diagnosis Coal workers’ pneumoconiosis must be distinguished histologically from anthracosis, due to cigarette smoking and urban

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Emphysema and chronic bronchitis, and carcinoma of the lung

Figure 28. Ferruginized coal bodies.

living, graphite worker’s lung and carbon electrode-maker’s pneumoconiosis. Some anthracotic pigmentation can be found in the lungs of nearly all adults in an industrialized society. Although pigment accumulation is related both to age and amount of cigarette smoking, there is considerable individual variation with regard to efficiency of particle clearance.195 Simple anthracosis, resulting from smoking and urban living, appears as pigment-laden macrophages within the peribronchovascular interstitium, beneath the pleura, and within hilar nodes. It differs from CWP in both the degree of pigmentation and absence of carbon-laden macrophages consolidating alveolar spaces. Detection of ferruginous coal bodies with a broad, black carbonaceous core (mimicking asbestos bodies) (Figure 28) may also be observed in CWP.32 Graphite miners may show the same pathological changes observed in silicosis and CWP, as graphite dust may contain crystalline carbon, quartz and other silicates. Graphite miners’ lungs may also contain giant cells within alveolar spaces. Carbon electrode makers are exposed to a dust containing crushed coke and anthracite. The lungs of these workers may show pathological changes indistinguishable from simple CWP and, occasionally, PMF.196

Diffuse interstitial fibrosis Diffuse interstitial fibrosis has also been described as a pathological response to inhaled coal dust, with severe cases demonstrating classic honeycomb changes, in addition to black pigmentation of lung tissue.197,198 Usual interstitial pneumonia has been described in coal miners at autopsy.199 The incidence of idiopathic pulmonary fibrosis in coal miners is as high as 18% reported in selected autopsy series. This has led to a potential role for silica, silicates and coal dust in the development of diffuse interstitial fibrosis.200

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Patients with simple CWP may show histopathological changes of emphysema or bronchitis. There is evidence for the development of chronic airflow limitation in the setting of such pathologies, even in the absence of CWP and controlling for cigarette smoking.186 The effects of cigarette smoking and dust exposure appear additive in the production of the obstructive changes. Those who develop clinically significant chronic obstructive lung disease are almost invariably miners who smoke.201,202,197 Coal mining is reportedly associated with an increased risk of gastric cancer, but lung cancer is less common among these workers than with comparable cohorts.203,204 The lung cancers in these patients are no different histologically from those seen in smokers otherwise employed.205

Pneumoconioses associated with non-asbestos silicates (silicatoses) Silicates is a generic term for a common class of minerals that consists of silicon dioxide (SiO2), often complexed with metallic cations, such as magnesium, aluminum and calcium. These are ubiquitous as a significant proportion of the earth’s rocks and soil, and in fact all mineral species, consist of silicates. The forms of commercial importance with the most potential for clinical relevance are typically non-fibrous sheet silicates, such as talc, kaolin and mica. The fibrous silicates include the zeolites, wollastonite and attapulgite (fuller’s earth). Vermiculite is non-fibrous. As common airborne dusts in both urban and rural settings, profuse quantities of silicates may be identified in human lung tissue at autopsy, with hundreds of millions of such particles detected using analytic techniques.206

Talcosis (talc pneumoconiosis) Talc is a hydrated magnesium silicate that occurs in platy, granular and fibrous forms. It is formed by two sheets of silica tetrahedral, separated by a magnesium hydroxide layer. Quartz and the non-commercial varieties of amphibole asbestos, such as tremolite, and anthophyllite are important contaminants. Exposure to talc, which is used in a number of manufacturing processes due to its lubricant properties, occurs in a variety of ways. Occupational exposure may result from mining and milling. Other occupations associated with exposure to talc include the leather, rubber, paper and textile industries; manufacture of ceramics, cosmetics, paints, pharmaceuticals, soaps, toiletries, and refractory and roofing materials; plate casting in which molds are dusted with talc before pouring; and dusting of life rafts with talc.60,207 Substantial personal exposure may occur in individuals who use cosmetic talc excessively. Experimental animal studies demonstrated the amphibole contaminants of talc, tremolite and anthophyllite are markedly

Chapter 14: Occupational lung disease Figure 29. (a) Interstitial granulomas following chronic inhalation of cosmetic talc. (b) Details of granuloma with giant cells containing asteroid bodies. (c) Birefringent talc crystals. (d) Brightly birefringent plate-like and needle shape of talc in pleurodesis specimen.

fibrogenic, whereas “pure talc” following milling and purification produces primarily a cellular macrophage response with granuloma formation.208 Exposure to pure talc may still be injurious to humans.209 Talcosis develops primarily in individuals with prolonged heavy exposure to talc dust and manifests with dyspnea and the typical clinical features of mild interstitial fibrosis. Cough and wheezing, due to chronic bronchitis, have also been reported,210 the latter possibly resulting in part from airway irritation and mucosal desiccation.211 Radiographically, numerous small opacities affecting all lung zones, due to granulomatous pneumonitis, may be observed on plain chest films. Diffusely distributed centrilobular and subpleural 1–2 mm nodules may be observed on high resolution CT scans.148,212,213 Pleural plaque has also been described213 but this is probably due to contaminating asbestos. These findings have not been documented to result from talc alone.210 Pleural thickening and dense pleural adhesions are described in many cases. Parietal pleural plaques were first described in talc miners, and probably result from contaminating tremolite and anthophyllite asbestos. Macroscopically the lung may contain tiny discrete palpable nodules or show diffuse interstitial fibrosis.207 The histological appearance of talcosis includes interstitial fibrosis, poorly defined fibrotic nodules, and foreign-body granulomas (Figure 29a–c).209,214 The degree of fibrosis appears to correlate with duration of exposure and dust content of the lung parenchyma. Mild lesions consist of dust-laden macrophages and connective tissue in the peribronchiolar and perivascular interstitium. Prolonged exposure to high concentrations of talc dust is associated with diffuse alveolar septal fibrosis, and even cases

of PMF. Variable numbers of multinucleated giant cells are seen. Birefringent particles are observed with polarized light microscopy within giant cells, within macrophages, or free in the interstitium.209 These brightly birefringent, needle-shaped particles, 0.5–10 µm in length, represent plates of talc viewed on edge (Figure 29d). Talc has also been used as filler for oral medicine preparations, and is one of several types of particles that may embolize to the lung, when oral medications are crushed and injected intravenously (see Chapter 16).215 Patients with intravenous drug abuse have a unique histological response to embolized talc particles. Intravascular and interstitial granulomas may be associated with variable degrees of fibrosis.216 Giant cells within the granulomas contain birefringent talc particles, many of which are too large to have been inhaled.217 Pulmonary hypertension and cor pulmonale may be the initial presentations in these individuals. Non-necrotizing foreign-body granulomas may resemble those seen in sarcoidosis, but can be distinguished by the presence of birefringent platy particles (Figure 29d). The latter are more numerous and larger than the occasional small birefringent calcific particles observed in giant cells in sarcoidosis (see Chapter 13).218 Progressive massive fibrosis, following both inhalational and intravenous exposure to talc, manifests as circumscribed, bilateral, irregular gray to gray-black masses with a firm, often gritty consistency.218 These lesions involve the central portions of the upper lobes and superior portions of the lower lobes and may cavitate.213 As is the case for silicosis and CWP, the factors responsible for development of PMF in talcosis are complex and similar, and are likely to be most influenced by cumulative dust burden within the lung.

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An increase in lung cancer risk for talc miners was reported.219 However, pulmonary tumors have not been produced in experimental animals exposed to pure talc158 and the lung cancer excess in the talc miners was probably attributable to contamination of the talc dust with non-commercial amphibole asbestos. No increased lung cancer mortality has been described in a review of talc miners and millers heavily exposed to talc not containing asbestiform minerals, except when there was an association with other known carcinogens.220

Kaolin pneumoconiosis Kaolinite, a hydrated aluminum silicate, is a member of the group of sheet silicates known as phyllosilicates. Kaolinite is the major component of kaolin (china clay), and is mined in the state of Georgia in the United States, as well as in Europe, Egypt, Japan and China. It has a worldwide distribution. Mined kaolinite contains small amounts of mica and quartz. Kaolinite has also been identified on tobacco leaves and in cigarette smoke, as well as in smoker’s alveolar macrophages.221 Kaolin’s in vitro cytotoxic potential for peritoneal and alveolar macrophages has been reported222 but the fibrogenicity of kaolinite appears to be minimal. Pulmonary fibrosis induced by kaolinite is not observed in animal models.223,224 Kaolin is used in the manufacture of paper products, refractory materials and ceramics, and as filler in paints, plastics and rubber. The most intense exposures, causing pneumoconiosis, have been reported in kaolin miners and processing plant workers.225,226 Radiographic findings are generally those of small irregular opacities in the mid-zones and are not typically associated with significant clinical symptoms or ventilatory impairment, although cases of PMF have been reported. Kaolin exposure rarely produces symptomatic changes, except in advanced disease.227,228 The lungs show gray-brown discoloration in the form of non-palpable macules, and numerous 0.5 cm to 12 cm firm, rubbery gray-brown nodules, which replace large portions of parenchyma. The hilar lymph nodes are often enlarged, secondary to accumulation of masses of dust-laden macrophages.226 Microscopically, the macular lesions of kaolin pneumoconiosis have the appearance and distribution of the lesions seen in CWP. They show intra- and extracellular deposits of fine golden-brown particulates, located primarily in a peribronchiolar distribution. Particle-laden macrophages distend the interstitium (Figure 30) and in cases with recent exposure are found in large numbers within alveolar spaces. The nodular masses consist largely of dust deposits, traversed by scattered randomly distributed bands of collagen. Whorled, dense collagenous deposits have been described in some cases, but such lesions should be attributed to contaminating quartz, rather than kaolinite. Progressive massive fibrosis may occur in individuals exposed concomitantly to significant amounts of

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Figure 30. Kaolin macule. Fine golden brown particles within macrophages elicit parenchymal fibrosis.

quartz, as in firebrick manufacture or in individuals with superimposed tuberculosis.226

Mica pneumoconiosis Mica comprises a group of sheet silicates whose crystalline structure allows for cleavage into very thin and transparent leaves. The transparent forms have been used historically in the doors of lanterns and stoves (isinglass). Currently, mica finds usage in thermal and electrical insulation. Mica exposure is often sustained in the setting of exposure to other minerals, such as quartz and feldspar; however, it is apparent that exposure to mica alone may result in pneumoconiosis.229,230 The radiographic findings resemble asbestos-related pleuropulmonary disease, with reticulonodular opacities, parenchymal fibrosis, pleural plaques and calcification.231 These authors have observed a case of endstage pulmonary fibrosis, clinically and pathologically similar to the other silicatoses, due to mica inhalation in a mica quarry worker in North Carolina. The pathological features of mica pneumoconiosis may include extensive parenchymal fibrosis (Figures 31 and 32).

Zeolites Zeolites are a complex group of hydrated aluminum silicates that include some 40 distinct mineral species used in the petrochemical industry, waste water treatment, water filtration, cement production and in animal litter.232,233 Natural deposits of zeolites are found in the western United States and in Turkey. Mordenite and erionite are two forms that occur as

Chapter 14: Occupational lung disease Figure 31. Lung from mica quarry worker removed at autopsy. Note extensive upper lung zone fibrosis.

Figure 32. Mica pneumoconiosis. Dust accumulations within macrophages. Chronic inflammation and fibrosis follow. Inset: abundant crystalline material observed under polarized light.

bronchoalveolar lavage fluid from individuals in the general population.238,239

Vermiculite

predominantly fibrous species. Erionite is found naturally in volcanic tuff in some areas of Turkey, where it is used as a construction material and is ubiquitous (see Chapter 36). It has physical characteristics that closely resemble amphibole asbestos.234 Most erionite fibers have diameters of less than 0.25 µm and it is a potent inducer of mesothelioma in animal models. The carcinogenic and fibrogenic effects of fibrous erionite are similar to, if not actually greater than, those of asbestos.235,236 However, most of the zeolites used commercially are non-fibrous with a likely minimal pathogenic potential. Interest in the pathological effects of zeolites can be largely attributed to the discovery of an epidemic of malignant pleural mesothelioma in two small villages in the Anatolian region of Turkey.237,238 These villages, situated on volcanic tuffs rich in fibrous erionite, have a combined population of only several thousand and the highest rates of mesothelioma of any population yet encountered. Surveys of the villages indicate a high prevalence of pleural calcification, diffuse pleural fibrosis and plaque formation, as well as diffuse interstitial fibrosis.233,237,238 Fibrous erionite is associated with a range of pathological changes similar to those that occur in individuals exposed to asbestos. The mineral has been identified in lung tissue from villagers suffering from mesothelioma, as well as in

Vermiculite is the mineralogical term for a group of hydrated, laminar, aluminum-iron-magnesium-silicates, which form accordion-shaped granules with numerous airspaces upon rapid heating. This imparts a light-weight and high insulation value when used commercially. The mineral finds utility as loose-fill thermal insulation and fire production, aggregate in cement, wallboards and plasters, as well as a soil additive and bulking agent in animal feeds. It is also used as a carrier for various industrial chemicals.240–242 Prior to its mine closure in 1990, 80% of the world’s supply of vermiculite came from Libby, Montana. Other deposits of this mineral are found elsewhere in the USA and in South Africa. Contamination with tremolite, actinolite or anthophyllite asbestos fibers raised concerns regarding the potential health effects of vermiculite exposure.241 Vermiculite from the Libby mine was heavily contaminated with tremolite asbestos, related asbestiform amphiboles, such as winchite and richterite, as well as crystalline silica and talc.240,242 Pleural abnormalities among vermiculite workers are probably related to contamination of vermiculite by asbestiform minerals. Vermiculite miners have an increased risk of pleural mesothelioma and carcinoma of the lung. Analysis of lung tissue samples revealed large numbers of high-aspect-ratio tremolite asbestos fibers.133 Workers in the Libby vermiculite mine showed an increased risk of pleural mesothelioma and lung cancer, as well as other non-malignant respiratory disease, chiefly chronic obstructive pulmonary disease. In addition, nearly 7% of Libby community residents, without occupational or

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para-occupational exposure, had radiographic evidence of asbestos-related disease.240 The catastrophe attendant to the Libby mine caused the US EPA to declare its first public health emergency, spawning extensive civil litigation, multimillion dollar expenditures to provide clean-up and medical assistance to the local population, as well as the largest environmental crime prosecution in US history. The health effects of pure vermiculite are largely unknown. Some animal studies show no tumorigenic potential for vermiculite, following intrapleural injection.101,243

Diseases from metals Beryllium Beryllium, from the mineral beryl (beryllium aluminum silicate), is a naturally occurring metal whose physicochemical characteristics allow numerous applications in a broad range of industries. As the second lightest known metal, beryllium is more rigid than steel, and an excellent conductor of heat and electricity.244 Historically, the principal industrial use of beryllium was in the manufacture of fluorescent lights. The element is often alloyed with other metals, such as copper and aluminum. Modern usage of beryllium has shifted to the hightechnology and aerospace industries, where it is used in structural materials, guidance systems, optical devices, rocket motor parts and heat shields. It is also used in the electronics industry in ceramic parts, in the manufacture of thermal couplings and crucibles, in nuclear reactors, as well as in dental prosthetics and even in some bicycle frames and golf clubs.245,246 The great utility of beryllium is offset by the potential human toxicity of the element and all its associated compounds. A safe alternative has not been discovered. The manufacturing of these products results in exposure not only to beryllium silicate but also to beryllium oxide, hydroxide, fluoride, chloride and sulfate.246 Exposure also occurs during mining and extraction of beryllium ores. The first ambient air quality standard ever established was that set in 1949 for beryllium, which preceded all others by some 25 years.247,248 Hundreds of thousands of workers have been exposed to beryllium. There is also the potential for bystander exposure in individuals who work in facilities that use beryllium but do not handle the metal itself. In addition exposure can occur in those who live in the vicinity of such facilities, or who handle the clothing of beryllium workers.245,248,249 Both acute and chronic beryllium disease has largely been brought under control as a result of the establishment of air quality standards, limiting exposure and discontinuance of beryllium in fluorescent lights.249 Low levels of exposure may result in the development of clinical disease, and a significant number of individuals remain at risk of developing beryllium lung disease.245,248 Despite strict regulation, an estimated 2–16% of exposed individuals may develop chronic beryllium lung disease and new cases will probably continue to be reported.245

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Exposure to beryllium results in sensitization, i.e., the development of a beryllium-specific cell-mediated immune response, in 2–19% of exposed individuals. Up to 50% of screened workers show evidence of sensitization. Sensitization follows the recognition of the element by CD4 T-cells, as either an antigen or hapten. This is the mechanism for the beryllium lymphocyte proliferation test, used clinically as a biomarker. It may be performed using lymphocytes from the peripheral blood, or those obtained using bronchoalveolar lavage. Following T-cell activation, inflammatory cytokine release, including interleukins and TNFa, leads to macrophage aggregation, further elaboration of pro-inflammatory cytokines and ultimately granuloma formation and fibrosis.245 The development of chronic parenchymal lung disease varies within different exposed occupational groups. Machinists appear to have the highest incidence of sensitization and chronic beryllium disease. In some series 10% of the sensitized workers will develop chronic beryllium disease annually. Some population-based studies show chronic disease will eventually develop in 2–16% of such cases.249 The overall low prevalence of the disease implies that host susceptibility is an important factor.245,248 A genetic predisposition to the development of chronic beryllium disease is suggested in both animal models and humans, with an association of the HLA-DPB1 Glu 69 allele.245 Currently, the clinical application of genetic markers as screens for those at risk of developing chronic beryllium disease is limited by the low prevalence of beryllium sensitization and beryllium disease itself, and the low specificity of the markers.249 Berylliosis, the term used for pulmonary disease resulting from exposure to beryllium, occurs in both acute and chronic forms. Acute berylliosis is a form of acute pneumonitis resulting from short-term exposure to high levels of soluble salts of beryllium (> 25 µg/m3), with symptoms beginning a few hours to several days after exposure. This has been reported in those engaged in the primary production of beryllium metal, or those exposed to beryllium-containing compounds in the fluorescent light industry. Acute beryllium pneumonitis shares the clinical features of other acute inhalational injuries, with nasopharyngeal irritation, as well as dyspnea, cough and other symptoms of lower airway irritation.247 These patients may be quite ill and febrile, with hypoxia and diffuse bilateral pulmonary infiltrates on chest X-ray. In fatal cases of acute berylliosis, the lungs are wet, heavy and congested with the nonspecific histological findings of diffuse alveolar damage, including edema, alveolar epithelial injury and scattered inflammatory cells within the interstitium and alveolar spaces. This may be accompanied by nasopharyngitis, tracheobronchitis and dermatitis.247 Approximately 10% of individuals with acute berylliosis will develop the chronic form of the disease, which may also occur in individuals with no history of acute berylliosis.245,250 Acute beryllium pneumonitis is largely of historical interest.

Chapter 14: Occupational lung disease

Figure 34. Berylliosis. This compact granuloma features several multinucleated giant cells.

Figure 33. Berylliosis. Interstitial compact non-necrotizing granulomas resemble sarcoidosis.

Chronic beryllium disease was described in fluorescent lamp workers, as well as other beryllium workers and their household contacts. It is also seen in individuals living in areas that surround sites manufacturing beryllium-containing products.251 In contrast to acute disease, this process is a subacute and systemic disease, mediated by a delayed-type hypersensitivity reaction to beryllium. Individuals present with nonspecific constitutional symptoms and dyspnea. Chronic beryllium disease may be indolent and asymptomatic or patients may have insidious onset of dyspnea. This may first become manifest 15 or more years after initial exposure and follow a variable clinical course. Mortality rates may be as high as 38% due to chronic fibrotic lung disease. In chronic berylliosis, the lungs are small, fibrotic, and may show honeycomb changes. Bilateral hilar lymphadenopathy can be present.252 Histological examination shows a pattern similar to pulmonary sarcoidosis, with interstitial lymphocytic infiltration and non-necrotizing granulomatous inflammation (Figures 33 and 34). Severe cases may progress to a diffuse but nonspecific pattern of interstitial fibrosis with development of honeycombing. Granulomas may be inconspicuous or absent in some cases. Giant cells often contain inclusions, such as Schaumann bodies, which are basophilic, laminated calcospherites, or asteroid bodies (see Chapters 2 and 13).152,253 In cases where lymphocytic interstitial infiltrates are prominent and granulomas are scattered and poorly formed, berylliosis may be difficult to distinguish from hypersensitivity pneumonitis (see Chapter 12). When there are abundant non-necrotizing granulomas, berylliosis must be distinguished from sarcoidosis (see Chapter 13).

In patients suspected of having beryllium lung disease, a beryllium lymphocyte proliferation test is undertaken. Lymphocytes from peripheral blood or bronchoalveolar lavage fluid are evaluated to demonstrate in vitro beryllium-induced lymphocyte proliferation. Such is indicative of beryllium hypersensitivity, but not necessarily disease.254 The pathological diagnosis depends on the finding of one of the previously described histological patterns, together with an appropriate exposure history or elevated tissue levels of beryllium. As beryllium is gradually cleared from tissue and excreted in the urine, it may not be detected in all cases of chronic beryllium disease.245,255 Beryllium has classically been identified in lung tissue by wet chemical spectrographic techniques. Individual particles have also been detected in situ by means of laser microprobe, ion microprobe mass spectrometry or electron energy-loss spectrometry. The low atomic number of beryllium poses problems for the identification of the element using conventional microprobe analysis. These authors have recently described the detection of beryllium using atmospheric thinwindow energy-dispersive X-ray analysis.255 This technique is advantageous since it is non-destructive and may be performed using routine formalin-fixed, paraffin-embedded sections.255 Energy-dispersive X-ray analysis is a potentially useful tool to complement a detailed review of exposure history, as a number of metals including gold, copper, cobalt, titanium and zirconium demonstrate similar morphological findings similar to beryllium, including granulomas.256,257 Exposure to beryllium probably results in an increased risk of lung cancer.258,259

Hard-metal (cobalt) lung disease (tungsten carbide pneumoconiosis) Hard metal is the generic term for a group of synthetic materials composed predominantly of tungsten carbide, with properties of extreme strength, rigidity and heat resistance. The hardness of this compound is similar to diamond; it is

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Figure 36. Hard metal lung disease. Bronchoalveolar lavage preparation showing “cannibalistic” giant cells. Wright’s stain

Figure 35. Hard metal lung disease. Patchy ground-glass opacities are seen on chest radiographs.

therefore useful in the manufacture and application of cutting tools, drilling equipment, armaments, alloys and ceramics. The occupational use of hard metal implements is associated with lower exposures to cobalt or hard metal dust than the maintenance and sharpening of such tools, or the manufacture of the hard metal itself. The solid and polycrystalline form of hard metal is produced in a metallurgic process, known as sintering, where tungsten and carbon are heated and cemented in the presence of a binder, typically cobalt. The fine dust particles generated during this process are less than 2.0 µm in diameter and thus in the respirable range. The finished product contains 5–25% cobalt, by weight. There is also a very high proportion of cobalt in bonded diamond tools. This is critical to the development of lung disease. Exposure to respirable hard metal dust may cause interstitial lung disease, known as giant cell interstitial pneumonia (GIP) in the original Liebow classification of idiopathic interstitial lung disease.260 Hard metal lung disease (HMLD) differs from many pneumoconioses, as the development of parenchymal lung disease is probably more dependent on host factors and individual susceptibility, rather than an accumulation of dust.261 The low prevalence of HMLD supports variable susceptibility to the development of HMLD. Balmes concluded the development of sensitization to hard metal dust, rather than the dust burden per se, provides the basis of the disease.262 The pathogenesis of HMLD is incompletely understood, but cobalt, rather than tungsten or tungsten carbide, is believed to be the critical etiological agent. Cobalt exposure alone has not been reported to result in significant parenchymal lung disease, but its toxicity is enhanced by other metallic carbides. Animal studies show that intratracheal instillation of tungsten or tungsten carbide is innocuous, whereas mixtures of

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tungsten carbide and cobalt result in a pneumonitis.263 Cobalt has pro-oxidant properties and a role for the transition metal in the genesis of reactive oxygen species and free radicals leading to lung injury has been suggested.264,265 It has been further proposed that CD163(þ) macrophages and cytotoxic T cells may act in concert to form the fibrotic lesions of HMLD following the phagocytosis of tungsten.266 HMLD can develop after brief exposures, also suggesting that individual susceptibility may be important in the pathogenesis of the disease. The HLA-DPglu69 allele, associated with susceptibility to chronic beryllium disease, is reportedly associated with susceptibility to cobalt-related interstitial lung disease.267 Hard metal lung disease most often presents as an obstructive airways disease, resembling asthma, hypersensitivity pneumonia or chronic fibrosing interstitial lung disease. Extrapulmonary manifestations of hard metal exposure include contact dermatitis. Interstitial lung disease in tungsten carbide production workers is associated with elevated peak air concentrations of cobalt in excess of 500 µg/m3,268 although some cases have occurred following exposures of less than 50 µg/m3. Interstitial lung disease occurred in less than 1% of individuals at risk in cross-sectional studies of current workers. The obstructed airways syndrome in tungsten carbide workers has also been correlated with cobalt exposure, and occurs in approximately 10% of workers at risk.269 The radiographic features of HMLD are nonspecific and varied, including patchy lobular ground-glass opacities (Figure 35).270,271 Bronchoalveolar lavage may provide useful information in the diagnostic evaluation. Cytology preparations feature giant cells of probable macrophage origin, with bizarre and “cannibalistic” features (Figure 36).272 Cells recovered from lavage fluid can also be analyzed to detect the presence of tungsten carbide and cobalt.273 Lung biopsies at early stages of the disease show a giant cell interstitial pneumonitis (Figure 37).274 In some cases, the microscopic appearance of airway-centered

Chapter 14: Occupational lung disease

inflammation with giant cells resembles hypersensitivity pneumonitis or desquamative interstitial pneumonitis (Figure 38). Late-stage disease may feature diffuse and severe interstitial fibrosis, mimicking UIP. Epithelial hyperplasia may be exuberant (Figure 39). Hard metal lung disease must be distinguished histologically from the hypersensitivity and fibrosing interstitial pneumonitides. The distinction is often based on the exposure history and the detection of tungsten carbide particles in lung tissue. Giant cell interstitial pneumonitis is almost

pathognomonic of hard-metal lung disease, requiring only a confirmatory occupational exposure history.274 Polykaryocytic giant cells may be present in HIV and other viral infections, as well as other systemic illnesses, but immunohistochemistry and correlation with the clinical and microbiological data discriminate the processes. Cobalt is only detected in approximately 10% of lung tissue samples from patients with HMLD, due to the solubility of the element in body fluids.275 Tungsten particles may be identified in formalin-fixed, paraffin embedded biopsy material in cases of HMLD (Figure 40). Coates and Watson276 identified tungsten carbide, cobalt and titanium in lung tissue of tungsten carbide workers with interstitial fibrosis by mass spectroscopy. The detection of cobalt in tissues of these individuals probably depends on both the sensitivity of the technique employed and the time since one’s last exposure.

Siderotic lung disease: iron, hematite miner’s lung

Figure 37. Hard metal lung disease. Interstitial pneumonitis with (inset) prominent intra-alveolar giant cells.

(a)

Occupational exposure to iron occurs in the mining of iron ore, welding, smelting, foundry work, manufacture of steel and metal alloys and the use of abrasives containing iron oxide. Iron oxide is considered inert, and the inhalation of iron oxide dust causes siderosis. Siderosis is of minimal clinical and pathological significance, although radiographic abnormalities and the development of interstitial fibrosis have been described following intense exposures.254,277 Iron oxide “fibers” may form ferruginous bodies with dark cores (Figure 41). Hematite, or iron sesquioxide (Fe2O3), is a type of iron ore mined in western Europe and in the Great Lakes area of the (b)

Figure 38. Hard metal lung disease. (a) At low magnification panlobular involvement resembles desquamative interstitial pneumonia. (b) Intra-alveolar macrophages appear to mold to the contours of the inflamed and fibrotic alveolar walls.

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Chapter 14: Occupational lung disease

Figure 39. Hard metal lung disease. Alveolar septa with epithelial hyperplasia and prominent giant cells. Figure 40. Hard metal lung disease. Note giant cell. Refractile silicates and iron observed using polarized microscopy with (inset) confirmed presence of tungsten using SEM and EDXA.

Figure 41. Digest of lung tissue from iron foundry worker showing ferruginized iron oxide “fiber”.

United States. The ore contains iron oxide and varying amounts of silica and silicates. Thus, miners exposed to hematite may develop a form of siderotic lung disease, similar to welders (see below). Although iron oxide is considered inert, the presence of crystalline silica may result in varying degrees of fibrosis, a process sometimes referred to as siderosilicosis (Figure 42). The complications of exposure to hematite, similar to those seen with coal dust, include cor pulmonale in patients with massive fibrosis and tuberculosis. An unexpected increased prevalence of lung cancer has been observed in hematite miners in west Cumberland, United Kingdom, where the mines are contaminated with radon gas. The resulting radiation exposure may offer a partial explanation for the occurrence of lung cancer in these miners. Severe pulmonary hypertensive vasculopathy, following inhalation of silicohematite dust,278 has been reported with hematite dust deposition. The changes are within intima and adventitia of veins, leading to fibrotic occlusion and recanalization.

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Figure 42. Siderosilicosis. Accumulations of iron oxide around areas of interstitial fibrosis and microscopic silicotic nodule.

Silicon carbide pneumoconiosis Silicon carbide (carborundum) is a very hard synthetic abrasive. It is used for abrasive wheels and in the manufacture of refractory materials for boilers and foundry furnaces. Silicon carbide is made by fusing high-grade sand, finely ground carbon, common shale and wood dust at high temperature. The fused product is then crushed and milled to remove impurities. Silicon carbide is thought to be inert in humans, and does not produce fibrosis in experimental animals. When regional fibrosis (Figure 43) or silicosis are described in silicon carbide workers, it is attributable to the use of quartz dust in the production process.279

Rare earth pneumoconiosis Rare earth pneumoconiosis is caused by the inhalation of lanthanides, the 14 elements with atomic numbers 58 to 71

Chapter 14: Occupational lung disease

Figure 43. Silicon carbide worker. Minimal interstitial fibrosis and a prominent alveolar macrophage response.

Figure 45. Dental technician’s pneumoconiosis. Chest radiograph showing upper lung zone fibrosis.

in the periodic table. Rare earth metals are commonly used industrial materials, principally in the manufacture of catalytic converters, mirrors and optical lenses, including sunglass lenses.280 Poorly characterized and infrequently described, the clinical and pathological findings in the few cases of rare earth pneumoconiosis are variable, with the common histopathological observations of granulomatous inflammation and interstitial fibrosis (Figure 44). The lanthanide most frequently implicated is cerium, owing to its prominence among the rare earths in industrial usage. A hypersensitivity response to the element leading to interstitial fibrosis over time, akin to the hypersensivity response as established in chronic berylliosis, seems probable. Cytotoxic injury, resultant from lanthanide exposure, has been demonstrated in vitro.280,281 This provides another possible mechanism for the development of interstitial fibrosis in these cases. In addition to interstitial fibrosis, birefringent particles of cerium oxide may be demonstrated using polarized microscopy.

Figure 44. Rare earth pneumoconiosis. Diffuse interstitial fibrosis in an optical lens grinder.

Dental technicians’ pneumoconiosis Dental laboratory workers are exposed to a dusty environment that may include a number of different particles potentially injurious to the lung. Precious metals, porcelain and alloys are used to make crowns and bridges. These dental appliances may also contain chromium, molybdenum, nickel, cobalt, as well as beryllium and aluminum.282,283 Prosthetic devices made of metal alloys are polished with high-speed abrasive wheels that generate respirable silica or silicon carbide. In addition, asbestos molds are used in the process of dental gold casting. Exposure to substantial levels of aerosolized asbestos fibers may also occur when these molds are dismantled. Recent studies suggest that chromium-cobalt-molybdenum alloys may be found in the lungs of some dental technicians and may be an important factor in the development of pneumoconiosis.283 These alloys cleave into elongated fragments that subsequently become coated with iron, to produce a type of non-asbestos ferruginous body (see below). Other investigators have suggested a pathogenic role for acrylic resins used in the preparation of dental prostheses,284 or alginate impression powder in dentists with pneumoconiosis.282–285 The radiographic features of dental technician’s pneumoconiosis may include significant upper lung zone opacities and bulky nodular masses on chest X-ray (Figure 45). Histological examination of lung tissue in such workers may demonstrate dense interstitial fibrosis and polarizable particulate matter, the latter shown to consist of silica and silicon carbide, and metals such as aluminum, gold, tin and titanium (Figure 46). The authors have also seen an unusual example of silica-associated histiocytosis and parenchymal fibrosis without typical silicotic nodules in the lung of a denture maker undergoing lung transplantation for severe pulmonary fibrosis (Figure 47).

Benign pneumoconioses/nuisance dusts Titanium pneumoconiosis Titanium dioxide is a white powder extensively used in the dye industry. Grinding titanium oxide generates 0.5–1.0 µm

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The pneumoconiosis related to tin exposure is called stannosis. The greatest exposure occurs during bagging of processed tin ore or during smelting. Accumulation of tin in the lung causes striking interstitial opacities, visible by chest radiography because of its high atomic number (Z ¼ 50). As in welders’ siderosis, the radiographic changes are caused by the dust itself, rather than by fibrosis. The lungs of tin workers show gray-to-black, 2 to 5 mm macules, which are distributed fairly evenly throughout their lungs. Secondary lobular septa may stand out in bold relief as a result of dust accumulation; massive fibrosis does not occur. As much as 3 g of tin dioxide may be recovered from individual lungs. Histology shows dust-laden macrophages in a perivascular and peri-bronchiolar location, subpleurally, within both secondary lobular septa and hilar lymph nodes.5 The dust particles do not elicit a fibrous reaction and are brightly birefringent.

Barium pneumoconiosis (baritosis)

Figure 46. Dental technician’s pneumoconiosis. (a) Dense and confluent interstitial fibrosis. (b) Birefringent material corresponding to silica, silicon carbide, gold, tin, chromium, titanium and aluminum.

particles. Non-fibrogenic in animal models, titanium dioxide is considered an inert nuisance dust. Inspection of the lungs of former titanium workers shows deposits of white pigment that may have a greenish hue.286 Fibrosis is not observed. Microscopic examination shows dust-laden macrophages in a perivascular and peribronchiolar distribution (Figure 48a). The particles are birefringent, when viewed with polarized light (Figure 48b). Rutile is a form of titanium dioxide that may occur in a fibrous form in association with silica. These fibers may become coated with iron to form pseudoasbestos bodies with dark central cores.287 Titanium carbide is used in the hard-metal industry (see above), and like tungsten carbide is considered to be biologically inert.

Stannosis Tin is a silver-white metal of industrial importance, because of its pliability and ability to readily form alloys with other metals. It is mined principally as tin dioxide ore (cassiterite), and deposits are often closely associated with quartz-containing rock. In the past, miners of tin ore often developed silicosis but the risk has decreased greatly with the introduction of wet drilling procedures.

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Barium sulfate (BaSO4, barite) is a relatively insoluble salt of barium, mined in Mississippi, Nevada and Georgia, USA,288 as well as Peru, Colombia, China, Turkey, Pakistan, Hong Kong, Thailand and India. It is often found in combination with other minerals, such as calcite, fluorite and silica (quartz). Barium sulfate is used in the manufacture of paint, vulcanized rubber, glass and radiographic contrast media. Exposure to barium may occur during mining or during its industrial application. Radioopaque and insoluble, it is useful as a radiographic contrast medium. It may be aspirated following upper gastrointestinal imaging (see Chapter 17). Barium accumulation in the lung results in striking opacities, visible on chest radiographs because of its high atomic number (Z ¼ 56). On gross inspection of the lung, white patches may be observed. Microscopically, most of the particles occur in alveolar spaces, with few in the interstitium. They are refractile and brightly birefringent when viewed with polarized light. A few particles may assume a fibrous shape. Many particles are phagocytized by alveolar macrophages. Little, if any, fibrosis occurs, and its presence should prompt a search for a contaminating fibrogenic agent, such as quartz. When barium sulfate is aspirated, simultaneous aspiration of other materials may be found in the form of foreign-body giant cells, or particulate food matter.

Lung diseases caused by metal fumes Fumes are composed of particles less than 1 µm and as small as 0.1 µm in diameter. Many particles, including aluminum, cadmium and mercury, behave like vapors or gases, and hence penetrate readily into the air-exchanging regions of the lung. They rapidly enter the alveoli, and, if irritative, can lead to a chemical pneumonitis that may result in diffuse interstitial fibrosis. The initial chemical pneumonitis presents as acute respiratory distress syndrome with pathology of diffuse alveolar damage.

Chapter 14: Occupational lung disease

Figure 47. Dental technician’s pneumoconiosis. (a) Extensive bilateral interstitial fibrosis is noted. (b) Nodular histiocytic aggregates replace lung parenchyma. (c) Refractile acicular crystalline material, viewed using polarized microscopy. (d) Energy dispersive X-ray analysis showing silicon.

Welders’ pneumoconiosis (WP) Welding is the process of joining metals by softening with electrically generated heat. Welders are a heterogeneous group of workers, working indoors and out, as well as underwater, in different jobsite ventilatory conditions. Welding materials are typically alloy mixtures of different metals. Welders are exposed to various fumes, derived from the consumable electrode wire and noxious oxidant gases, whose composition varies with the type of welding.289 Welding fumes contain vaporized and condensed particles in the range 0.1–1 µm in diameter. They are typically a variety of metal oxides,

including iron, titanium, manganese and aluminum, together with various silicates and carbonates. The nature of the pulmonary reaction to welding fumes depends on the duration and intensity of exposure, as well as the predominant component of the fumes. Chronic exposure to high concentrations of fumes from aluminum arc welding may result in accumulation of significant amounts of aluminum oxide. This has been associated with a variety of tissue changes, including severe interstitial fibrosis. In contrast, titanium and iron oxides appear to be relatively inert. Metal fume fever is the most common acute respiratory complaint among welders. This self-limited febrile illness is

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Figure 49. Welders’ pneumoconiosis. Interstitial macular accumulations of iron are seen.

Figure 48. Titanium worker’s lung. (a) Dust-laden macrophages are in a predominantly peribronchovascular distribution. (b) Examination under polarized light shows nodular birefringent material.

caused by exposure to welding fumes containing zinc, copper, magnesium and/or cadmium. Small numbers of heavily exposed welders suffer from decreased lung function, but pulmonary function within welders with siderosis has not been demonstrated to differ significantly from matched, nonwelding controls.294 The issue of obstructive lung disease caused by welding is controversial, and complicated by smoking. Emphysema has been described as the predominant radiographic abnormality in welders,271 but there is no evidence to suggest the welding process leads to significant airflow obstruction.290 Radiographically, WP demonstrates diffusely distributed, ill-defined micronodules. Chest radiographs may show prominent interstitial markings, often mistaken for interstitial fibrosis. These interstitial markings result from extensive metal deposits. On gross inspection, the lungs involved with WP are deeply pigmented, but usually there is little or no fibrosis. In fact, the identification of pleural thickening or interstitial fibrosis macroscopically suggests exposure to an additional agent, such as asbestos in shipyard welders. Short-term and low-level exposures to welding fumes produce little histopathological change in lung tissue. Chronic exposure to welding fumes may lead to the development of

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Figure 50. Lung tissue digest from welder. Non-asbestos ferruginous bodies are found.

siderotic lung disease, which is a “benign” form of pneumoconiosis. Microscopically, WP features interstitial macrophages filled with dust. Little or no fibrosis is seen. The dust consists of dark brown to black spherical particles, often occurring as macular aggregates, in a peribronchiolar or perivascular distribution (Figure 49). The dust is principally iron, with a golden-brown outer layer and dark centers in some particles. This probably represents in situ conversion of iron oxide to iron hydroxide (hemosiderin). Various sheet silicates may also be found in the lungs, and may be partially coated with iron, to form pseudoasbestos bodies or non-asbestos ferruginous bodies (Figure 50). Of note, there are rare reports of extensive interstitial fibrosis, resulting from severe exposure to welding fumes occurring in poorly ventilated job sites.277 Aluminum fume-induced pneumoconiosis among aluminum arc welders is infrequent. A metallic sheen, resembling tarnished aluminum, has been described in the lungs of such workers, and diffuse fibrosis may occur with exposure to aluminum oxides (Figure 51).291 In addition to diffuse

Chapter 14: Occupational lung disease Figure 51. Lung specimen from an arc welder. Note interstitial fibrosis and gray sheen within hilar lymph nodes.

Figure 52. Aluminum worker. Interstitial and airspace macrophages containing aluminum.

Figure 53. Alveolar proteinosis in aluminum worker.

interstitial fibrosis, granulomatous inflammation291–293 and a desquamative interstitial pneumonitis-like morphology have also been described in aluminum arc welders.294 Despite extensive study, assessment of lung cancer as an occupational risk among welders is difficult.295 Welding fumes are listed as “possibly carcinogenic” by the IARC.296 Some studies suggest welders have an increased risk for developing bronchogenic carcinoma.289 A study from the Danish Welding Institute in Copenhagen indicated the excess lung cancer risk for welders can be accounted for by cigarette smoking, asbestos (e.g. in shipyard welders) and hexavalent chromium exposures (e.g. in stainless steel welders).297–299

Aluminum lung disease Aluminum is the most abundant metal in the earth’s crust and has many commercial and industrial applications. Occupational exposures may occur during the refining and production of aluminum and aluminum-containing compounds. This causes pneumoconiosis, granulomatous lung disease and

interstitial fibrosis.265 The prevalence of disease is probably low, in view of the large number of workers exposed to alumina. The pathogenesis of lung disease related to such exposure is not completely understood. Bauxite (aluminum ore) is mined in open pits, and electrolysis converts bauxite to alumina. The latter process is undertaken in large pots. The pot room environment may contain alumina dust, metal fumes and fumes from other organic and inorganic compounds, leading to the development of asthma, bronchitis, as well as parenchymal lung disease. Pulmonary fibrosis may result from exposure to fumes generated during the manufacture of aluminum abrasives.300 Corundum, a synthetic form of crystalline aluminum oxide made by finely grinding bauxite, iron and coke and fusing the mixture in an electric furnace, is chiefly responsible. The resulting fumes also contain amorphous silica, ferric oxide and traces of titanium oxide and other constituents. Individuals exposed to these fumes may become dyspneic and develop radiographic changes as early as 3 months following exposure. Pneumothorax can occur. In fatalities, the lungs are heavy, gray-black, with scattered dense fibrotic areas. Pleural adhesions and large subpleural emphysematous bullae can be seen and may lead to a spontaneous pneumothorax. Microscopically, there is diffuse alveolar septal fibrosis, associated with aggregates of brown-black dust particles (Figure 52). Churg et al. described a case of alveolar proteinosis in an aluminum rail grinder, and these authors have also demonstrated the presence of increased pulmonary aluminum content in an exposed worker with alveolar proteinosis (Figure 53).301 Progression to diffuse fibrosis with obliteration of alveolar spaces is prognostically ominous. Analyses of lungs show the same constituents that are present in bauxite fumes. Interstitial fibrosis is also described among aluminum smelters, aluminum arc welders and aluminum polishers. This process generally manifests as upper lung zone reticulonodular fibrosis.271,300,302,303

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Cadmium pneumonitis Cadmium is used in the manufacture of alloys and alkaline accumulators and in the control of atomic reactors. Cadmium oxide fumes can be generated in electroplating, alloy and solder production, and also during welding of steel with cadmium, used as an anticorrosive.5 However, the most likely environmental source of cadmium exposure is tobacco smoke. It is estimated each cigarette contains 2 µg.304 Given the metal’s low boiling point and high vapor pressure, industrial usages may give rise to toxic concentrations of cadmium in exposed workers. Such exposures may result in a chemical pneumonitis and severe acute alveolar injury.265 Symptoms may begin within 8 hours of exposure, starting with throat irritation, followed by pulmonary edema and death in approximately 16% of cases. In acutely fatal cases, the lungs are heavy and microscopically show diffuse alveolar damage. Chronic cadmium exposure is associated in epidemiological studies with emphysema and interstitial fibrosis.265 While edema, perivascular fibrosis and chronic lymphocytic infiltration are described, these are nonspecific findings. Diagnosis requires correlation with occupational history and/or analysis of lung tissue for cadmium.

Mercury pneumonitis Mercury is chiefly used in the electronics industry, as well as in the production of explosives and metallurgy. Inhalation is the main route for exposure to elemental mercury. Organic mercury compounds and mercury salts, by contrast, are absorbed through the gastrointestinal tract. Mercury vapors are produced during the volatilization of mercury. Exposure to these vapors has been reported to produce a chemical tracheobronchitis that may progress to pulmonary edema.305,306 Diffuse alveolar damage has been described in fatal cases.306

Lung disease caused by non-asbestos mineral fibers A fiber is generally defined as a mineral particle with an aspect (length-to-diameter) ratio of 3:1 or greater and roughly parallel sides. A variety of non-asbestos mineral fibers have been identified in the human lung (Table 9).307,308,312 Some of these have been discussed in previous sections, including silica and other silicates (e.g. talc, kaolin), rutile (titanium dioxide) and barium sulfate. Others, such as apatite, feldspar, gypsum, illite, mullite, pyroxene, pyrophyllite, sericite and sillimanite, are identified in human lung tissue but not implicated in disease. The remaining non-asbestos mineral fibers are the subject of this section. Most of these minerals occur in both fibrous and non-fibrous forms. A wide range of pathogenic potentials is associated with these mineral fibers, and the mechanisms of tissue injury are incompletely understood.

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Table 9 Non-asbestos mineral fibers that may be found in human lung tissue

Apatite

Richterite

Attapulgite

Rock wool

Carbon

Rutile

Erionite

Sericite

Feldspar

Silica

Fibrous glass

Sillimanite

Gypsum

Talc

Illite

Vermiculite

Kaolinite

Winchite

Mullite

Wollastonite

Pyrophyllite

Zeolite

Refractory ceramic fibers

Man-made fibers, mineral and synthetic Man-made mineral fibers (MMF) possess the qualities of high tensile strength and resistance to heat, cold and chemicals. On account of these properties, MMF have to a considerable extent replaced asbestos in industrial applications, including ceramics, insulation, textiles, structural materials and filters. This group is composed of cylindrical amorphous silicates, and includes fibrous glass (fiber glass), rock wool and refractory ceramic fibers (RCF). Fibrous glass is manufactured from borosilicate and low alkaline silicate glasses mixed with various amounts of silica, soda, lime, aluminum, and titanium.309 Rock wool is made by melting and drawing it out into a fibrous form from naturally occurring rock. RCF are made from raw materials, such as kaolin and alumina/silica. These materials tend to fall within the respirable size range, and may have greater biodurabiltity than other MMF.310 They may form ferruginized pseudoasbestos bodies (Figure 6). Lengthy employment in the RCF industry has been associated with the development of pleural plaques on chest X-rays without pulmonary interstitial fibrosis (see Chapter 36).311,312 Man-made fibers have a wide range of fiber diameters depending on the particular industrial application. They vary from microfibers 0.05 µm in diameter to coarse glass fibers 254 µm in diameter.232 Similarly, the lengths of individual fibers may range from less than a micrometer to many hundreds of micrometers. Man-made fibers tend to break perpendicularly to their length, rather than separating longitudinally, as happens with asbestos fibers. In view of the known risks caused by inhalation of asbestos fibers, MMFs are well studied both experimentally and epidemiologically. The IARC considers glass, rock and slag wools unclassifiable as human carcinogens while RCF are classified as possible human carcinogens.313–316 Dimensional considerations are very important in any analysis of the biological activity of fibers. Because of the

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tendency of fibers to align with their long axes parallel to the air flow, the aerodynamic behavior of fibers is relatively independent of fiber length. Fibers with a diameter of 3.5 µm or less may penetrate to the lung periphery, whereas larger-diameter fibers impact mostly in the upper airways. Fibers with lengths ranging well over 200 µm can penetrate into respiratory bronchioles and alveolar ducts, provided they have an appropriately thin diameter. Experimental studies with fibrous glass show long fibers are more fibrogenic than shorter ones.317 In addition, long (> 8 µm) and thin (< 1.5 µm) glass fibers are the most carcinogenic in an animal model of pleural mesothelioma.318 Thus, long and thin fibers are particularly dangerous because of their access to distal airways and biological activity. Physicochemical characteristics seem to confer a low degree of biopersistence of MMF in human lung.319 Fibrous glass is relatively brittle, especially in comparison with asbestos, and fibers appear soluble in vivo. Long glass fibers tend to fragment into shorter fibers, which are then transported to regional lymph nodes. Further, fibrous glass dissolves in tissues, particularly in an alkaline milieu.319 These tendencies probably account to some extent for the relative lack of fibrogenicity of fibrous glass, compared with asbestos fibers of comparable dimensions.313 Most epidemiological studies indicate man-made mineral fibers do not produce chronic respiratory disease in exposed workers.232,319–321,323–325 However an excess of non-malignant respiratory disease in a group of fibrous glass workers was reported in one study.322 Gross et al.323 found no significant difference in the fiber content of lungs from individuals who had worked for up to 30 years in the fibrous glass industry, compared to controls. A sole epidemiological study showing a slightly increased risk of mesothelioma in fibrous glass workers has not been confirmed.101 Despite concerns for carcinogenicity, a recent multi-center European case-control study did not demonstrate a significantly increased risk of lung cancer in cohorts exposed to MMF and RCF.313 Non-mineral synthetic fibers, by contrast, consist of a large number of non-naturally occurring organic polymers, whose physical characteristics allow for respirability. The organic fibers with potential for causing respiratory disease include polyamides, aramids and polyolefins. Some members of this class of organic fibers, e.g. polyvinyl alcohols and some types of carbon fibers, may not be respirable owing to size. Ghio et al. described the development of interstitial lung disease in patients exposed to synthetic textile fibers.324 Interestingly, the lung biopsies in these cases demonstrated accumulations of iron, similar in appearance to ferruginous bodies. Energy-dispersive X-ray analysis showed the fiber cores were carbonaceous, similar in terms of appearance and composition to free fibers collected at the textile mill. This indicates that such fibers may sequester and complex iron and generate oxidative stress within tissue.101

Nylon flock worker’s lung Nylon comprises a group of synthetic condensation polyamide polymers and plastics. The strength of nylon allowed for its original use as replacement for natural fibers in fashion and textiles. A powder of short fibers, such as cut nylon and other synthetic fibers, is termed “flock”. This powder is applied (“flocked”) to fabric and used in the creation of plush upholstery surfaces. Certain techniques in the manufacture of flock may lead to the creation of respirable-sized particles. Outbreaks of respiratory illness among workers exposed to flock are occasionally reported.325–327 While nylon fiber size may inhibit respirability, the particulate-sized and fragmented sheared ends of the fibers generated during the rotary cutting of flock are considered pathogenic.327 Clinically, nylon flock workers’ lung is characterized by dyspnea and cough, which may be accompanied by rales. Ground-glass and reticular opacities are observed on highresolution CT scans. The typical histopathological appearance of flock worker’s lung is a bronchiolocentric interstitial pneumonitis. The histopathology of nylon flock worker’s interstitial lung disease is further described as a lymphocytic bronchiolitis with lymphoid hyperplasia (Figure 54). This may mimic the interstitial lung disease that complicates such entities as Sjögren syndrome or lymphoid interstitial pneumonia. It lacks the granulomatous response seen with hypersensitivity pneumonia and the extensive monotonous infiltrate of atypical lymphocytes and angiocentricity identified in some pulmonary lymphomas.328 Desquamative interstitial pneumonia and bronchiolitis obliterans organizing pneumonia have also been described in the spectrum of histopathological findings observed in flock worker’s lung.327

Miscellaneous fibers and agents: carbon, non-asbestos ferruginous bodies and diacetyl carbon Carbon particles occasionally occur in a fibrous form and have been described in coal workers144 and in individuals with no known occupational exposure.329 Ramage et al. described a unique case of an elderly woman exposed to woodstove smoke, who developed dyspnea and radiographic interstitial changes.329 Numerous black fibers (some coated with iron) were recovered by bronchoalveolar lavage and open lung biopsy showed patchy interstitial fibrosis, associated with numerous black fibers (Figure 55a,b). Digestion studies of the biopsy specimen demonstrated more than 2 million black fibers per gram of lung tissue, and energy-dispersive X-ray analysis showed these contained no elements with atomic number greater than or equal to 11. The black fibers included lathe-like and grid-like structures, as well as bizarre forms, some of which were coated with iron (Figure 55c,d). The composition and appearance of these particles fits that described in woodstove dust.330 Studies regarding the cytotoxicity of certain carbon fiber composites indicated that some of these fibers are not inert. They cause irreversible injury to

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(a)

(b)

Figure 54. Nylon flock worker’s lung. (a) Bronchiolocentric interstitial fibrosis with focally dense lymphoid infiltrates, including nodules, are seen. (b) Peribronchiolar lymphocytes distort the airway.

Figure 55. Carbon particulate matter in lung tissue from patient with chronic exposure to woodstove smoke. (a) Interstitial histiocytes and fibrosis along with black fibers are seen. (b) Scanning electron micrograph of bronchoalveolar lavage showing broad carbon fragments. (c) Bizarre forms. (d) Iron-coated fibers produce so-called pseudoasbestos bodies.

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alveolar macrophages and large increases in airway cells and neutrophils in rat lungs, as well as the development of nodular interstitial fibrosis.331,332 Carbon nanotubes, the product of an emerging science involving materials at the nanometer scale, are coaxial layers of graphite. The physicochemical characteristics of the nanotubes include high mechanical strength and electrical conductivity. These features allow for their use in many biomedical and industrial applications, including in vivo delivery of drugs.333 They are synthesized to form seamless or stacked cylinders and feature extremely high aspect ratios.334 Catalytic materials, such as iron, cobalt and nickel, may be present within or on the nanotubes. These minerals, along with silica and alumina, may be important sources of reactive oxygen species.335 Carbon nanotubes, along with platy silicates and asbestos, have been identified in lung tissues of responders to the 2001 World Trade Center disaster who subsequently developed pulmonary interstitial fibrosis.336 It is suspected the high temperatures generated in the combustion of fuel and development of the complex dust cloud at the World Trade Center generated large numbers of the carbon nanotubes.336 While promising technologically, the structural aspects and low aqueous solubility of carbon nanotubes have prompted concerns over the potential for toxic properties akin to asbestos. When compared with larger respirable-sized particles of equivalent mass, ultrafine particles in general have a greater pulmonary deposition, and may result in exacerbations of chronic obstructive lung disease. Additional concerns over occupational exposure to carbon nanotubes exist. Animal models indicate carbon nanotube toxicity exceeds that of quartz, following deposition in the lung.337 Experimental pulmonary lesions include acute

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Popcorn worker’s/food flavorer’s lung disease Workers engaged in the processing and packaging of microwave popcorn and its flavoring agents have been reported to suffer excess rates of lung disease. They may develop fixed airways obstruction. The disease has been reported in both the USA and Europe.339–342 The investigators and NIOSH concluded workers in microwave popcorn plants are at risk of the development of “occupational bronchiolitis obliterans”, due to the inhalation of volatile butter flavoring agents, principally 2,3-butanedione (diacetyl). A similar clinical picture has been described in a worker, who manufactured flavorings for potato crisps.343 Animal studies suggest diacetyl’s potential to result in airway and pulmonary epithelial injury, following its inhalation.344–346 While it is apparent a subset of these workers suffers from significant respiratory illness, the diagnosis of bronchiolitis obliterans in these cases has been made predominantly on clinical and radiographic grounds and the histopathological descriptions are incomplete. In several cases of suspected popcorn worker’s lung, where surgical lung biopsy was undertaken, the findings were believed to be consistent with constrictive bronchiolitis obliterans (CBO), but contained granulomas to suggest alternative diagnoses. In two studies some cases had no findings of CBO, possibly representing sampling error at biopsy.340,346

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third follow up. 1989–1995. J Occup Environ Med 1999;41:294–303. 300. Shaver CG, Riddell AR. Lung changes associated with the manufacture of aluminum abrasives. J Ind Hyg Toxicol 1947;29:145–57. 301. Miller R, Churg AM, Hutcheon M, Lam F. Pulmonary alveolar proteinosis and aluminum dust exposure. Am Rev Respir Dis 1984;130:312–15. 302. Abramson MJ, Wlodarcyk JH, Saunders NA, Hensley MJ. Does aluminum smelting cause lung disease? Am Rev Respir Dis 1989;139:1042–57. 303. Gilks B, Churg A. Aluminum-induced pulmonary fibrosis: do fibers play a role? Am Rev Respir Dis 1987;136:176–9. 304. Newman-Taylor AJ. Cadmium, Chapter 70. In Rom WN, ed. Environmental and Occupational Medicine, 3rd ed. Philadelphia: Lippincott Raven, 1998. pp. 1011–21. 305. Kanluen S, Gottlieb CA. A clinical pathological study of four adult cases of acute mercury inhalational toxicity. Arch Pathol Lab Med 1991;115:56–60. 306. Rowens B, Guerrero-Bettancourt D, Gottlieb CA, Boyes RJ, Eichenhorn MS. Respiratory failure and death following acute inhalation of mercury vapor. Chest 1991;99:185–90. 307. Churg A. Non-asbestos pulmonary mineral fibers in the general population. Environ Res 1986;31:189–200. 308. Roggli VL. Non-asbestos mineral fibers in human lungs. In Russell PE, ed. Microbeam Analysis – 1989. San Francisco: San Francisco Press, 1989. pp. 57–9. 309. Baker D, Kupke KG, Ingram P, Roggli VL, Shelburne JD. Microprobe analysis in human pathology. In Johari O, ed. Scanning Electron Microscopy, vol. II. Chicago: SEM, 1985. pp. 659–80. 310. Lemasters GK, Lockey J, Levin LS, et al. An industry-wide pulmonary study of men and women manufacturing refractory ceramic fibers. Am J Epidemiol 1998;148:910–19. 311. Lemasters GK, Lockey J, Rice C, et al. Radiographic changes among workers manufacturing refractory ceramic fibre and products. Ann Occup Hyg 1994;38 (suppl 1):745–51.

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312. Lockey J, Lemasters G, Rice C, et al. Refractory ceramic fiber exposure and pleural plaques. Am J Resp Cit Care Med 1996;154:1405–10. 313. Carel R, Olsson A, Zaridze D, et al. Occupational exposure to asbestos and man-made mineral fibers and risk of lung cancer: a multicentre case-control study in Europe. Occup Environ Med 2007;64:502–8. 314. Kjaerheim K, Boffetta P, Hansen J, et al. Lung cancer among rock and slag wool production workers. Epidemiology 2002;13:445–53. 315. Pohlabeln H, Jockel KH, BruskeHohlfeld I, et al. Lung cancer and exposure to man-made vitreous fibers: results from a pooled case-control study in Germany. Am J Ind Med 2000;37:469–77. 316. Man-made Vitreous Fibres/IARC working group on the evaluation of carcinogenic risks to humans. IARC monographs on the carcinogenic risks to humans; 81. Lyon: IARC, 2002. 317. Wright GW, Kuschner M. The influence of varying lengths of glass and asbestos fibres on tissue response in guinea pigs. In Walton WH, ed. Inhaled Particles IV. Oxford: Pergamon, 1977, pp. 455–74.

324. Ghio AJ, Funkhouser W, Pugh CB, et al. Pulmonary fibrosis associated with exposure to synthetic fibers. Toxicol Pathol 2006;34:723–9. 325. Eschenbacher WL, Kreiss K, Lougheed MD, et al. Nylon flock-associated interstitial lung disease. Am J Respir Crit Care Med 1999;159:2003–8. 326. Kern DG, Crausman RS, Durand KT, et al. Flock workers lung: chronic interstitial lung disease in the nylon flocking industry. Ann Intern Med 1998;129:261–72. 327. Kern DG, Kuhn C, Ely EW, et al. Flock workers lung: broadening the spectrum of clinicopathology, narrowing the spectrum of suspected etiologies. Chest 2000;117:251–9. 328. Boag AH, Colby TV, Fraire AE, et al. The pathology of interstitial lung disease in nylon flock workers. Am J Surg Pathol 1999;23:1539–45. 329. Ramage JE Jr., Roggli VL, Bell DY, Piantadosi CA. Interstitial lung disease and domestic wood burning. Am Rev Respir Dis 1988;137:1229–32.

336. Wu M, Gordon RE, Herbert R, et al. Case report: lung disease in World Trade Center responders exposed to dust and smoke: carbon nanotubes found in the lungs of World Trade Center patients and dust samples. Envrion Health Perspect 2010;118:499–504. 337. Lam C-W, James JT, McCluskey R, Hunter RL. Pulmonary toxicity of single-wall carbon nanotubes in mice 7 and 90 days after intratracheal instillation. Toxicol Sci 2004;77:126–34. 338. Donaldson K, Aitekne R, Tran L, et al. Carbon nanotubes: a review of their properties in relation to pulmonary toxicology and workplace safety. Toxicol Sci 2006;92:5–22. 339. Akpinar-Elci A, Travis WD, Lynch DA, Kreiss K. Bronchiolitis obliterans syndrome in popcorn production workers. Eur Resp J 2004;24:298–302. 340. Kreiss K, Gomaa A, Kullman G, et al. Clinical bronchiolitis obliterans in workers at a microwave popcorn plant. N Engl J Med 2002;347:330–7. 341. Kanwal R. Bronchiolitis obliterans in workers exposed to flavoring chemicals. Curr Opin Pulm Med 2008;14:141–6.

318. Morgan A, Holmes A, Davison W. Clearance of sized glass fibres from the rat lung and their solubility in vivo. Ann Occup Hyg 1982;25:317–31.

330. McCrone WC, ed. The Particle Atlas, vols V and VI. 2nd ed. Ann Arbor: Ann Arbor Science, 1980. pp. 1336–634.

342. Kanwal R, Kullman G, Piacitelli C, et al. Evaluation of flavorings-related lung disease risk at six microwave popcorn plants. J Occup Env Med 2006;48:149–57.

319. McDonald JC, Case BW, Enterline PE, et al. Lung dust analysis in the assessment of past exposure of manmade mineral fibers. Ann Occup Hyg 1990;324:427–41.

331. Martin TR, Meyer SW, Luchtel DR. An evaluation of the toxicity of carbon fiber composites for lung cells in vitro and in vivo. Environ Res 1989;49:246–61.

343. Hendrick DJ. “Popcorn workers lung” in Britain in a man making potato crisp flavoring. Thorax 2008;63:267–8.

320. Enterline PE, Marsh GM. Environment and mortality of workers from a fibrous glass plant. In Lemen R, Dement DM, eds. Dusts and Disease: Occupational and Environmental Exposures to Selected Fibrous and Particulate Dusts. Park Forest South: Pathotox, 1979. pp. 221–31.

332. Luchtel DL, Martin TR, Boatman ES. Response of the rat lung to respirable fractions of composite fiber-epoxy dusts. Environ Res 1989;48:57–69.

321. Wright GW. Proceedings of the Second Symposium on Occupational Exposure to Fibrous Glass. Washington DC: US Government Printing Office, 1976. p. 126. 322. Bayliss D, Dement J, Wagoner JK, Blejer HP. Mortality patterns among fibrous glass production workers. Ann NY Acad Sci 1976;271:324–35.

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323. Gross P, Tuma J, de Treville TP. Lungs of workers exposed to fiber glass: a study of their pathologic changes and their dust content. Arch Environ Health 1971;3:67–76.

333. Shedova AA, Kisin ER, Porter D, et al. Mechanisms of pulmonary toxicity and medical applications of carbon nanotubes: two faces of Janus? Pharamcol Therap 2009;121:192–204. 334. Simeonova PP. Update on carbon nanotube toxicity. Nanomedicine 2009;4:373–5. 335. Muller J, Huaux F, Lison D. Respiratory toxicity of carbon nanotubes: how worried should we be? Carbon 2006;44:1048–56.

344. Hubbs AF, Goldsmith WT, Kashon ML, et al. Respiratory toxicology of inhaled diacetyl in Sprague-Dawley rats. Toxicol Pathol 2008;36:330–44. 345. Fedan JS, Dowdy JA, Fedan KB, Hubbs AF. Popcorn workers lung: in vitro exposure to diacetyl, an ingredient in microwave popcorn butter flavoring, increases reactivity to methacholine. Toxicol Appl Pharmacol 2006;215(1):17–22. 346. Van Rooy FG, Rooyackers JM, Prokop M, et al. Bronchiolitis obliterans syndrome in chemical workers producing diacetyl for food flavorings. Am J Respir Crit Care Med 2007;176:498–504.

Chapter

15

Eosinophilic lung disease Henry D. Tazelaar, Joanne L. Wright and Jay H. Ryu

Introduction There are numerous diseases associated with pulmonary eosinophilia.1,2 They represent a heterogeneous group of diseases that, except for the presence of eosinophils, sometimes bear little clinical relationship to one another. The most common disease associated with eosinophilia is asthma, and the pathologist’s role in the diagnosis is non-existent, except when there are complications. The most common diffuse disease associated with eosinophilia is chronic eosinophilic pneumonia, which may or may not be associated with asthma. These and other less common diseases are discussed in this chapter. Pulmonary Langerhans’ cell histiocytosis (formerly known as eosinophilic granuloma) and hypersensitivity pneumonitis (extrinsic allergic alveolitis, which is rarely associated with tissue eosinophilia) are discussed in Chapters 34 and 12, respectively.

Asthma Introduction Asthma is one of the commonest respiratory disorders and is characterized by airway hyperresponsiveness and inflammation. Over the past few decades, the prevalence of asthma has increased and affects as many as 300 million persons worldwide.3

Epidemiology and genetics Asthma probably results from complex interactions between multiple genes and environmental factors. Occupational asthma accounts for approximately 10 to 15% of cases of adult asthma.4 Although new pharmacological therapies have become available, asthma continues to cause substantial morbidity and mortality.

Clinical manifestations Asthma causes recurrent episodes of wheezing, breathlessness, chest tightness and coughing. These symptoms can be associated with specific triggers such as allergens, environmental

irritants and workplace exposures. Physical examination commonly yields wheezing audible on auscultation of the lungs. With severe airflow obstruction, expiratory slowing, hyperexpansion of the chest and accessory muscle use are seen.

Radiological findings Chest radiography usually yields normal results unless there are comorbid conditions or complications, e.g. pneumonia. High-resolution computed tomographic (HRCT) findings may include bronchial wall thickening, mucoid impaction, focal or diffuse decreased lung attenuation (due to air trapping), centrilobular opacities and bronchial dilatation (Figures 1 and 2).5 Additional findings may be identified on CT when associated disorders, e.g. chronic eosinophilic pneumonia, or complications, e.g. pneumomediastinum, are present.

Macroscopic pathology The autopsy table is the most common place where a pathologist will be asked to consider whether asthma has significantly contributed to a patient’s illness. However, bronchial biopsies, surgical lung biopsies and cytological specimens may also have significant abnormalities which could, in the correct clinical context, be helpful in the diagnosis of asthma. It is difficult to definitively outline the pathology of a condition which is difficult to define clinically, and may not represent a single disease entity.6 This is complicated by an apparent tissue effect relating to severity of the condition (mild, moderate, severe; or fatal asthma versus a subject with asthma who is alive or who has succumbed to other factors). It is also complicated by the fact the pathological changes are not uniform throughout the bronchial tree. The individual features of asthma will be described in the following sections, noting the majority are neither specific nor sensitive (Table 1). Nevertheless, a combination of several of the features described below would be sufficient to at least suggest a diagnosis of asthma, particularly in the right clinical context. As a consequence of an asthma attack, mucous plugs result in areas of hyperinflation alternating with areas of atelectasis

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 15: Eosinophilic lung disease Table 1 Pathological features in asthma and related airway diseases

Gross pathology in patients with asthma Atelectasis Hyperinflation Bronchiectasis Mucus plugs Microscopic pathology Mucous plugs Goblet cell metaplasia Airway wall thickening Muscular hyperplasia

Other diseases in which the same pathology may be found Idiopathic bronchiectasis COPD Idiopathic bronchiectasis COPD COPD COPD, bronchiectasis COPD, bronchiectasis COPD

(Figures 1 and 2). In patients with fatal asthma, the gas trapping can be so severe that the lungs do not deflate when the chest is opened at autopsy (Figure 3). Mucus plugs are seen throughout the airways, most marked in cases of fatal asthma, but are also found in subjects with asthma who have died of other causes. The larger airways have thickened walls which are better appreciated radiologically than grossly; bronchiectasis complicating asthma can be recognized both radiologically and grossly.7–10 Radiological bronchial dilatation (cylindrical bronchiectasis) can be seen in 30–50% of asthmatics,9 while varicose or cystic bronchiectasis has been identified in approximately 3% of asthmatics, all of whom have severe symptoms.8

Figure 1. Asthma. CT image demonstrating thickening of the airway walls with areas of gas trapping seen as increased radiolucency. (Courtesy of Dr Jonathon Leipsic, St Paul’s Hospital, Vancouver, B.C., Canada.)

Histopathology Abnormalities have been described in all of the airway compartments (lumen, epithelium and wall) with considerable variation between large and small airways.

Airway lumen The airway mucus in asthma contains an admixture of epithelial and inflammatory cells, which are generally scattered throughout the mucus rather than present in layers (Figure 4). Charcot-Leyden crystals can be found. In fatal asthma, mucus can be found in airways of all sizes.11–13 It is also increased in the small airways in subjects with non-fatal asthma.13 Pathologists should be cautious about using this as a definite criterion for asthma as mucus plugs are also found in the airways of patients with severe chronic obstructive pulmonary disease (COPD).14 They are also sometimes present as a nonspecific response to injury in other conditions.

Airway epithelium There is a controversy in the literature as to whether there is true epithelial sloughing in asthmatic patients, whether the epithelium is fragile and sloughs during biopsy or lavage, or whether the entire concept of epithelial sloughing is an artifact.15–21 Electron microscopic changes including epithelial vacuolization, cilial blebs and cilial loss, suggest the epithelium

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Figure 2. Asthma. CT image demonstrating areas of poor ventilation with mucus plugs, associated with thickening of the airway walls. (Courtesy of Dr Jonathon Leipsic, Vancouver, B.C., Canada.)

is damaged.18,19 Interestingly, nerve profiles close to the airway lumen have also been described, which may explain correlations between epithelial sloughing and airway reactivity.20

Chapter 15: Eosinophilic lung disease Figure 3. Asthma. Lung at autopsy from a patient who died of status asthmaticus. Note that the lung has not collapsed, and the lumen of the mainstem bronchus is occluded with a mucus plug. (Reprinted from Thurlbeck’s Chronic Airflow Obstruction with permission from B.C. Decker publishers.)

Figure 4. Asthma, cartilaginous airway. Note mucus within the airway lumen. There is thickening of the subepithelial compartment, a marked increase in thickness of the muscle layer, and prominent bronchial glands.

Basement membrane

Figure 5. Asthma. Airway epithelium showing marked goblet cell metaplasia. The underlying basement membrane is thickened. Note also the dilated vessels in the subepithelial compartment.

In the older literature, goblet cell metaplasia was considered to be one of the classic features of asthmatic airways (Figure 5). Morphometric studies, comparing the central airways in asthmatics with control airways, have been generally, but not totally, supportive of this contention.13,16,22 There is evidence of increased Muc5AC mRNA and protein, as well as increased stored and secreted mucin in subjects with moderate asthma.17 Goblet cell metaplasia is a very nonspecific finding, as it is increased in both the large and small airways in cigarette smokers.23,24

The epithelial basement membrane is divided into two components: the “true” basement membrane (lamina densa) which is formed of collagen IV, collagen VII, laminin-entactin and proteoglycans;25 and the reticular basement membrane which is formed of collagen I, III, and V.26 It is the reticular basement membrane which is thickened in asthma (Figure 5), and although the data in the literature are highly variable, the asthmatic basement membrane is generally greater than 10 mm in thickness, compared to approximately 4–8 mm in the control population.20,21,25–29 An important point for the pathologist to recognize is that basement membrane thickening is largely confined to the central airways; in the non-cartilaginous airways the basement membrane is unobtrusive and is not thickened, even in fatal asthma.22,30 While thickening of the basement membrane in the larger airways forecasts remodeling in the larger airways, it has no positive predictive value for small airways remodelling.30 However, in the larger airways, an increased basement membrane thickness may have sufficient sensitivity and specificity to separate patients with severe asthma from controls, but not from those with mild asthma or COPD.31 Evaluation of the collagen deep to the reticular basement membrane has demonstrated a different ultrastructural pattern in asthma, with thinner fibers and fewer banded collagen fibers.29 Analysis of the different types of collagen in this area has shown

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disparate results, with one study32 finding increased Type III and Type V collagen, and another33 detecting no differences.

Inflammatory infiltrate Eosinophils have long been considered the hallmark inflammatory cell of asthma. Many studies have demonstrated they are increased in bronchial biopsies from central airways in asthmatics17,28,34–37 and increased in the central38–40 and peripheral12,22,39,40 airways of subjects who have died with or because of their asthma. Interestingly, in subjects who have died a short time after the onset of an asthmatic attack, neutrophils may predominate over eosinophils (Figure 6).41,42 This suggests the relative numbers of neutrophils and eosinophils are a function of the duration of asthmatic symptoms. Although eosinophils and neutrophils are part of the infiltrate, mast cells, and particularly those that are degranulated, are increased in the subepithelial compartment and the airway smooth muscle in many studies.21,28,37,43–46 Some,28 but not all,47 studies found correlations between mast cell numbers and airway responsiveness. However, mast cells are also noted to be increased in subjects who have died with anaphylaxis,38 and must not be considered as specific for asthma. In normal airways, the lymphocyte is the most common inflammatory cell, and can be identified throughout the airway wall in small numbers (Figure 7). T lymphocytes are thought to be responsible for the initiation and regulation of the normal immune response. As asthma has a major immunological component, pathologists might expect to find an increase in T lymphocytes, and this has been found in studies which examined the segmental and subsegmental airways.35,39,48 There is an increase in lymphocytes (not otherwise categorized) in airways of all sizes in fatal asthma, and in the smaller airways in non-fatal asthma, compared to controls.40 In fatal asthma, the number of T-cells is greater in the proximal airways as compared to in the distal airways, with CD8 T-cell predominance.49 There also appears to be an increase in the numbers of peribronchial CD8 T-cells in airways from patients with fatal asthma, with expression of interleukin (IL)-4 and interferon-g.50 There may also be reduced apoptosis of the memory T-cells in the large airways of patients with either fatal or non-fatal asthma.51

Vessels The blood supply to the airways is systemic in nature, and is provided by the bronchial arterial system. A morphometric study of the larger vessels present in the outer airway wall in fatal and non-fatal asthmatics found intimal thickening, fragmentation and duplication of the internal elastic lamina, calcification of the elastica and luminal obliteration. However, these changes were also present in cigarette smokers, with smoking acting as an additive factor in asthmatics.52 In the inner wall of the lower trachea, there is increased vascular density in people with asthma compared to COPD or normal. This feature can be recognized using a

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Figure 6. Increased neutrophils in the airway wall of a subject with fatal asthma. Immunohistochemical stain for myeloperoxidase. (Image courtesy of Drs Alan James and John Elliot, Perth, Australia.)

Figure 7. Asthma. Cartilaginous airway with lymphoid aggregate and a diffuse increase in lymphocytes within the airway wall. Note also mucin within the airway lumen and a thickened basement membrane.

bronchovideoscope,53 even in stable asthma. In the larger airways, some studies found an increased proportion of the vascular area and an increased number of vessels in asthmatics (Figure 5).43,54–56 One study failed to find differences between control, non-fatal and fatal asthma in any size, but examined only vascular number.40,57 There is also disagreement as to whether there is increased vascular endothelial growth factor (VEGF) immunoreactivity in the vessels.43,54 The increase in vascularity may be reversible in response to high-dose steroids.44 Vascular alterations have also been described at the level of membranous bronchioles, with asthmatic airways having an increased proportion of vascular area compared to control or those with COPD. There is a gradation of change between fatal and nonfatal asthma.58

Chapter 15: Eosinophilic lung disease

Mucous glands Many asthmatics, particularly those who have died of their asthma, have an increased proportion of bronchial glands in the airway wall (Figure 4).13,15,46,59,60 This finding is neither sensitive nor specific as increased bronchial glands are also present in chronic bronchitis, and in emphysema without chronic bronchitis.60,61

Wall thickness High-resolution computed tomography (HRCT) scans demonstrate thickened airway walls (Figure 1). Histologically, overall thickening of the airway wall with increased muscle thickness has been a hallmark alteration of asthma, but is nonspecific (Figure 4). In the central airways, the total airway wall is thickened in fatal asthmatics,15 and there is an increased proportion, and an absolute increase, of muscle,15,30,59–61 in those who have died because of, or with, asthma. However, an absolute increase in muscle has also been identified in chronic bronchitis.62 Total airway wall is also thickened in the small airways in fatal asthma, and this is accompanied by increased smooth muscle area.13,15 In general, smooth muscle is also increased in the membranous bronchioles, but this is not a specific finding, as it is also increased in the airways of subjects with COPD.58 There may be two different structural phenotypes in asthma, with one type demonstrating muscular thickening confined to the large airways, and the second in which the muscular thickening is present over the entire range of airways.63,64 A consequence of all of the above is that there is thickening of the submucosa between the basement membrane and the smooth muscle58,65 in patients with asthma and COPD. This remodeling has physiological effects. Modeling65–67 studies suggest that for any significant degree of muscle shortening, an increase in wall thickness leads to a much greater degree of airway narrowing, and thus a greater degree of airway resistance. Thickening of the adventitial fibrous tissue in the small airways, also found in asthma,58 has an important theoretical physiological effect. It may “uncouple” airway-parenchymal interdependence and all the smooth muscle to shorten excessively, thus increasing airway narrowing.

Cytology of asthma Regardless of whether the epithelium is fragile and easily removed by trauma or whether sloughing is a true component of asthma, cohesive clusters of epithelial cells are found in the sputum or bronchial washings in patients with asthma. In filter preparations, the cells form three-dimensional balls known as Creola bodies or “epithelial cell balls” (Figure 8). The cells often retain their cilia. In cell blocks, the epithelium is normally accompanied by abundant mucus. Within the mucus, there are inflammatory cells with eosinophils and the crystalline eosinophil protein structures, known as Charcot-Leyden crystals (Figure 9). Curschmann’s spirals are elongated and spiral mucus plugs with entrapped epithelial cells, and can be

Figure 8. Asthma. Creola body consisting of an epithelial sheet which has formed into a three-dimensional figure (Papanicolaou stain).

Figure 9. Charcot-Leyden crystals. These needle-shaped crystals are formed from eosinophil proteins. Note the bilobed eosinophil nuclei in the background (Papanicolaou stain).

found in filter preparations and cell blocks (Figure 10). The mucus of asthma can be distinguished from the “allergic mucus” of allergic bronchopulmonary fungal disease with mucoid impaction. The latter condition has a dense appearance, with layers of compacted eosinophils and epithelial cells.68

Laboratory findings Pulmonary function testing is crucial in the diagnosis of asthma but also for assessing severity of disease and monitoring ongoing care. The documentation of reversible airways obstruction is the basis for the diagnosis of asthma. For patients suspected of asthma and normal pulmonary function results, provocative testing (most commonly with inhaled methacholine) is used to demonstrate airway hyperresponsiveness.

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Histopathology The pathology of eosinophilic bronchitis is very similar to that seen in patients with asthma and includes eosinophilia in sputum, bronchial wash fluid and in bronchial walls. Lymphocytic inflammation (T-cell rich) and basement membrane thickening are also characteristic. In contrast to asthma, however, the number of mast cells in the smooth muscle layers is not increased, although the number of mast cells in the submucosa is similar in both diseases.

Prognosis and natural history

Figure 10. Curschmann spiral. Dense mucus with entrapped epithelial cells take the form of a small airway (Papanicolaou stain).

Prognosis and natural history The reversibility of the anatomic changes in asthma with therapy (usually corticosteroids) appears to vary considerably with the study population and the anatomic compartment.69 It is quite possible that there are dose and time-scale effects in addition to individual variations in response. Overall, there does appear to be a reduction of epithelial fragility, reticular basement membrane thickening, airway vascularity and airway smooth muscle alterations with steroid regimes. Collagen deposition appears to be resistant. The use of HRCT to monitor the effects on treatment appears promising, with one study showing a treatment effect in the bronchial wall thickening of the subsegmental (but not segmental) airways.70

Non-asthmatic eosinophilic bronchitis Introduction This condition is thought to account for 10 to 30% of patients presenting to pulmonologists for evaluation of chronic cough.5,71–80

Clinical manifestations Non-asthmatic eosinophilic bronchitis is a somewhat controversial entity, characterized as a form of chronic cough in nonsmokers. The disease typically affects adults. Chronic cough lasts > 8 weeks in the absence of obvious lung disease by clinical or radiographic evaluation. Non-asthmatic eosinophilic bronchitis is chronic cough in patients with sputum eosinophilia without airflow obstruction or hyperresponsiveness. Some use the presence of eosinophils > 3% non-squamous cells as the cut off.81 Some cases of eosinophilic bronchitis may be associated with occupational exposures, e.g. baking, or inhaled allergens.74

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Anti-inflammatory therapy, which may include inhaled corticosteroids, and avoidance of potential environmental triggers are the usual treatment strategies. Most patients appear to respond to treatment, but occasionally oral steroids may be necessary. Contrary to earlier reports, this condition is generally not self-limiting. Follow-up data on 32 patients at > 1 year indicate two-thirds of patients are still symptomatic from their airway eosinophilia, approximately 10% of patients develop asthma, and 16% develop fixed airway obstruction. Only one patient was asymptomatic and off all therapy.82

Chronic eosinophilic pneumonia Introduction Carrington and colleagues are generally recognized as reporting the first large series of patients with idiopathic chronic eosinophilic pneumonia (CEP).83,84

Clinical manifestations The clinical diagnosis is usually based on the presence of respiratory symptoms > 2 weeks duration, alveolar or blood eosinophilia, pulmonary infiltrates, usually peripheral, and exclusion of known causes for eosinophilia. The peak incidence of CEP is in the fifth decade, and it occurs predominantly in women (2:1).85 The disease is characterized by an insidious onset and the average duration of symptoms before diagnosis is approximately 7 months. The commonest symptoms include cough, fever, dyspnea and weight loss. These patients often feel sick for months. Asthma has been reported in 50% of patients.86 Respiratory failure is rare but has been reported in accelerated cases and cases where the diagnosis has been delayed.86–90 Bronchoalveolar lavage characteristically shows eosinophilia.

Radiographic findings Chest imaging usually demonstrate infiltrates in the outer twothirds of the lung fields.91,92 Infrequently, nodular densities, infiltrates (Figure 11) and cavitation may be observed.92 Rarely, the foci of consolidation may be predominantly in the lower lobes.92 The radiographic finding of dense, extensive, bilateral peripheral infiltrates (the so-called “photographic negative” of pulmonary edema) has been said to be virtually

Chapter 15: Eosinophilic lung disease

(a)

(b)

Figure 11. High-resolution CT scans from a patient with chronic eosinophilic pneumonia. (a) Prior to treatment bilateral areas of parenchymal consolidation are seen. (b) Two weeks after steroid therapy the infiltrates have completely cleared.

diagnostic for chronic eosinophilic pneumonia, but is noted in only 25% of patients.88 Mediastinal adenopathy may be seen on CT scanning in about one-half of patients.

Histopathology Chronic eosinophilic pneumonia is characterized by intraalveolar accumulations of eosinophils and macrophages in varying proportions (Figures 12 and 13). Smaller numbers of lymphocytes and plasma cells may also be intermixed. An interstitial infiltrate characteristically accompanies the intraalveolar changes. The alveolar septa may be thickened by eosinophils, lymphocytes, plasma cells, and may be associated with alveolar lining cell hyperplasia (Figures 13–16). The density of the infiltrate in alveolar spaces and in the interstitium sometimes makes it impossible to tell exactly where the cells are located. This gives the tissue a very “dense” appearance at low magnification. In fact this histological pattern can be a “tip off” that the diagnosis of chronic eosinophilic pneumonia should be considered and prompt a careful look at the cellular composition of the infiltrate. In many cases the inflammatory infiltrate is associated with intra-alveolar edema and a proteinaceous exudate, which is often colloid-like (Figure 15) and sometimes fibrinous (Figure 17). There may also be foci of intra-airway and intraalveolar organization (bronchiolitis obliterans organizing pneumonia-like foci) (Figures 16 and 18), eosinophilic microabscesses, focal intra-alveolar necrosis, sarcoid-like granulomas and giant cells (Figure 19). Giant cells were present in 100% of

Figure 12. Chronic eosinophilic pneumonia. The alveolar space is flooded by an admixture of macrophages and eosinophils. Many of the eosinophils have degranulated and Charcot-Leyden crystals as well as eosinophilic granules can be readily identified.

the cases initially described by Carrington et al.83 CharcotLeyden crystals may also rarely be identified in the giant cells. A mild non-necrotizing infiltration of vessel walls by eosinophils and mononuclear cells may also occasionally be seen (Figure 20). True vasculitis is rare and when present suggests the possibility of concomitant Churg-Strauss syndrome (see Chapter 19). The number of eosinophils is extremely variable from case to case and from field to field in the same case. There is no absolute number required for the diagnosis. In some cases,

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Figure 13. Chronic eosinophilic pneumonia. Transbronchial biopsy. Macrophages and eosinophils fill airspaces. In this particular example the eosinophils are less numerous than seen in Figure 12.

Figure 14. Chronic eosinophilic pneumonia. The lung shows several characteristic features including an interstitial pneumonia, flooding of alveolar spaces by eosinophils and macrophages, and several alveoli filled with protein-rich, colloid-like edema fluid.

Figure 15. Chronic eosinophilic pneumonia. Higher-power image of Figure 14 showing colloid-like edema fluid with prominent scalloping as well as interstitial thickening and alveolar space eosinophils and macrophages. Figure 16. Chronic eosinophilic pneumonia. Numerous eosinophils fill many of the alveolar spaces. The process is associated with foci of organizing pneumonia.

there is a massive outpouring of eosinophils, making the diagnosis fairly straightforward. However, in most cases, the number of eosinophils is variable. It may only be the presence of marked eosinophilia in less than 10% of the affected tissue that suggests the diagnosis. Tissue eosinophilia is extremely sensitive to systemic corticosteroid therapy and just one dose prior to biopsy may make confirmation of a suspected diagnosis impossible on morphological grounds. Recently treated cases may look like cases of cryptogenic organizing pneumonia.

Differential diagnosis Figure 17. Fibrin-rich alveolar exudate containing eosinophils and cholesterol clefts.

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There are two ways to consider the differential diagnosis of chronic eosinophilic pneumonia, clinical and pathological. “Carrington’s pneumonia” is the idiopathic form of the

Chapter 15: Eosinophilic lung disease

Figure 18. Chronic eosinophilic pneumonia. Prominent foci of organizing pneumonia can be seen at low magnification as well as marked interstitial thickening (interstitial pneumonia).

Figure 19. Chronic eosinophilic pneumonia with multinucleated giant cells.

Table 2 Associations and causes of chronic eosinophilic pneumonia

Allergic bronchopulmonary fungal disease109 Simple pulmonary eosinophilia90,91 Systemic infection (parasites, fungi)90,91 Connective tissue disease, esp. rheumatoid arthritis2,131 Radiation (ipsilateral or contralateral)132 Cigarette smoking, esp. at initiation or with change of brands133 Drugs, esp. salicylates, antibiotics134 Churg-Strauss syndrome135 Hodgkin lymphoma Inflammatory bowel disease136 Lung cancer137 Figure 20. Chronic eosinophilic pneumonia with vascular wall infiltration. No true necrosis is present.

disease, but there are multiple etiologies for the histology of chronic eosinophilic pneumonia. The most common clinical associations are shown in Table 2. With the exception of additional histological features of allergic bronchopulmonary fungal disease as discussed below and Churg-Strauss disease, cases of CEP associated with or caused by another process are similar histologically to cases of idiopathic CEP (Figures 21 and 22). This is also likely to be true for the histology associated with simple pulmonary eosinophilia (Löffler syndrome). Patients with this disease present with fever and peripheral blood eosinophilia usually in the setting of parasitic infection. Eosinophils are present in sputum. But, as the disease is self -limiting, biopsy descriptions are rare, but come closest to the histology of “chronic” eosinophilic pneumonia.

Inhalation of nickel carbonyl vapor138

The pathological differential diagnosis is shown in Table 3.93–96 The three histological manifestations of allergic bronchopulmonary fungal disease include chronic eosinophilic pneumonia, bronchocentric granulomatosis and mucoid impaction of bronchi.97,98 If features of either of these latter two diseases (see below) are present in addition to CEP, the diagnosis of allergic bronchopulmonary disease should be strongly suggested. A histological pattern of chronic eosinophilic pneumonia can also be seen in association with infections, most notably Cryptococcus99 and Histoplasma. Therefore, special stains should be performed in all cases in which a diagnosis of CEP is considered. Deaths have resulted from steroid therapy given for presumed idiopathic CEP, when the etiology was pulmonary fungal infection with an eosinophilic pneumonia reaction.

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(a)

(b)

Figure 21. Chronic eosinophilic pneumonia with lung cancer. (a) The transbronchial biopsy shows an invasive non-small cell carcinoma. (b) Another fragment of tissue from the same biopsy shows prominent interstitial eosinophils.

(a)

(b)

Figure 22. Eosinophilic pneumonia superimposed on usual interstitial pneumonia. (a) The low-magnification field shows marked peripheral interstitial fibrosis with some central sparing. Several fibroblast foci can be readily identified. (b) Foci of eosinophilic pneumonia were also present.

Eosinophilic pneumonia is also one of the histological components of Churg-Strauss syndrome (CSS). Churg-Strauss syndrome has several phases and vasculitis may not be seen histologically in the early stages of the disease. Tissue eosinophilia alone in the appropriate clinical context may be enough to help establish the diagnosis. When CEP occurs in a patient with asthma, CSS is always in the differential diagnosis. Clinical correlation is critical to establishing the diagnosis of CSS. Histologically, patients with acute eosinophilic pneumonia (see below) appear to have evidence of more acute lung injury in the form of diffuse alveolar damage (DAD) in either the

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acute or organizing phase. The inflammatory cell infiltrate is usually mixed and includes lymphocytes, macrophages, small numbers of plasma cells and prominent collections of eosinophils. Hyaline membranes have not been described in cases of chronic eosinophilic pneumonia. Due to the frequent occurrence of organizing pneumonia (OP) in CEP, the differential diagnosis of CEP includes cryptogenic organizing pneumonia, formerly known as idiopathic BOOP.100 Colloid-like edema fluid is rare in OP and the tissue eosinophilia should alert one to the diagnosis of CEP.

Chapter 15: Eosinophilic lung disease Table 3 Pathological differential diagnosis of chronic eosinophilic pneumonia

Table 4 Histological differences between DIP and CEP

Allergic bronchopulmonary fungal disease Pulmonary infection, esp. fungal incl. histoplasmosis and coccidioidomycosis99 Churg-Strauss syndrome Acute eosinophilic pneumonia Organizing pneumonia Langerhans cell histiocytosis Desquamative interstitial pneumonia Usual interstitial pneumonia Eosinophilic variant of Wegener granulomatosis139,140 Reactive eosinophilic pleuritis

a

DIP

CEP

Macrophages

Contain light brown granular pigment

No pigmenta

Airspace fibrin and edema

None

Common

Uniform interstitial fibrosis

Usually

None

Organizing pneumonia

None

Common

Lymphoid hyperplasia

Common

Usually none

Except in cases where EP may be related to cigarette smoking.

141–145

Pulmonary Langerhans cell histiocytosis (LCH) is also characterized by a combination of histiocytes and eosinophils, but in this condition the infiltrate is mainly interstitial rather than alveolar. On low magnification, the lesions in LCH tend to be discrete, bronchiolocentric and associated with stellate zones of interstitial scarring. The histiocytic and eosinophilic infiltrate tend to be present in a loose fibroblastic background (sometimes OP-like). In LCH the histiocytes have the grooved nuclei, characteristic of Langerhans cells. In difficult cases immunoperoxidase stains (S100, langerin, CD1a) may be helpful in distinguishing LCH from CEP (see Chapter 34). Desquamative interstitial pneumonia (DIP) is characterized by the presence of numerous macrophages in airspaces, a feature also seen in most cases of CEP (see Chapter 10).101 Eosinophils are also frequently seen scattered among the macrophages in DIP. Chronic eosinophilic pneumonia typically does not have the uniform interstitial fibrosis seen in DIP and the macrophages in the latter condition are usually lightly pigmented. Desquamative interstitial pneumonia also lacks the fibrin deposition and edema fluid commonly seen in CEP, giving CEP a very dense appearance. Table 4 summarizes the histological differences between these diseases. Rarely, eosinophilic pneumonia may occur in a background of usual interstitial pneumonia (UIP).102 Such a finding should suggest the possibility of a drug reaction, either for the eosinophilic pneumonia reaction or for both patterns. The prognosis in these cases is that of UIP (see Chapter 10).

Natural history and prognosis Less than 10% of patients will have spontaneous resolution of their illness, and deaths from chronic eosinophilic pneumonia have been reported.85,88 The disease is remarkably steroidresponsive, and most patients will have a dramatic response within 24–48 hours.85,88,91 Relapses are common if corticosteroids are discontinued in the first 6 months, an issue recognized by Carrington et al.83 Up to 50% of patients may need long-term steroid therapy.88,91 Unfortunately, there are no histological or clinical features which identify this subset of patients.

Table 5 Organisms associated with allergic fungal disease109

Candida albicans

Stemphylium lanuginosum

Bipolaris sp.

Penicillium rubrum

Aspergillus sp.

Pseudallescheria boydii

Geotrichum helminthosporium

Torulopsis glabrata

Fusarium vasinfectum

Drechslera hawaiiensis

Curvularia sp.

Allergic bronchopulmonary fungal disease Introduction Allergic bronchopulmonary fungal disease is a syndrome that mainly affects asthmatic patients. It consists of fleeting pulmonary opacities, eosinophilia, elevation of serum IgE and evidence of fungal hypersensitivity.103–109 While patients with asthma constitute the largest group of patients, it may also complicate cystic fibrosis.110–112 While Aspergillus sp. are the most notorious fungi to cause this allergic phenomenon (allergic bronchopulmonary aspergillosis), multiple other fungi can cause the disease (see Table 5).109

Clinical manifestations The disease occurs over a broad age range from children to adults and both genders can be affected.109 Diagnostic criteria include the presence of six, including the first five, of the following:113,114
history of asthma or cystic fibrosis (10–15% of cases)
immediate skin reactivity to fungal (usually Aspergillus) extract
precipitating antibodies to fungi (usually Aspergillus)
elevated total serum IgE (> 1000 ng/ml)
elevated IgE and/or IgG to fungi, usually Aspergillus fumigatus antigens

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(a)

(b)

Figure 23. Allergic bronchopulmonary aspergillosis. (a) High-resolution CT scan showing marked central bronchiectasis. (b) The resection specimen from the same patient demonstrates green/gray mucus filling dilated airways.


central bronchiectasis
recurrent pulmonary infiltrates 3
peripheral eosinophilia (> 500/mm ). Clinical features of allergic bronchopulmonary fungal disease are dominated by those of asthma, along with expectoration of brownish mucous plugs.

Radiographic findings Chest imaging usually demonstrates abnormal parenchymal findings reflecting the presence of bronchiectasis, mucoid impaction (including the “gloved finger” sign) and atelectasis (Figure 23a). The “gloved finger” sign is characterized by branching tubular or finger-like soft tissue densities.

Histopathology The diagnosis is usually made clinically, but occasionally it will be established initially by pathology, and can even be suggested on transbronchial biopsy specimens. Resections may also be performed to alleviate symptoms in those who fail medical therapy. The pathological changes in patients with allergic bronchopulmonary fungal disease condition include a complex constellation of findings. These include, in various combinations, bronchiectasis, mucoid impaction of bronchi (MIB), bronchocentric granulomatosis (BCG), eosinophilic bronchiolitis and CEP. In areas of lung away from the allergic response, airway changes may reflect the presence of asthma.

Bronchiectasis and mucoid impaction of bronchi Identifying bronchiectasis histologically requires the presence of bronchi in the pathological material, i.e., airways with cartilage or submucosal glands. Patients with proximal bronchiectasis may also have distal airway dilatation, i.e.,

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Figure 24. Allergic bronchopulmonary aspergillosis – mucoid impaction of bronchi. Marked bronchiectasis with a majority of airways are filled with greenish mucus.

bronchiolectasis. The diameter of the airways should be approximately the same as that of the accompanying vessel. The airways in allergic bronchopulmonary fungal disease are often filled with tenacious mucous plugs and casts of variable color from green-gray, to yellow to white (Figures 23–26). Microscopically, the mucus may resemble the lamellated mucus seen in other allergic syndromes of the respiratory tract, such as allergic fungal sinusitis (Figures 26–29). Alternatively it may be composed of more particulate blue-gray debris. In individual cases, the make-up of the mucin may vary among airways. In the lamellated mucus, pink to blue acellular mucin irregularly alternates with mucin rich in eosinophils, in various stages of degeneration. They may even appear hyperchromatic and resemble degenerating neoplasm. In these areas in particular, Charcot-Leyden crystals can usually be identified (Figures 28 and 29). These are elongate bi-pyramidal needle-shaped crystals

Chapter 15: Eosinophilic lung disease

Figure 26. Mucoid impaction of bronchi. Dilated airway with lamellated mucus characteristic of so-called “allergic mucin”. Figure 25. Mucoid impaction of bronchi. Dilated airway filled with abundant mucus and degenerating inflammatory cells.

Figure 28. Mucoid impaction of bronchi with numerous degenerating eosinophils and Charcot-Leyden crystals. Figure 27. Mucoid impaction of bronchi. Higher-power image of allergic mucin showing pink mucus, and degenerating inflammatory cells in various stages of degeneration. There are large clusters of degenerating eosinophils.

that can vary greatly in size and are composed of lysophospholipase from degenerated eosinophils. The bronchial walls are frequently thinned and usually contain a predominantly chronic inflammatory cell infiltrate. Reactive lymphoid nodules, follicular bronchitis or bronchiolitis may also be seen. Fungal hyphae can often be identified within the mucus (Figure 30) and in the central areas of necrosis seen in bronchocentric granulomatosis (see below), but, in contrast to a fungal infection, the number of organisms is relatively few and they are frequently fragmented.

Bronchocentric granulomatosis (BCG) BCG is a usually exquisitely bronchiolocentric process. It is extremely rare and most cases of possible BCG are in fact BCG-like reactions, associated with true infection.115 In BCG,

Figure 29. Mucoid impaction of bronchi. At high magnification one appreciates degenerating eosinophils, mucus and Charcot-Leyden crystals.

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(a)

(b)

Figure 30. Mucoid impaction of bronchi. (a) Gomori methenamine silver stains (GMS) showing fungal hyphae consistent with aspergillus. (b) The hyphae are often very fragmented and difficult to speciate.

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Figure 31. Bronchocentric granulomatosis with airway completely replaced by necrotizing granulomatous inflammation containing a central focus of necrosis with numerous degenerating inflammatory cells.

Figure 32. Allergic bronchopulmonary aspergillosis. The biopsy showed foci of bronchocentric granulomatosis (Figure 31) as well as areas of eosinophilic pneumonia.

the airways, usually bronchioles, appear to be replaced by necrotizing granulomas (Figures 31–33). The foci of necrosis are usually centrally located within the airway and there is a thick rim of epithelioid histiocytes surrounding the necrosis. Portions of bronchiolar wall may help in the recognition that the inflammation is airway-centered. Sometimes the location must be inferred by the presence of an adjacent pulmonary artery. As noted above, fungal hyphae may be identified in the necrotizing granuloma, but they should be rare, and noninvasive. It is often very difficult to speciate the organisms in BCG because they are so fragmented and degenerate. Airways adjacent to those with BCG may contain an exudate not unlike that described above in the context of mucoid impaction of bronchi. Bronchocentric granulomatosis is most common in asthmatics, where it is usually associated with mucoid impaction of

bronchi, eosinophilic pneumonia and changes of asthma. In idiopathic disease, these features are lacking. The differential diagnosis for both forms of BCG mainly includes infection, but similar changes have been reported in patients with Wegener granulomatosis and rheumatoid arthritis. Chronic cavitary aspergillosis is distinguished from BCG on the basis of a larger number of organisms and the presence of invasion of the fungi into the substance of the granuloma.

Natural history and prognosis Traditionally, patients have been treated with corticosteroids. Recent studies suggest that the addition of anti-fungal agents, e.g. itraconazole, may improve outcome. Total serum IgE level correlates well with disease activity and is useful in monitoring clinical course and response to therapy. Many patients have

Chapter 15: Eosinophilic lung disease

relapses and the natural history of the disease is not well understood.109 Long-term complications in patients with recurrent exacerbations include irreversible airway obstruction, development of aspergilloma and respiratory failure.

Acute eosinophilic pneumonia (AEP) Introduction

the disease. Patients typically present with an acute illness of 1–5 days duration accompanied by myalgia, pleuritic chest pain and hypoxemic respiratory failure, often requiring mechanical ventilation.91,123 Physical examination has been notable for fever (often high), respiratory distress and bibasilar or diffuse crackles on auscultation. Criteria for diagnosis are shown in Table 6.

In 1986, Davis et al.116 reported a case of acute respiratory failure associated with marked eosinophilia in bronchoalveolar lavage (BAL) specimens. This was followed by other reports of similar cases, many in the Japanese literature. In 1989, the disease was dubbed “acute eosinophilic pneumonia” and had as one of its prime features reversibility with steroid therapy.117–122

Table 6 Criteria for diagnosis of acute eosinophilic pneumonia

Acute febrile illness (days, rarely weeks duration) Hypoxemic respiratory failure Diffuse alveolar/mixed alveolar interstitial radiographic changes BAL eosinophils > 25% or biopsy confirmation of eosinophilic lung infiltrate

Clinical manifestations

No identifiable infection

Patients with acute eosinophilic pneumonia may be of any age and either gender.91,123 Teenagers have been reported with (a)

Prompt and complete response to corticosteroids (b)

(c)

Figure 33. Bronchocentric granulomatosis. (a) The granuloma appears adjacent to a bronchiole which contains an exudative bronchiolitis. (b) The accompanying artery indicates that the focus of granulomas inflammation is probably centered on the airway in a different plane. (c) The center portion of the necrotizing granuloma contains degenerating eosinophilic material.

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A rapid (< 1 week) response to steroid therapy has been reported but in our experience this is not always the case. A relapse-free course is characteristic (Figure 34).121–123 Similar to patients with CEP, there is both a clinical and a pathological differential diagnosis. There is an idiopathic form of the disease. However, numerous patients with AEP have been reported with unusual exposures, as well as more common exposures, especially cigarettes (see Table 7).124–128

Radiographic manifestations Imaging typically shows an extensive mixed alveolar and interstitial infiltrate involving all lobes (Figure 34). Unlike chest radiographs in chronic eosinophilic pneumonia, they rarely show peripheral-based infiltrates at presentation.91,123 Small to moderate-sized pleural effusions are frequent and usually bilateral.

Histopathology The main change in the lung biopsies is acute and organizing Diffuse alveolar damage (DAD) with marked numbers of interstitial and lesser numbers of alveolar eosinophils (Figures 35–37).129 Eosinophilic hyaline membrane-like structures are evident, with some suggesting they are different from the hyaline membranes of ordinary DAD.130 Interstitial widening by a combination of edema, proliferating fibroblasts and inflammatory cells characteristic of the organizing phase of DAD is also seen in most cases. Most cases also show hyperplasia of type II pneumocytes and interstitial lymphocytes. Perivascular and intramural inflammation without necrosis may be present, but granulomas are not present. Acute fibrinous pleuritis may also be seen.

Peripheral blood eosinophilia is uncommon at presentation but becomes elevated during the course of the illness.91,123 In contrast, a very high percentage of BAL eosinophils is characteristic, with an average of 54% reported in one series.121 Some suggest the diagnosis of acute eosinophilic pneumonia can be

578

Differential diagnosis The main differential diagnosis includes DAD and CEP, but other diseases with tissue eosinophilia enter the differential occasionally. The progressive alterations in lung architecture associated with DAD have been divided into the exudative (acute) and proliferative (organizing) phases (see Chapters 9 and 10). Both stages may be observed in AEP. Inflammatory cells, most notably neutrophils, lymphocytes and plasma cells, are seen most often in the organizing phase of DAD. Eosinophils, however, have not been mentioned in association with DAD or acute interstitial pneumonia. For this reason, cases of DAD Table 7 Associations and causes of acute eosinophilic pneumonia

Spelunking (caving or potholing)122,146 Plant repotting122 Wood pile moving122 Smokehouse cleaning122 Working with mulch World Trade Center dust exposure147 Firework smoke exposure148 Tobacco smoke

Laboratory findings

(a)

made on the basis of clinical findings and the results of BAL alone.121 Others caution that transbronchial or wedge lung biopsy should be performed to exclude the possibility of an infectious etiology, which could prove fatal with corticosteroids.122

(b)

133,149

Drug exposures, particularly crack cocaine, minocycline, fludarabine, BCG vaccination91 Infection, particularly aspergillus and coccidioidomycosis91 Deployment in or near Iraq (78% recently began to smoke cigarettes)123 Figure 34. Acute eosinophilic pneumonia. (a) At presentation extensive bilateral airspace opacities are noted. (b) After approximately 2 weeks of treatment with steroids the infiltrates are almost completely resolved.

Chapter 15: Eosinophilic lung disease

(a)

(b)

(c)

Figure 35. Acute eosinophilic pneumonia. (a) There is a diffuse process characterized by interstitial widening and pulmonary edema. Hyaline membranes can be seen. (b) Inflammatory cells are present, but in contrast to chronic eosinophilic pneumonia, the alveolar spaces appear less filled with cells. (c) Hyaline membranes and fibrin fill alveoli associated with numerous eosinophils.

(a)

(b)

Figure 36. Acute eosinophilic pneumonia probably related to cigarette smoking.

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Figure 37. Acute eosinophilic pneumonia. In this focus eosinophils are not prominent, but the diffuse alveolar damage features are apparent, with interstitial thickening, hyaline membranes and reactive type II pneumocytes.

with eosinophils should be considered possible cases of AEP. How many eosinophils are necessary in a case of DAD to suggest a diagnosis of AEP? Since they are so rare in typical cases of DAD and acute interstitial pneumonia, and the disease has a relatively good prognosis with steroid therapy, we recommend that small groups of 5–10 eosinophils scattered in the interstitium or alveolar spaces in cases of DAD should alert the pathologist to suggest this diagnosis. Pathologically, CEP is characterized by interstitial and intra-alveolar accumulations of eosinophils and macrophages in varying proportions, as well as smaller numbers of lymphocytes and plasma cells. It is characteristically

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associated with an intra-alveolar proteinaceous exudate. There may be intra-airway organization associated with eosinophilic microabscesses and focal intra-alveolar necrosis, sarcoid-like granulomas and giant cells. Hyaline membranes are not usually present. Cases of AEP lack the proteinaceous, colloid-like, alveolar exudate and prominent admixture of macrophages and eosinophils characteristic of CEP. In addition AEP has more prominent interstitial organization and hyaline membranes similar to cases of acute and organizing DAD. Cases of AEP are more likely to be confused with cases of DAD than CEP. In most cases of patients with eosinophilic lung disease, a combination of history and pathology allows a pretty clean separation between AEP and CEP. The distinction may be most difficult on transbronchial biopsy. Rare patients with AEP in the literature have had asthma; none has had other clinical features to suggest the diagnosis of CSS.91 An eosinophil-rich variant of Wegener granulomatosis exists and mild vascular involvement can be observed in cases of AEP. However, DAD is a rare manifestation of Wegener granulomatosis and patients with AEP lack other evidence of this disease (see Chapter 19). Infection as a cause of the lung disease can usually be excluded on the basis of either culture results or special stains.

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23. Wright JL, Lawson LM, Pare PD, Wiggs BJ, Kennedy S, Hogg JC. Morphology of peripheral airways in current smokers and ex-smokers. Am Rev Respir Dis 1983;127(4):474–7. 24. Wright JL, Lawson LM, Pare PD, et al. The detection of small airways disease. Am Rev Respir Dis 1984;129(6):989–94. 25. Paulsson M. Basement membrane proteins: structure, assembly, and cellular interactions. Crit Rev Biochem Mol Biol 1992;27(1–2):93–127. 26. Roche WR, Beasley R, Williams JH, Holgate ST. Subepithelial fibrosis in the bronchi of asthmatics. Lancet 1989;1(8637):520–4. 27. Roche WR. Fibroblasts and asthma. Clin Exp Allergy 1991;21(5):545–8. 28. Brightling CE, Bradding P, Symon FA, et al. Mast-cell infiltration of airway smooth muscle in asthma. New Engl J Med 2002;346(22):1699–705. 29. Saglani S, Molyneux C, Gong H, et al. Ultrastructure of the reticular basement membrane in asthmatic adults, children and infants. Eur Respir J 2006;28(3):505–12. 30. James AL, Maxwell PS, Pearce-Pinto G, Elliot JG, Carroll NG. The relationship of reticular basement membrane thickness to airway wall remodeling in

36. Lacoste JY, Bousquet J, Chanez P, et al. Eosinophilic and neutrophilic inflammation in asthma, chronic bronchitis, and chronic obstructive pulmonary disease. J Allergy Clin Immunol 1993;92(4):537–48. 37. Beasley R, Roche WR, Roberts JA, Holgate ST. Cellular events in the bronchi in mild asthma and after bronchial provocation. Am Rev Respir Dis 1989;139(3):806–17. 38. Perskvist N, Edston E. Differential accumulation of pulmonary and cardiac mast cell-subsets and eosinophils between fatal anaphylaxis and asthma death: a postmortem comparative study. Forensic Sci Int 2007;169(1):43–9. 39. Azzawi M, Johnston PW, Majumdar S, Kay AB, Jeffery PK. T lymphocytes and activated eosinophils in airway mucosa in fatal asthma and cystic fibrosis. Am Rev Respir Dis 1992;145(6):1477–82. 40. Carroll N, Cooke C, James A. The distribution of eosinophils and lymphocytes in the large and small airways of asthmatics. Eur Respir J 1997;10(2):292–300.

41. Sur S, Crotty TB, Kephart GM, et al. Sudden-onset fatal asthma. A distinct entity with few eosinophils and relatively more neutrophils in the airway submucosa? Am Rev Respir Dis 1993;148(3):713–9. 42. Carroll N, Carello S, Cooke C, James A. Airway structure and inflammatory cells in fatal attacks of asthma. Eur Respir J 1996;9(4):709–15. 43. Chetta A, Zanini A, Foresi A, et al. Vascular endothelial growth factor upregulation and bronchial wall remodelling in asthma. Clin Exp Allergy 2005;35(11):1437–42. 44. Chetta A, Zanini A, Foresi A, et al. Vascular component of airway remodeling in asthma is reduced by high dose of fluticasone. Am J Respir Crit Care Med 2003;167(5):751–7. 45. Bradding P, Walls AF, Holgate ST. The role of the mast cell in the pathophysiology of asthma. J Allergy Clin Immunol 2006;117(6):1277–84. 46. Carroll NG, Mutavdzic S, James AL. Distribution and degranulation of airway mast cells in normal and asthmatic subjects. Eur Respir J 2002;19(5):879–85. 47. Liesker JJ, Ten Hacken NH, Rutgers SR, et al. Mast cell numbers in airway smooth muscle and PC20AMP in asthma and COPD. Respir Med 2007;101(5):882–7. 48. Vignola AM, Chanez P, Campbell AM, et al. Airway inflammation in mild intermittent and in persistent asthma. Am J Respir Crit Care Med 1998;157(2):403–9. 49. Faul JL, Tormey VJ, Leonard C, et al. Lung immunopathology in cases of sudden asthma death. Eur Respir J 1997;10(2):301–7. 50. O’Sullivan S, Cormican L, Faul JL, et al. Activated, cytotoxic CD8(þ) T lymphocytes contribute to the pathology of asthma death. Am J Respir Crit Care Med 2001;164(4):560–4. 51. Lamb JP, James A, Carroll N, et al. Reduced apoptosis of memory T-cells in the inner airway wall of mild and severe asthma. Eur Respir J 2005;26(2):265–70. 52. Green FH, Butt JC, James AL, Carroll NG. Abnormalities of the bronchial arteries in asthma. Chest 2006;130(4):1025–33. 53. Tanaka H, Yamada G, Saikai T, et al. Increased airway vascularity in newly

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diagnosed asthma using a highmagnification bronchovideoscope. Am J Respir Crit Care Med 2003;168(12):1495–9. 54. Barbato A, Turato G, Baraldo S, et al. Epithelial damage and angiogenesis in the airways of children with asthma. Am J Respir Crit Care Med 2006;174(9):975–81. 55. Salvato G. Quantitative and morphological analysis of the vascular bed in bronchial biopsy specimens from asthmatic and non-asthmatic subjects. Thorax 2001;56(12):902–6. 56. Li X, Wilson JW. Increased vascularity of the bronchial mucosa in mild asthma. Am J Respir Crit Care Med 1997;156(1):229–33. 57. Carroll NG, Cooke C, James AL. Bronchial blood vessel dimensions in asthma. Am J Respir Crit Care Med 1997;155(2):689–95.

66. James A, Pare P, Hogg J. The mechanics of airway narrowing in asthma. Am Rev Respir Dis 1989;139(1):242–6. 67. Wiggs BR, Bosken C, Pare PD, James A, Hogg JC. A model of airway narrowing in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1992;145(6):1251–8. 68. Jelihovsky T. The structure of bronchial plugs in mucoid impaction, bronchocentric granulomatosis and asthma. Histopathology 1983;7(2):153–67. 69. Ward C, Walters H. Airway wall remodelling: the influence of corticosteroids. Curr Opin Allergy Clin Immunol 2005;5(1):43–8.

58. Kuwano K, Bosken CH, Pare PD, et al. Small airways dimensions in asthma and in chronic obstructive pulmonary disease. Am Rev Respir Dis 1993;148(5):1220–5.

70. Capraz F, Kunter E, Cermik H, Ilvan A, Pocan S. The effect of inhaled budesonide and formoterol on bronchial remodeling and HRCT features in young asthmatics. Lung 2007; 185(2):89–96.

59. Takizawa T, Thurlbeck WM. A comparative study of four methods of assessing the morphologic changes in chronic bronchitis. Am Rev Respir Dis 1971;103(6):774–83.

71. Gibson PG, Dolovich J, Denburg J, Ramsdale EH, Hargreave FE. Chronic cough: eosinophilic bronchitis without asthma. Lancet 1989;1(8651):1346–8.

60. Dunnill MS, Massarella GR, Anderson JA. A comparison of the quantitative anatomy of the bronchi in normal subjects, in status asthmaticus, in chronic bronchitis, and in emphysema. Thorax 1969;24(2):176–9.

72. Gibson PG, Fujimura M, Niimi A. Eosinophilic bronchitis: clinical manifestations and implications for treatment. Thorax 2002;57(2):178–82.

61. Hossain S. Quantitative measurement of bronchial muscle in men with asthma. Am Rev Respir Med 1973;107:99–109. 62. Hossain S, Heard BE. Hyperplasia of bronchial muscle in chronic bronchitis. J Pathol 1970;101(2):171–84. 63. Ebina M, Takahashi T, Chiba T, Motomiya M. Cellular hypertrophy and hyperplasia of airway smooth muscles underlying bronchial asthma. A 3-D morphometric study. Am Rev Respir Dis 1993;148(3):720–6. 64. Ebina M, Yaegashi H, Chiba R, et al. Hyperreactive site in the airway tree of asthmatic patients revealed by thickening of bronchial muscles. A morphometric study. Am Rev Respir Dis 1990;141(5 Pt 1):1327–32. 65. Pare PD, Wiggs BR, James A, Hogg JC, Bosken C. The comparative mechanics

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73. Ayik SO, Basoglu OK, Erdinc M, et al. Eosinophilic bronchitis as a cause of chronic cough. Respir Med 2003;97(6):695–701. 74. Brightling CE. Chronic cough due to nonasthmatic eosinophilic bronchitis: ACCP evidence-based clinical practice guidelines. Chest 2006;129(1 Suppl):116S–21S. 75. Birring SS, Berry M, Brightling CE, Pavord ID. Eosinophilic bronchitis: clinical features, management and pathogenesis. Am J Respir Med 2003;2(2):169–73. 76. Brightling CE, Pavord ID. Eosinophilic bronchitis: an important cause of prolonged cough. Ann Med 2000;32(7):446–51. 77. Brightling CE, Pavord ID. Eosinophilic bronchitis – what is it and why is it important? Clin Exp Allergy 2000;30(1):4–6.

78. Brightling CE, Symon FA, Birring SS, et al. Comparison of airway immunopathology of eosinophilic bronchitis and asthma. Thorax 2003;58(6):528–32. 79. Brightling CE, Ward R, Goh KL, Wardlaw AJ, Pavord ID. Eosinophilic bronchitis is an important cause of chronic cough. Am J Respir Crit Care Med 1999;160(2):406–10. 80. Brightling CE, Ward R, Woltmann G, et al. Induced sputum inflammatory mediator concentrations in eosinophilic bronchitis and asthma. Am J Respir Crit Care Med 2000;162(3 Pt 1):878–82. 81. Belda J, Leigh R, Parameswaran K, et al. Induced sputum cell counts in healthy adults. Am J Respir Crit Care Med 2000;161(2 Pt 1):475–8. 82. Berry MA, Hargadon B, McKenna S, et al. Observational study of the natural history of eosinophilic bronchitis. Clin Exp Allergy 2005;35(5):598–601. 83. Carrington C, Addington W, Goff A, et al. Chronic eosinophilic pneumonia. N Engl J Med 1969;280(15):787–98. 84. Liebow A, Carrington C. The eosinophilic pneumonias. Medicine (Baltimore) 1969;48(4):251–85. 85. Alam M, Burki NK. Chronic eosinophilic pneumonia: a review. South Med J 2007;100(1):49–53. 86. Marchand E, Reynaud-Gaubert M, Lauque D, et al. Idiopathic chronic eosinophilic pneumonia. A clinical and follow-up study of 62 cases. The Groupe d’Etudes et de Recherche sur les Maladies “Orphelines” Pulmonaires (GERM“O”P). Medicine (Baltimore) 1998;77(5):299–312. 87. Allen J, Davis W. Eosinophilic lung diseases. Am J Respir Crit Care Med 1994;150(5 Pt 1):1423–38. 88. Jederlinic P, Sicilian L, Gaensler E. Chronic eosinophilic pneumonia. A report of 19 cases and a review of the literature. Medicine (Baltimore) 1988;67(3):154–62. 89. Marchand E, Cordier JF. Idiopathic chronic eosinophilic pneumonia. Semin Respir Crit Care Med 2006;27(2):134–41. 90. Alberts WM. Eosinophilic interstitial lung disease. Curr Opin Pulm Med 2004;10(5):419–24. 91. Cottin V, Cordier JF. Eosinophilic pneumonias. Allergy 2005; 60(7):841–57.

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92. Johkoh T, Müller NL, Akira M, et al. Eosinophilic lung diseases: diagnostic accuracy of thin-section CT in 111 patients. Radiology 2000;216(3):773–80. 93. Travis W, Colby T, Koss M, et al. Nonneoplastic disorders of the lower respiratory tract. In: Atlas of Nontumor Pathology. First Series, Fascicle 2 ed. Washington DC: American Registry of Pathology and the Armed Forces Institute of Pathology, 2002. 94. Churg A, Myers J, Tazelaar H, Wright J, eds. Thurlbeck’s Pathology of the Lung, 3rd ed. New York: Thieme Medical, 2005. 95. Katzenstein A, Askin F. Surgical Pathology of Non-neoplastic Lung Disease, 4th ed. Philadelphia: W.B. Saunders, 2006. 96. Leslie KO, Wick MR. Practical Pulmonary Pathology: a diagnostic approach. Philadelphia: Churchill Livingstone, 2005. 97. Bosken C, Myers J, Greenberger P, et al. Pathologic features of allergic bronchopulmonary aspergillosis. Am J Surg Pathol 1988;12(3):216–22. 98. Katzenstein AL, Liebow AA, Friedman PJ. Bronchocentric granulomatosis, mucoid impaction, and hypersensitivity reactions to fungi. Am Rev Respir Dis 1975;111(4):497–537. 99. Lombard CM, Tazelaar HD, Krasne DL. Pulmonary eosinophilia in coccidioidal infections. Chest 1987;91(5):734–6. 100. Ryu JH, Myers JL, Swensen SJ. Bronchiolar disorders. Am J Respir Crit Care Med 2003;168(11):1277–92. 101. Carrington C, Gaensler E, Coutu R, et al. Natural history and treated course of usual and desquamative interstitial pneumonia. New Engl J Med 1978;298(15):801–9. 102. Yousem SA. Eosinophilic pneumonialike areas in idiopathic usual interstitial pneumonia. Mod Pathol 2000;13(12):1280–4. 103. Elliott MW, Newman Taylor A J. Allergic bronchopulmonary aspergillosis. Clin Exp Allergy 1997;27 Suppl 1:55–9. 104. Franquet T, Muller NL, Gimenez A, et al. Spectrum of pulmonary aspergillosis: histologic, clinical, and radiologic findings. Radiographics 2001;21(4):825–37. 105. Franquet T, Muller NL, Oikonomou A, Flint JD. Aspergillus infection of the

airways: computed tomography and pathologic findings. J Comput Assist Tomogr 2004;28(1):10–6. 106. Khan AN, Jones C, Macdonald S. Bronchopulmonary aspergillosis: a review. Curr Probl Diagn Radiol 2003;32(4):156–68. 107. Wark PA, Gibson PG. Allergic bronchopulmonary aspergillosis: new concepts of pathogenesis and treatment. Respirology 2001; 6(1):1–7. 108. Wardlaw A, Geddes DM. Allergic bronchopulmonary aspergillosis: a review. J R Soc Med 1992;85(12):747–51. 109. Agarwal R. Allergic bronchopulmonary aspergillosis. Chest 2009; 135(3):805–26. 110. Kraemer R, Delosea N, Ballinari P, Gallati S, Crameri R. Effect of allergic bronchopulmonary aspergillosis on lung function in children with cystic fibrosis. [see comment] Am J Respir Crit Care Med 2006;174(11):1211–20. 111. Mroueh S, Spock A. Allergic bronchopulmonary aspergillosis in patients with cystic fibrosis. Chest 1994;105(1):32–6. 112. Simmonds EJ, Littlewood JM, Evans EG. Cystic fibrosis and allergic bronchopulmonary aspergillosis. Arch Dis Child 1990;65(5):507–11. 113. Greenberger PA, Patterson R. Diagnosis and management of allergic bronchopulmonary aspergillosis. Ann Allergy 1986;56(6):444–8. 114. Cockrill BA, Hales CA. Allergic bronchopulmonary aspergillosis. Annu Rev Med 1999;50:303–16. 115. Myers JL. Bronchocentric granulomatosis. Disease or diagnosis? Chest 1989;96(1):3–4. 116. Davis WB, Wilson HE, Wall RL. Eosinophilic alveolitis in acute respiratory failure. A clinical marker for a non-infectious etiology. Chest 1986;90(1):7–10. 117. Allen J. Acute eosinophilic pneumonia. Semin Respir Crit Care Med 2006;27(2):142–7.

pneumonia. Intern Med 1992;31(9):1139–43. 120. Umeki S, Soejima R. Acute and chronic eosinophilic pneumonia: clinical evaluation and the criteria. Intern Med 1992;31(7):847–56. 121. Philit F, Etienne-Mastroianni B, Parrot A, et al. Idiopathic acute eosinophilic pneumonia: a study of 22 patients. Am J Respir Crit Care Med 2002;166(9):1235–9. 122. Pope-Harman AL, Davis WB, Allen ED, Christoforidis AJ, Allen JN. Acute eosinophilic pneumonia. A summary of 15 cases and review of the literature. Medicine (Baltimore) 1996;75(6):334–42. 123. Shorr AF, Scoville SL, Cersovsky SB, et al. Acute eosinophilic pneumonia among US military personnel deployed in or near Iraq. JAMA 2004;292(24):2997–3005. 124. Al-Saieg N, Moammar O, Kartan R. Flavored cigar smoking induces acute eosinophilic pneumonia. Chest 2007;131(4):1234–7. 125. Miki K, Miki M, Nakamura Y, et al. Early-phase neutrophilia in cigarette smoke-induced acute eosinophilic pneumonia. Intern Med 2003;42(9):839–45. 126. Nakajima M, Manabe T, Sasaki T, Niki Y, Matsushima T. Acute eosinophilic pneumonia caused by cigarette smoking. Intern Med 2000;39(12):1131–2. 127. Shiota Y, Kawai T, Matsumoto H, et al. Acute eosinophilic pneumonia following cigarette smoking. Intern Med 2000;39(10):830–3. 128. Watanabe K, Fujimura M, Kasahara K, et al. Acute eosinophilic pneumonia following cigarette smoking: a case report including cigarette-smoking challenge test. Intern Med 2002;41(11):1016–20. 129. Tazelaar H, Linz L, Colby T, et al. Acute eosinophilic pneumonia: histopathologic findings in nine patients. Am J Respir Crit Care Med 1997;155(1):296–302.

118. Badesch DB, King TE Jr, Schwarz MI. Acute eosinophilic pneumonia: a hypersensitivity phenomenon? Am Rev Respir Dis 1989;139(1):249–52.

130. Mochimaru H, Kawamoto M, Fukuda Y, Kudoh S. Clinicopathological differences between acute and chronic eosinophilic pneumonia. Respirology 2005;10(1):76–85.

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144. Coax WA. Reactive eosinophilic pleuritis and pleural effusion. Arch Pathol Lab Med 1977;101(10):555. 145. Askin FB, McCann BG, Kuhn C. Reactive eosinophilic pleuritis: a lesion to be distinguished from pulmonary eosinophilic granuloma. Arch Pathol Lab Med 1977; 101(4):187–91.

140. Yousem S, Lombard C. The eosinophilic variant of Wegener’s granulomatosis. Hum Pathol 1988;19(6):682–8.

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Chapter

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Drug- and therapy-induced lung injury Mary Beth Beasley and Glenn A. Rudner

Introduction Drug-induced lung disease represents one of the more challenging areas of pulmonary medicine from both a clinical and pathological standpoint. Over 300 agents are associated with adverse pulmonary reactions and the list is continually expanding.1 Clinically, drug-induced pulmonary disease may manifest as an acute, subacute or chronic reaction. An acute reaction occurs within minutes or hours following exposure. Anaphylaxis, bronchospasm and pulmonary edema are examples of such acute reactions. Subacute reactions occur days to weeks following administration and chronic reactions manifest months to years after initiation of a particular drug. Complicating the picture are drug reactions presenting as an acute lung disease in patients who have taken a drug for years.1,2 Additionally, drug reactions may occur after discontinuation of a drug, most notably exemplified by bischloroethyl nitrosourea (BCNU) therapy, in which the drug has been implicated in pulmonary disease years after discontinuation.3 Clinical diagnoses of drug toxicity require a high index of suspicion, given that presenting symptoms are not specific and overlap with numerous other pulmonary diseases. Further confounding the picture, a patient may have multiple factors which could potentially contribute to the development of pulmonary disease (e.g. a patient with rheumatoid arthritis on methotrexate or an immunosuppressed patient on chemotherapy). Radiographic findings similarly tend to reflect the wide range of histological findings associated with drug toxicity and are typically not specific by themselves. Specific laboratory findings have also remained elusive. Krebs von den Lunge-6 (KL-6) is a high molecular weight glycoprotein expressed by type II pneumocytes, and elevated serum KL-6 levels have been reported in association with druginduced pneumonitis. Unfortunately, elevated KL-6 levels are only seen in approximately half of drug-induced pneumonitis cases and are also encountered in idiopathic interstitial pneumonitis, pneumonitis related to connective tissue disease and hypersensitivity pneumonitis. However, since elevated levels have not been observed in infectious pneumonia, aspergillosis,

asthma, idiopathic eosinophilic pneumonia or organizing pneumonia, this test may be of some value.4,5 Ultimately, a diagnosis of a drug reaction, much like interstitial lung disease in general, requires careful clinical, radiographic and pathological correlation. Criteria for diagnosis of a pulmonary drug reaction include correct identification of the drug in question, exclusion of other primary or secondary lung diseases, an appropriate temporal relationship, a characteristic reaction pattern to a specific drug, and remission of symptoms with removal of drug. Confirmation would ideally include recurrence of symptoms with rechallenge but in reality this is seldom done.6 In some cases, the temporal relationship between administration of the drug and the onset of symptoms, or the presence of a clinical finding associated with a particular drug, such as peripheral blood eosinophilia, will point to a diagnosis of drug reaction clinically. Therefore a lung biopsy will not be required. In other clinical situations, where the association is less clear cut or the patient has other potential diagnostic considerations, biopsy may be performed. The pathologist’s role in interpretation of a biopsy from a suspected drug reaction is somewhat limited, given a precise diagnosis is not often made from histology alone. The pathologist’s role is essentially two-fold. First, he/she should identify the histological pattern of disease, which may exclude other diagnoses. For example, eosinophilic pneumonia in a patient with suspected toxicity, related to sulfasalazine, and lacking other potential causes of eosinophilic pneumonia supports a clinical diagnosis of drug toxicity. The second major role of the pathologist is to exclude other potential causes of lung disease, especially infections. As with most diffuse lung diseases, a wedge biopsy or large tissue sample provides the most information with regard to classification of the histological pattern of disease. Transbronchial biopsy may be useful to exclude infection or malignancy. It is possible on such a small biopsy to identify patterns of disease, such as eosinophilic pneumonia, organizing pneumonia, macrophages characteristic of amiodarone exposure, or diffuse alveolar damage (DAD). Finding non-necrotizing

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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granulomas may support a drug reaction in the setting of a potential reaction to an agent known to produce a sarcoid-like reaction, such as interferon-a, but otherwise should raise suspicion for infection. It is essential the pathologist remembers a small biopsy may not be representative.7 Similarly, cytology specimens such as bronchoalveolar lavage (BAL) play a limited role and are primarily useful in identifying infection or malignancy. Eosinophilia or amiodarone-associated alveolar macrophages may be detected. Inverted CD4/CD8 ratios have been reported in hypersensitivity reactions and might have some utility.8,9 The aim of this chapter includes an overview of the pathogenesis of pulmonary drug toxicity, a review of the most commonly encountered patterns of injury, a specific review of reactions associated with selected drugs, radiation-induced pulmonary disease, transfusion-related acute lung injury and drug reactions associated with illicit drug use. Drug-induced pulmonary disease in the broadest sense also includes secondary infections and malignancies, which are covered elsewhere in the text.

Pathogenesis of drug-induced pulmonary disease The mechanism of drug-induced pulmonary injury is poorly understood. It is probably multifactorial and dependent on the specific agent in question. Additionally, pre-existing lung diseases, such as pulmonary fibrosis or chronic obstructive pulmonary disease (COPD), have been associated with an increased incidence of pulmonary drug toxicity. Some drugs may have an additive effect when administered simultaneously. Most therapeutic drugs reach the lungs through the bloodstream via intravenous or enteric intake, although some may be administered by inhalation. Nontherapeutic and/or illicit drugs may be introduced through either mechanism. Ocular and topical medications may also reach the lungs. Several theories have been suggested regarding the development of pulmonary toxicity. First, a drug may cause direct injury to pneumocytes or capillary endothelium resulting in release of cytokines and recruitment of inflammatory cells. A second theory is that a drug may cause a systemic release of cytokines, which circulate and injure pneumocytes and endothelium. Cell-mediated lung injury may also play a role as elevated CD4/CD8 ratios have been found in BAL fluid. Oxygen free radicals have also been suggested as a potential cause. Targeted agents have the potential to incite pulmonary injury by their own unique mechanisms. For example, epidermal growth factor receptors (EGFR) are expressed on type II pneumocytes, which are involved in alveolar wall repair. Drugs which act as EGFR inhibitors or EGFR tyrosine kinase inhibitors may impair alveolar repair mechanisms and potentially enable injury due to other causes, such as infection or other medications.1,2,10,11

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Histological patterns associated with drug toxicity Drug-induced lung disease is not characterized by a single or specific histological pattern of lung disease. Few if any drug toxicities are characterized by unique histological findings. A single drug may be associated with multiple different microscopic patterns of pulmonary disease. Conversely, the finding of a particular histological pattern, such as DAD, may be produced by myriad drugs or other etiologies. Additionally, retrospective review of the literature is complicated by the use of outdated or inappropriate nomenclature. Given these facts, the commonest manifestations of drug-induced lung disease include DAD, “chronic interstitial pneumonitis”, organizing pneumonia, eosinophilic pneumonia, pulmonary edema, pulmonary hypertension, alveolar hemorrhage, pulmonary venoocclusive disease and granulomatous inflammation. Table 1 summarizes the most common histological patterns of disease and the associated drugs. Additionally, some drug toxicities have been associated with a presentation of radiographic nodules, mimicking malignancy. While some of these reactions represent nodular foci of organizing pneumonia or granulomas, unique findings, such as those discussed in the section on amiodarone, also occur. As the list of drugs with pulmonary reactions is constantly expanding, the reader is referred to the excellent website www.pneumotox.com for a continued update on the most recently described pulmonary drug reactions.12 In this chapter, the histological patterns of disease will be discussed briefly below and the reader is referred to the appropriate sections of the text for more detailed information on the histopathological features of each pattern. Pleural disease associated with drug toxicity, while less frequently encountered than parenchymal, will also be discussed. Drug reactions associated with selected drugs of frequent clinical inquiry will follow.

Diffuse alveolar damage Diffuse alveolar damage is the most frequently described histological pattern associated with pulmonary drug toxicity. Chemotherapeutic agents account for the majority of cases of drug-induced DAD, with busulfan, cyclophosphamide, BCNU and bleomycin among the most commonly implicated (see Table 1).2,13–15 The histological pattern of DAD is not specific for druginduced lung disease and may be seen in association with myriad other underlying etiologies. Diffuse alveolar damage is discussed in detail in Chapter 9. The histological findings depend on the stage of disease. In the acute phase, the interstitium exhibits edematous widening, type II pneumocyte hyperplasia and hyaline membranes. In the organizing phase, the hyaline membranes subside and the interstitium contains myxoid fibrous tissue (Figure 1). Type II pneumocyte hyperplasia usually remains prominent and the cells may

Chapter 16: Drug- and therapy-induced lung injury Table 1 Drugs associated with the most common patterns of drug-induced lung injury12,14,18,5

Diffuse alveolar damage: Amiodarone, amitriptyline, azothioprine, BCNU, bleomycin, busulfan, CCNU, cocaine, colchicine, cyclophosphamide, cytosine arabinoside, deferoxamine mesylate, erlotinib, etoposide, everolimus, gefitinib, gemcitabine, gold salts, heroin, imatinib, infliximab, irinotecan, melphalan, methotrexate, mitomycin, nitrofurantoin, paraquat, penicillamine, piritrexim, procarbazine, streptokinase, sulfathiazole, taxanes (paclitxel, docetaxel), temiposide, temozolomide, tocainide, topotecan, trastuzumab, vinblastine, vindesine, zinostatin Chronic interstitial pneumonia: Amiodarone, BCNU, busulfan, CCNU, chorambucil, chlorozotocin, cocaine, cyclophosphamide, fluoxetine, gefitinib, gold salts, ifosfamide, melphalan, methotrexate, nilutamide, nitrofurantoin, oxaliplatin, phenytoin, pindolol, procarbazine, quinidine, sulfasalazine, taxanes (paclitxel, docetaxel). temsirolimus, tocainide, uracil mustard Organizing pneumonia: Amiodarone, bleomycin, chlorozotocin, cocaine, cromolyn sodium, cyclophosphamide, doxorubicin, gold salts, hemamethonium, interferon, mecamylamine, methotrexate, mitomycin, mitozantrone, milutamide, nitrofurantoin, penicillamine, phenytoin, sulfasalazine, tocainide, topotecan

Granulomatous inflammation: Acebutol, cocaine, cromolyn sodium, fluxetine hydrochloride, interferon-a, interferon-b, methotrexate, nitrofurantoin, procarbazine, pentazocine, sirolimus, tripelennamine Lung nodules: Amiodarone, bleomycin, carbamazepine, fludarabine, minocycline, phenytoin, propylthiouracil, ticopidine, vinblastine Pleural disease: Effusion: acyclovir, amiodarone, bleomycin, bromocriptine, clozapine, cyclophosphamide, dantrolene, D-penicillamine, granulocyte-macrophage colony-stimulating factor, IL-2, imatinib, isotretinoin, itraconazole, methotrexate, methysergide, mesalamine, minoxidil, mitomycin, nitrofurantoin, proctolol, propylthiouricil, procarbazine, simvastin, sodium morrhuate/ absolute alcohol, taxanes, valproic acid Pleural thickening: ergoline drugs (methysergide, bromocriptine) Lupus-like pleuropericarditis: procainamide, hydralazine, chlorpromazine, isoniazid, mesalamine, methyldopa, penicillamine, quinidine

Eosinophilic pneumonia: Non-steroidal anti-inflammatory agents, acetaminophen, ampicillin, bleomycin, captopril, carbamazepine, chlorproamide, cocaine, cromolyn sodium, hydralazine, imipramine, mephenesin, nabumetone, naproxen, nitrofurantoin, oxaliplatin, penicillin, phenylbutazone, procarbazine, prontosil, propanolol, pyrimethamine, streptomycin, sulfasalazine, tetracycline, trazodone Pulmonary edema: Albuterol, aspirin, buprenorphine, chlordiazepoxide, cocaine, codeine, cytosine arabinoside, epinephrine, ethchlorvynol, haloperidol, heroin, hydrochlorothiazide, isoxsuprine, lidocaine, magnesium sulfate, methadone, methamphetamine, methotrexate, mitomycin, nalbuphine, naloxone, nifedipine, paraldehyde, paraquat, penicillin, propoxyphene, propanolol, radiocontrast material, ritodrine, salbutamol, salicylates, sulindac and terbutaline Pulmonary hemorrhage: Amphotericin B, amiodarone, bevacizumab, cocaine, cyclophosphamide, gefitinib, haloperidol, hydralazine, leukotriene antagonists, mitomycin, mitrofurantoin, paraquat, penicillamine, propylthiouracil, sulfonomides, streptokinase and urokinase Pulmonary hypertension: Mitomycin, aminorex, fenfluramine combined with phenteramine (fenphen), methamphetamine Pulmonary veno-occlusive disease: BCNU, bleomycin, cyclophosphamide, etoposide, mitomycin, zinostatin

Figure 1. Organizing diffuse alveolar damage. The alveolar septa are expanded by myxoid fibroconnective tissue and type II pneumocyte hyperplasia is prominent. A residual hyaline membrane is also present. This case was clinically felt to be secondary to cyclophosphamide.

exhibit marked cytological atypia. Squamous metaplasia may also be evident.16,17

Chronic interstitial pneumonitis/nonspecific interstitial pneumonia

The term “chronic interstitial pneumonitis” is often encountered in association with drug-induced lung disease (Table 1). Most cases appear to correspond to a histological pattern of nonspecific interstitial pneumonia (NSIP).2 A histological

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Figure 2. Nonspecific interstitial pneumonia, cellular pattern. Diffuse infiltration of the alveolar septa by mild to moderate chronic inflammatory infiltrates is present without associated fibrosis. This pattern is seen in association with numerous drugs and probably represents the findings in cases of “chronic interstitial pneumonitis” reported in older literature.

Figure 3. Organizing pneumonia. Organizing fibroblastic tissue is present within alveolar spaces in a patchy distribution centered on bronchioles. The interstitium in these areas contains mild lymphocytic infiltrates.

pattern mimicking usual interstitial pneumonia (UIP) is much less common.14,18 Nonspecific interstitial pneumonitis is characterized by relatively uniform parenchymal involvement by diffuse fibrosis, mild to moderate lymphocytic infiltrates or a combination of both (Figure 2). The entity is discussed in detail in Chapter 10.19,20

Organizing pneumonia Organizing pneumonia with the histological pattern previously referred to as bronchiolitis-obliterans organizing pneumonia has been increasingly recognized in association with pulmonary drug toxicity (Table 1).21 This pattern is usually associated with a subacute onset of symptoms and patchy ground-glass opacities radiographically. A pattern of large nodules of organizing pneumonia, resembling metastatic disease radiologically, has been reported in association with bleomycin.22–24 Histologically, organizing pneumonia is characterized by patchy involvement of the lung parenchyma by intra-alveolar plugs of organizing fibroblastic tissue, which may also involve bronchiolar lumina. The associated interstitium typically exhibits mild lymphocytic infiltrates which dissipate away from the areas of alveolar involvement (Figure 3).17,25 Organizing pneumonia is described in detail in Chapter 10.

Eosinophilic pneumonia Drug toxicity should always be a consideration in a patient with acute or chronic eosinophilic pneumonia. The term “pulmonary infiltrates with eosinophilia” may be encountered in older literature. Eosinophilic pneumonia is discussed in depth in Chapter 15, and a summary of drugs associated with this histological pattern is found in Table 1.26 Acute eosinophilic pneumonia may present with respiratory failure and most

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Figure 4. Eosinophilic pneumonia. Intra-alveolar fibrin is admixed with large numbers of eosinophils. Eosinophils are also present in alveolar septa, which also show mild pneumocyte hyperplasia. This case was clinically felt to be secondary to sulfasalazine.

patients lack peripheral blood eosinophilia. Patients with chronic eosinophilic pneumonia typically have an indolent presentation and usually show peripheral blood eosinophilia. Histologically, both acute and chronic forms feature intraalveolar eosinophils and oftentimes microabscesses. In addition, acute eosinophilic pneumonia features hyaline membranes along with intra-alveolar fibrin and macrophages, while chronic eosinophilic pneumonia rarely demonstrates hyaline membranes (Figure 4). It is important to note that eosinophils clear rapidly from pulmonary tissue after steroid therapy has been administered. This should be taken into account in patients with a high clinical suspicion for eosinophilic pneumonia.27–30

Chapter 16: Drug- and therapy-induced lung injury

Pulmonary edema Pulmonary edema is characterized by intra-alveolar proteinaceous material and is usually not difficult to recognize histologically. Pulmonary edema may represent a very early manifestation of DAD, although distinction between edema and very early DAD may not always be possible. This is especially the case if more classic features of acute DAD, such as hyaline membranes, are absent. Drugs associated with pulmonary edema are summarized in Table 1.13 Pulmonary edema should also be distinguished from pulmonary alveolar proteinosis (PAP), characterized by coarsely granular intra-alveolar material, which may contain cholesterol clefts (see Chapter 10). While PAP is not typically associated with drug toxicity, there has been at least one case report of PAP associated with leflunomide, an anti-rheumatological agent.31 Pulmonary alveolar proteinosis has been reported in patients with chronic myelogenous leukemia (CML) who have received busulfan. As PAP may be seen in association with underlying hematological malignancies, the association of PAP with busulfan is more tenuous.32

Pulmonary hemorrhage/vasculitis Pulmonary hemorrhage may occur secondary to drug reactions (Table 1).33 Pulmonary hemorrhage is classically characterized by the accumulation of intra-alveolar macrophages containing coarse hemosiderin. Fresh blood and fibrin are often present, and the presence of hemosiderin indicates the pathological nature of the blood rather than representing a procedural artifact. Hemosiderin generally takes 48 hours to form and may not be seen in extremely acute cases of hemorrhage, a situation requiring clinical correlation to avoid misinterpretation of abundant fresh blood. Similar to pulmonary hemorrhage syndromes, discussed in Chapter 19, druginduced pulmonary hemorrhage may be associated with neutrophilic capillaritis.33 Medium vessel vasculitis is, in contrast, infrequently encountered in drug reactions. However, cases of drug-induced Churg-Strauss syndrome have been reported in association with leukotriene antagonists such as pranlukast, zafirlukast and montelukast.34

Pulmonary hypertension/veno-occlusive disease Pulmonary hypertension associated with drug toxicity has most recently received attention due to association with anorexigens, particularly fenfluramine combined with phenteramine (so-called “fenphen”), but has also been reported in association with other drugs (Table 1).35–39 The vascular changes in drug-induced pulmonary hypertension are essentially identical to those due to other causes. The findings range from intimal and medial thickening to plexiform and angiomatoid lesions.36,37 Pulmonary veno-occlusive disease (PVOD) is a rare entity, which has been reported in association with drug toxicity in a small number of cases, namely in association with bleomycin,

busulfan, carmustine (BCNU) contraceptives (oral), lomustine (CCNU) and nitrosoureas.40–46 Pulmonary veno-occlusive disease is characterized by obstruction of small pulmonary veins as well as evidence of pulmonary arterial hypertension and occlusive change in pulmonary arteries. Associated hemorrhage and proliferation of capillaries in the alveolar septa may also be present. Pulmonary hypertension and PVOD are discussed in Chapter 18.

Granulomatous inflammation Granulomatous inflammation may occasionally represent a manifestation of a pulmonary drug reaction (Table 1). Such reactions may consist of poorly formed granulomas, similar to those seen in hypersensitivity pneumonitis, or well-formed granulomas comparable to those encountered in infection or sarcoidosis. The presence of granulomas should always raise the question of infection, and necrosis should particularly point to infection over other causes. Sarcoidosis-like reactions have been reported in association with interferon-a and interferon-b administration in particular, while small granulomas are more frequently seen in association with etanercept, leflunomide, mesalamine and methotrexate.2,14,18,47–49 Both necrotizing and non-necrotizing granulomas are documented in association with sirolimus.50–52 Sarcoidosis is discussed in detail in Chapter 13.

Pleural disease Drug-induced pleural disease may occur in isolation or in association with underlying parenchymal disease. Isolated drug-induced pleural disease is much less common than drug-induced parenchymal disease. Pleural disease may include asymptomatic pleural effusion, acute pleuritis or pleural thickening. In most cases, the mechanism is speculative but postulated theories include hypersensitivity reaction, direct toxic effect, increased oxygen free radical production, suppression of anti-oxidant defenses and chemical-induced inflammation. In addition, a number of drugs have been associated with drug-induced lupus-associated pleuritis. A summary of drug induced pleural disease is given in Table 1.53–55 Drugs most commonly associated with pleural disease include cardiovascular agents, ergoline drugs, sclerotherapy agents and chemotherapeutic agents. Pathologically the findings are often nonspecific. The pleural fluid is usually an exudate. Pleural fluid eosinophilia, defined as greater than 10% eosinophils, may provide a clue to a drug reaction. This finding has been associated with valproic acid, propylthiouracid, isotrentinoin, nitrofurontoin, bromocriptine, dantrolene, glicazide and mesalamine. Peripheral blood eosinophilia may or may not be present. Pleural fluid eosinophilia is not specific for drug reaction; however, and has been associated with other entities including pneumothorax, benign asbestos pleural effusion, fungal and parasitic infections and pulmonary infarction.7,53,55,56 Drug-induced lupus has been reported in association with numerous drugs but has only been reported to be strongly

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Figure 5. Amiodarone toxicity. Intra-alveolar macrophages contain vacuoles characteristic of amiodarone exposure. The case was otherwise consistent with organizing diffuse alveolar damage and the background in this field exhibits interstitial widening and pneumocyte hyperplasia.

associated with those in Table 1. Pleuropulmonary involvement is very common in drug-induced lupus, occurring in slightly over half of all patients. Pleural fluid characteristics of both drug-induced and systemic lupus are essentially identical and are exudative with normal pH and glucose. Pleural fluid antinuclear antibodies are usually higher than corresponding serum values and the titre is usually 1:250 or higher. Cytological evaluation may reveal so-called LE cells representing neutrophils, which have ingested the nuclear material of a degenerating cell resulting in a large eosinophilic inclusion.53,56

Pulmonary drug toxicity associated with selected agents Amiodarone Amiodarone-related pulmonary toxicity occurs following administration a few months up to several years of therapy. The prevalence ranges from 10 to 15% of patients and most are diagnosed after an average of 2 months of treatment. An increased risk of respiratory complications has been noted in patients older than 60 years of age and those on maintenance doses of 400 mg or greater per day. From a clinical standpoint, CT scans may show nonspecific findings, usually ground-glass infiltrates; however, a pattern of peripheral high-attenuation consolidation, accompanied by increased attenuation in the liver or spleen, is relatively specific for amiodarone toxicity. This pattern has been attributed to deposition of iodine-containing amiodarone metabolites.57–59 A spectrum of pulmonary disease has been reported in association with amiodarone toxicity; however, the most common manifestations are DAD and chronic interstitial pneumonitis, resembling NSIP. In all cases, a characteristic finding is the presence of prominent foamy histiocytes with

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Figure 6. Amiodarone toxicity. Electron micrograph of a macrophage in a case of amiodarone toxicity showing membrane-bound, whorled lysosomal inclusion bodies. (Image courtesy of E. Mark, MD, Boston, MA, USA.)

relatively uniform vacuoles, corresponding to membranebound, whorled, lysosomal inclusion bodies by electron microscopy (Figures 5 and 6). Such macrophages are also encountered in patients on amiodarone in the absence of clinical lung toxicity. Vacuolization may also occur in endothelial cells and type II pneumocytes.59–61 An unusual pattern of multiple lung nodules mimicking malignancy has also been reported, although histological findings are limited.57,58 A series of four patients with lung nodules and central basophilic necrosis, surrounded by either a rim of eosinophilic necrosis or solid inflammatory cells, is documented (Figure 7). These areas were in turn surrounded by vacuolated histiocytes, which showed characteristic features associated with amiodarone by electron microscopy. Some cases demonstrated areas of necrotizing bronchiolitis and abscess formation with features simulating those of infection, or in some Wegener granulomatosis, but no giant cells or palisaded histiocytes were present. Cultures and special stains were negative for microorganisms and the totality of the findings corroborated that the histological findings were secondary to amiodarone.62

Methotrexate Methotrexate-related pulmonary disease is most frequently encountered in patients receiving low-dose methotrexate for rheumatoid arthritis. The incidence is approximately 5%. Patients typically present within the first year after starting therapy and experience a subacute febrile illness, with almost 20% having a peripheral blood eosinophilia. Biopsies most frequently show the cellular pattern of NSIP, with up to half

Chapter 16: Drug- and therapy-induced lung injury

(a)

(b)

Figure 7. Amiodarone toxicity presenting as pulmonary nodules. (a) The nodules consist of zones of basophilic necrosis surrounded by a rim of inflammatory cells. (b) At higher magnification vacuolated histiocytes are seen. (Images courtesy of E. Mark, MD, Boston, MA, USA).

(a)

(b)

Figure 8. Methotrexate toxicity. (a) In this example of methotrexate toxicity there is an interstitial pattern resembling cellular nonspecific interstitial pneumonia. (b) Occasional poorly formed granulomas are also seen.

of cases also having associated eosinophils. Poorly formed granulomas may be encountered, similar to those in hypersensitivity pneumonitis (Figure 8).2,63–67

Busulfan Busulfan is an alkylating agent used for the treatment of leukemias, soft tissue sarcomas and other malignancies. The incidence of pulmonary toxicity appears to be dose-related and it occurs in less than 5% of patients. Concurrent radiation therapy may exacerbate toxicity. An interesting aspect of busulfan-related pulmonary toxicity is that it may follow a long latent period, with the average onset exceeding 4 years. The prognosis is typically poor.15,68,69

Histologically, busulfan-related lung disease is essentially that of acute and/or organizing DAD and is associated with diffuse interstitial fibrosis. Type II pneumocytes frequently show marked atypia, as may bronchiolar epithelium (Figure 9), although this finding is not pathognomonic.14,15,18,55 A pattern of injury resembling alveolar proteinosis has also been reported.32

Bleomycin Bleomycin is a widely recognized agent of pulmonary toxicity. It has served as an experimental animal model to study pulmonary fibrosis for potential application to human disease. Bleomycin toxicity occurs in approximately 10% of treated patients and

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Figure 9. Organizing diffuse alveolar damage attributable to busulfan toxicity. Type II pneumocyte hyperplasia is particularly marked. Although not pathognomonic, the marked atypia seen in type II pneumocytes is a characteristic finding in busulfan toxicity.

is dose-related. As with other therapies, pre-existing underlying lung disease and use of other concurrent chemotherapeutic drugs and/or radiation are thought to increase the risk of pulmonary toxicity. The clinical presentation may be fulminate or indolent. Radiographs can demonstrate ground-glass opacities or consolidation. Nodular densities mimicking metastatic disease are reported.22,23,68,70–73 Most cases of bleomycin toxicity show acute or organizing DAD. Diffuse fibrosis, which may be progressive and exhibit honeycomb change, may occur. Cases of bleomycin toxicity associated with hypersensitivity pneumonitis, eosinophilic pneumonia, NSIP, organizing pneumonia, veno-occlusive disease and pleuritis have also been reported.14,15,18,55,68,72

Nitrofurantoin

particularly used with renal transplants. Temsirolimus, a related drug, has promise as an anti-neoplastic agent, especially against renal cell carcinoma. Everolimus has also been used for immunosuppression following organ transplantation and holds promise as an anti-neoplastic agent. All three drugs have been associated with the development of interstitial pneumonitis. Reported pathology includes lymphocytic interstitial infiltrates, organizing pneumonia and alveolar hemorrhage.11,78 Necrotizing and non-necrotizing granulomas have also been reported (Figure 10).50

Nitrofurantoin is used primarily for treatment and prophylaxis of urinary tract infections. Symptoms include dyspnea, rash, fever and peripheral blood eosinophilia. These usually occur within the first month of therapy. Prognosis is generally favourable, with symptoms resolving following discontinuation of the drug. A small number of patients develop chronic toxicity with progressive pulmonary infiltrates and fibrosis.74,75 A variety of histological findings have been reported in association with nitrofurantoin toxicity including DAD, organizing pneumonia, hypersensitivity pneumonitis and a pattern resembling cellular NSIP. The chronic form has been primarily reported as a fibrosing pneumonitis with either a UIP or NSIP pattern.14,18,21,76 Cases associated with a giant cell interstitial pneumonic pattern have also been reported.77

Monoclonal antibodies and targeted inhibitors

Rapamycin and rapamycin analogs Sirolimus (rapamycin, Rapamune®) is an immunosuppressive

Monoclonal antibodies Bevacizumab (Avastin®) is

agent used in preventing organ transplant rejection and is

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Figure 10. Granulomatous pneumonitis clinically attributable to Sirolimus toxicity. A loosely formed non-necrotizing granuloma is present in the center of the field. The parenchyma is also remarkable for intra-alveolar macrophage and fibrin accumulation. (Image courtesy of D. Flieder, MD, Philadelphia, PA, USA).

Novel therapeutic agents have emerged in recent years which utilize direct targeting of specific cellular antigens or receptors. The majority of these agents are used as anti-neoplastic agents. Others are used as anti-virals or immune modulating agents for treatment of rheumatoid arthritis, other autoimmune disorders or following organ transplantation. Some agents are effective in treating macular degeneration and other ocular disorders.11 Pulmonary toxicity associated with these novel agents is rare, and reports are mainly clinical with limited morphological descriptions of the histopathology. The discussion below represents reported findings in the more commonly encountered agents.

a monoclonal antibody directed against vascular endothelial growth factor and is used

Chapter 16: Drug- and therapy-induced lung injury

primarily for treatment of colorectal, pulmonary and breast carcinomas. Pulmonary hemorrhage with significant hemoptysis has been reported in lung cancer patients only. Furthermore, this adverse effect may be a therapy effect rather than a drug effect since only lung cancer patients with central squamous cell carcinomas appear to be at risk. While tumor location rather than histology may be responsible for the hemorrhage, bevucizumab use in lung cancer patients is currently reserved for those with non-squamous histology.11,79–81 Trastuzumab (Herceptin ) is a monoclonal antibody that selectively binds EGFR (HER-2) and is used for the treatment of breast carcinomas with overexpression of HER-2 protein. Pulmonary toxicity associated with trastuzamab is very rare and consists of isolated reports of acute respiratory failure and organizing pneumonia.11,82,83 Rituximab (Rituxin ) is a monoclonal antibody directed against CD20 and is used primarily for treatment of certain CD20-positive non-Hodgkin lymphomas. It has also been used for treatment of certain autoimmune disorders, such as idiopathic thrombocytopenic purpura, systemic lupus erythematosus and autoimmune hemolytic anemia. Reported pulmonary toxicities include clinical descriptions of “interstitial pneumonitis” and “interstitial fibrosis” as well as alveolar hemorrhage and respiratory failure.84 Infliximab (Remicade ) is a tumor necrosis factor-alpha (TNFa) inhibitor used primarily in the treatment of inflammatory bowel disease, as well as autoimmune disorders, such as rheumatoid arthritis. Reported pulmonary complications consist primarily of opportunistic infections. Nonspecific interstitial pneumonia and eosinophilic pleural effusions have been reported, as well as clinical cases of respiratory failure. A case of infliximab toxicity with an NSIP pattern and an associated eosinophilic and histiocytic component has also been reported (Figure 11).85,86 Cetuximab (Erbitux ) is a monoclonal antibody directed against the epidermal growth factor receptor and is used primarily in the treatment of colon cancer. Cases of DAD attributable to Cetuximab have been reported.87

®

®

®

®

Tyrosine kinase inhibitors Erlotinib (Tarceva®) and, although no longer FDA-sanctioned, Gefitinib (Iressa®) are inhibitors of the tyrosine kinase domain

of EGFR. One to 2% of patients treated with gefitinib suffer drug toxicities including interstitial pneumonitis, DAD, diffuse alveolar hemorrhage and pulmonary fibrosis. The incidence of pulmonary toxicity is higher in Japanese studies, suggesting a possible genetic predisposition. All cases were reported within the first 90 days of treatment. Pulmonary toxicity due to erlotinib has been reported in 0.8–1% of patients, with reports describing interstitial pneumonitis. Most cases were fatal.11,88–90 Imatinib (Gleevec ) is a tyrosine kinase inhibitor used primarily in the treatment of chronic myelogenous leukemia and gastrointestinal stromal tumors. Imatinib specifically

®

Figure 11. Infliximab-related lung disease. This example exhibits chronic interstitial inflammation with a pattern reminiscent of cellular NSIP. Alveolar macrophages and eosinophils are also present. (Image courtesy of E. Mark, MD, Boston, MA, USA.)

targets the tyrosine kinase domains of the abl proto-oncogene, c-kit and platelet-derived growth factor (PDGF). Pulmonary disease is largely reported in patients receiving treatment for CML but is still estimated to occur in only 0.2–1.3% of patients. Symptomatic pulmonary disease secondary to imatinib is most often due to pulmonary edema. This complication probably develops due to prolonged inhibition of PDGF, which plays a role in fluid regulation and homeostasis. Chronic interstitial pneumonitis, fibrosis and pleural effusion have also been reported.91–94

Radiation Radiation pneumonitis Radiation pneumonitis develops as a consequence of thoracic irradiation, usually given for carcinomas of the lung, esophagus, thyroid and breast, as well as hematological malignancies. Radiation pneumonitis secondary to non-therapeutic radiation/environmental exposure also occurs.95–97 The pathogenesis of radiation pneumonitis involves both direct and indirect cellular injury. Pneumocytes and alveolar capillary endothelial cells are most directly affected. Cellular injury results in damage to DNA and/or cellular membranes, either through direct injury or via intermediate damage by generation of oxygen free radicals or finally activation of a variety of cytokines and growth factors.95–97 Radiation-induced pneumonitis secondary to therapeutic radiation occurs in approximately 5–10% of patients. The risk of developing radiation-induced pneumonitis is dependent on

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dose and size of exposure field. Increased risk has also been associated with pre-existing underlying lung disease, patient age and type of the radioisotope. A history of prior irradiation or use of anti-neoplastic agents such as bleomycin, cyclophosphamide or doxorubicin also probably increases the risk of developing radiation pneumonitis.95–97 Radiation pneumonitis may be divided into acute and chronic forms both clinically and pathologically. Acute radiation pneumonitis typically develops 2–3 months after radiation therapy but may occur as late as 6 months following therapy. Most cases are subclinical and resolve without therapy. Symptoms include dyspnea, cough and fever, which is often spiking. Radiographic changes are observed more frequently than clinical symptoms. They include ground-glass changes or consolidation, which is typically confined to the radiated field. Extensive radiation exposure, such as that encountered in massive environmental exposure, may result in clinical acute respiratory distress syndrome. This typically develops days to weeks after exposure and is typically rapidly fatal. Chronic radiation pneumonitis may follow the acute phase but may rarely develop in the absence of clinically apparent acute disease. Chronic radiation pneumonitis typically develops 6 months or longer after therapy and generally stabilizes after 2 years. Symptoms are similar to those of other chronic lung diseases. Like the acute form, the radiographic changes of chronic radiation pneumonitis are typically confined to the radiation field. Occasionally the changes may extend beyond the field and may involve the contralateral lung.95–100 Histologically, acute radiation pneumonitis resembles DAD. Hyaline membranes may be present in the acute phase. In the organizing phase, type II pneumocyte proliferation occurs and the pneumocytes may have marked cytological atypia, including binucleation and large nucleoli. Atypical interstitial fibroblastic cells and vascular foam cells may be present. The fibroblasts have enlarged hyperchromatic nuclei and basophilic perhaps vacuolated cytoplasm. Blood vessels often exhibit plump endothelial cells and hyalinization. In the chronic form, there is dense fibrosis and scar formation, typically confined to the radiation field. The fibrosis is generally more prominent around vessels and airways. Atypical fibroblasts and type II pneumocytes may be present and vessels show myointimal proliferation and hyalinization (Figure 12).14,18,96,98,100 “Radiation recall pneumonitis” is an interesting phenomenon occurring in patients who receive radiation therapy and subsequent chemotherapy. In such cases, patients receiving chemotherapy develop pulmonary infiltrates in the same field as the previous radiation. Progression to bilateral diffuse disease may occur in severe cases. The precise mechanism is unknown but it is thought that the prior radiation perhaps induces subclinical lung injury and subsequent chemotherapy produces an additive “second hit” effect. The anti-neoplastic agents which have been reported in association with this phenomenon

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Figure 12. Radiation pneumonitis. Interstitial fibrosis containing sparse inflammation and occasional atypical fibroblasts. Pneumocytes are hyperplastic and exhibit atypical features and focal vacuolization.

include adriamycin, carmustine, doxorubicin, etoposide, gefitinib, gencitabine, paclitaxel and trastuzumab.101

Chemotherapeutic and radiation effects on pulmonary carcinomas Administration of neoadjuvant therapy prior to the resection of pulmonary carcinomas has become more commonplace and is particularly aimed at stage IIIA carcinomas. AJCC/UICC staging guidelines recommend assigning post-treatment staging using a “y” prefix.102 Initial studies suggest that tumors exhibiting a complete histological response or those that show extensive response, with less than 10% of the gross tumor mass containing histologically viable tumor, have a survival advantage.103,104 The percentage of tumor response may be estimated by comparing the largest cross-sectional area of residual tumor to the cross-sectional areas of the identifiable fibrous/inflammatory mass.103 Histological evidence of tumor response consists of coagulative or infarct-like necrosis, fibrosis, foam cell and/or giant cell infiltration and mixed inflammatory cell infiltrates (Figure 13). Typically a region of necrosis is surrounded by a rim of foamy macrophages and inflammatory cells. Cholesterol clefts may be present and a giant cell reaction may occur, which should not be mistaken for granulomatous inflammation. Residual tumor cells tend to have greater cytological atypia and pleomorphism in comparison with corresponding pre-treatment specimens (Figure 14). The residual tumor has the same histological type as the pretreatment specimens. The pattern of response present does not appear to be related to the type of neoadjuvant therapy administered (i.e. chemotherapy plus radiation therapy versus chemotherapy alone), or with the type of chemotherapy administered. Squamous carcinomas tend to show a significantly greater degree of response than adenocarcinomas.103–105 Interestingly, in one study, the residual tumor present in many

Chapter 16: Drug- and therapy-induced lung injury

(a)

(b)

Figure 13. Adenocarcinoma following pre-operative radiation and chemotherapy. (a) A focus of residual adenocarcinoma is present on the right while the adjacent tissue shows hemorrhage, fibrosis, inflammatory infiltrates and occasional foamy macrophages. (b) Elsewhere, viable tumor is absent and only foamy macrophages and necrosis are seen.

Radiofrequency ablation Radiofrequency ablation (RFA) is most often used in patients with hepatocellular carcinoma but holds treatment potential for primary and secondary pulmonary malignancies in patients who are poor surgical candidates. At the time of writing, it is currently being investigated at the phase 2 trial level. Reports of histological changes in the lung are limited. Owing to the differing thermal and electrical conduction properties of the lung, it cannot necessarily be assumed that the histological findings will be the same as those in the liver. A prospective study evaluating surgically resected tumors 3 days after RFA treatment describes a zone of hemorrhage at the outer rim of the ablation zone, surrounded by interstitial and alveolar edema. While tumor cells in the vicinity of this region exhibited increased cytoplasmic eosinophilia and homogenization of cytoplasm, tumor in the interior of the zone was preserved. Ultrastructural examination demonstrated changes consistent with progression to apoptosis and DNA fragmentation by transferase-mediated nick-end-labeling (TUNEL). This finding suggests a more significant degree of tumor ablation than was evident from a histological standpoint.106 Figure 14. Carcinoma following neoadjuvant therapy. The viable tumor often shows marked cytologic atypia and cellular pleomorphism as in this example of adenocarcinoma. (Image courtesy of D. Flieder, MD, Philadelphia, PA, USA.)

Transfusion-related acute lung injury

adenocarcinomas diagnosed as high-grade on pre-treatment biopsies exhibited a lepidic growth pattern. Perhaps this pattern represents a group of non-responders, although the authors cautioned that their findings should not be considered conclusive. Although the degree of fibrosis tends to correlate with radiographic evidence of size reduction, radiological evidence of size regression does not seem to significantly correlate with the degree of histological tumor response.105

Transfusion-related acute lung injury (TRALI) is a clinical syndrome associated with transfusion of plasma containing blood components. Symptoms typically begin within 2 hours of transfusion but can appear up to 6 hours later. TRALI is usually associated with antibodies to white blood corpuscles in transfused blood components. It has also been reported in association with infusion of biologically active lipids in stored blood cell components. Implicated antibodies include HLA class I and II antibodies, granulocyte antibodies and monocyte antibodies.

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TRALI is believed to occur in approximately 1 in 5000 transfusions, but is probably underrecognized. Symptoms range from mild dyspnea to fulminant respiratory failure. This condition is fatal in 5–10% of cases and is the third most common cause of transfusion-related mortality in the United States and the second most common reported cause in the United Kingdom. Patients present with dyspnea, which may progress to severe hypoxemia, and fever. Radiographs demonstrate diffuse lung infiltrates, like those seen in acute respiratory distress syndrome patients. In most patients, symptoms resolve within 96 hours with fluid and oxygen support. Since TRALI is typically a clinical diagnosis, reports of pathological findings are few and generally restricted to fatal cases. As would be expected, several cases report findings identical to those of DAD with classic hyaline membranes. Other reports demonstrate only pulmonary edema with neutrophil accumulation within alveolar capillaries. Some cases also reported neutrophils within alveolar spaces. The lack of hyaline membranes in many reported cases suggests a differing pathological mechanism from DAD.107–109

Pulmonary pathology associated with illicit drug use Given that illicit drug abuse is common and not infrequently fatal, much of what is known about the pathology of drug abuse is derived from autopsy information. From the forensic pathologist’s viewpoint, a fatal overdose as a cause of death is often established by gross and toxocological findings, and the microscopic examination portion of the autopsy is often secondary. However, the histological examination of autopsy cases has unequivocally contributed greatly to our understanding of these particular disease processes. The major classes of drugs with ensuing pulmonary manifestations are not coincidentally amongst the most commonly abused of all illicit substances, namely, cocaine, the cannabinoids and heroin. The corresponding injury patterns depend upon many factors including the route of delivery, the concentration of the drug, the chronicity of use and any additives or filler substances with the drug. In order to understand their pharmacology and pathology, it is necessary to appreciate how these drugs are manufactured and commonly consumed.

Cocaine The social implications of cocaine abuse are widely understood, and as a result the general population is familiar with some of the terminology associated with its abuse. Cocaine is easily extracted from the leaves of the coca plant, which is mostly harvested in the Far East and South America. Cocaine hydrochloride is readily converted to cocaine base, which can then be cut with a knife or broken into small pieces. Cocaine base is ingested by smoking through a glass pipe. Cocaine hydrochloride is consumed either by inhalation or

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Figure 15. Alveolar hemorrhage secondary to cocaine abuse. Alveolar macrophages are filled with coarse hemosiderin granules.

intravenously. Numerous adulterants, such as benzocaine, procaine and diluents such as mannitol, are added to both the base and hydrochloride forms of the drug throughout its processing. This yields cocaine hydrochloride concentrations ranging from 20% to greater than 95%, and cocaine base concentrations ranging from 30% to greater than 95%. Crack cocaine is the popular street name for the bicarbonate formed base of cocaine that derives it name from the crackling sound produced when it is heated and smoked. Crack cocaine has the advantages of being relatively simple to manufacture, does not require freezing to store and tends to be stable.110,111 The toxicity of cocaine is primarily a function of the vasoconstrictor effects exerted upon the coronary vessels, which may clinically lead to angina and acute myocardial infarction. Cocaine also impairs cardiac conduction, leading to arrhythmias. Other mechanisms of action by cocaine include increased blood pressure as well as an increased incidence of intracranial hemorrhage or rupture of a pre-existing cerebral artery aneurysm. Clinically, inhalational use of cocaine is associated with a reduction in pulmonary diffusion capacity for carbon monoxide as well as acute exacerbation of asthma in chronic asthmatics.110,111 There are no specific autopsy findings in patients with acute cocaine toxicity, but post-mortem studies examining the lungs of individuals with recognized histories of chronic cocaine use frequently report pulmonary congestion and pulmonary edema, along with acute and chronic alveolar hemorrhage (Figure 15). Less common findings include DAD, eosinophilic pneumonia and bronchiolitis obliterans/organizing pneumonia. Hemosiderin-laden macrophages were noted in 35% of victims, along with pulmonary artery medial hypertrophy in 20% of victims.112,113 The etiology of the edema is probably secondary to acute cardiogenic failure, as opposed to a direct pulmonary toxic effect. It is not uncommon to find lungs weighing in excess of 1000 g.

Chapter 16: Drug- and therapy-induced lung injury

(a)

(b)

Figure 16. Macrophages associated with marijuana use. (a) The macrophages contain coarse dark brown pigment. (b) The contents are negative on an iron stain. (Images courtesy of D. Hwang, MD, Toronto, Ontario, Canada.)

The proposed mechanisms for cocaine-induced alveolar hemorrhage include capillary endothelial damage with resultant reperfusion as a consequence of vasoconstriction, and direct cocaine toxicity upon the alveolar-capillary membrane.114 A bronchoalveolar lavage study in a non-fatal case of pulmonary edema following freebase cocaine use demonstrated an elevated (four times normal) protein level, implying the edema was due to altered capillary permeability.115 Prolonged inhalation associated with the smoking process probably increases the pulmonary carbon load.111 As crack cocaine is consumed, a dark residue of concentrated drug deposits within the inside of the pipe bowl. This is frequently reheated and resmoked, which is most likely the source of the increased carbon load. Barotrauma may occur during cocaine use. Increased intraalveolar pressure results from several drug culture practices. Both increased frequency of prolonged Valsalva techniques and the forceful blowing of cocaine into a partner’s lungs may precipitate alveolar rupture and pneumomediastinum. The deep inspiratory effects of these forms of barotrauma may also cause pneumothorax, rupture of a visceral pleural bleb, or emphysematous bullae.116 While rarely fatal, barotrauma is an infrequent complication of inhalational cocaine use that is also seen in marijuana smokers.110

Marijuana and related substances The hemp plant Cannabis sativa gives rise to marijuana, the most widely abused illicit drug in the world. Marijuana is a Schedule 1 controlled substance, and the plant is cultivated throughout the world both for cloth and for its fiber. The major psychoactive substance of the plant is 9-tetrahydrocannabinol (THC), which is found within the leaves, resin and flowering tops of the plant. Both marijuana and hashish, which is the resin extract of the flowering tops of the plant, and

contains a higher concentration of THC, are typically smoked, but both may be consumed orally as well.110,111 The pulmonary effects of marijuana smoking are similar to those of tobacco use, and, much akin to inhalational cocaine use, factors such as depth of inhalation and increased Valsalva maneuvers are thought to play a role in the pathology. Interestingly, no difference in the prevalence of chronic cough, sputum production or wheezing was noted in marijuana or tobacco smoking cohorts.117 Heavy and daily marijuana smoking may result in increased airway resistance. As there tends to be a fair amount of overlap in the pathological response to smoking marijuana and tobacco, separating the two can be difficult. Marijuana abusers may develop paraseptal bullae, in contrast to centrilobular emphysema seen more commonly in exclusively tobacco smokers.118 This may in part explain the relatively high frequency of spontaneous pneumothorax in cannabis smokers. Mucosal biopsies with video bronchoscopy and bronchoalveolar lavage studies on smokers of tobacco or marijuana demonstrated variously elevated levels of submucosal edema, vascular hyperplasia, inflammatory cell infiltration, significant airway inflammation and goblet cell hyperplasia, compared with non-smokers.119 Other studies also demonstrated mucosal changes, such as basal/goblet cell hyperplasia, basement membrane thickening and squamous metaplasia in chronic marijuana smokers.116 Additionally, cannabis smokers may develop cystic and irregularly dilated blebs and bulla along with large numbers of pigmented alveolar macrophages. Macrophages are relatively evenly distributed throughout the lung in a pattern reminiscent of desquamative interstitial pneumonia (see Chapter 10). In contrast to the finely granular pigment encountered in typical “smokers’ type” macrophages, those seen in association with cannabis use contained a very coarse brown pigment, which is negative with iron stains (Figure 16). This finding appears unrelated to whether a “joint” or “water bong” method

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of smoking is used.120 Interestingly, alveolar macrophages from marijuana smokers demonstrate decreased ability to phagocytose S. aureus and kill tumor cells.121 Aspergillus species within the plant may also cause human disease including allergic bronchopulmonary aspergillosis (see Chapter 15).116 Immunocompromised patients, including bone marrow transplant individuals, may be at particularly high risk.122 As a corollary to the pulmonary manifestations of marijuana use, the herbicide paraquat also deserves mention. Paraquat was first produced for commercial purposes in 1961, and is internationally amongst the most commonly used herbicides. It is quick-acting, non-selective, and kills the green marijuana plant on contact. In the United States, paraquat is available primarily as a liquid in various strengths, and it is very fast acting. It is classified as “restricted use”, which means that it can be used only by licensed applicators. In Europe, paraquat was banned in 2007. In its pure form, ingested paraquat is highly toxic to mammals, including humans, and there are no antidotes. However, fuller’s earth or activated charcoal are effective treatments, if administered promptly. During the late 1970s, a controversial program sponsored by the US government sprayed paraquat on marijuana fields in Mexico. Since much of this marijuana was subsequently smoked by Americans, the US government’s “Paraquat Pot” program stirred much debate. From a forensic standpoint, both accidental and suicidal overdoses of the drug are not uncommon. Such an overdose can occur within several days to several weeks following the initial dose, making a careful history important. Most famously, the demise of the late English international style icon Isabella Blow was officially attributed to an intentional paraquat overdose.110,111 Paraquat accumulates in the lungs at concentrations six to ten times higher than those in serum. The polyamine transport system is particularily sensitive to the agent and epithelial cell death is probably secondary to oxidative stress.123 Paraquat causes DAD along with extensive pulmonary fibrosis (Figure 17). Of 188 cases reported to the London Centre of the British National Poisons Information Service, 57 died acutely within 7 days, and 12 died later with evidence of pulmonary fibrosis.124 One study reported that persistent but patchy hemorrhage occurred within 16 hours, intra-alveolar edema with fibrin, phagocytes and hyaline membranes after 6 days, and both interstitial and intra-alveolar fibrosis thereafter.125 Electron microscopy of a lung biopsy taken 22 days after the ingestion of paraquat showed collapsed alveoli lined by type II pneumocytes and filled with proteinaceous fluid. The interstitium was widened, endothelial cells were abnormal, and there was patchy disruption of the capillary basement membrane.126 Other biopsies from fatal cases of paraquat overdose have demonstrated medial hypertrophy of the pulmonary arterioles.127

Heroin Of the opioids, medical examiners have the most experience with heroin-related deaths. The drug has been in use since the

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Figure 17. Paraquat poisoning. Tremendous interstitial edema and hyaline membranes are noted in this autopsy lung. No viable pneumocytes are seen. (Image courtesy of D. Flieder, MD, Philadelphia, PA, USA.)

late nineteenth century, when it was initially given to patients suffering from tuberculosis. Throughout much of the late nineteenth century extending through the late twentieth century, heroin was the most commonly abused illicit drug throughout the United States, and it was sold as an elixir in over-the-counter medicines until its addiction potential was accepted. After the drug was officially outlawed in the United States in the early 1900s, an illicit market with strong criminal ties developed for distribution and use of the drug, which continues today. The concentration, form and purity of the drug have diverse geographic variability, which is why addicts generally prefer a stable drug source. Heroin is manufactured by acetylating morphine with acetic acid to produce diacetylmorphine, or heroin. Heroin is more potent than pure morphine, a fact that was not recognized when the drug was initially advertised as a substitute for morphine addiction. Heroin is usually sold as a fine white powder wrapped in aluminum foil. The end product is either intravenously injected or emptied into a cooker and inhaled, after being heated over an open flame, referred to as “chasing the dragon”. Before injecting heroin intravenously, the user must dissolve the crystalline powder in water; the mixture is then usually heated to facilitate solubility. The drug is then filtered through any of a number of crude filters, such as a cotton ball, aspirated within a syringe and injected into a peripheral vein. Infrequently, the drug is snorted by addicts with needle phobias, but the euphoric effect is minimal when snorted. The drug is not taken orally.110,111 Pulmonary findings in heroin abusers depend on the drug route of administration, as well as how long the addict has been

Chapter 16: Drug- and therapy-induced lung injury

using the drug. Pulmonary edema and congestion are wellknown nonspecific complications of both acute and chronic heroin abuse. In fatal cases of acute heroin intoxication, a characteristic “cone of foam” is often noted in which the decedent is observed to have a frothy exudate of gray-white proteinrich foam centered on the nasal and oral orifices as a consequence of severe pulmonary edema. This exudate is sometimes the first clue to the cause of death. Proposed mechanisms of heroin-induced edema have included anoxic injury, direct toxicity/hypersensitivity of the alveolar membrane or aspiration. Post-mortem pulmonary findings in acute heroin overdoses frequently demonstrate lungs that collectively weigh in excess of 2000 g. exuding a frothy fluid when cut. Histologically, this edema fluid has an eosinophilic pattern, which expands the alveolar spaces and is rich in neutrophils.116 Eosinophils are usually sparse, leading one to doubt an allergic basis for the untoward effect. Aspiration is also seen in conjunction with the decreased central respiratory effect of heroin, and, with prolonged survival following aspiration, bronchopneumonia and purulent bronchitis may result.111

morphological characteristics in tandem with their histological and polarization patterns provide ready identification of the more common filler substances (Figure 18). In general, the starches with their easily recognized Maltese cross pattern are less frequently associated with granulomatous inflammation, as compared to the reaction caused by talc, which is more irritating to pulmonary tissue and may cause thrombosis with occlusion of arterioles and capillaries.128 Crospovidone has a distinct morphology worthy of special mention. Crospovidone is a water-soluble, cross-linked polymer from the monomer N-vinylpyrrolidone. This binder is completely inert to humans and simply passes through the body when taken orally. When injected, and seen within the lungs, it has a very characteristic non-birefringent coral-like pattern (Figure 19).129 Table 2 Histological characteristics of common tablet filler substances116,136

Filler type

Morphology Size Polarization Other staining qualities

Manifestations of intravenous drug abuse

Talc

Needle-like or plate-like

Intravenous drug abusers will often crush oral medications and subsequently intravenously inject themselves with the now aqueous drug. The insoluble filler material within the ground-up pills causes vascular complications including foreign-body embolism, thrombosis, granulomatous disease and fibrosis. Birefringent foreign material and large interstitial fibrotic masses containing foreign particles surrounded by multinucleated giant cells and granulomas are often seen. The usual filler materials seen in the lungs include talc, methylcellulose and other starches (Table 2). The former may persist in the lungs while the latter are usually degraded. The

Cornstarch

Polyhedral or 8–12 Yes-Maltese round µm cross

(a)

Microcrystalline Rod or cellulose needle shaped Crospovidone

5–12 Yes µm

25– 200 µm

Coral-shaped 100 Globular µm

N/A PAS, GMS positive

Yes

PAS, GMS, Congo Red positive

Nonpolarizable

Basophilic with H&E Congo Red positive

GMS, Gomori methenamine silver; PAS, periodic acid Schiff.

(b)

Figure 18. Methylcellulose in the wall of a pulmonary artery, secondary to intravenous drug use. (a) The substance is refractile on H&E stains and is associated with foreign-body giant cells. (b) Polarization reveals the birefringent nature of the substance. (Images courtesy of T. Sporn, MD, Durham, NC, USA.)

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(a)

(b)

Figure 19. (a) Crospovidone is characterized by a distinctive basophilic “coral-like” pattern. (b) In contrast to the methylcellulose also present in this picture, crospovidone is not birefringent under polarized light. (Images courtesy of Dr J. Yi, MD, Rochester, MN, USA.)

Radiologically, material-rich granulomas often appear as diffuse nodules. Restrictive lung disease and possibly pulmonary hypertension and thrombosis ensue. Thromboembolic outcomes include recanalization and partial thrombus reabsorption while concentric medial hypertrophy through to plexogenic angiomatoid lesions can be seen.130–133 Distant particle emboli within other organs have also been noted by the authors. Other pulmonary complications of intravenous drug abuse also deserve mention. An unusual form of panacinar emphysema has been described amongst intravenous drug abusers who inject methylphenidate (Ritalin). Histologically, there are varying degrees of talc granulomatosis along with lower lobe bullae. This pattern may mimic the emphysema seen in alpha1-antitrypsin deficiency (see Chapter 17). Septic thromboemboli from endocarditis can also be responsible for pneumonias and pulmonary abscesses.110 Finally, embolized foreign material may rarely resemble mineral dust pneumoconiosis including but not limited to progressive massive fibrosis.134

Other miscellaneous drugs The phenethylamine-derived drugs are generally considered either stimulants or hallucinogens. These synthetically produced drugs are often produced in clandestine home laboratories and frequently have an amphetamine backbone to which a methyl group is added, producing compounds such as methamphetamine. Many underground chemists previously used over-the-

References 1. Camus P, Rosenow EC, III. Iatrogenic lung disease. Clin Chest Med 2004;25: XIII–XXIX. 2. Camus P, Bonniaud P, Fanton A, et al. Drug-induced and iatrogenic infiltrative

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counter ephedrine, found within cold tablets, to serve as a methamphetamine precursor by reductive dehalogenation utilizing either sulfuric or phosphoric acid. Ecstasy is a popular clandestinely produced semi-synthetic hallucinogenic compound that is derived from the parent phenethylamine homolog through the addition of an N-methyl analog to an amphetamine backbone. The ensuing powders or crystals are usually heated to vapor and the fumes are inhaled. Most of the pulmonary manifestations of the phenethylamine-derived drugs yield nonspecific findings including pulmonary edema, plentiful intra-alveolar macrophages and rarely polarizable foreign material.110,111 Volatile inhalants are less commonly abused than the above-mentioned categories of drugs but are a particular problem amongst adolescents. Inhalation of drugs, gases, aerosol propellants and other vapors is especially common amongst teenagers, due to the ease of obtaining these substances. Numerous household and industrial products contain volatiles, such as butanes, fluorocarbons, trichloroethane, toluene and nitrous oxides. Generally, the product is placed within a container and the fumes are inhaled. Hypoxia and drowsiness may develop while cardiac arrest and sudden death are attributed to arrhythmias. Chronic inhalation of volatile hydrocarbons leads not only to thermal injury but also to emphysema. Inhalation of toluene has been linked with panacinar emphysema and explosive and burn injuries are seen with gasoline and butane inhalation.135

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80. Johnson DH, Fehrenbacher L, Novotny WF, et al. Randomized phase II trial comparing bevacizumab plus carboplatin and paclitaxel with carboplatin and paclitaxel alone in previously untreated locally advanced or metastatic non-small-cell lung cancer. J Clin Oncol 2004;22(11):2184–91. 81. Sandler A, Gray R, Perry MC et al. Paclitaxel-carboplatin alone or with bevacizumab for non-small-cell lung cancer. N Engl J Med 2006;355 (24):2542–50. 82. Vahid B, Mehrotra A. Trastuzumab (Herceptin)-associated lung injury. Respirology 2006;11(5):655–8. 83. Radzikowska E, Szczepulska E, Chabowski M, Bestry I. Organising pneumonia caused by transtuzumab (Herceptin) therapy for breast cancer. Eur Respir J 2003;21(3):552–5. 84. Wagner SA, Mehta AC, Laber DA. Rituximab-induced interstitial lung disease. Am J Hematol 2007;82(10):916–9. 85. Villeneuve E, St-Pierre A, Haraoui B. Interstitial pneumonitis associated with

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infliximab therapy. J Rheumatol 2006; 33(6):1189–93. 86. Wiener CM, Muse VV, Mark EJ. Case records of the Massachusetts General Hospital. Case 33–2008. A 63-year-old woman with dyspnea on exertion. N Engl J Med 2008;359(17):1823–32. 87. Leard LE, Cho BK, Jones KD, et al. Fatal diffuse alveolar damage in two lung transplant patients treated with cetuximab. J Heart Lung Transplant 2007;26(12):1340–4. 88. Vahid B, Esmaili A. Erlotinib-associated acute pneumonitis: report of two cases. Can Respir J 2007;14(3):167–70. 89. Sumpter K, Harper-Wynne C, O’Brien M, Congleton J. Severe acute interstitial pneumonia and gefitinib. Lung Cancer 2004;43(3):367–8. 90. Ieki R, Saitoh E, Shibuya M. Acute lung injury as a possible adverse drug reaction related to gefitinib. Eur Respir J 2003;22(1):179–81. 91. Isshiki I, Yamaguchi K, Okamoto S. Interstitial pneumonitis during imatinib therapy. Br J Haematol 2004;125(4):420. 92. Krug LM, Crapanzano JP, Azzoli CG, et al. Imatinib mesylate lacks activity in small cell lung carcinoma expressing c-kit protein: a phase II clinical trial. Cancer 2005;103(10):2128–31. 93. Ma CX, Hobday TJ, Jett JR. Imatinib mesylate-induced interstitial pneumonitis. Mayo Clin Proc 2003; 78(12):1578–9. 94. Ohnishi K, Sakai F, Kudoh S, Ohno R. Twenty-seven cases of drug-induced interstitial lung disease associated with imatinib mesylate. Leukemia 2006; 20(6):1162–4. 95. Abratt RP, Morgan GW, Silvestri G, Willcox P. Pulmonary complications of radiation therapy. Clin Chest Med 2004;25(1):167–77. 96. Abratt RP, Morgan GW. Lung toxicity following chest irradiation in patients with lung cancer. Lung Cancer 2002; 35(2):103–9. 97. Rosiello RA, Merrill WW. Radiationinduced lung injury. Clin Chest Med 1990;11(1):65–71. 98. Abid SH, Malhotra V, Perry MC. Radiation-induced and chemotherapyinduced pulmonary injury. Curr Opin Oncol 2001;13(4):242–8. 99. Abratt RP, Morgan GW, Silvestri G, Willcox P. Pulmonary complications of

radiation therapy. Clin Chest Med 2004;25(1):167–77. 100. Movsas B, Raffin TA, Epstein AH, Link CJ, Jr. Pulmonary radiation injury. Chest 1997;111(4):1061–76.

Investigation of Death. Guidelines for the Application of Pathology to Crime Investigation. 3rd ed. Springfield: Charles C. Thomas, 1993. pp. 733–66.

101. Thomas PS, Agrawal S, Gore M, Geddes DM. Recall lung pneumonitis due to carmustine after radiotherapy. Thorax 1995;50(10):1116–8.

112. Murray RJ, Smialek JE, Golle M, Albin RJ. Pulmonary artery medial hypertrophy in cocaine users without foreign particle microembolization. Chest 1989;96(5):1050–3.

102. Greene FL, Page DL, Fleming D, et al. Lung. AJCC Cancer Staging Manual. 6th ed. New York: Springer, 2002. pp. 165–77.

113. Murray RJ, Albin RJ, Mergner W, Criner GJ. Diffuse alveolar hemorrhage temporally related to cocaine smoking. Chest 1988;93(2):427–9.

103. Junker K, Langner K, Klinke F, Bosse U, Thomas M. Grading of tumor regression in non-small cell lung cancer: morphology and prognosis. Chest 2001;120(5):1584–91.

114. Bailey ME, Fraire AE, Greenberg SD, Barnard J, Cagle PT. Pulmonary histopathology in cocaine abusers. Hum Pathol 1994;25(2):203–7.

104. Junker K, Thomas M, Schulmann K, et al. Tumour regression in non-smallcell lung cancer following neoadjuvant therapy. Histological assessment. J Cancer Res Clin Oncol 1997; 123(9):469–77. 105. Liu-Jarin X, Stoopler MB, Raftopoulos H, et al. Histologic assessment of nonsmall cell lung carcinoma after neoadjuvant therapy. Mod Pathol 2003;16(11):1102–8. 106. Clasen S, Krober SM, Kosan B, et al. Pathomorphologic evaluation of pulmonary radiofrequency ablation: proof of cell death is characterized by DNA fragmentation and apoptotic bodies. Cancer 2008;113(11):3121–9. 107. Danielson C, Benjamin RJ, Mangano MM, Mills CJ, Waxman DA. Pulmonary pathology of rapidly fatal transfusion-related acute lung injury reveals minimal evidence of diffuse alveolar damage or alveolar granulocyte infiltration. Transfusion 2008;48(11):2401–8. 108. Kopko PM, Popovsky MA. Pulmonary injury from transfusion-related acute lung injury. Clin Chest Med 2004; 25(1):105–11. 109. Toy P, Popovsky MA, Abraham E, et al. Transfusion-related acute lung injury: definition and review. Crit Care Med 2005;33(4):721–6. 110. Bell MD. Pathology of drug abuse-lung disease. In Karch SB, ed. Drug Abuse Handbook. Boca Raton: CRC Press, 1998. pp. 129–35. 111. Stephens BG. Investigation of deaths from drug abuse. In Spitz WU, ed. Spitz and Fisher’s Medicolegal

115. Cucco RA, Yoo OH, Cregler L, Chang JC. Nonfatal pulmonary edema after “freebase” cocaine smoking. Am Rev Respir Dis 1987;136(1):179–81. 116. Tomashefski JF Jr, Felo J. The pulmonary pathology of drug and illicit substance abuse. Current Diagnostic Pathology 2004;10(5):413–26. 117. Tashkin DP, Coulson AH, Clark VA, et al. Respiratory symptoms and lung function in habitual heavy smokers of marijuana alone, smokers of marijuana and tobacco, smokers of tobacco alone, and nonsmokers. Am Rev Respir Dis 1987;135(1):209–16. 118. Johnson MK, Smith RP, Morrison D, Laszlo G, White RJ. Large lung bullae in marijuana smokers. Thorax 2000; 55(4):340–2. 119. Roth MD, Arora A, Barsky SH, et al. Airway inflammation in young marijuana and tobacco smokers. Am J Respir Crit Care Med 1998; 157(3 Pt 1):928–37. 120. Gill A. Bong lung: regular smokers of cannabis show relatively distinctive histologic changes that predispose to pneumothorax. Am J Surg Pathol 2005;29(7):980–2. 121. Baldwin GC, Tashkin DP, Buckley DM, et al. Marijuana and cocaine impair alveolar macrophage function and cytokine production. Am J Respir Crit Care Med 1997;156(5):1606–13. 122. Hamadeh R, Ardehali A, Locksley RM, York MK. Fatal aspergillosis associated with smoking contaminated marijuana, in a marrow transplant recipient. Chest 1988;94(2):432–3. 123. Dinis-Oliveira RJ, Duarte JA, Sanchez-Navarro A, et al. Paraquat

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poisonings: mechanisms of lung toxicity, clinical features, and treatment. Crit Rev Toxicol 2008;38: 13–71. 124. Higenbottam T, Crome P, Parkinson C, Nunn J. Further clinical observations on the pulmonary effects of paraquat ingestion. Thorax 1979; 34(2):161–5. 125. Rebello G, Mason JK. Pulmonary histological appearances in fatal paraquat poisoning. Histopathology 1978;2(1):53–66. 126. Dearden LC, Fairshter RD, McRae DM, et al. Pulmonary ultrastructure of the late aspects of human paraquat poisoning. Am J Pathol 1978; 93(3):667–80. 127. Levin PJ, Klaff LJ, Rose AG, Ferguson AD. Pulmonary effects of contact exposure to paraquat: a clinical and experimental study. Thorax 1979; 34(2):150–60.

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128. Fischman MW, Schuster CR, Resnekov L, et al. Cardiovascular and subjective effects of intravenous cocaine administration in humans. Arch Gen Psychiatry 1976;33(8):983–9. 129. Ganesan S, Felo J, Saldana M, et al. Embolized crospovidone (poly[Nvinyl-2-pyrrolidone]) in the lungs of intravenous drug users. Mod Pathol 2003;16(4):286–92. 130. Tomashefski JF Jr, Hirsch CS. The pulmonary vascular lesions of intravenous drug abuse. Hum Pathol 1980;11(2):133–45. 131. Waller BF, Brownlee WJ, Roberts WC. Self-induced pulmonary granulomatosis. A consequence of intravenous injection of drugs intended for oral use. Chest 1980;78(1):90–4. 132. Arnett EN, Battle WE, Russo JV, Roberts WC. Intravenous injection of talc-containing drugs intended for oral use. A cause of pulmonary

granulomatosis and pulmonary hypertension. Am J Med 1976; 60(5):711–8. 133. Zeltner TB, Nussbaumer U, Rudin O, Zimmermann A. Unusual pulmonary vascular lesions after intravenous injections of microcrystalline cellulose. A complication of pentazocine tablet abuse. Virchows Arch A Pathol Anat Histol 1982;395(2):207–16. 134. Crouch E, Churg A. Progressive massive fibrosis of the lung secondary to intravenous injection of talc. A pathologic and mineralogic analysis. Am J Clin Pathol 1983;80(4):520–6. 135. Wolff AJ, O’Donnell AE. Pulmonary effects of illicit drug use. Clin Chest Med 2004;25(1):203–16. 136. Kringsholm B, Christoffersen P. The nature and occurrence of birefingement material in different organs in fatal drug addiction. For Sci Inter 1987; 34(1):53–62.

Chapter

17

Chronic obstructive pulmonary disease and diseases of the airways Wim Timens, Hannie Sietsma and Joanne L. Wright

Introduction Airflow obstruction, due to chronic obstructive lung disease, and parenchymal and airways disease due to other etiologies are major causes of morbidity and mortality. They account for a very large percentage of healthcare costs. The airways are in open communication with the direct environment. Anything inhaled will come into direct contact with the airways. Many disorders of large and small airways are therefore associated with inhaled substances, such as cigarette smoke, atmospheric pollution and other exposures related to work or hobbies. Even in airway diseases of unknown etiology, environmental factors affect the histopathology. Disorders of the bronchioles show similar changes to those observed in larger airways, caused by inhalation of cigarette smoke and other noxious materials. In addition small airways can show changes that are more often associated with alveolar disease. This chapter will discuss diseases originating in the airways or notably affecting airway function. The different components of chronic obstructive pulmonary disease (COPD), namely chronic bronchitis/bronchiolitis and emphysema, will be discussed separately. Pulmonary and systemic diseases that feature airway involvement, such as asthma and connective tissue diseases, are described elsewhere in the textbook (see Chapters 15 and 21).

Chronic obstructive pulmonary disease in general Introduction Several distinct pathological changes in the lung having a common clinical presentation are recognized under the heading chronic obstructive pulmonary disease (COPD). Pathological changes are present in different parts of the lung, with the main distinction between large and small airways and parenchyma.1–5 In COPD three components can be discerned in any patient in varying percentages: chronic bronchitis, bronchiolitis and emphysema. A broader definition includes changes in the bronchial tree, such as bronchiectasis.6,7

However, this process is usually considered a separate disease entity. Exposure to tobacco smoke is the main etiological factor, along with environmental influences, such as atmospheric pollution and combustion (cooking) gases in poorly ventilated housing.2 The prototypical forms of the various disorders within COPD, namely, chronic bronchitis, emphysema and small airways disease, have distinct anatomical and clinical characteristics. Patients with predominantly pulmonary emphysema and those with principally chronic bronchitis are considered specific clinical categories.2,8,9 Most entities have overlapping features and each individual combination determines the clinical severity of bronchial obstruction.

Epidemiology of COPD Epidemiological data reflect the worldwide burden of disease, including the economic and social burden, in addition to the morbidity and mortality related to COPD. Chronic obstructive pulmonary disease is a disease that for a long time was not properly recognized, often misdiagnosed as “asthma”, and therefore underdiagnosed. This led to significant underreporting and until relatively recently it was difficult to quantify prevalence, morbidity and mortality.10 COPD is frequently found in patients presenting with other respiratory and non-respiratory symptoms.11 The prevalence of COPD is higher in people who smoke or have smoked, compared to never smokers. It is higher in men than in women, and more significant in the “over 40s”.9 A family history of COPD also appears to be important,12 indicating at least some degree of genetic susceptibility. The prevalence and gender distribution of COPD varies markedly around the world, depending upon cigarette smoking, pollutant exposure, and biomass exposure.9 As reviewed in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) document,9 the worldwide prevalence of COPD is estimated as 9.34/1000 men and 7.33/1000 women, with the lowest prevalence in men in the Middle Eastern Crescent. This latter finding is unusual as there is a consistently high level of cigarette smoking in the adult male population in this area. The lowest prevalence in women is in a region

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Prevalenece per 1000 population

Figure 1. COPD prevalence in various populations.

Females

Males

30

20

10

grouped as “Other Asia and Islands” (including Indonesia, Papua New Guinea, Nepal, Vietnam, Korea, Hong Kong and the small island countries) (Figure 1). Prevalence is affected by age distribution and susceptibility genes in the populations studied. As an example, in China, the overall prevalence of COPD is 12.4% in men and 5.1% in women. The incidence is higher in rural residents, elderly patients, smokers, those with poor kitchen ventilation, exposure to occupational dusts or biomass fuels, and in those with a family history of pulmonary diseases.13 In the United States,14 United Kingdom,15 France,16 Spain17 and the Netherlands18 smoking is the chief risk factor, and several studies also indicate an increasing prevalence in women. However, there is some evidence from Spain19 and Finland20 that the prevalence of COPD is stabilizing. Worldwide, approximately 8% of people over the age of 40 develop COPD.21 In North America, approximately 20% of the population currently smoke cigarettes,22 and over 10 million adults have been diagnosed with COPD. COPD is a major cause of “loss of work”, and is currently the fourth leading cause of death in the US population.23,24 It is projected to become the third commonest cause of death worldwide by 2020.9,21,25,26

Genetics Clinical and pathological heterogeneity in COPD is intriguing. It is unknown why a common etiology, smoking, leads to destruction of parenchyma and/or in fibrosis of the respiratory bronchiolar walls in only a subset of tobacco abusers. Specific genetic factors may play an important role in disease susceptibility.26–28 This is supported by the fact that the risk of developing COPD is increased in the families of COPD patients, a feature which has been known for a long time.29 Surprisingly, airway wall thickening and emphysema

606

a Ame ric Latin

resce nt ern C East

Afric a

Othe r Asia n

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India

Rotte rdam

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World

0

contribute independently to COPD, as aggregations of these characteristics are found within families of subjects with COPD.30 The genetic risk of developing COPD is most obvious in alpha-1-antitrypsin (A1AT) deficiency, involving the SERPINA1 gene (formerly PI-gene). This gene codes for the alpha1-antitrypsin-serine protease inhibitor. The gene product protects against neutrophil elastase (see below).31 However, A1AT deficiency only accounts for around 2% of COPD patients. In recent years many polymorphisms have been linked to determinants of COPD, or to important features underlying COPD. These polymorphisms are not always specific for this disease.28,32–36 The search for significant polymorphisms has not identified consistent gene candidates.37 Differences in emphysema distribution appear to be explained by the effects of different genes.38 A recent report identifies a nicotinic acetylcholine receptor gene variant at 15q24/25 as a risk factor for emphysema as well as lung carcinoma.39 Implicated genes are often associated with the three recognized main mechanisms of COPD pathogenesis: disturbed protease-anti protease balance, oxidant-antioxidant balance, and continuous, abnormal inflammation (Table 1).40,29 Genes for matrix metalloproteinase 9 (MMP9), glutathione S-transferase P1, and tumor necrosis factor alpha (TNFa) are reportedly associated with emphysema. Transforming growth factor beta 1 (TGFb1) was associated with airway disease, and Serpin E2 was associated with both emphysema and airways disease. However, a recent meta-analysis failed to demonstrate associations between candidate genes and COPD,28 and significant results were seen only for a few genes. Three TGFb1 polymorphisms were associated with COPD, although these results were only based on a few studies. The interleukin-1 receptor antagonist gene variable number tandem repeat (IL1RN VNTR) polymorphism increases the risk of COPD. However,

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Table 1 COPD candidate genes investigated in emphysema

Genes

Functional class

Variants

Matrix metalloproteinase 9 (MMP 9)

Metalloprotease: type IV and V collagenase

rs 3918242(C-1562 T)

Microsomal epoxide hydrolase (EPHX1)

Xenobiotic metabolism: first-pass metabolism/detoxification of highly reactive species

rs 1051740(T 113 C) rs 2234922(A 139 G)

Heme oxygenase 1 (HMOX1)

Antioxidant: against heme- and non-heme-mediated oxidant damage

(GT)n repeats at 50 end

Glutathione S-transferase P1 (GST P1)

Xenobiotic metabolism: conjugates hydrophobic/electrophilic compounds

rs 1695(A 105 G)

Vitamin D binding protein (GC)

Immune response: in addition to binding vitamin D

1F allele

β2-Adrenergic receptor (ADRβ2)

β2-Adrenergic receptor

rs 1042713(A 16 G)

TNFa (TNF)

Proinflammatory cytokine

rs 1800629(G 308 A)

Transforming growth factor-β1 (TGFβ1)

Autocrine peptide involved in proliferation/apoptosis and differentiation

Transforming growth factor-β receptor-3 (TGFβR3)

Glycoprotein receptor for transforming growth factor-β

Modified from29,40

the TNFa-promoter region 308 G/A polymorphism increases this risk only in Asian populations. Some polymorphisms, such as in ADAM-33 (a disintegrin and metalloproteinase domain 33), were first found to be associated with accelerated lung function decline35 and also with an increased risk of developing COPD.35,41,42 Recently a polymorphism in a minor allele of the matrix metalloprotease 12 gene was associated with a reduced risk of COPD in adult smokers.43,44 Considering the heterogeneity of COPD and the fact that it appears genetically multifactorial, further genetic studies are needed.45

Table 2 Classification of airflow obstruction according to Gold criteria

Clinical manifestations Cough and shortness of breath are the relatively nonspecific clinical manifestations of COPD. If the cough is productive, and meets the definition described in the next section, a diagnosis of chronic bronchitis can be made. An exacerbation of COPD is suggested if there are increasing symptoms of cough and shortness of breath.46–50 Correctly identifying an exacerbation is important, as these episodes are poor prognostic indicators.51 Although the triggers of exacerbation are not definitively known, bacterial and viral agents have been suggested.49,50,52 The frequency of exacerbations also appears to be related to the severity of baseline lung function.53 However, host factors and environmental pollution play roles in exacerbations. Clinical phenotypes in COPD show considerable overlap between emphysema, chronic bronchitis, and even asthma (see Chapter 15). These “overlap” phenotypes may be important in clinical management.54 The classic phenotypic differentiation of “pink puffer” versus “blue bloater” was described in the 1950s.55 The pink puffer was thought to be an older patient with clear clinical evidence of emphysema, muscle wasting and severe dyspnea, while the blue bloater had the clinical signs of

Gold Stage 1

FEV1/FVC < 70% and FEV1  80% predicted. With or without chronic symptoms of cough or sputum production

Gold Stage 2

FEV1/FVC < 70% and FEV1 < 80% and  50% predicted. With or without chronic symptoms of cough or sputum production

Gold Stage 3

FEV1/FVC < 70% and FEV1 < 50% and  30% predicted. With or without chronic symptoms of cough or sputum production

Gold Stage 4

FEV1/FVC < 70% and FEV1 < 30% or FEV1  50% predicted plus chronic respiratory failure

Modified from9

chronic bronchitis and usually also of right heart failure. Pink puffers are thought to be able to maintain reasonably normal blood gas tensions because of a relatively better match between ventilation and perfusion, but at the expense of severe breathlessness, probably related to stimulation of the respiratory centers to maintain pCO2 at normal levels.56,57 Blue bloaters, on the other hand, have progressive arterial hypoxemia and hypercapnia leading to pulmonary hypertension and right heart failure. Both clinical patterns may be clinically indistinguishable, especially when there is acute respiratory insufficiency. Airflow obstruction is usually classified by its severity, based on the GOLD criteria (Table 2).9 This system proposes a grading system based in large part on pulmonary function test findings. Respiratory failure, a part of Gold stage 4, is the only clinical feature considered in the scheme.9 Pulmonary function tests are influenced by both loss of recoil (from any existing emphysema) and airways disease with

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(a)

(c)

(b)

(d)

Figure 2. CT scans of different forms of emphysema. (a) Mild centrilobular emphysema. (b) Panlobular emphysema. (c) Paraseptal emphysema. (d) Severe confluent emphysema. (Images courtesy of Dr J. Verschakelen, Leuven, Belgium)

consequent turbulent flow and increased resistance. There may be associated decreases in diffusing capacity. Obstructive lung disease is diametrically opposite in nature to restrictive lung disease, in which there is increased lung elastic recoil. While both conditions may have decreased vital capacities, obstructive lung disease will have a decreased FEV1/FVC ratio, and a pure restrictive lung defect will have an increased ratio. Unfortunately, many restrictive diseases, such as sarcoidosis and hypersensitivity pneumonia (see Chapters 12 and 13), have a combination of obstructive and restrictive defects. In addition, it is now known that emphysema and idiopathic pulmonary fibrosis (IPF) can co-exist, usually with emphysema in the upper lobes and varying degrees of fibrosis in the lower lobes.58 In emphysema, but also in IPF, smoking plays an etiological role.59,60 Clinical work-up must also include detailed history and physical examination, in addition to appropriate radiological assessment.

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Radiographic findings At present radiology does not play a main role in the diagnosis of COPD.26 The main value of chest X-ray is exclusion of alternative diagnoses and/or detection and establishment of important comorbidities, in particular cardiac disease. Only in obvious bullous disease is an abnormal X-ray in keeping with a diagnosis of COPD. There is no place in a routine diagnostic work-up for CT scanning.9,26 However, CT scanning plays a role in determining suitability for lung volume reduction surgery, as the distribution of emphysema must be taken into consideration.9,61,62 While not routine, recent advances in high-resolution CT scanning have led to improved recognition of disease distribution and airway changes. These might allow more precise phenotyping of COPD patients (Figure 2).63–65,66 The airway pathology and emphysema determined in this way show independent aggregation of COPD

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

patients in families, indicating that these pathologies may have different genetic factors.30

Pathophysiology Physiological abnormalities can occur via two main processes, which may be independent or concurrent. Emphysematous lung destruction results in loss of the elastic recoil of the lung. This leads to increased lung compliance and alterations of the pressure–volume curve. Structural and dynamic alterations of the airway lead to increased airflow resistance, dynamic hyperinflation during exercise, and early airway closure during expiration leading to gas trapping and increased residual volume. Destruction of the peribronchiolar alveoli leads to loss of both airway tethering and airway-parenchymal interdependence, the latter being referred to as “uncoupling”.67,68 These changes result in a relative collapse of the airways. Irreversible components of these processes include fibrosis and narrowing of airways, loss of elastic recoil due to destruction, and destruction of airway alveolar support. Components which are potentially reversible by therapy include mucus and inflammatory cells in airways, smooth muscle contraction in airways and dynamic hyperinflation during exercise. The increased thickness of the smooth muscle layer acts to decrease the effective lumen of the airway, but also alters its mechanical behavior. As the muscle constricts, the airway lumen is narrowed to an even greater degree. Yet airways resistance is increased more than would normally be expected from the smooth muscle shortening alone.69 The smooth muscle is functionally altered, with an increased ability to generate force and therefore narrow the airways to a greater degree. Airway smooth muscle cells may also generate proinflammatory cytokines, thus perpetuating airways inflammation (reviewed in70). Airway fibrosis is an important component of the remodeling process in COPD, with resultant airway obliteration causing increased airflow resistance.71 Analysis of small airway mRNA suggests a direct relationship to the inflammatory and immune cascade.71

Pathology of COPD Large airways

The presence of bronchial wall inflammation is a nonspecific finding observed in many diseases, but is consistently identified in chronic bronchitis (see below). Goblet cell and squamous cell metaplasia of the epithelium can be identified and the basement membrane may be altered. The airway wall may show remodeling with changes in the muscle, fibrous tissue and bronchial gland components. Oncocytic metaplasia in the bronchial glands may be observed but is a nonspecific finding (see Chapter 2). Mild cartilaginous changes, including metachromatic staining and chondrocyte degeneration, are also nonspecific.72 The inflammatory process also involves adventitial/peribronchial tissue to varying degrees.73

Small airways Small airways are arbitrarily defined as those < 2 mm in diameter. Luminal obstruction and increased rigidity of the small airways are the most important pathophysiological contributions to chronic airway obstruction.74,75 As in the large airways, small airway mucosa features goblet cell hyperplasia, squamous cell metaplasia, and to a lesser degree dysplasia. Small airway mucus plugs are more viscous than those in large airways and are largely responsible for obstruction (Figure 3). As noted above, smooth muscle changes also play a large role in obstruction. The normal membranous and respiratory bronchioles contain small slips of smooth muscle, arranged in a spiral fashion. In COPD, remodeling of the small airways with increasing muscularization is much more prominent than in the larger airways (Figure 4). Increased amounts of smooth muscle are reported in many studies.75–80 There is a suggestion that smooth muscle alteration may be a dynamic process, being prominent in people with more severe obstruction.70,75,79,80

Pathogenesis of bronch(iol)itis Bronchial inflammation with edema, mucus production and hypertrophy of smooth muscle all play roles in the development and severity of obstruction. The inflammatory process in chronic bronchitis/bronchiolitis takes the form of a nonspecific immune response, most probably as a reaction to an irritant. Epithelial changes in chronic bronchitis/bronchiolitis are due to smoke exposure and subsequent inflammation.71,81,82 These changes constitute a basis for airway colonization with microorganisms, which may partly explain the inflammatory response.83,84 In smokers there is an association between the degree of airway wall inflammation and the severity of the airway obstruction.85 Accompanying lung parenchymal destruction, i.e., emphysema, releases matrix fragments with the chemotactic capacity to attract inflammatory cells.86,87 Because these inflammatory cells contribute to inflammation and destruction, a vicious cycle is created.84 This phenomenon may partly explain the ongoing inflammation in COPD patients, years after tobacco cessation.84,88–90 An increase in alveolar macrophages is a hallmark of cigarette smoking76,91 and the metalloproteolytic secretions from these macrophages are important in emphysematous lung destruction (see below). However, cigarette smoke exposure also increases a variety of pro-inflammatory cytokines, increasing the numbers of neutrophils and activating the innate immune response.92 In smokers and in patients with COPD, histone deacetylase 2 (HDAC2) expression and activity are reduced in both the peripheral lung and alveolar macrophages, resulting in amplification of the inflammatory response.93 Since cigarette smoke is mutagenic, it has been postulated that smoke-induced (i.e. acquired) somatic mutations in airway cells contribute to persisting inflammation94,95 in addition to the increased risk of lung cancer (see Chapter 23).

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(a)

(b)

(c)

Figure 3. Mucus plugging. (a) Bronchioloectatic airway with consolidated mucus (periodic acid-Schiff stain). (b and c) Sticky mucus with cellular constituents.

(a)

(b)

Figure 4. Bronchiole with muscular hypertrophy. (a) Prominent smooth muscle is seen. (b) A smooth-muscle actin immunohistochemical stain highlights the muscle layer.

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(a)

(b)

Figure 5. Small airway with lymphoid follicles. (a) The infiltrate is predominantly adventitial. (b) Submucosal expansion is also seen.

In patients with COPD, an increase in pro-inflammatory mediators of epithelial origin, such as monocyte chemoattractant protein and interleukin 8, is observed,96 with a correspondingly reduced production of anti-inflammatory mediators.97 Interstitial fibroblasts also show upregulation of inter-cellular adhesion molecule 1 (ICAM1),98 which contributes to local inflammatory cell recruitment and leads to persistent inflammation. The inflammatory infiltrate in both large and small airways is nonspecific. Greater numbers of tobacco smoke-induced CD8þ T cells are found in the submucosa.99–103 There may also be a B cell response in large and small airways (Figure 5),79,104–106 and there are increased numbers of plasmacytoid dendritic cells in the pulmonary lymphoid follicles.107 This humoral response seems to be antigen-specific, and auto-antigens are also present.106,108,109 This auto-immune component probably plays a role in pathogenesis and progression of COPD. It has also been suggested that the immune system alterations arising in childhood as a result of infection, genetic mutation or prematurity may be important factors in COPD development.110 Increased numbers of macrophages and neutrophils populate the airways.73,99 In the large airways neutrophils are found in the mucus glands and their factors probably contribute to the proliferation and increased production of mucus by the glands.111 In COPD exacerbations, a marked increase in airway wall neutrophils and eosinophils is described.111–114 The greater the eosinophilic influx, the better is the response to systemic, but not inhaled, corticosteroids.115 Effects on disease progression and mortality are unknown.115 Besides local effects on the airway wall, inflammation also increases the stretching, and ultimately the loss, of alveolar septal attachments to the small airways. Initially, this is caused by the potentially reversible inflammation. If destruction in this area has already occurred, as in emphysema (Figure 6) or

Figure 6. Small airway with thickened wall and pronounced inflammation often leads to extensive peribronchiolar alveolar destruction.

chronic bronchitis with a fibrotic component, there will be, at the most, partial reversibility.

Course and prognosis Morbidity due to COPD increases with age and is influenced by other processes, either related to cigarette smoking (cardiovascular disease) or other diseases (e.g. diabetes and musculoskeletal disease).9,26 Progressive respiratory failure accounts for approximately 30% of COPD-related mortality.116 Contributing factors include number of exacerbations, presence of pulmonary hypertension,117,118 degree of diaphragmatic dysfunction,119 and body mass.120 A recent analysis of the Framingham offspring cohort revealed several important principles. In both males and

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females, smoking increases the rate of lung function decline. There is a wide range of susceptibility to cigarette smoke. Symptoms may be a marker of susceptibility, and smoking cessation at any time has a beneficial effect, but the effect is more pronounced in those who quit at an earlier time.121 A biomarker which could be utilized as a surrogate marker for disease and response to therapy remains elusive.122

Treatment Treatment of COPD can be divided into several separate sections: smoking cessation; those devised to reduce inflammation; those devised to augment airflow; those designed to reduce exacerbations; those designed to support or rehabilitate the patient as a whole; and surgical therapies. Smoking cessation is effective,123,124 especially in those who quit before age 30.125 Pharmacological therapy usually includes b agonists and/or anticholinergics with variable success in ameliorating airflow obstruction.123 Although inhaled or systemic corticosteroids are the mainstay as anti-inflammatory drugs, prostaglandin E4 inhibitors and anti-TNF inhibitors have also been utilized. Treatment of exacerbations usually involves both anti-inflammation therapy and airflow-augmenting agents, and may also include antibiotics. Rehabilitation126 in combination with nutritional advice127 improves patient well-being. Oxygen therapy is a mainstay therapy, improving survival, exercise and cognitive performance in hypoxemic patients.127 Finally, surgical (or other invasive) methods to remove lung tissue and improve ventilation-perfusion match have some short-term benefits.127 Lung transplantation is a possibility, particularly for patients with alpha-1-antitrypsin deficiency (see Chapter 20).

Chronic bronchitis in COPD Definition Chronic bronchitis is defined on clinical grounds alone, as the presence of chronic productive cough for 3 months in 2 successive years, in the absence of any other explanation for this symptom.128,129

Epidemiology It is often difficult to separate the incidence of chronic bronchitis from COPD in the literature, as they are often grouped together, and chronic bronchitis, like COPD, is underdiagnosed.12 Diagnosis is important, especially in younger patients, as in one study chronic bronchitis was able to predict subsequent airflow obstruction and mortality in subjects less than 50 years of age.130 Chronic bronchitis is seen worldwide, with significant mortality, which is up to 80 per 100 000 male adults in Poland.131 Epidemiological surveys suggest that the incidence is affected by recurrent respiratory infections in childhood, industrial pollution and cigarette smoking (reviewed in131). Interestingly, livestock farmers have an increased risk of chronic bronchitis compared to crop farmers, perhaps due to increased exposure

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to dusts and fumes.132 Indoor pollution from cooking fires may be important in developing countries.133 Infective exacerbations are a major cause of mortality.134 Haemophilus influenzae is the most frequently isolated pathogen, but Moraxella catarrhalis and Streptococcus pneumoniae account for approximately one-third of isolates.131

Pathophysiology Normal airway mucus functions to trap and remove pathogens and particulate matter from the airway via the mucociliary escalator. The mucins are of two major families: secreted, as for example MUC5AC, MUC5, MUC2, MUC8 and MUC19, and membrane-associated, such as MUC1, MUC4, MUC11, MUC13, MUC15 and MUC20. Membrane-associated mucins localize to the apical surfaces of epithelial cells, and MUC5AC is expressed in goblet cells. In the submucosal glands, MUC5B is expressed in the mucous cells, while MUC7 is present in serous cells. Mucin glycoprotein overproduction and hypersecretion are important in disease, and their clearance can be altered by cilial damage or alterations of sputum components, such as proteoglycans, biofilm and DNA (discussed in135,136). Mucins are upregulated by inflammatory mediators such as the interleukins and TNFa,135,137 but can be directly upregulated by microorganisms activating the nuclear factor (NF)kB via cell surface receptors. Airway proteases upregulate MUC5AC expression.135 However, the signal transduction cascades that lead to upregulation of gene expression and hence mucin biosynthesis are different from the mechanisms that regulate mucin secretion.138,139 There may also be different mechanisms for storage and secretion of the different MUC sub-types.140 This knowledge is important in the development of therapies directed towards reduction of production and enhanced clearance of mucin.

Pathology In chronic bronchitis, large airway changes are generally related to mucus hypersecretion. This hypersecretion is accompanied by a marked multiplication in goblet cells and an increase in the size of tracheal and bronchial submucosal mucus glands (Figure 7).99,141 Submucosal fibrosis, sometimes edema, and a mixed inflammatory infiltrate with lymphocytes, plasma cells and macrophages are commonly noted (Figure 8).99,142 In pure chronic bronchitis, granulocytes are less evident in the wall of the airways, while a significant increase in mucus gland number is observed.111 With respect to the epithelium, goblet-cell hyperplasia is the most common change but squamous metaplasia and dysplasia are observed (Figure 9). The associated pronounced reduction in presence and functionality of cilia causes a reduction in mucociliary clearance and consequently less effective mucus transport to the oral cavity.75,143,144 In contrast to patients with asthma (see Chapter 15), subjects with only chronic bronchitis have a normal-appearing basement membrane in lobar and subsegmental bronchi.145 Studies evaluating the basement membrane in COPD have not

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

shown concordant results (reviewed in146). A recent study demonstrated an increased basement membrane thickness in COPD, with values comparable to those in asthmatics.146 However, the basement membrane in asthmatics had greater staining for collagen I and laminin, and less staining for collagen IV when compared to those from COPD patients. There is also a condition called “eosinophilic bronchitis” in which patients have productive cough, but without the hyperresponsiveness and variable airflow obstruction found in asthmatics. In this rare entity, the basement membrane is thickened to values approximating those in asthma.57 One of the main features in chronic bronchitis is the formation of bronchial pits along the inferior margins of the lobar bronchi, usually adjacent to bifurcations. Pits develop from distorted bronchial gland ducts, and often contain inspissated mucus (Figure 10). These pits are often identified between slips of hypertrophied smooth muscle. The degree of smooth muscle hypertrophy is variable, and even if present there appears to be a wide range of severity from site to site, and from patient to patient.

Figure 7. Bronchus with enlarged bronchial mucus glands.

Figure 9. Bronchus with goblet cell metaplasia (right) and squamous metaplasia (left)

Figure 8. Bronchus wall with goblet cell hyperplasia and submucosal lymphoplasmacytic infiltrates.

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Figure 10. Bronchial pits. (a) bronchial wall gland pit in normal airway and (b) bronchial wall gland pit in chronic bronchitis.

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Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Figure 11. Reid index. (a and b) Portions of bronchial wall used to determine the Reid index.

(a) Epithelium

Basement Membrane

Mucous Gland

Perichondrium

Cartilage (b) Epithelium Basement Membrane Mucous Gland

Perichondrium Cartilage

There are several methods available to quantify changes in the tracheobronchial seromucinous glands. Point counting the various components of the glands or the entire bronchial wall provides data on glands (including the ratio of serous to mucinous acini), muscle and cartilage.147–149 Absolute measurements of glands and acini can be performed, so long as proper stereological methods are used. The most common and simplest method is the Reid index (Figure 11).150 This requires a section of a designated bronchus (as the index varies, depending on bronchial segments) with a cartilaginous plate without artifact or inflammation. A line is cast from the basement membrane to the perichondrium over the maximum thickness of the bronchial gland, and the ratio of gland thickness to line length is calculated. In general, the mean value of Reid index is increased in chronic bronchitics (52, SD 0.1) compared to non-bronchitics (smokers, as well as non-smokers: 44, SD 0.1), but there is considerable overlap of the distribution curves of subject groups.151

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Emphysema in COPD Definition Traditionally, the definition of emphysema is based on pathology.6,7,152,153 The American Thoracic Society128 and the European Respiratory Society154 follow this historical model. The definition states that emphysema comprises “changes of the lung, characterized by an abnormal, permanent enlargement of airspaces, distal to the terminal bronchiole and accompanied by destruction of alveolar septa without a pronounced fibrotic component”. Lack of fibrosis was included in the definition to clearly differentiate between emphysematous airspace enlargement and the enlarged airspaces of fibrotic interstitial lung disease, which are secondary to remodeling. This issue is made more complicated by the realization that emphysema can co-exist with fibrotic lung disease.58 Moreover, clinically occult fibrosis can even be found in smokers without COPD.155 The clinical relevance of this fibrosis is uncertain.

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Table 3 Classification of emphysema types

Emphysema type

Site

Characteristics

Proximal acinar Centrilobular (centriacinar) Mineral dust associated (so-called focal emphysema)

Upper lobe predominant Involves center of lobule in area of terminal respiratory bronchioles and alveolar ducts i.e. involving all three orders of RB

Found characteristically in cigarette smokers or related to inhalation of mineral dusts

Panlobular (panacinar)

Lower lobe predominant in A1AT deficiency only Involves the entirety of the lobule

Found characteristically in alpha-1antitrypsin deficiency

Distal acinar (paraseptal)

Subpleural in position Found adjacent to lobular septa

Most often found with centrilobular emphysema Found in tall, thin subjects in whom it is associated with spontaneous pneumothorax

Irregular (scar)

Adjacent to scars, old granulomata, apical caps, foci of Langerhans cell histiocytosis, etc.

Generally has no clinical significance

Classification There are four main types of emphysema: centriacinar, panacinar, paraseptal, and irregular emphysema.7,99,156 An overview of the different types is given in Table 3. Only centriacinar and panacinar emphysema cause clinically significant airway obstruction. This distinction provides an insight into the development of the abnormalities (see below). Centriacinar and panacinar emphysema may coexist, and the distinction between the two may be difficult, especially in collapsed lung or in severe disease (Figure 12). Centriacinar (centrilobular) emphysema is the commonest form and affects respiratory bronchioles with relative sparing of the distal alveoli. This type of emphysema is more common in heavy smokers and is often associated with chronic bronchitis. In some cases an association may also be seen with air pollution, and even pneumoconioses. The upper lobes of lung are preferentially involved, especially the apices (Figure 13). There is no adequate explanation for the observed upper lobe predominance, although it has been hypothesized that it may be related to blood flow or airway dimensions, which preferentially distribute tobacco smoke to this location. Carbon pigment accumulates in the walls of the emphysematous areas, and large amounts can be seen around bronchioles and vessels (Figure 14). Inflammation is frequently present, both diffusely in the alveolar septa and in the peribronchiolar or peribronchial parenchyma. There may be increased fibrous tissue,157,158 but this is not associated with parenchymal remodeling. Focal emphysema is a specific type of centriacinar emphysema. This pattern is often associated with mineral dust exposure. It affects all three orders of respiratory bronchioles with associated proximal bronchiolar occlusion (see Chapter 14).159 In panacinar (panlobular) emphysema, the enlarged acini are uniformly distributed from the respiratory bronchioles to the terminal alveoli. The frequency of panacinar emphysema varies widely, probably because of measuring techniques.

When identified, it occurs more frequently in the lower lobes and anterior lung margins, and is more severe toward the lung bases (Figure 15).160 The basal distribution is even more marked in subjects with A1AT (see below). In many cases, a distinction from centriacinar emphysema is difficult, as many A1AT individuals also smoke. In both centriacinar and panacinar emphysema the walls of the dilated airspaces are thin, yet wisps of residual parenchyma cross the airspaces in panacinar disease. This finding distinguishes emphysematous lung from honeycomb lung, which has fibrotic walls. In paraseptal emphysema, also called distal acinar emphysema, almost the entire proximal part of the acinus is normal while distal alveolar ducts and sacs are abnormal. This form of emphysema often shows great variations in sizes of airspaces. Abnormalities parallel to the pleura and along the lobular septa are most obvious (Figure 16). These abnormalities are particularly prevalent in the upper parts of the lungs, often bordering areas with fibrosis and atelectasis.161 Paraseptal emphysema is frequently found in association with centriacinar emphysema.160 Bullous emphysema is a descriptive term for any form of emphysema that manifests with airspaces measuring greater than 1 cm (Figure 17). It most commonly coexists with panacinar and centriacinar emphysema. These bullae usually develop from the fusion of small emphysematous areas. Rupture of bullae often leads to pneumothoraces. There is a common misconception that blebs are separated from bullae by a size criterion. This is completely erroneous. Blebs are collections of air within the visceral pleura that probably result from rupture of subpleural alveoli. They represent a form of interstitial emphysema, as opposed to alveolar destruction, leading to bullae. Emphysema with blebs is often seen in young adults with spontaneous pneumothoraces (see Chapter 36). Irregular emphysema is a descriptive term for dilated alveoli associated with parenchymal scars (Figure 18). This

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(c)

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(a)

TB

TB

TB

RB RB

RB

AD

AD AD A

A

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A

(e)

TB

TB

RB

RB

AD AD A A

Figure 12. Schematic representation of the changes seen in different types of emphysema compared to the normal airways and parenchyma. (a) Normal lung. (b) Panacinar emphysema. (c) Proximal acinar emphysema. (d) Distal acinar emphysema. (e) Irregular emphysema. TB, terminal bronchiole; RB, respiratory bronchiole; AD, alveolar duct; A, acinus.

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Figure 13. Centrilobular emphysema. (a) Gough section. (b) Macroscopic appearance.

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Figure 14. Carbon pigment accumulation in centriacinar emphysema. (a) Heavily pigmented bronchus. (b) Muscularized airway features extensive depositions of carbon pigment (smooth muscle actin immunohistochemical stain).

(b)

(b)

(c)

Figure 15. Panacinar emphysema. (a) Macroscopic appearance. (b) The Gough section demonstrates uniformly enlarged airspaces throughout the lobule. (c) Histologically similar changes are noted.

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(a)

(b)

Figure 16. Paraseptal emphysema. (a) Distal lung is involved. (b) Subpleural lung features the most striking change.

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(b)

Figure 17. Bullous emphysema. (a and b) Bullae are partially deflated. Apical disease is common but these lesions are not pleural blebs.

type is associated with prior inflammatory processes. There is usually no relationship with COPD, and in general this pattern has no clinical significance.

Estimation of severity Evaluation of a cut surface of a sectioned inflated lung can provide information on emphysema type, distribution and severity, particularly if the slice is subjected to barium sulfate impregnation.161 The Dunnill point count162 methodology (adapted from Weibel and Vidone’s method163) uses a

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transparent grid to measure the extent of involvement of the lung by emphysema, while the Ryder grid164 uses a grading system and the grid to assess extent and severity. Papermounted (Gough) sections are an excellent method of evaluating the extent and severity of gross emphysema and provide a permanent record. The final paper section is compared to a grading standard to produce an overall severity estimation.165 This method is not commonly utilized due to cost and possible medico-legal issues relating to the collection and preservation of post-mortem material.

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Figure 18. Irregular emphysema. Enlarged airspaces are associated with prior inflammation and scarring.

An overall estimation of the severity of emphysema can be performed on histological slides if the lung is distended (but not necessarily to a standard degree).166 When the histological image is compared to a standard set of grading photographs, the data correlate reasonably well with those obtained from evaluation of paper-mounted sections. Indirect measurements of emphysema, such as airspace size, surface to volume ratio and alveolar surface area, require standardized morphometric techniques. These include proper distension to a known pressure, randomized sampling and appropriate morphometric tools.167 The destructive index168 involves quantification of alveolar wall breaks and emphysematous destruction. Emphysema severity can also be computed using algorithms derived from high-resolution CT scans.169,170

Emphysema related to alpha-1-antitrypsin deficiency Alpha-1-antitrypsin deficiency is a genetic disorder characterized by decreased to low serum levels of A1AT, the main neutrophil elastase inhibitor in human serum. Unchecked, neutrophil elastases digest alveolar walls. Although the disorder is most common in individuals of Scandinavian descent,171 A1AT deficiency afflicts every major racial group in the world.172 173 The overall prevalence of A1AT deficiency in western Europe and the United States is estimated at approximately 1 in 3000. When only newborns are considered, the prevalence is approximately doubled (see Chapter 3).174,175 A1AT is a 52 kDa, 394 amino acid globular glycoprotein molecule produced by hepatocytes and released into the blood. It is formed by nine a helices and three b-pleated sheets (Figure 19). The protein is encoded by SERPINA1, located on the long arm of chromosome 14. There are a large number

Figure 19. Line drawing – AAT molecule. Structure of alpha-1-antitrypsin molecule with configuration likened to a mousetrap. Neutrophil elastase is inactivated by movement from the upper to the lower pole of the protein. The protease attaches the reactive center loop (yellow) of A1AT with the active serine of the protease (small red side chain) forming a link to the amino acid at the base of the reactive center (small green side chain) of A1AT. The resulting cleavage of the reactive loop allows it to snap back into the B sheet (red ribbons with arrows) of the A1AT. This spring-like movement flings the tethered protease to the opposite end of the A1AT molecule, distorting its active site (inset) and altering its structure so that it can be destroyed. Reprinted with permission from Carrell RW, Lomas DA. Alpha1-antitrypsin deficiency – a model for conformational diseases. N Engl J Med 2002;346:45–53.

of different alleles, expressed in a co-dominant fashion, each of which produces a different phenotypic variant.176 The alleles are named according to their migration characteristics on gel electrophoresis, with the fastest variant labeled A, and the slowest Z; the normal molecule has a medium rate of

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migration and is known as M. The Z allele is associated with very low levels of A1AT, and in its homozygous expression is responsible for 95% of cases of A1AT deficiency. The normal serum concentration ranges between 20 and 48 µM but, as A1AT is an acute-phase reactant, levels may vary depending on the presence of other stimuli. The Z protein can form polymers, which trap A1AT within the rough endoplasmic reticulum of the hepatocytes. This leads to reduced serum levels. A1AT-associated emphysema results from the imbalance between elastase and anti-elastase and manifests as a lower lobe predominant panlobular (panacinar) type of emphysema. Spirometry is normal for the first two decades of life,176 but can be influenced by cigarette smoking. Beyond this, there is an annual decrease in FEV1 of 70 ml per year in non-smokers, and up to 100 ml in smokers. Up to 43% of patients have chronic bronchitis, and these patients have greater airflow obstruction.177 Bronchiectasis may also be found.177 In general, emphysema accounts for approximately 70% of deaths in subjects with A1AT deficiency, with a mortality of approximately 3% per year. However, there also appears to be disease modification by other genetic variants, such as SNP in the nitric oxide synthase genes.178 Diagnosis of A1AT deficiency disease is often delayed. Diagnosis depends upon measurement of serum protein level, protein phenotyping and patient (and family) genotyping. After diagnosis, all patients are also monitored for liver disease. A1AT augmentation therapy is available for patients with emphysema, but its effectiveness is not certain.175

Pathogenesis of emphysema Only 10–15% of smokers develop clinically relevant emphysema, irrespective of the degree of tobacco smoke exposure. This fact illuminates the complex nature of emphysema. The pathogenesis of emphysema is not straightforward, with individual genetic factors and many interrelated mechanisms involved.32,33 Major mechanisms include proteolytic/antiproteolytic imbalance and oxidant damage, disruption of the lung’s homeostatic maintenance and repair system, and immunological derangements.

Emphysema as a consequence of proteolytic/antiproteolytic imbalance and oxidant damage Parenchymal destruction and inadequate tissue repair are responsible for the development of emphysema. Oxidants from cigarette smoke (oxygen radicals), as well as oxidants and proteases released from neutrophils and macrophages induced by cigarette smoke, destroy lung tissue. This inflammatory onslaught is inadequately compensated for by antioxidants and antiproteases.8,179 Oxidants inactivate A1AT, thus potentiating the effect of serine proteases, and activate proMMPs, thus increasing the metalloproteinase burden. Lipid peroxidation products are also elevated.180 The net effect is destruction of lung parenchyma.

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There is longstanding evidence of an increase in neutrophils and macrophages in the lungs of cigarette smokers (reviewed in181,182). This increase is persistent, even after smoking cessation,183 resulting in a continued increase in proteolytic enzymes, even after the putative inducing agent has been removed.84 The traditional inflammatory cell implicated in emphysema is the neutrophil, which produces neutrophil elastase. Other neutrophil proteases, including cathepsin G and matrix metalloproteinase (MMP) 9, as well as macrophage-derived proteases, such as cathepsins, MMP 2, 9 and 12, are also implicated in the development of emphysema. A large number of animal model investigations provide valuable information relating to the potential role of proteases and antiproteases in the genesis of emphysema (reviewed in184,185). Over-expression of cytokines, such as interferon-g and interleukin 13, also plays a role.186,187 Tumor necrosis factor alpha probably recruits inflammatory cells; however, a recent clinical trial involving TNFa inhibition was negative.188

Emphysema as a result of disruption of the lung’s homeostatic maintenance and repair system There are several excellent reviews of this subject.189–191 In the normal lung, vascular endothelial growth factor (VEGF) is central to lung homeostasis, and is produced primarily by vascular endothelial cells, but also by Type II pneumocytes.189 VEGF enhances phagocytosis of apoptotic cells,189 which in turn, increases VEGF secretion.192 This increases expression of bcl-2 which decreases apoptosis. Early evidence also suggests that VEGF also enhances cell proliferation.193 Obviously, disruption of this system could lead to disease. Animal models suggest disruption of the VEGF system is associated with increased apoptosis.194–196 Cigarette smoke exposure is associated with increased apoptosis in rat alveolar septal cells (not identified as either epithelial or endothelial),197 and there is increased oxidative stress in smoke exposed mice.198 Interestingly, A1AT suppresses apoptosis.199 Other animal studies have shown changes in bulk lung mRNA with continued cigarette smoke exposure, consistent with a failure to repair alveolar parenchyma.82 Increased apoptosis has also been found in the alveolar septa (with no distinction as to type of cell involved) of emphysema patients,200–202 although largely restricted to lungs with severe disease. In one study196 VEGFR2 protein expression levels were similar in the lungs of smokers and nonsmokers, although there was decreased expression of VEGFR2 in smokers with COPD. Another investigation203 demonstrated decreased VEGF and VEGFR2 protein, but only in severe emphysema. A recent hypothesis proposes that insufficient repair of lung tissue damage plays a role in disease development.67,204,205 The repair response after injury, as well as the damage itself, is related to inflammation and its sequelae. These processes are modulated by cell-cell and cell-matrix

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

interactions between inflammatory cells, fibroblasts and epithelial cells along with the mediators produced by these cells.205,206 The fibroblast is the main producer of extracellular matrix proteins and is the central cell in the restoration and maintenance of connective tissue. An altered composition of the extracellular matrix, especially with regard to proteoglycans, probably contributes to reduced tissue integrity. In the lungs of patients with COPD with severe emphysema, decreased amounts of peribronchiolar decorin and biglycan are noted.67,207,208 These proteoglycans strengthen other connective tissue structures, such as collagen fibers and bind, release and regulate fibrogenic and pro-inflammatory cytokines including TGFb, basic fibroblast growth factor and interleukin-8.67,205 TGFb is one of the important growth factors in lung connective tissue remodeling and is aberrantly regulated in COPD.208–212 Elastin and elastin fiber-associated molecules, such as fibrillin-1, are also reduced in alveolar walls and small airways.213,214 Such derangements contribute to decreased tissue integrity.

Emphysema as an immunological process Recent work suggests an autoimmune component to emphysema pathogenesis.110,215–218 An adaptive immune response may be involved in the genesis or perpetuation of the disease.216,217,219 Expansion of both the dendritic cell and lymphocyte subsets is noted. The severity of the inflammatory infiltrate appears to roughly correlate with the severity of emphysema,220 and also parallels the level of apoptosis.221 Many studies find increased numbers of T lymphocytes in emphysematous lung parenchyma.215,220–223 CD4 and CD8 T cells are increased, while oligoclonal CD4 T cell and B cell populations are identified in severe emphysema.106,224 Inflammatory cell infiltrates persist for years following smoking cessation.183 Tobacco smoke is a complex xenobiotic amalgam of proteins, polysaccharides and free radicals, which could all act as antigens to induce or perpetuate an immune response. Smoke could also damage the lung and expose or alter lung proteins, which in turn could act as targets, resulting in loss of tolerance to self-epitopes. Alternatively, the apoptotic cells may form a recognition ligand that promotes an inflammatory response.193,225 It is possible that colonization of the airways by bacteria, or persistence of viral antigens, such as adenovirus, represents a continued source of new antigens to perpetuate the inflammatory reaction.217

Pulmonary vascular disease in COPD Pulmonary hypertension (PHT) is an important complication of COPD, a significant predictor of mortality, and a major cause of morbidity (see Chapter 18).118,226–230,231 Pulmonary arterial pressure is mildly to moderately elevated, with a resting pressure between 20 and 35 mmHg in the majority of patients.226,230,232–237 There is a subset of patients with very high pressures.238

Figure 20. Pulmonary vascular disease in COPD. Muscle extends into precapillary vessels (smooth-muscle actin immunohistochemical stain).

The etiology of the pulmonary hypertension in this population is controversial, and is probably multifactorial.237 Likely factors include a direct effect of smoke on the vessels, hypoxic stimulation and inflammation with upregulation of vasoactive cytokines and induction of endothelial dysfunction. These all lead to vascular remodeling. Remodeling affects small and precapillary arteries with longitudinal extension of muscle into their usually poorly muscularized walls (Figure 20). Muscular pulmonary arteries have intimal and medial thickening as well as adventitial inflammation.80,222,239–244 Although these features are identified when pulmonary hypertension is present, it is controversial (and probably too simplistic an explanation) as to whether they are the etiology of the increase in pressures. At least some degree of separation between dynamic and structural vascular characteristics is a significant probability. There appears to be no change in muscular artery structure after treatment with oxygen, although there may be regression of the precapillary remodeling.245 Although most studies on vascular remodeling have been performed on resected lung tissue or tissue obtained at autopsy, recent advances in computed tomography have enabled assessment of the magnitude of the vascular alterations.246

Systemic effects of COPD Right ventricular changes

Cor pulmonale is defined as “right ventricular hypertrophy, dilatation, or both as a result of pulmonary hypertension caused by pulmonary disorders involving the lung parenchyma, impaired pulmonary bellows function, or altered ventilatory drive”. It is a recognized complication of COPD.247,248 Right ventricular dysfunction in COPD patients is multifactorial. Chronic hypoxia by direct action on the myocardium, pulmonary and systemic inflammation and increased

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workload due to pulmonary arterial remodeling and pulmonary hypertension are responsible.249 Altitude exposure, with consequent chronic hypoxia, carries no identified risk of myocardial ischemia in healthy subjects but has to be considered as a potential stress in patients with previous cardiovascular conditions.250 Right ventricular hypertrophy is rarely pure, and is usually associated with left ventricular hypertrophy, even in the absence of systemic hypertension. Indeed, there also appears to be a relationship between aortic atherosclerosis and small pulmonary artery remodeling.251 Chronic intermittent hypoxia-mediated left ventricular global dysfunction is associated with myocyte hypertrophy and apoptosis at the cellular level in rats.252 The effects of acute hypoxia on right ventricular function are controversial.253 Nevertheless, treatment of COPD exacerbations, even in patients without pulmonary hypertension, improves systolic and diastolic function.253 As cor pulmonale develops, there is a decrease in ventricular volume relative to its mass, with the ventricle losing its crescentic shape and becoming more concentric.249 Pathological assessment of right ventricular hypertrophy is best performed by examining the weight of the right ventricle, particularly as a ratio to that of the left ventricle and septum (LV&S).254 The suggested criteria for right ventricular hypertrophy are a right ventricular weight of more than 80 g, and a ratio of LV&S to RV of less than 2.0. Obviously, this ratio would change in the presence of left ventricular hypertrophy, and the absolute numbers probably vary according to sex and size. Right ventricular wall thickness is an imprecise measurement affected by tangential sectioning or sectioning through trabecular muscle.

Panniculitis Panniculitis is an unusual complication of A1AT deficiency, generally found in the PiZZ variant, but also described in several emphysema variants.255 The association is uncommon, with a prevalence of 1:1000.255 Unexplained panniculitis is one of the clinical features which should prompt suspicion of A1AT deficiency in an undiagnosed patient.176 Clinically, the lesions are widely disseminated, but are commoner in the lower extremities. Skin defects can be large, and can extend down to muscle. Histologically, the panniculitis is classified as a neutrophilic septal panniculitis in its acute phase, with foamy histiocytes and giant cells in the chronic phase. A variety of pathogenetic mechanisms have been proposed, but the strongest evidence supports unopposed elastolysis, with polymeric A1AT acting as a pro-inflammatory stimulus.176

Small vessel vasculitis Antiproteinase 3 (cANCA, PR3-ANCA) associated vasculitis has a strong relationship with A1AT deficiency variants (see Chapter 19).176 Although approximately 17% of patients with Wegener granulomatosis may have A1AT deficiency, there is no direct association. The main inhibitor of proteinase 3 is A1AT and it is possible that the absence of A1AT alters the antigenicity of PR3. A recent study found genetic linkage disequilibrium in

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the serpin-associated gene cluster.256 This suggests the pathogenesis may be much more complex and related to gene-associated regulation of the inflammatory process.

Liver abnormalities Liebermeister grooves are grooves on the superior and anterior surface of the right lobe of the liver, which correspond to the muscle arrangements of the diaphragm and intercostal spaces. They are found more commonly in middle age, and are generally, but not always, associated with hyperinflation, thoracic deformity and a raised abdomino-thoracic pressure gradient, thus explaining their presence in COPD.257 The grooves have no association with liver dysfunction. In patients with A1AT deficiency, liver dysfunction is the second most common manifestation, affecting more than 30% of PiZZ individuals,176 but liver dysfunction is also present in PiMZ and PiSZ patients.175 Polymerized protein in the hepatocytes leads to hepatocyte injury, cell death and eventually cirrhosis. The protein appears as cytoplasmic, periodic acid Schiff-positive, diastase-resistant globules within hepatocytes. An acute response to the polymerized proteins can manifest as a neonatal hepatic syndrome with hyperbilirubinemia and abnormal liver enzymes.174 Additional hepatic pathology seen in COPD is due to right heart failure, and includes centrilobular congestion and necrosis, as well as fine fibrosis. Cirrhosis may develop depending upon the severity and chronicity of the heart failure.

Osteoporosis Glucocorticoid therapy does not account for the decreased bone mineral density and increased prevalence of osteoporosis found in patients with (usually severe) COPD.258,259 A study260 found the number of smoking years was associated with decreased bone mineral density, even after adjusting for age, height, weight and number of cigarettes smoked daily. Interestingly, smoking does not affect biochemical marker serum values for bone turnover. The etiology of the osteoporosis appears multifactorial, and includes decreased mobility, poor nutritional status and decreased levels of anabolic steroids. The importance of osteoporosis is not confined to an increased risk of fractures, but includes deformation of the thoracic cavity with consequent decreases in vital capacity.261

Body mass wasting and skeletal muscle dysfunction Weight loss and low body mass in COPD is associated with increased morbidity and mortality, independent of muscle weakness and general quality of life.120,259,262 The mechanisms are multifactorial. There may be increased energy expenditure by respiratory muscles, compounded by poor nutritional intake. Elevated cytokines, such as TNFa, as a part of the systemic inflammatory reaction, are also found in people with weight loss. There is increasing evidence with regard to the importance of leptin in COPD, both dependent and independent of its metabolic parameters.263,264 Finally genetic

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susceptibility may also play a role in the degree of muscle wasting and dysfunction.265 Muscle dysfunction is extremely common in COPD, particularly in the lower extremities, and separate from deconditioning effects due to immobility.259 There appears to be a shift in twitch fiber types from slow to fast types.266 In addition, there is altered metabolism from oxidative to glycolytic,267 thus leading to early lactic acidosis during exercise.

Diaphragmatic dysfunction Diaphragmatic dysfunction is a major contributor to overall inspiratory muscle weakness in patients with COPD. Increased morbidity and mortality are noted in those who have low diaphragmatic mobility.119 A shift in muscle types toward Type I slow twitch, fatigue-resistant fibers is a relatively early change.268 In severe COPD, however, there is generalized reduction of the cross-sectional areas of fibers with a shift in molecular signaling pathways.268,269 No consistent microscopic changes have been identified.

Systemic inflammation An increased systemic inflammatory reaction is observed in all COPD patients.270 Increased CRP, IL-6, IL-8 and TNFa serum levels are noted in all patients.271,272 It is suggested the inflammation is associated with coagulation cascade activation. Such may be responsible for increased episodes of deep vein thrombosis and thrombo-emboli.273–275 COPD patients also have higher stroke and myocardial infarction rates. Tobacco use is certainly a large factor though.

Other forms of emphysema (Table 4) Non-destructive emphysema (hyperinflation) Emphysema is sometimes used as a descriptive term for excessive inflation of mostly peripheral lung without alveolar septal destruction. Such over- or hyperinflation may represent a

compensatory effect after surgical removal of lung segments, or may be associated with more proximal airway obstruction or “air trapping”. Etiological agents include central tumors, foreign bodies, and bronchomalacia in young children.

Development-related airspace enlargement The lungs in children with Trisomy 21 or Leprechaunism have enlarged airspaces and widened alveolar ducts. These findings are considered manifestations of inadequate alveolarization during pulmonary development.276–278

The “senile” lung With aging, there is a progressive loss of alveolar surface area of approximately 4% per decade after the age of 30.279 Grossly, the lungs have an increase in antero-posterior distance.280 These changes are secondary to a progressive dilatation of the alveolar ducts, so called alveolar duct ectasia (Figure 21).281,282

Starvation Severe starvation with loss of, or failure to attain, greater than 35% normal body weight is associated with morphometric airspace enlargement in rats.283–286 Although biochemical analysis of these lungs shows decreased amounts of collagen and elastin,285,287 this may be due to lack of growth rather than actual depletion. Histology demonstrates abnormal elastic fibers with short, irregular and non-uniform courses along the alveolar walls. This raises the possibility that there is increased turnover of the connective tissue elements with abnormal production of elastic fibers, similar to those seen in centriacinar emphysema.286,288 A mouse model showed the lungs were stiffer with increased resistance and a decreased lung capacity. Histology showed no changes in alveolar cord lengths with calorie restriction.289 Thus, in these two strains of

Table 4 Non-emphysematous airspace enlargement

Type

Site

Characteristics

Development-related

All lobes affected

Enlarged airspaces Alveolar duct ectasia Inadequate alveolarization

Congenital lobar overinflation

Upper lobe dominant

Lobe increased in volume Normal number of alveoli Overdistention of airspaces

Compensatory overinflation

Post-pneumonectomy

Modest increase in volume Alveoli increased only in childhood Overdistention of airspaces

Obstructive overinflation

Variable

Affected area increased in volume Gas trapping on expiration

Aging (senile) lung

All lobes affected

Alveolar duct ectasia

Starvation

All lobes affected

Simple airspace enlargement In childhood – decreased numbers of alveoli

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Figure 21. Senile lung. (a) Young adult lung, gross (bar ¼ 0.2 cm). (b) Young adult lung, micro (bar ¼ 200 mm). (c) Octogenerian lung, gross (space bar ¼ 0.2 cm). (d) Octogenarian lung, micro (space ¼ 200 mm).

mice with genetically determined differences in alveolar size, neither the mechanics nor the histology showed any evidence of emphysema-like changes with this severe caloric insult. In humans, calorie restriction appears to lead to airspace enlargement.289 This study confirms the historical data obtained from the Warsaw ghetto during World War II.290,291 Physiological changes may be seen in individuals with simple airspace enlargement. As a practical consideration, they are mild and do not cause the severity of airflow alterations seen in chronic obstructive pulmonary disease. For the pathologist, this means that if the patient had clinical COPD, and only simple airspace enlargement is seen on gross examination, careful attention should be given to histology of the small airways. He/she should search for evidence of small airways disease, or other pathologies such as bronchiolitis obliterans or asthma.

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Localized giant bullous emphysema: Birt-Hogg-Dubé syndrome The Birt-Hogg-Dubé (BHD) syndrome is a rare autosomal dominant multi-organ disease first described in 1977. The most prominent findings are cutaneous fibrofolliculomas and renal tumors, particularly chromophobe and hybrid oncocytic/ chromophobe renal cell carcinoma.292 Lung disease manifests with thin-walled pulmonary cysts and spontaneous pneumothorax before the fifth decade of life.293,294 Over 40% of BHD patients experience at least one spontaneous pneumothorax.295 Folliculin gene mutations are not associated with severe COPD.296 The genetic changes in this syndrome are found at chromosome 17p12q11 with several different mutations in the gene.293,294 The gene in this region has already been cloned

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

Figure 23. Birt-Hogg- Dubé syndrome. An excised cyst wall is lined by bland cuboidal cells. (Reprinted from: Adley BO, Smith ND, Nayar R, et al. Birt-Hogg-Dubé syndrome: clinicopathologic findings and genetic alterations. Arch Pathol Lab Med 2006;130:1865–70, with permission from Archives of Pathology & Laboratory Medicine. Copyright 2006, College of American Pathologists.) Figure 22. CT scan of Birt-Hogg- Dubé syndrome. Bilateral cysts and bullae are seen. (Reprinted from: Coplin RE. Birt–Hogg–Dubé syndrome. The Internet Journal of Pulmonary Medicine, 2008;9(2), with permission from The Internet Journal of Pulmonary Medicine.)

but while the function of the gene product, folliculin, is unknown, it is considered a tumor suppressor gene.294,297 Surprisingly folliculin gene mutations have been identified in families with histories of spontaneous pneumothorax, lacking other features of the syndrome.297–299 Such families have been considered a forme fruste.298 In one study, exon location of the BHD mutation was associated with the number of HRCT-detected pulmonary cysts.300 One research group suggests haploinsufficiency of folliculin may cause deranged alveolar development.301 Radiographically 14 to 83% of BHD patients have pulmonary cysts or bullae.295 CT scans demonstrate lesions ranging from subcentimeter air-filled cysts to 15 cm bullae rimmed by smooth, thin non-enhancing walls (Figure 22).295,297 The pulmonary pathology in BHD syndrome is confined to pulmonary and pleural cysts leading to pneumothorax. A basilar location is noted.302 Histologically, intraparenchymal and intrapleural cysts (i.e., bullae and blebs) have a thin wall usually lined by cuboidal epithelium (Figure 23). No other structures are present in the walls and the cysts are filled with air. Some investigators report emphysematous changes adjacent to bullae.297 The morphological differential diagnosis includes many other causes of spontaneous pneumothorax including bullous emphysema, pulmonary lymphangioleiomyomatosis (PLAM), pulmonary Langerhans cell histiocytosis (PLCH), Ehlers-Danlos syndrome, Marfan syndrome and idiopathic spontaneous pneumothorax (see Chapters 11 and 36). The presence of skin lesions should suggest the diagnosis of BHD, but in the absence of other syndromic manifestations, BHD cysts are predominantly basilar. This is in distinct contrast to the apical location seen in bullous emphysema, Ehlers-Danlos syndrome,

Figure 24. CT scan of placental transmogrification. A giant left upper lobe bulla is the only finding. (Reprinted from Cavazza A, Lantuejoul S, Sartori G, et al. Placental transmogrification of the lung: clinicopathologic, immunohistochemical and molecular study of two cases, with particular emphasis on the interstitial clear cells. Hum Pathol 2004;35:517–21, © 2004, with permission from Elsevier.)

Marfan syndrome, PLCH and idiopathic spontaneous pneumothorax. Pulmonary LAM cysts are lined by scattered HMB45-positive smooth muscle cell proliferations, rather than having thin fibrous walls. Families studied do not appear to develop progressive respiratory insufficiency, although they are at increased risk of recurrent pneumothoraces. Pleurodesis is often required.302

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Figure 25. Placental transmogrification. (a) Right upper lung lobe revealing severe emphysema with bullous disease, bronchiectasis, and peribronchial fibrosis. (b) Right lower lung lobe with panacinar emphysema. (c) Large cysts may be surrounded by condensed lung tissue. (Panels a and b reprinted from: Marchevsky AM, Guintu R, Koss M, et al. Swyer-James (MacLeod) syndrome with placental transmogrification of the lung: a case report and review of the literature. Arch Pathol Lab Med 2005;129:686–9, with permission from Archives of Pathology & Laboratory Medicine, © 2005, College of American Pathologists.)

Placental transmogrification Pulmonary placental transmogrification (PT) is a rare condition presenting as localized, often large bullae in young to middle-aged patients. Clinically, PT was initially described in patients with severe emphysema and has been associated with cigarette smoking, congenital bullous emphysema and fibrochondromatous hamartomas of the lung. It has incidentally been described presenting as Swyer-James-MacLeod syndrome (unilateral hyperlucency of the lung) (see below).303 PT was

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considered a variant of giant bullous emphysema but it probably represents a benign proliferation of immature interstitial clear cells with secondary cystic change.304 Chest X-ray and CT are not distinctive (Figure 24). In general giant bullae are bilateral while the unaffected lung parenchyma is normal without emphysema. Macroscopically one sees well-demarcated lesions with papillary tissue, cysts and fibrosis (Figure 25). One or more giant bullae contain epithelial-lined papillary structures. These structures resemble placental villi. The centers of these villi are

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

(a)

(b)

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Figure 26. Placental transmogrification. (a–c) Placentoid nodules admixed with adipose tissue contain bland cells with clear cytoplasm.

edematous or fibrotic, and vessels, inflammatory cells and fat are present together with distinctive bland cellular structures with a clear cytoplasm (Figure 26).304,305 The peculiar interstitial clear cells are positive for CD10 and are focally positive for vimentin but negative for smooth muscle actin, desmin, S100, HMB-45, CD68, CD34, CD117, TTF1, chromogranin, synaptophysin, estrogen and progesterone receptors.304 Electron-microscopy shows no specific differentiation. The interstitial cells are thought to have an immature mesenchymal phenotype.304 The initial theory of PT as a variant of emphysema contrasts with the young age of the patients, the very localized nature of the disease plus the lack of COPD-like changes in the remaining lung. Cavazza et al. suggested the interstitial clear cell proliferation represents the primary event and the cystic changes are likely secondary. 304 Several pulmonary lesions may show a growth pattern similar to PT. A papillary/microcystic pattern can been seen in emphysema, sclerosing hemangioma, solitary fibrous tumor

and pulmonary “hamartoma.”304 However, most of these diseases have sufficiently distinctive features to enable easy differentiation from PT. Conservative bullectomy is the recommended treatment and patients have an excellent prognosis after removal.

Bronchiectasis Definition Bronchiectasis is defined morphologically as abnormal, irreversible dilatation of bronchi and bronchioles.306,307 Although often associated with airflow obstruction, bronchiectasis is considered as a disease entity separate from COPD, with many etiologies and many pathogenetic pathways. Bronchiectasis was described by Laennec 308 in patients who had died with chronic catarrh, and especially after “hoping” (sic) cough. He noted that the bronchial tubes had areas of dilatation and stenosis.

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Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Table 5 Etiologies of bronchiectasis

Idiopathic Congenital Tracheobronchomegaly (Mounier-Kuhn syndrome) Williams-Campbell syndrome Primary or secondary immune defects Neutrophil abnormalities Panhypogammaglobulinemia Hyper-IgE syndrome (Job syndrome) IgA deficiency

Relapsing polychondritis Post-intubation tracheomalacia Related to other medical conditions Inflammatory bowel disease Rheumatoid arthritis Miscellaneous Post-chemotherapy Post-irradiation Asthma

IgG subclass deficiencies Ataxia-telangiectasia Yellow nail syndrome HIV disease Ciliary dysfunction/dyskinesias Primary cilial dyskinesia Kartagener syndrome Young syndrome Cystic fibrosis Inhalation Injury Smoke

Bronchiectasis can be classified according to clinical, anatomic or quasi-etiological schemes.

Clinical This corresponds to the degree of sputum production. In wet bronchiectasis, the sputum is abundant, and contains inflammatory cells, inflammatory debris and desquamated epithelium. Although this classification does have a relative correlation with pathology, it provides no etiological relevance.

Acrolein

Gross anatomic

Mustard gas

Depending upon the site of disease, bronchiectasis can be classified into proximal or distal. For example, proximal bronchiectasis is one of the common manifestations of allergic bronchopulmonary aspergillosis (see Chapter 15). Another classification utilizes the appearance of the dilated bronchi309 to divide bronchiectasis into cylindrical, varicose or saccular. Uniform, elongated dilatation characterizes the varicose type, alternating dilatation and constriction patterns the cylindrical type, and progressive dilatation towards the periphery the saccular form. This classification is of limited value since most cases have intermediate or mixed appearances.

Ammonia Sulfuric acid Hydrocarbons Aspiration Infections Viral Measles Adenovirus Fungal Aspergillus (invasive or allergic related) Coccidioidomycosis Bacterial Whooping cough (pertussis) Tuberculosis (M. tuberculosis or Mycobacterium avium intracellulare) Actinomyces Post-obstructive Foreign bodies Malignancies Middle lobe syndrome Bronchial cartilage alterations Amyloidosis Tracheobronchopathia osteochondroplastica

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Classification

Microscopic anatomic This classification divides bronchiectasis into follicular, saccular, and atelectatic forms.310 Follicular bronchiectasis is characterized by the presence of prominent lymphoid follicles in the walls of affected bronchi, and was hypothesized to occur as a sequel to viral infections. Saccular bronchiectasis includes extensive destruction of the bronchial wall, normality of alveoli around the abnormal bronchi, and the presence of proliferative bronchiolitis (see below) in the proximal airways. Squamous metaplasia is identified frequently. Atelectatic bronchiectasis appears to describe bronchiectasis related to chronic atelectasis. This classification has limited usefulness since there does not appear to be any clinical or radiological relevance to the different groups.

Quasi-etiological (Table 5) This is probably the most functional classification as it provides some degree of clinical relevance and thus is useful when

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

airway-associated clustered thick-walled cysts, sometimes with air-fluid levels.316,317 These are clearly different from emphysema in that they have thick, not thin, fibrous walls. For the differential diagnosis of lung cysts, which are usually single, the reader is referred to Chapter 3.

Pathology

Figure 27. HRCT of bronchiectasis. Saccular enlargement of large and small airways extend to the periphery. (Image courtesy of Dr Jonathon Leipsic, Vancouver, BC, Canada.)

considering therapies. However, up to 50–80% of bronchiectasis may be idiopathic.311

Epidemiology Bronchiectasis represents a major global disease burden, often related to childhood infections.312 Immunization, better housing and nutrition and greater access to healthcare with access to antibiotics in developed nations have greatly reduced the incidence and prevalence of this disease. In societies with limited access to adequate healthcare, the prevalence of bronchiectasis is still high.313–315,311

Clinical The clinical presentation is mainly related to the underlying cause and/or to the consequences of mucostasis and chronic infections. In general, non-cystic fibrosis-associated bronchiectasis is commoner in women. Although it is found across all ages, the disease is slightly more frequent in the middle-aged and elderly. Cough with mucopurulent sputum is characteristic, but it may be dry and non-productive.311 Dyspnea and fever are nonspecific symptoms. Hemoptysis, due to bleeding from bronchopulmonary anastomoses, may occur and can be impressive. Physical findings are also nonspecific, but may include crackles and wheezes; chronic bronchiectasis is associated with digital clubbing (see Chapter 24).

Radiology Radiological studies are usually diagnostic. Chest X-ray and bronchograms have been superseded by HRCT (Figure 27). In mild disease, diagnosis depends upon an increased ratio of the diameter of the airway to the adjacent pulmonary artery, the so called “signet ring sign”,315 and a lack of bronchial tapering on sequential CT slices. In more severe disease, findings include

Distribution of disease varies, depending upon the etiology of the disease, but, overall, bronchiectasis is often a bilateral lower lobe disease, with a patchy distribution in the vertical parts of the airways. There is often an increase in severity towards the periphery. Irregularly dilated airways approach the visceral pleura and do not taper toward the periphery. The dilatation can be substantial, sometimes up to four times the normal airway diameter. One of the most important pathological findings is obliteration of bronchi and bronchioles beyond the dilated bronchi (Figure 28).309,310,318 These lesions are probably physiologically important and are responsible for airflow obstruction in bronchiectasis. (see below). They are always present and are often extensive. Mucus with or without pus in the airways compounds the obstruction. Specific histological features again vary depending upon the etiology, but the general changes are nonspecific (Figure 29). Extensive destruction of the bronchial wall occurs; in severe bronchiectasis all components (cartilage, muscle and bronchial glands) may be lost and the wall may be deformed by fibrous tissue. Destruction of the bronchial wall is less apparent in other cases,319 but loss of elastic tissue and muscle with replacement fibrosis are common. Varying degrees of acute inflammation are present in the bronchial walls. Chronic inflammatory cells are usual, and lymphoid aggregates or follicles are often present. Occasionally inflammation is surprisingly slight,320 and instead fibrosis appears to be the dominant pathology. Ulceration and squamous metaplasia of the bronchial epithelium may be seen, as well as goblet cell metaplasia. Inflammatory debris and/or mucus often at least partially fills airway lumina. Secondary neuroendocrine cell hyperplasia is common (Figure 30).

Pathophysiology Bronchiectasis probably evolves through a variety of different mechanisms. Most likely it results from a gradual process, as a reaction to an injury which impairs the mucociliary escalator, leading to mucostasis and infection.315 There is a cycle of infection, inflammation and bronchial wall distortion which spirals into progressive disease. Analysis of sputum from bronchiectatics shows the expected inflammatory mediators interleukin-8, tumor necrosis factor-a and prostanoids in combination with increased elastases (reviewed in311). A role for the adaptive immune system is suggested by genetic associations with HLA-DR1, DQ5.321

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Figure 28. Bronchiectasis. (a, b and c) Dilated and fibrotic airways lead to destruction of alveolar parenchyma and secondary infections. (d–f) Bronchiectasis and bronchioloectasis in a patient with cystic fibrosis also features airway obliteration (arrows in panel d) (panel f: elastic stain). (Panels d, e and f images courtesy of Dr A. Churg, Vancouver, BC, Canada.)

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(a)

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(f)

Figure 29. Bronchiectasis and bronchioloectasis. (a and b) Inflammatory infiltrates surround dilated small airways. Mucostasis is apparent. (c) Bronchiectasis with brisk inflammatory infiltrates and mucosal ulceration. (d) Collapsed bronchiectasis with extensive fibrosis and inflammation in the surrounding parenchyma. (e and f) Dilated airway with extensive chronic lymphoplasmacytic infiltrate and mucosal/submucosal infoldings.

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Figure 30. Carcinoid tumorlet in bronchiectatic lung. Airway distortion and destruction affects remaining airways. Neuroendocrine cell hyperplasia and neuroendocrine tumorlets may develop.

Course and prognosis Prognosis relates largely to etiology, but the clinical course of non-cystic fibrosis bronchiectasis is highly variable (reviewed in311,312). There may be exacerbations of disease and decline of pulmonary function with progressive airflow obstruction.322 An accelerated decline is associated with colonization with Pseudomonas aeruginosa, more frequent severe exacerbations and more systemic inflammation.322 The long-term outcome, however, is generally regarded as good. However, there is still a significant reduction in life expectancy in patients with bronchiectasis.

Therapy Therapy is mainly aimed at reduction of symptoms and prevention of exacerbations.311,312,315,323 In children, optimization of normal growth and development is a priority. This includes treatment of the underlying condition. Bronchodilators and inhaled corticosteroids may have a role in specific situations where there is a bronchoconstrictive or allergic component. Inhaled hyperosmolar agents and mucolytic agents may reduce mucus viscosity and allow increased expectoration, and physiotherapy may aid drainage. Various new antibiotic regimes have shown progress.324 Surgical resection of affected segments may be necessary as a last resort to control bleeding or to prevent recurrent infections.306 Transplantation is a final option.312

Special entities Aspiration Aspiration is defined as the misdirection of oropharyngeal or gastric contents into the larynx and lower respiratory tract. A distinction must first be made between aspiration pneumonitis and aspiration pneumonia.325,326 Aspiration pneumonitis,

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also known as Mendelson syndrome, results from the aspiration of gastric contents, which may include bile. This causes a chemical injury with diffuse alveolar damage as its characteristic pathology. Bile can be identified in the alveoli by its green/ brown color. This substance is very toxic to the lung. Aspiration pneumonia is an inflammatory, and often infectious, process within the lung in reaction to pathogenic bacteria contained within the aspirated oropharyngeal material. Bronchiectasis usually results from the pneumonia. Evaluation of serum, sputum and bronchoalveolar lavage biomarkers may aid in the diagnosis of aspiration.327 Aspiration is causal or associated with bronchiectasis in approximately 5–15% of subjects.328,329 Aspiration of foreign bodies and aspiration of gastric or oral contents associated with reflux disease is common in both children and adults (reviewed in312). A large range of different foreign-body materials have been reported.330 In children, foreign materials are usually toys or money, while in adults, foods are common.330 Patients with neurological dysfunction may be particularly prone to aspiration. Hospitalized patients postextubation are also at high risk.326 Aspiration can lead to bronchiectasis through several mechanisms. First, a foreign body can obstruct the airway and lead to post-obstructive bronchiectasis. Secondly, the inflammatory reaction to the foreign material may alter the airway wall structure. Thirdly, the infectious material within the aspirated material may induce a destructive and inflammatory process. As noted below, aspiration is a known cause of bronchiolitis, and is probably clinically331 and pathologically332 underrecognized. The characteristic pathological response to aspirated material is a granulomatous inflammatory reaction (Figure 31). Aspirated material can often be identified. Striated muscle fibers can be found and vegetable materials show thick cell walls and are sometimes in a honeycomb or compartmentalized configuration. Pharmaceutical materials include talc, microcrystalline cellulose and crospovidone (see reference333 for discussion) (see Chapter 16). Aspiration of lipid material was reasonably common when various oils were used as a vehicle for pharmaceutical agents, such as nose drops or laxatives.334 A pneumonic pattern is more common than pure bronchiectasis, and the term “exogenous lipid pneumonia” has been utilized to indicate that the reaction is due to lipidbased foreign material.

Middle lobe syndrome Middle lobe syndrome (MLS) is a clinical disorder representing persistent or recurrent right middle lobe or lingular atelectasis or infiltrates. Children and adults may be affected. The most frequent symptoms are chronic cough, hemoptysis, chest pain, dyspnea and/or fever. Chest radiographs and CT scans may show consolidation, variable patchy middle lobe infiltrates or bronchiectasis.

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

(a)

(d)

(g)

(b)

(c)

(e)

(f)

(h)

(i)

Figure 31. Aspiration. (a) Anterior-posterior chest X-ray following right upper lobectomy for lung adenocarcinoma. Lung fields are free of infiltrates. (b) Same patient 24 hours later following witnessed aspiration. Bilateral diffuse airspace disease is striking. (Image courtesy of K. Edwards, MD, Philadelphia, PA, USA.) (c and d) Secondary actinomycosis following aspiration in a patient with bronchiectasis. (e) Chronic mineral oil aspiration produces nodular infiltrates. (f) Various-sized vacuoles with fibrosis replace alveolar parenchyma. (g) Multinucleated giant cells ingest aspirated vegetable material. (h) Pine needle aspiration elicits a brisk inflammatory response leading to bronchiectasis. (i) Granulomatous pneumonitis secondary to crospovidone aspiration. The pill filler is insoluble (arrow).

Given its narrow width, sharp take-off from the main bronchus, long tubular length and the presence of a group of adjacent lymph nodes, this airway is vulnerable to both intrinsic and extrinsic obstruction.335 Other etiologies include recurrent infection. Allergic bronchopulmonary aspergillosis and endobronchial silicosis336,337 have been described, and Mycobacterium avium intracellulare is often isolated. MAI middle lobe infection is commonly seen in elderly women and may be

related to a decreased cough reflex. These cases have been referred to as the Lady Windermere syndrome.338 (To those who have read Oscar Wilde’s Lady Windermere’s Fan, this eponym is highly irritating, as Lady Windermere’s refusal to shake hands with Lord Darlington was because she was annoyed with him, not because she was being fastidious!) Although enlarged peribronchial lymph nodes were once believed to be the major cause of airway obstruction, poor

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drainage of middle lobe lymphatics may play a large role.338 Weak collateral ventilation from upper and lower lobes may also contribute as it makes the lobe susceptible to atelectasis.339,340

Pathological findings are varied (Table 6) but one usually finds bronchiectasis at macroscopic examination (Figure 32). Microscopic findings include bronchiolitis, organizing pneumonia and abscesses (Figure 33). Granulomatous inflammation is not an infrequent finding. Lobar resection specimens rarely feature a definitive etiology, since one cannot be certain whether an infection is the cause or a secondary finding. Surgical resection is a definitive treatment.341

Table 6 Pathological findings in middle lobe syndrome

Conducting airways (bronchi, bronchioles) Bronchiectasis Bronchiolitis with lymphoid hyperplasia (“follicular bronchiolitis”)

Broncholithiasis

Proliferative bronchiolitis

Broncholithiasis is the result of impingement on and subsequent erosion of calcified lymph nodes into airways (Figure 34).342

Broncholithiasis Enlarged peribronchial lymph nodes Distal airspaces, interstitium Atelectasis Organizing pneumonia Granulomatous inflammation (both necrotizing and non-necrotizing) Abscess formation Hemosiderin Interstitial fibrosis Honeycomb change Pleura Fibrosis Modified from340

(a)

Figure 32. Middle lobe syndrome. Bronchiectasis is apparent. There is hardly any residual alveolar parenchyma.

(b)

Figure 33. Middle lobe syndrome. (a) Whole mount demonstrating bronchioloectasis and peribronchiolar inflammation. (b) Peribronchial lymphoplasmacytic infiltrates and fibrosis are noted.

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(b)

(c)

Figure 34. Broncholithiasis. (a) This CT mediastinal window demonstrates a left lung central calcification. Lung windows confirmed the endobronchial location. (b) At bronchoscopy a white lesion appears attached to the bronchial sidewall. (c) A yellow-brown calficied nodule is noted in a bronchiectatic cavity (arrow). A small calcified lymph node is also seen (arrowhead). (Panel b courtesy of Dr P. Wilcox, St Paul’s Hospital, Vancouver; panel c reproduced from: Seo JB, Song KS, Lee JS, et al. Broncholithiasis: review of the causes with radiologic-pathologic correlation. Radiographics 2002;22 Spec No:S199–S213, with permission of the Radiological Society of North America.)

This process induces anatomical distortion of the airways, irritation and/or ulceration of the bronchial epithelium, as well as middle lobe syndrome (see above). This can cause chronic cough, hemoptysis, lithoptysis (coughing up the calcified airway structure) or recurrent pneumonia.343 In severe cases, a fistula may develop between airways and mediastinal structures, including the esophagus. On CT, broncholithiasis is characteristically represented by bronchial obstruction, along with endobronchial or peribronchial calcified structures.344 The diagnosis is most often made at bronchoscopy. The calcified lymph nodes are usually the result of either granulomatous lymphadenitis (e.g. mycobacterial or silicosis) or fungal infections (e.g. Histoplasmosis capsulatum).343,344 Therapy depends on whether the broncholith is mobile or fixed.342,343,345 If it is mobile, extraction by bronchoscopy usually suffices; when the broncholith is fixed, operative repair is necessary. In complicated cases, with bronchiectasis, hemoptysis, fistulae or pneumonitis, surgical management is required.345

Plastic bronchitis

Plastic bronchitis is a rare disease, first described by Galen.346 Other names for plastic bronchitis are pseudomembranous bronchitis or fibrinous bronchitis. The main hallmark is the formation of large gelatinous or sometimes rigid branching casts (Figure 35).346,347 The casts can be large and are more extensive than those observed in simple mucus plugging. These casts can be expectorated spontaneously or removed at bronchoscopy. In severe cases they cause significant airway obstruction.348 The prevalence of plastic bronchitis is unclear and many patients go undiagnosed. The main clinical symptoms are dyspnea, wheezing, pleural pain and fever. Auscultation reveals absence of breath sounds or wheezing. On radiology, lung

Figure 35. Plastic bronchitis. Expectorated cast from a 19-year-old woman. (Reprinted from Madsen P, Shah SA, Rubin BK. Plastic bronchitis: new insights and a classification scheme. Paediatr Respir Rev 2005;6:292–300, © 2005, with permission from Elsevier.)

collapse with associated hyperinflation can be seen but the disease can also present with bilateral variable or whole lung consolidation. Confirmation of the diagnosis is usually obtained by demonstrating the casts either at bronchoscopy or by expectoration. Plastic bronchitis can be idiopathic but is often associated with difficult diagnostic problems, such as treatment-refractory asthma.347 In atopy or asthma, the casts are inflammatory and contain variable numbers of eosinophils and CharcotLeyden crystals (see Chapter 15).346,347 Eosinophilic casts may also be seen without associated asthma or atopy. Primary treatment of this condition involves removal of central casts, before providing further therapy.347,348

Tracheobronchopathia osteochondroplastica Tracheobronchopathia osteochrondoplastica was originally described by Aschoff.349 It is a relatively frequent condition of the trachea and major bronchi, characterized by cartilaginous and bony nodules in the subepithelial space (submucosa)

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Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Figure 36. Tracheobronchopathia osteochondroplastica. At bronchoscopy bulging submucosal nodules distort the tracheal lumen. (courtesy of Dr T. Shaipanich, Vancouver, BC, Canada).

Figure 37. Tracheobronchopathia osteochondroplastica. Bone spicules with adipose tissue expand the submucosa.

expiration with decreased inspiratory flows, indicative of extrathoracic airway obstruction. The nodules can also be seen on tracheal tomograms, or CT scans (Figure 38);357 and postmortem X-rays can be striking.

Small airway diseases/bronchiolitis Definition and history Figure 38. CT scan of tracheobronchopathia osteochondroplastica. Tracheal mucosal irregularity with calcification is noted in this mediastinal window. (Courtesy of Dr Jonathon Leipsic, Vancouver, BC, Canada)

which ultimately produce rigidity and narrowing of the airways. High-resolution CT scans and bronchoscopic evaluations of the airways have increased the ability of the physician to recognize lesions. The frequency varies from approximately 1 in 400 autopsies350 to 1 in 1000 bronchoscopies in patients older than age 50.351 Patients may present with repeated infections, stridor, hoarseness and/or cough, depending on the extent and severity of the lesions. On bronchoscopy, multiple hard nodules or plaques are observed on the anterior and lateral tracheal walls (Figure 36). Histology demonstrates submucosal cartilaginous nodules with partial or complete ossification. Bone marrow elements may be seen (Figure 37).352 A previous suggestion that tracheobronchopathia osteochondroplastica represented a subtype of tracheobronchial amyloidosis353 is probably in error, although the two conditions can co-exist.354,355 The nodules probably represent a nonspecific response to injury rather than multiple osteochondromas. Lung function tests demonstrate an obstructive pattern,356 and flow-volume loops may show a peak flow “cut-off” during

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Both small airways diseases and bronchiolitis refer to diseases involving non-cartilaginous airways (membranous and respiratory bronchioles) measuring less than 2 mm in diameter. The notion that the small airways are important arose from work which demonstrated that, in normal individuals, airflow resistance in airways less than 3 mm in diameter contributes approximately 25% of total lung resistance, but in individuals with COPD this figure is 80%.358 This work is complemented by a study demonstrating a direct relationship between bronchiolar diameter and airways resistance.359

Clinical features Clinical symptoms are nonspecific. Patients complain of shortness of breath with or without an associated cough, which may be productive or non-productive. Pulmonary function tests demonstrate airflow limitation with obstruction, sometimes in combination with restriction if the condition includes an interstitial component (see below). While a plain chest radiograph may show various findings from normal to hyperinflation to nodules or reticular infiltrates, HRCT provides more detailed information. Three CT patterns are described; centrilobular nodules/branching linear opacities (the latter is also known as “tree in bud”) (Figure 39),360 air-trapping and mosaic attenuation (Figure 40), and poorly defined centrilobular nodules (Figure 41).361 A mosaic pattern results when hypoventilated and therefore

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Figure 39. Small airway disease. High-resolution CT demonstrating branching centrilobular nodular opacities. (Courtesy of Dr J. Verschakelen, Leuven, Belgium.)

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Figure 40. Constrictive bronchiolitis. (a) This high-resolution CT inspiration film demonstrates normal parenchyma. (b) The expiration CT demonstrates mosaic attenuation with low-density areas of air trapping. (Courtesy of Dr J. Verschakelen, Leuven, Belgium.)

hypoperfused areas show decreased attenuation in contrast to unaffected areas with normal or increased attenuation.362 Affected bronchioles often have larger diameters than normal airways.363,362 This feature is best recognized in the subpleural aspects of the lung.361 Poorly defined centrilobular nodules result when the disease process involves adjacent lung parenchyma.361

Classification

Figure 41. Bronchiolitis. High-resolution CT image demonstrating poorly defined centrilobular nodules. (Courtesy of Dr Jonathon Leipsic, Vancouver, BC, Canada.)

Small airway diseases can be classified in several different ways. A broad etiological classification is offered in Table 7. While this classification is easily understood by clinicians, it does not offer any pathological approach to the diagnosis. A pathological approach is outlined in Table 8, and represents an adaptation of several pathological approaches.364–372 Many conditions may manifest different pathological patterns as the bronchiolitis evolves. In most cases the histological diagnosis

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Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Table 7 Classification of small airways disease based upon etiological mechanisms

Table 8 Classification of small airways disease based upon pathological patterns

Diseases of the bronchioles: direct injury Infections Bacterial (especially TB and mycoplasma) Fungal Viral Toxic fume Inhalation Aspiration

Alterations of epithelium þ/ lumen Infections

Diseases of the bronchioles: immunological associations Asthma Allergic reactions Eosinophilic pneumonia Hypersensitivity pneumonia Drug reactions Connective tissue disease Follicular bronchiolitis Constrictive bronchiolitis Lung transplant rejection Graft vs. host disease Inflammatory bowel disease Vasculitis Wegener granulomatosis Churg-Strauss syndrome Sarcoidosis Diseases of the bronchioles: smoking/dust associations Tobacco-associated bronchiolitis Respiratory bronchiolitis – interstitial lung disease Crack users’ bronchiolitis Mineral dust-associated bronchiolitis

of bronchiolitis is nonspecific. Not unlike the approach espoused for interstitial lung diseases (see Chapter 10), correlating histological bronchiolitis with clinical and radiographic data is essential to enable a reasonable clinicopathological diagnosis. Appropriate therapy relies on such an approach.

Acute bronchiolitis Acute bronchiolitis is recognized by its extensive alteration of the epithelium, with epithelial cell sloughing and neutrophilia (Figure 42), accompanied by airway wall edema and lymphoplasmacytic infiltrates. Acute bronchitis/bronchiolitis is mainly due to infections, seen most commonly in early infancy and childhood. Although most cases are mild and resolve, infectious bronchiolitis is the most frequent cause of respiratory failure and admission to pediatric intensive care units. Viruses are the main causative agents, especially respiratory syncytial virus (RSV),373 adenovirus, influenza, parainfluenza and recently new discovered viruses including H1N1, metapneumovirus, bocavirus and coronavirus (severe acute respiratory syndrome (SARS).374–378 Viral cytopathic effects with inclusions can be recognized in many of the viral induced

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Inhalational injury Asthma Transplant rejections Alterations of basement membrane Asthma Proliferative bronchiolitis (luminal polyps) with or without organizing pneumonia Organizing diffuse alveolar damage Organizing pneumonia or bronchiolitis Infective Obstructive Aspiration Toxic fumes and gases Connective tissue disease Gastrointestinal-related (inflammatory bowel disease, celiac disease) Hypersensitivity pneumonia Drug reaction Transplant-associated Idiopathic (with or without organizing pneumonia) Constrictive bronchiolitis Scarring from previous injury Viral/bacterial infections Inhalational injury Hypersensitivity pneumonia Idiopathic Airway centered ILD (? chronic HP) Connective tissue disease Transplant-associated Drug reactions Gastrointestinal-related (inflammatory bowel disease, celiac disease) Diffuse idiopathic neuroendocrine hyperplasia/neuroendocrine cell hyperplasia of infancy Radiation effect Vasculitis Bronchiolocentric with or without adjacent parenchymal disease Macrophage-rich Pigmented Smokers’ bronchiolitis/respiratory bronchiolitis interstitial lung disease Crack users’ bronchiolitis Langerhans’ histiocytosis Hard metal disease

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

Non-pigmented Diffuse panbronchiolitis MAI Hypersensitivity pneumonia Metabolic (Gaucher) Lymphocyte-rich Follicular bronchiolitis Viral and mycoplasma infection Transplant reaction Toxic fumes or gases Transplant-associated GI-related (inflammatory bowel disease, celiac disease) Eosinophil-rich Eosinophilic bronchiolitis Asthma Granulomas Infections Foreign body (aspiration) Sarcoid Bronchocentric granulomatosis Hypersensitivity pneumonia GI-related (inflammatory bowel disease) Fibrosis only Mineral dust Langerhans’ histiocytosis Post-infective Chronic hypersensitivity pneumonia Peribronchiolar fibrosis and bronchiolar metaplasia (Lambertosis)

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bronchiolitides (see Chapter 5). Bacteria including mycoplasma and fungi can cause or complicate bronchitis/ bronchiolitis. A minority of patients, usually those with adenovirus infection, progress to constrictive bronchiolitis with fibrous obliteration. This results in a unilateral hyperlucent lung.379 Other viral pneumonias, as well as mycoplasma and pertussis, have been implicated.380,381 This complication is referred to as Swyer-James (MacLeod) syndrome (post-obstructive constrictive bronchiolitis).382,383 Swyer-James (MacLeod) syndrome, or unilateral hyperlucent lung, usually presents with dyspnea on exertion, hemoptysis and chronic cough secondary to recurrent lung infections and/or bronchiectasis.384,385 The process may be localized, unilateral or even bilateral. Chest radiographs demonstrate lobar or unilateral hyperlucent lung with loss of lung volume. Normal or reduced lung volume is noted on full inspiration, while severe airway obstruction is detected during expiration. CT scan demonstrates a diminished pulmonary vascular bed with decreased blood flow in the affected area (Figure 43). Unilateral perfusion loss is also seen on technetium-99c scans.386 The final size of the lung in this syndrome depends on the age of the patient at the time of the infection. If early in life, development is affected and the lung is smaller than the uninvolved lung. If late in childhood, the lung can be of normal size. Histopathological descriptions include emphysematous changes with obliteration of the pulmonary capillary bed, bronchitis, bronchiolitis, bronchiectasis and honeycomb lung

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Figure 42. Acute bronchiolitis. (a) Whole mount demonstrating the exquisitely airway-centered disease. (b) At higher magnification the bronchiolar mucosa is overrun by neutrophils with sparing of surrounding alveolar parenchyma.

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with pulmonary artery hypertrophy.381,382,387 A case of placental transmogrification accompanied by centriacinar, panacinar and bullous emphysema has also been reported.371 In adults, infectious acute bronchiolitis leading to physician consultation mainly presents in immunocompromised patients or in those with COPD or asthma. Influenza,

rhinoviruses and respiratory syncytial virus are usually implicated.388 H1N1 influenza virus causes acute bronchiolitis with epithelial necrosis and in some cases also hemorrhage and diffuse alveolar damage (see Chapter 5).389

Obliterative bronchiolitis Obliterative bronchiolitis is generally divided into two types: proliferative and constrictive. Proliferative bronchiolitis is known to evolve into constrictive bronchiolitis, but may also resolve completely.367

Proliferative bronchiolitis

Figure 43. Swyer-James syndrome. High-resolution CT image demonstrating right lung hyperlucency due to severe bullous and panacinar emphysema that displaces the mediastinum to the left side and compresses the contralateral lung. (Reprinted from: Marchevsky AM, Guintu R, Koss M, et al. Swyer-James (MacLeod) syndrome with placental transmogrification of the lung: a case report and review of the literature. Arch Pathol Lab Med 2005;129:686–9, with permission from Archives of Pathology & Laboratory Medicine. © 2005 and 2006. College of American Pathologists.)

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Proliferative bronchiolitis, regardless of etiology, is characterized by a repair reaction consisting of polypoid tufts of granulation tissue within the membranous and respiratory bronchioles, often extending into the alveolar ducts (Figure 44). There may be associated airspace disease, termed organizing pneumonia. This combination was originally termed “bronchiolitis obliterans organizing pneumonia (BOOP)”. In early lesions, ulceration of the epithelium may be identified, and this necrosis precedes, and probably induces, the granulation tissue repair reaction. Interestingly, the proliferation rate of fibroblasts is similar to that found in cutaneous keloids, but lower than that found in diffuse alveolar damage or in usual interstitial pneumonia.390 Bone morphogenetic protein is also increased in post-transplant proliferative bronchiolitis, suggesting an etiological role for activating myofibroblasts with subsequent increases in extracellular matrix.391 There may also be an immunomodulatory component, as a decreased percentage of CD4þ FoxP3þ cells in bronchoalveolar lavage of transplants correlates with the development of obliterative bronchiolitis.392

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Figure 44. Proliferative bronchiolitis. (a) Whole mount demonstrating a bronchiolocentric disease. (b) A bronchiolar lumen is filled with edematous fibromyxoid connective tissue and fibrin. Airway neutrophils represent the residual acute bronchiolitis.

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Figure 45. Constrictive bronchiolitis (a) Concentric submucosal fibrosis can be subtle. Inflammatory infiltrates are sparse. (b) Only wisps of bronchial wall smooth muscle remain. (c) Partial airway obliteration is highlighted with an elastic tissue stain. (d) In time the airway is entirely obliterated. (e) An elastic stain demonstrates the scarred airway adjacent to the accompanying pulmonary artery.

Constrictive bronchiolitis Constrictive bronchiolitis has a variety of etiologies, and depending on the underlying cause there may be periods with superimposed acute bronchiolitis (Table 8).393 In general, constrictive bronchiolitis has a patchy and focal distribution throughout both lungs. The disease is characterized by varying degrees of concentric, submucosal fibrosis with reduction of the bronchiolar lumina and ultimately their disappearance (Figure 45).368,394,395 In the early stages, inflammatory infiltrates can be prominent, but later this is often not obvious. Many cases show subepithelial fibroblastic proliferation with collagen deposition and sparing of the epithelium. In late stages, the bronchiolar lumina disappear and their remnants are difficult to discern. Elastic stains usually identify small airway remnants adjacent to paired small arteries.

Constrictive bronchiolitis related to neuroendocrine hyperplasia Constrictive bronchiolitis in diffuse neuroendocrine cell hyperplasia is due to bronchiolar wall neuroendocrine cell proliferations just below the airway basement membrane. Lesions range from several cells to < 5 mm nodules, the latter being termed tumorlets (Figure 46).396–399 Patients have symptoms of constrictive bronchiolitis or even obstructive disease. The process is associated with the development of carcinoids,

both typical and atypical, and can be part of the multiple neuroendocrine neoplasia syndrome (see Chapter 31).396 Neuroendocrine cell hyperplasia of infancy is a rare, recently described disorder similar to diseases in adults. Large numbers of neuroendocrine cells in the bronchial epithelium narrow the luminal diameter and alter airflow. Tachypnea and hypoxemia are the manifestations even before the development of interstitial lung disease (see Chapter 3).400,401

Constrictive bronchiolitis related to ingestion of toxic agents Sauropus androgynus ingestion is a particular cause of constrictive and obstructive bronchiolitis. Raw leaf or extracted juice consumption was a common Malaysian weight loss method/fad. Outbreaks were first reported in 1994 in Taiwan in young to middle-aged women, after at least 10 weeks of ingesting uncooked S. androgynus juice.402–405 Individuals present with dyspnea and cough, and in severe cases progressive respiratory failure within a few months of S. androgynus consumption. Pulmonary function tests demonstrate obstructive lung disease with no improvement following bronchodilators. Chest X-rays may be normal while HRCT demonstrates bronchiectasis along with patchy low attenuation of lung tissue with mosaic perfusion.402,406 Lack of homogeneous aerosol distribution and increased lung

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Figure 46. Constrictive bronchiolitis secondary to neuroendocrine cell hyperplasia. (a) The bronchiolocentric pattern is obvious. (b) At first neuroendocrine cells fill the airway lumen. (c) Eventually airway lumens are obliterated. Fibrosis may be prominent.

epithelial permeability were also reported with technetium 99m-labeled diethylenetriaminepentaacetate (DTPA) radioaerosol lung scans.407,408 Histopathology from biopsy and explant specimens demonstrates striking bronchiolar disease, while most bronchi larger than 0.5 cm are unaffected (Figure 47).409–411 Changes range from bronchiolar inflammation and fibrosis to marked submucosal fibrosis with cicatricial luminal obliteration.

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Lymphocytes are prominent but eosinophils are also noted. Segmental ischemic necrosis of bronchioles at the bronchial and pulmonary circulation watershed areas was demonstrated by one group. They also observed that proximal airways showed fibrosis and bronchial wall atrophy, while distal bronchioles were either obstructed or dilated.409 Bronchial artery fibromuscular intimal sclerosis was also noted but considered a minor finding.411

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

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Figure 47. Constrictive bronchiolitis after Sauropus androgynus ingestion. (a) Bronchiolitis spills out into adjacent alveolar spaces. Small airway branches are not spared (arrows). (b) Submucosal edema and inflammation compress the airway lumen. (c) The inflammation eventually gives rise to fibrosis and oblieration of the airway.

T-cell-mediated immunity may play an etiological role in the disease.402 Patients with S. androgynus constrictive bronchiolitis progress to respiratory failure. Given the morphological findings it is not surprising that neither high-dose corticosteroids, cytotoxic agents nor even plasmaphoresis provide a positive response. Lung transplantation is the only effective therapy.409,412,413

Diseases of the bronchioles with or without adjacent parenchymal disease This group of pathologies is characterized by a definite type of inflammatory reaction, mostly related to the etiologies of the condition. However, there is still significant overlap of diseases between some of the morphological categories. In many of the

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conditions, the inflammation is not confined to the airways themselves, but also involves adjacent lung parenchyma.

Smokers’ bronchiolitis with or without adjacent interstitial lung disease Respiratory bronchiolitis is usually an incidental histological finding in lung samples from smokers undergoing procedures for other reasons (see Chapter 10).414 Respiratory bronchiolitis features pigmented macrophages in the lumina of the respiratory bronchioles.415,416 Macrophages usually extend into adjacent alveolar ducts and sacs, as well as into the more proximal membranous bronchioles. Finely granular light brown intracytoplasmic particles stain faintly with an iron stain and are referred to as smoker’s macrophages. The histological abnormalities may persist many years after smoking cessation.414 Interestingly, similar pathological and radiological features can be found in crack cocaine smokers (see Chapter 16).

Diffuse panbronchiolitis Diffuse panbronchiolitis (DPB) is an inflammatory airway disease of unknown etiology.417 It is a progressive suppurative and obstructive airway disease that often leads to bronchiectasis. This disease is largely restricted to Japan but DPB may also be rarely seen in Westerners.418 DPB is usually found in the second to fifth decades, with twice the incidence in males compared to females. Two-thirds of patients are non-smokers. Most patients with DPB have a history of chronic sinusitis. Although the etiology is still unclear, the finding of DPB in East Asians and Ashkenazi Jews indicates a genetic predisposition. A particular association is noted with the HLA-Bw54 antigen and is only found in Japanese, Chinese and Koreans.419 A positive association is also found with the HLA class 1 antigen HLA-A11. It has been suggested there may be a major disease susceptibility gene for DPB between the HLA-A and HLA-B loci.419,420,421 Inflammatory cells play a central role in the pathogenesis of this disease.421 The key players, neutrophils, CD8 T lymphocytes, interleukin 8 and macrophage inflammatory protein 1 (MIP1), however, are nonspecific and present in most airway diseases. The aggressive inflammation that causes destruction of the epithelium leads to a vicious cycle of inflammation and subsequent fibrosis. Reduced apoptosis of neutrophils may also play a role in the persistent inflammation.422 A very characteristic laboratory finding associated with DPB is a drastic elevation of serum cold agglutinins in the absence of raised mycoplasma antibody titers.423 Serum IgA is usually elevated and rheumatoid factor may be positive. In many cases, patients are colonized with Hemophilus influenzae or Pseudomonas aeruginosa.421 Neutrophilia is noted on bronchioloalveolar lavage specimens. Most gross findings of DPB come from autopsies. Gross abnormalities are more pronounced in the lower lobes, where extensive hyperinflation and bronchiectasis are noted. Yellow nodules (0.1 to 0.4 cm) associated with small airways are also

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seen. Microscopically a diagnosis of DPB rests on the finding of the so-called panbronchiolitis unit lesion. This is an exquisitely bronchiolocentric inflammatory process and one should only consider this diagnosis when distal centrilobular foam cells and lymphoplasmacytic infiltrates involve the full thickness of respiratory bronchioles and extend into adjacent alveolar ducts and sacs (Figure 48). Airway lumina may be filled with neutrophils and mucus, but not in the early stages of the disease. Lymphoid hyperplasia may accompany the bronchiolitis. Dendritic cells may also be numerous.424 Bronchial epithelium is often destroyed by the inflammation, whereas the alveoli are usually unaffected. However, post-obstructive lipoid pneumonia may develop. Scarring and associated narrowing of respiratory bronchioles occurs and in time results in more proximal bronchiectasis and peripheral air-trapping. The differential diagnosis for DPB includes many diseases (Table 9).417 However, in virtually all instances these processes feature only focal panbronchiolitis-like lesions.421 Similarities between DPB and bronchiolitis associated with human T-cell lymphotropic virus type I (HTLV-1) has led some to suggest DPB may be a manifestation of the latter disease.425

Follicular bronchiolitis Follicular bronchiolitis pertains to small airway disease featuring lymphoid aggregates and germinal centers within airway walls (see Chapter 34).426,427 It is associated with immune disorders, frequently in the context of connective tissue disease, especially rheumatoid arthritis and Sjögrens syndrome.428–430 It is also seen in other acquired immune disorders, such as AIDS or congenital immunodeficiency diseases.431 It is observed in diseased lungs with bronchiectasis and middle lobe syndrome. Finally, the histological pattern may not have an identifiable associated disease. Histologically, enlarged submucosal lymphoid follicles with germinal centers disrupt the bronchiolar mucosa and luminal diameter (Figure 49). The B-cell-rich polyclonal follicles distort and compress bronchiolar lumina into irregular shapes, and rare T cells wander beyond the follicles into adjacent alveolar septa. The respiratory epithelium overlying the hyperplastic nodules is attenuated and permeated by lymphocytes but lymphoepithelial lesions are not seen. A concentric ring of lymphocytes and plasma cells may cuff the airways and rare non-necrotizing granulomas may be seen.

Eosinophilic bronchiolitis Eosinophilic bronchitis/bronchiolitis is usually associated with asthma or allergic bronchopulmonary aspergillosis (see Chapters 7 and 15).432–436 Eosinophilic bronchiolitis is more commonly a secondary finding in interstitial lung disease, eosinophilic pneumonia, fungal and/or parasitic infections or drug reactions. An idiopathic case has also been described.437 Bronchoalveolar lavage features marked eosinophilia while lung biopsy demonstrates chronic bronchiolitis with prominent eosinophils.

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

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Figure 48. Diffuse panbronchiolitis (DPB). (a) This disease is predominantly bronchiolocentric but also involves alveolar parenchyma. (b) Foam cells admixed with lymphocytes and plasma cells expand the alveolar interstitium. (c) As airways are compromised neutrophilic infiltrates, mucus and cellular debris fill lumens.

Granulomatous bronchiolitis Granulomatous bronchiolitis is a morphological diagnosis with many possible etiologies.438–440 The clinical and radiographic findings depend on the cause. Mycobacterial, fungal and parasitic infections often manifest with necrotizing granulomas, while non-necrotizing lesions also raise the possibility of sarcoidosis, aspiration pneumonia or pulmonary involvement with associated inflammatory bowel disease, Wegener granulomatosis, or bronchocentric granulomatosis.441–443 Poorly defined peribronchiolar granulomas without necrosis suggest the possibility of hypersensitivity pneumonia and/or

drug reaction. In all instances one observes granulomas in bronchiolar walls (Figure 50). Larger airways may also be involved. Necrotizing lesions suggest infection, or rarely Wegener granulomatosis, while compact granulomas with minimal or no necrosis raise the possibility of sarcoidosis. Giant cells in the alveolar/bronchiolar lumens and not within the airway walls are more in keeping with aspiration pneumonia. Eosinophils are commoner in drug reactions, while an accompanying interstitial lymphoplasmacytic pneumonia suggests hypersensitivity pneumonitis. Treatment and outcome

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Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways Table 9 Differential diagnoses for diffuse panbronchiolitis

Infectious bronchiolitis Chronic bronchitis Follicular bronchiolitis Bronchocentric granulomatosis Bronchiectasis Primary ciliary dyskinesia Cystic fibrosis Aspiration pneumonia Rheumatoid arthritis-related bronchiolitis Inflammatory bowel disease-related bronchiolitis Idiopathic chronic bronchiolitis Hypersensitivity pneumonitis Hodgkin lymphoma Non-Hodgkin lymphoma Hypogammaglobulinemia Idiopathic thrombocytopenic purpura Adult T-cell leukemia Microscopic polyangitis Wegener granulomatosis Lambert-Eaton myasthenic syndrome Data from417 and421

are disease-dependent. Underlying entities are discussed elsewhere in the text.

Mineral dust disease Bronchiolitis can be caused by inhaled substances and mineral dust exposures, such as silicosis, asbestosis, iron oxide, and coal (see Chapter 14).444,445 Nonspecific histological features include acute bronchiolitis with bronchiolar necrosis (rarely seen at the time the patient comes to biopsy) as well as chronic bronchiolitis with or without severe fibrosis and honeycomb change. Mineral dust airways disease is suggested by extension of the fibrosis, in a linear pattern, into the subtending alveolar ducts and marked pigment deposition (Figure 51). Birefringent particles may be seen.

Peribronchiolar metaplasia (Lambertosis) This lesion is a nonspecific reaction to injury, which results in the extension of bronchiolar type epithelial cells along the alveolar walls adjacent to the airways (Figure 52). This is a very common finding in all tissue samples, including transbronchial biopsies. In some biopsy series, peribronchiolar metaplasia was the only identifiable abnormality.446

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Figure 49. Follicular bronchiolitis: a terminal bronchiole is distorted by lymphoid follicles with germinal centers.

Pathophysiology Tobacco-associated bronchiolitis was discussed above. Related to this, the clinical/physiological term “small airways disease” was introduced in 1968, and defined as “airway disease in patients with variably severe chronic airflow obstruction, characterized by loss of bronchioles, mucus plugging, inflammation and fibrosis”.358 Since correlations between pathological alterations of these airways and specialized physiological tests could be identified, it was hoped that early disease could be recognized and treated before the disease progressed to severe airflow obstruction. Unfortunately, the specialized tests were not predictive of progressive airflow obstruction, and the concept was not proved worthwhile.447 This does not, however, mean that the present clinical/ pathological usage of small airways disease or bronchiolitis implies the absence of physiological changes. Any airway remodeling will result in an increased resistance to airflow and airflow obstruction with decreases in the FEV1 or the FEV1 /FVC ratio. Gas trapping is frequent with increases in residual volume.448,449 The 6-minute walk test may prove helpful in assessing prognosis in lung transplant recipients with an obliterative bronchiolitis.450 These changes are due to several processes. Firstly, inflammatory mediators alter the constrictive/dilatation balance of the smooth muscle, leading to increased airway tone at rest. Secondly, intraluminal polyps alter airflow, making the usually laminar flow turbulent, thereby increasing resistance. Thirdly, increased fibrous tissue between the basement membrane and the smooth muscle narrows the airway lumen. Any further degree of smooth muscle contraction markedly increases airway resistance. Finally, inflammatory/ fibrotic infiltrates in the adventitia uncouple the airways from the adjacent parenchyma and allow increased contractility. With severe end-stage disease, there will be airway

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Figure 50. Granulomatous bronchiolitis. (a) Compact granulomas with a dense lymphoplasmacytic infiltrate expand the bronchiolar wall. An infectious etiology is likely. (b) Granulomatous inflammation may be mixed with neutrophils. This small airway is almost entirely obliterated. (c) Mucosal ulceration with secondary granulation tissuelike changes can be seen. This case is from a patient with inflammatory bowel disease. Figure 51. Mineral dust bronchiolitis. Long-term asbestosis exposure leads to small airway disease. Fibrosis extends into the alveolar duct. Carbon pigment is also noted.

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Figure 52. Peribronchiolar metaplasia. (a) Bronchiolar epithelium often extends through the canals of Lambert into adjacent alveolar parenchyma. This common finding may not be associated with any inflammation. (b) Significant small airway damage with fibrosis leads to pronounced extension of respiratory epithelium into alveolar tissue. Such lesions may be mistaken for glandular neoplasia.

dropout. Remaining airways have a rigid configuration with a densely fibrotic wall. Markedly increased airflow obstruction follows.

Treatment and prognosis Treatment of small airway disease focuses on the underlying disease. Therapies includes bronchodilators, corticosteroids and cytotoxic agents.448 Steroid responsiveness should

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383. Macleod WM. Abnormal transradiancy of one lung. Thorax 1954;9:147–53. 384. Schlesinger C, Koss M. Bronchiolitis: update 2001. Curr Opin Pulm Med 2002;8:112–6. 385. Panitch HB. Bronchiolitis in infants. Curr Opin Pediatr 2001;13:256–60. 386. Lucaya J, Gartner S, Garcia-Pena P, et al. Spectrum of manifestations of Swyer-James-MacLeod syndrome. J Comput Assist Tomogr 1998;22:592–7. 387. Morita K, Shimizu J, Kamesui T, et al. A case of surgical treatment of SwyerJames syndrome. Nippon Kyobu Geka Gakkai Zasshi 1994;42:1949–52. 388. Greenberg S. Respiratory viral infections in adults. Curr Opin Pulm Med 2002;8:201–8. 389. Mauad T, Hajjar L, Callegari G, et al. Lung pathology in fatal novel human influenza A (H1N1) infection. Am J Respir Crit Care Med 2010;181:72–9. 390. El-Zammar O, Rosenbaum P, Katzenstein A-LA. Proliferative activity in fibrosing lung diseases: a comparative study of Ki-67 immunoreactivity in diffuse alveolar damage, bronchiolitis, obliteransorganizing pneumonia, and usual interstitial pneumonia. Hum Pathol 2009;40:1182–58. 391. Jonigk D, Theophile K, Hussein K, et al. Obliterative airway remodelling in transplanted and non-transplanted lungs. Virchows Archives 2010;457:369–80. 392. Bhorade SM, Chen H, Molinero L, et al. Decreased percentage of CD4þ FoxP3þ cells in bronchoalveolar lavage from lung transplant recipients correlates with development of bronchiolitis obliterans syndrome. Transplantation 2010;90:540–6.

380. Stokes D, Sigler A, Khouri NF, et al. Unilateral hyperlucent lung (Swyer-James syndrome) after severe Mycoplasma pneumoniae infection. Am Rev Respir Dis 1978;117:145–52.

393. Angel L, Homma A, Levine SM. Bronchiolitis obliterans. Semin Respir Crit Care Med 2000;21:123–34.

367. Couture C, Colby TV. Histopathology of bronchiolar disorders. Semin Respir Crit Care Med 2003;24:489.

381. Trimis G, Theodoridou M, Mostrou G, et al. Swyer-James (MacLeod’s) syndrome following pertussis infection in an infant. Scand J Infect Dis 2003;35:197–9.

395. Cordier JF. Cryptogenic organising pneumonia. Eur Respir J 2006;28:422–46.

368. Popper HH. Bronchiolitis, an update. Virchows Archives 2000;437:471–81.

382. Swyer PR, James GC. A case of unilateral pulmonary emphysema. Thorax 1953;8:133–6.

365. Ryu JH, Myers JL, Swensen SJ. Bronchiolar disorders. Am J Respir Crit Care Med 2003;168:1277–92. 366. Camus P, Lombard JN, Perrichon M, et al. Bronchiolitis obliterans organising pneumonia in patients taking acebutolol or amiodarone. Thorax 1989;44:711–5.

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369. Beasley MB. Smoking-related small airway disease – a review and update. Adv Anat Pathol 2010;17:270–6.

394. Visscher D, Myers J. Bronchiolitis: the pathologist’s perspective. Proc Am Thorac Soc 2006;3:41–7.

396. Davies S, Gosney J, Hansell D, et al. Diffuse idiopathic pulmonary neuroendocrine cell hyperplasia: an under-recognised spectrum of disease. Thorax 2007;62:248–52.

Chapter 17: Chronic obstructive pulmonary disease and diseases of the airways

397. Aguayo SM, Miller YE, Waldron JA, et al. Brief report: idiopathic diffuse hyperplasia of pulmonary neuroendocrine cells and airways disease. N Engl J Med 1992;327:1285–8. 398. Armas OA, White DA, Erlandson RA, et al. Diffuse idiopathic pulmonary neuroendocrine cell proliferation presenting as interstitial lung disease. Am J Surg Pathol 1995;19:963–70. 399. Miller RR, Muller NL. Neuroendocrine cell hyperplasia and obliterative bronchiolitis in patients with peripheral carcinoid tumors. Am J Surg Pathol 1995;19:653–8. 400. Deterding R, Pye C, Fan L, et al. Persistent tachypnea of infancy is associated with neuroendocrine cell hyperplasia. Pediatr Pulmonol 2005;40:157–65. 401. Brody AS, Guillerman RP, Hay TC, et al. Neuroendocrine cell hyperplasia of infancy: diagnosis with highresolution CT. Am J Roentgenol 2010;194:238–44. 402. Lai RS, Chiang AA, Wu MT, et al. Outbreak of bronchiolitis obliterans associated with consumption of Sauropus androgynus in Taiwan. Lancet 1996;348:83–5. 403. Lin TJ, Lu CC, Chen KW, et al. Outbreak of obstructive ventilatory impairment associated with consumption of Sauropus androgynus vegetable. J Toxicol Clin Toxicol 1996;34:1–8. 404. Ger LP, Chiang AA, Lai RS, et al. Association of Sauropus androgynus and bronchiolitis obliterans syndrome: a hospital-based casecontrol study. Am J Epidemiol 1997;145:842–9. 405. Hsiue TR, Guo YL, Chen KW, et al. Dose-response relationship and irreversible obstructive ventilatory defect in patients with consumption of Sauropus androgynus. Chest 1998;113:71–6. 406. Yang CF, Wu MT, Chiang AA, et al. Correlation of high-resolution CT and pulmonary function in bronchiolitis obliterans: a study based on 24 patients associated with consumption of Sauropus androgynus. Am J Roentgenol 1997;168:1045–50. 407. Wu CL, Hsu WH, Chiang CD, et al. Lung injury related to consuming Sauropus androgynus vegetable. J Toxicol Clin Toxicol 1997;35:241–8.

408. Kao CH, Ho YJ, Wu CL, et al. Using 99mTc-DTPA radioaerosol inhalation lung scintigraphies to detect the lung injury induced by consuming Sauropus androgynus vegetable and comparison with conventional pulmonary function tests. Respiration 1999;66:46–51. 409. Chang YL, Yao YT, Wang NS, et al. Segmental necrosis of small bronchi after prolonged intakes of Sauropus androgynus in Taiwan. Am J Respir Crit Care Med 1998;157:594–8. 410. Chang H, Wang JS, Tseng HH, et al. Histopathological study of Sauropus androgynus-associated constrictive bronchiolitis obliterans: a new cause of constrictive bronchiolitis obliterans. Am J Surg Pathol 1997;21:35–42. 411. Wang JS, Tseng HH, Lai RS, et al. Sauropus androgynus-constrictive obliterative bronchitis/bronchiolitis – histopathological study of pneumonectomy and biopsy specimens with emphasis on the inflammatory process and disease progression. Histopathology 2000;37:402–10. 412. Luh SP, Lee YC, Chang YL, et al. Lung transplantation for patients with endstage Sauropus androgynus-induced bronchiolitis obliterans (SABO) syndrome. Clin Transplant 1999;13:496–503. 413. Wu CL, Hsu WH, Chiang CD. The effect of large-dose prednisolone on patients with obstructive lung disease associated with consuming sauropus androgynus. Zhonghua Yi Xue Za Zhi (Taipei) 1998;61:34–8. 414. Fraig M, Shreesha U, Savici D, et al. Respiratory bronchiolitis. Am J Surg Pathol 2002;26:647–53. 415. Craig PJ, Wells AU, Doffman S, et al. Desquamative interstitial pneumonia, respiratory bronchiolitis and their relationship to smoking. Histopathology 2004;45:275–82. 416. Niewoehner DE, Kleinerman J, Rice DB. Pathologic changes in the peripheral airways of young cigarette smokers. N Engl J Med 1974;291:755–8. 417. Iwata M, Colby TV, Kitaichi M. Diffuse panbronchiolitis: diagnosis and distinction from various pulmonary diseases with centrilobular interstitial foam cell accumulations. Hum Pathol 1994;25:357–63. 418. Randhawa P, Hoagland MH, Yousem SA. Diffuse panbronchiolitis in North America. Report of three cases and

review of the literature. Am J Surg Pathol 1991;15:43–7. 419. Sugiyama Y, Kudoh S, Maeda H, et al. Analysis of HLA antigens in patients with diffuse panbronchiolitis. Am Rev Respir Dis 1990;141:1459–62. 420. Keicho N, Tokunaga K, Nakata K, et al. Contribution of HLA genes to genetic predisposition in diffuse panbronchiolitis. Am J Respir Crit Care Med 1998;158:846–50. 421. Poletti V, Casoni G, Chilosi M, et al. Diffuse panbronchiolitis. Eur Respir J 2006;28:862–71. 422. Nishimaki K, Nawata J, Okada S, et al. Neutrophil survival-enhancing activity in sputum from patients with diffuse panbronchiolitis. Respir Med 2005;99:910–7. 423. Homma S, Sakamoto S, Kawabata M, et al. Comparative clinicopathology of obliterative bronchiolitis and diffuse panbronchiolitis. Respiration 2006;73:481–7. 424. Todate A, Chida K, Suda T, et al. Increased numbers of dendritic cells in the bronchiolar tissues of diffuse panbronchiolitis. Am J Respir Crit Care Med 2000;162:148–53. 425. Kadota J, Mukae H, Fujii T, et al. Clinical similarities and differences between human T-cell lymphotropic virus type 1-associated bronchiolitis and diffuse panbronchiolitis. Chest 2004;125:1239–47. 426. Howling SJ, Hansell DM, Wells AU, et al. Follicular bronchiolitis: thin-section CT and histologic findings. Radiology 1999;212:637–42. 427. Romero S, Barroso E, Gil J, et al. Follicular bronchiolitis: clinical and pathologic findings in six patients. Lung 2003;181:309–19. 428. Hayakawa H, Sato A, Imokawa S, et al. Bronchiolar disease in rheumatoid arthritis. Am J Respir Crit Care Med 1996;154:1531–6. 429. Sato A, Hayakawa H, Uchiyama H, et al. Cellular distribution of bronchus-associated lymphoid tissue in rheumatoid arthritis. Am J Respir Crit Care Med 1996;154:1903–7. 430. Kinoshita M, Higashi T, Tanaka C, et al. Follicular bronchiolitis associated with rheumatoid arthritis. Intern Med 1992;31:674–7.

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431. Exley CM, Suvarna SK, Matthews S. Follicular bronchiolitis as a presentation of HIV. Clin Radiol 2006;61:710–3.

439. Myers JL, Tazelaar HD. Challenges in pulmonary fibrosis: 6 – Problematic granulomatous lung disease. Thorax 2008;63:78–84.

432. Chetty A. Pathology of allergic bronchopulmonary aspergillosis. Front Biosci 2003;8:e110–14.

440. El Zammar OA, Katzenstein A-LA. Pathological diagnosis of granulomatous lung disease: a review. Histopathology 2007;50:289–310.

433. Agarwal R. Allergic bronchopulmonary aspergillosis. Chest 2009;135:805–26. 434. Zander D. Allergic bronchopulmonary aspergillosis: an overview. Arch Pathol Lab Med 2005;129:924–8. 435. Scott K, Wardlaw A. Eosinophilic airway disorders. Semin Respir Crit Care Med 2006;27:128–33. 436. Birring S, Berry M, Brightling C, et al. Eosinophilic bronchitis: clinical features, management and pathogenesis. Am J Respir Med 2003;2:169–73. 437. Takayanagi N, Kanazawa M, Kawabata Y, et al. Chronic bronchiolitis with associated eosinophilic lung disease (eosinophilic bronchiolitis). Respiration 2001;68:319–22. 438. Mukhopadhyay S, Gal A. Granulomatous lung disease: an approach to the differential diagnosis. Arch Pathol Lab Med 2010;134:667–90.

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441. Freeman H, Davis J, Prest M, et al. Granulomatous bronchiolitis with necrobiotic pulmonary nodules in Crohn’s disease. Can J Gastroenterol 2004;18:687–90. 442. Polychronopoulos V, Prakash U. Airway involvement in sarcoidosis. Chest 2009;136:1371–80. 443. Hutton Klein JR, Tazelaar HD, Leslie KO, et al. One hundred consecutive granulomas in a pulmonary pathology consultation practice. Am J Surg Pathol 2010;34:1456–64. 444. Antonini J, Taylor M, Zimmer A, et al. Pulmonary responses to welding fumes: role of metal constituents. J Toxicol Environ Health A 2004;67:233–49. 445. Churg A, Wright JL, Wiggs B, et al. Small airways disease and mineral dust exposure. Am Rev Respir Dis 1985;131:139–43.

446. Fukuoka J, Franks TJ, Colby TV, et al. Peribronchiolar metaplasia: a common histologic lesion in diffuse lung disease and a rare cause of interstitial lung disease. Am J Surg Pathol 2005;29:948–54. 447. Buist AS, Vollmer WM, Johnson LR, et al. Does the single-breath N2 test identify the smoker who will develop chronic airflow limitation. Am Rev Respir Dis 1988;137:293–301. 448. Epler GR. Diagnosis and treatment of constrictive bronchiolitis. F1000 Med Rep 2010;2:32. 449. Mattiello R, Mallol J, Fischer GB, et al. Pulmonary function in children and adolescents with postinfectious bronchiolitis obliterans. J Brasil Pneumol 2010;36:453–9. 450. Nathan SD, Shlobin OA, Reese E, et al. Prognostic value of the 6 min walk test in bronchiolitis obliterans syndrome. Respir Med 2009;103:1816–21. 451. Oonakahara K, Matsuyama W, Higashimoto I, et al. Outbreak of bronchiolitis obliterans associated with consumption of Sauropus androgynus in Japan – alert of food-associated pulmonary disorders from Japan. Respiration 2005;72:221.

Chapter

18

Pulmonary vascular pathology Katrien Gru¨nberg and Wolter J. Mooi

Introduction Pulmonary hypertension is defined as a sustained mean pulmonary arterial pressure at rest of
25 mmHg,1–3 i.e., elevated significantly above the upper limit of normal (20 mmHg). Typically, pulmonary arterial pressure increases during exercise. In pulmonary hypertension, this increase is disproportionately high. The pressure is measured by right heart catheterization or can be estimated by echocardiography. Pulmonary hypertension is a consequence of right ventricular adaptation to increased vascular resistance, increased pulmonary blood flow, or a combination of both. Initially, the symptoms of pulmonary hypertension are nonspecific and usually limited to dyspnea, particularly on exertion, or patients may be asymptomatic. As the pulmonary hypertension progresses, signs and symptoms of right-sided heart failure develop; namely peripheral edema, fatigue, abdominal fullness, angina pectoris and syncope. Depending on its cause, severity and possible treatment options, pulmonary hypertension may lead to death. Pulmonary hypertension is a feature of a heterogeneous group of disorders which differ in risk factor profile, initiating factors, response to treatment and prognosis (Table 1).4,5 Among the causal factors, left ventricular failure and mitral valve insufficiency are the commonest, followed by chronic thromboembolic disease.6 Globally, there are marked variations in prevalence of some important causes and risk factors, especially sickle cell disease, chronic schistosomiasis and HIV-AIDS. Approximately one-third of cases of pulmonary arterial hypertension (PAH) present with no apparent etiology or risk factor: these are referred to as “idiopathic” PAH (iPAH).7,8 Idiopathic PAH is a rare condition. The incidence of all PAH cases depends not only on the prevalence of risk factors, but also on the methods and inclusion criteria of registration, if present. Estimates of the annual incidence of PAH in Europe range from 2.4 cases in France7 to 7.1 in Scotland9 per million of adult inhabitants.

Table 1 Risk factors and associated conditions for pulmonary arterial hypertension

A. Drugs and toxins 1. Definite Aminorex (Menocil®) Fenfluramine (Ponderal®) Dexfenfluramine (Adifax®, Redux®) Fenfluramine-phentermine Toxic rapeseed oil 2. Likely Amphetamines Metamphetamines L-Tryptophan 3. Possible Cocaine Chemotherapeutic agents Phenylpropanolamine Hypericum perforatum (a.k.a. St. John’s Wort, Tipton’s weed or Klamath weed, a herbal treatment for depression) Selective serotonin reuptake inhibitors (SSRI) B. Diseases 1. Definite HIV infection Portal hypertension/liver disease Connective tissue diseases Sickle cell disease Chronic schistosomiasis Congenital or acquired systemic-pulmonary-cardiac shunts (left-to-right shunts) Atrial and/or ventricular septal defect Transposition of the great arteries with VSD Truncus arteriosus persistens Patent ductus arteriosus Aorto-pulmonary window Surgical shunt for tetralogy of Fallot 2. Possible Thyroid disorders

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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C. Genetic predispositions: genes involved BMPR2 ALK-1 (with or without clinically overt HHTa) Endoglin TGFb SMADs Hemorrhagic hereditary telangiectasia, Rendu-Osler Weber disease. ALK1, activin receptor-like kinase type I; TGFβ, transforming growth factor-β; SMAD, transcription factor, a homolog of both the drosophila protein MAD and the Caenorhabditis elegans protein, SMA; BMPR2, bone morphogenetic protein receptor type II. Adapted from.10,11 a

In this chapter, we concentrate on the major histological patterns of pulmonary vascular disease and discuss these findings in conjunction with the 2008 clinical classification.10,11 We review current thoughts regarding pathogenetic mechanisms of the various forms of pulmonary hypertension. Pulmonary vasculitis is only considered with respect to pulmonary hypertension (see Chapter 19) and persistent pulmonary hypertension in the newborn is discussed in Chapter 3.

Classification of pulmonary hypertension A number of histopathologically distinctive patterns of hypertensive pulmonary vascular disease are recognized. These provided a basis for the histopathological classification of pulmonary hypertensive vasculopathy.12,13 This is in contrast to the clinical classification of pulmonary hypertension, which is based largely on clinical criteria, including response to treatment, rather than on histology. This latter classification, which has evolved with new research (reviewed by van Wolferen et al.14), and updated after a WHO-supported consensus meeting in Dana Point, California, USA, in 2008, is referred to as the Dana Point classification (Table 2).11 It is important to note the histopathological and clinical approaches to classification result in an incomplete match. It is reasonable to assume that histopathological patterns of disease point to differences in etiology and pathogenesis.5 Knowledge of these is essential for rational design of therapeutic strategies. The term PAH has come to be reserved for the diseases in group 1 of the Dana Point clinical classification 2008 (Table 2).11 As a consequence, all the remaining types of pulmonary hypertension are now referred to by the tonguetwisting and cumbersome designation of “non-pulmonary arterial hypertension pulmonary hypertension” (non-PAH PH, formerly also known as “secondary pulmonary hypertension”). These terms have gained some acceptance among clinicians. However, the term PAH is a pleonasm, since isolated hypertension outside the arterial compartment does not exist and the term “non-PAH PH” invites confusion and misunderstanding. The term is also not strictly accurate, as

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Table 2 Revised clinical classification of pulmonary hypertension (Dana Point classification 2008)11

Group 1: Pulmonary arterial hypertension (PAH) 1.

Pulmonary arterial hypertension (PAH)

1.1.

Idiopathic PAH

1.2.

Hereditary

1.2.1. BMPR2 1.2.2. ALK1, endoglin (with or without hereditary hemorrhagic telangiectasia) 1.2.3. Unknown 1.3.

Drug- and toxin-induced

1.4.

Associated with:

1.4.1. Connective tissue diseases 1.4.2. HIV infection 1.4.3. Portal hypertension 1.4.4. Congenital heart diseases 1.4.5. Schistosomiasis 1.4.6. Chronic hemolytic anemia 1.5

Persistent pulmonary hypertension of the newborn

10

Pulmonary veno-occlusive disease (PVOD) and/or pulmonary capillary hemangiomatosis (PCH)

Group 2: Pulmonary hypertension owing to left heart disease 2.1.

Systolic dysfunction

2.2.

Diastolic dysfunction

2.3.

Valvular disease

Group 3: Pulmonary hypertension owing to lung diseases and/or hypoxia 3.1.

Chronic obstructive pulmonary disease

3.2.

Interstitial lung disease

3.3.

Other pulmonary diseases with mixed restrictive and obstructive patterns

3.4.

Sleep-disordered breathing

3.5.

Alveolar hypoventilation disorders

3.6.

Chronic exposure to high altitude

3.7.

Developmental abnormalities

Group 4: Chronic thromboembolic pulmonary hypertension (CTEPH) Group 5: Pulmonary hypertension with unclear multifactorial mechanisms 5.1.

Hematological disorders: myeloproliferative disorders, splenectomy

5.2.

Systemic disorders: sarcoidosis, pulmonary Langerhans cell histiocytosis: lymphangioleiomyomatosis, neurofibromatosis, vasculitis

Chapter 18: Pulmonary vascular pathology

5.3. 5.4.

Metabolic disorders: glycogen storage diseases, Gaucher disease and thyroid disorders Others: tumoral obstruction, fibrosing mediastinitis, chronic renal failure on dialysis

ALK1, activin receptor-like kinase type I.

congenital cardiac disease is included in Group 1 but has a cardiac cause for the pulmonary hypertension.

Handling of tissue specimens The histological evaluation of pulmonary vascular disease requires careful handling and processing of the lung tissue specimens (see Chapter 2). Compression and collapse of lung tissue produce various artifacts that compromise the assessment of many histological features. Such artifacts can be avoided by gently distending the tissue with formalin and allowing it to fix before cutting.15 This procedure works very well with open lung biopsies, when staples are still in place, but it can even be used if the cut surface is not stapled. Similarly, the quality of autopsy or explant specimens is greatly improved by lung infusion with formalin. This infusion should be slow and gentle, to avoid “washing out” macrophages, which may be hemosiderin-laden and of diagnostic relevance. Using too great a force may also result in overexpansion of lobules or interlobular septa. The latter will cause artifacts mimicking edema or even lymphangiectasia. Post-mortem, formalin can be injected directly into the lung via a transthoracic needle prior to opening the thoracic cage. This simple procedure can be done as soon as permission for autopsy is obtained, and results in well-fixed and expanded lung tissue devoid of autolysis. At post-mortem, both peripheral and central lung tissue should be sampled. The former allows easy identification of small veins, while the latter tissue allows examination of arteries and veins of both small and large caliber. In a lung biopsy, step sections should be routinely evaluated, in order not to miss focal abnormalities. For proper evaluation of the pulmonary blood vessels, an elastin stain, such as Elastic van Gieson (EvG), Masson trichrome combined with an elastin stain, or Movat pentachrome stain, in addition to the hematoxylin and eosin (H&E) stain, are indispensable.12,13 It is also advisable to include an iron stain, such as Perls’ Prussian Blue, to avoid overlooking mild hemosiderosis.12,13

Pulmonary vascular anatomy and histology Proper evaluation of pulmonary vascular lesions requires knowledge of the normal anatomy and microanatomy of the pulmonary vasculature (see Chapter 1).

Dual blood supply The lung has a dual blood supply, the pulmonary circulation, which receives the entire cardiac output, and a normally very minor systemic supply, provided by the bronchial circulation. The pulmonary circulation is derived from the pulmonary arterial trunk, while the bronchial arteries originate from the thoracic aorta. Bronchial arteries are accompanied by bronchial veins that reach the right atrium, via the azygos vein on the right, and the highest left intercostal or the accessory hemiazygos vein on the left.16 Bronchial veins have numerous anastomoses with pulmonary veins, so a considerable part of the – normally quite limited – bronchial venous outflow drains into the four pulmonary veins that terminate in the left atrium.13,16

Pulmonary arteries Like the systemic circulation, the pulmonary circulation accommodates the entire cardiac output but, in contrast, is a low-pressure system. This is reflected by the different vascular microanatomy. The larger pulmonary arteries, from the pulmonary trunk down to a diameter of approximately 0.5 mm, are elastic in type. Their media consists of smooth muscle cells and elastic lamellae, the latter being discontinuous in large vessels and more or less continuous in smaller ones (Figure 1a).13 These elastic arteries are conducting vessels and act as a “pressure reservoir” or “Windkessel”, transforming the pulsating bloodstream exiting the right ventricle to a more even pressure and flow curve in downstream, small arterial branches. Small arteries, down to a diameter of approximately 50–100 mm, are of the muscular type, with a thin media of smooth muscle cells sandwiched between distinct and continuous inner and outer elastic laminae (Figure 1b). The thickness of their muscle coat is a mere 5% (range 3–7%) of the external diameter of the vessel, as measured at the outer elastic laminae. Pulmonary arteries can also be divided according to their position, relative to airways. An axial pulmonary artery runs alongside an airway and divides dichotomously with it. The diameters of axial arteries gradually decrease with each subsequent airway generation, up to the terminal bronchioles. As a rule, the diameter of a normal axial artery is slightly smaller than the accompanying airway.13 In addition to axial arteries, there are so-called supernumerary arterial branches, which leave the bronchovascular bundle and enter the pulmonary parenchyma (Figure 1b,c). Arterioles have been variously defined by their size (smaller than 90–100 mm), localization (intra-acinar branches), or absence of a muscular media.13 The last descriptive term is most widely used. Most pulmonary arterial branches lose their muscular coat as they enter the alveolar parenchyma. This is a gradual rather than an abrupt change, and thus some small intra-acinar arteries with a muscular media may be identified. In this chapter, we use the term arteriole for small intra-acinar arterial branches. Some of these are located at the points where

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Chapter 18: Pulmonary vascular pathology

(a)

(c)

(b)

(d)

Figure 1. Normal pulmonary vasculature in adult. (a) Elastic artery: the elastic artery features a media of smooth muscle cells and discontinuous elastic lamellae (EvG stain). (b) Muscular artery: the muscular artery has a thin media of smooth muscle cells sandwiched between distinct inner and outer elastic lamellae. The thickness of this muscle coat is about 5% (range: 3–7%) of the external diameter of the vessel. A thin-walled supernumerary artery branches off the muscular artery (EvG stain). (c) Plastic cast from rabbit pulmonary artery. White arrows point at supernumerary arteries, red arrows point to dichotomous branches. (d) Corner vessel situated at the converging point of alveolar septa. This vessel has a single elastic lamina, no muscular media, and no recognizable adventitia (EvG stain).

alveolar septa converge. These, together with similarly located venules, are referred to as “corner vessels” (Figure 1d). As the lung expands during inspiration, alveolar septa are stretched. This temporarily impedes filling of the capillaries. The alveolar septa in turn exert their retractile force on convergence points of the septa, where corner vessels are situated. These corner vessels are stretched in all directions during inspiration, and hence, do not collapse in the expanded lung. Thus, these vessels provide a reservoir during inspiration, allowing quick shifts of blood volume during the respiratory cycle. Corner vessels have a single elastic lamina, separating the intima from the adventitia. In pulmonary hypertension pulmonary arterioles develop a muscular media and often a new elastic lamina internal to this

664

new muscular layer. This phenomenon has been designated “muscularization of arterioles”. Histologically, generalized dilatation of the axial arteries indicates increased blood flow, as may result from intracardiac left-to-right shunting. Paradoxically, decreased flow, as occurs in some congenital heart defects, such as tetralogy of Fallot, can also cause dilatation of these pulmonary axial arteries.

Supernumerary arteries

As briefly mentioned above, very small, so-called “supernumerary arteries” branch from the larger axial parent arteries and directly enter the alveolar parenchyma.17,18 These supernumerary arteries are typically much smaller in caliber

Chapter 18: Pulmonary vascular pathology

than their axial parent vessels, and branch, at roughly right angles, from the parent vessel (Figure 1b,c).13 They are far more numerous than conventional axial arteries and probably function as recruitment vessels, since they feature a V-shaped baffle or valve-like structure at their origin17 (reviewed by Stenmark and Abman19). Supernumerary arteries are more sensitive to 5-hydroxytryptamine (5-HT, serotonin), an important vascular smooth muscle constrictor and smooth muscle mitogen.20–22

Capillaries The densely anastomosing pulmonary alveolar capillary network consists of an attenuated layer of endothelium resting on a basal lamina. On one side, this basal lamina is shared with types I and II pneumocytes lining the alveolar lumen; on the other it is flanked by some collagen fibers of the alveolar interstitium. Changes in the thickness of the alveolar capillary membrane of any cause affect diffusion capacity, and hence oxygenation and exercise tolerance. Apart from interstitial distortion caused by, for example, fibrosis, the only light microscopic findings indicative of alveolar-capillary membrane pathology are indirect and nonspecific. Edema, intraalveolar exudation and hyaline membrane formation are all examples. Capillary congestion provides a diagnostic clue to the diagnoses of congestive vasculopathy and pulmonary venoocclusive disease (see below).

Pulmonary veins Pulmonary veins have an ill-defined media of smooth muscle cells with interspersed collagen and some elastin fibrils. Distinct inner and outer elastic laminae are normally absent. Larger pulmonary veins tend to be located in interlobular septa, i.e. away from the bronchovascular bundles containing the axial arteries (Figure 2). Interlobular septa are most distinctive in subpleural lung tissue, where they are connected to the pleura. Pulmonary venules are located in the alveolar parenchyma, and are devoid of a muscular media. They have a single elastic lamina, so they are indistinguishable from normal pulmonary arterioles. The only reliable way to identify these venules in tissue sections is to trace their connection to a recognizable vein.

Bronchial circulation Bronchial arteries are small systemic muscular arteries arising from the aorta. Bronchial arteries provide oxygen to hilar structures, large airways and the largest pulmonary arteries, the latter via “vasa vasorum”. They have a muscular wall sandwiched between two elastic laminae. The outer elastic lamina is often indistinct. The bronchial arteries travel together with the pulmonary arteries and airways in the bronchovascular bundles but can also be found in the pleura.13 Bronchial arteries are systemic vessels, hence their thicker muscular media. Occasionally, longitudinal smooth muscle

Figure 2. Vascular architecture at low magnification, in a case of congestive vasculopathy, due to mitral valve insufficiency. The axial muscular artery runs together with the bronchiole in the bronchovascular bundle. The vein, in the interlobular septum, is distended and hence easily visible. Normally, it is thin-walled and collapsed. The interlobular septum is easily identified in subpleural lung tissue by its connection to the pleura (EvG stain).

bundles can be seen in the intima. Bronchial arteries are usually much smaller in diameter than the pulmonary arteries and are therefore normally inconspicuous. Bronchial arterial enlargement may result from blood shunting between bronchial and pulmonary vascular anastomoses. Underlying causes include bronchiectasis, intralobular sequestration, tetralogy of Fallot and (segmental) atresia of a pulmonary artery.13 Both expansion of pulmonary-bronchial anastomoses and angiogenic sprouting of new bronchial arterial branches may occur as a consequence of pulmonary arterial obstruction. This establishes a bypass, feeding the capillary network (postobstructive vasculopathy).18,23

Lymphatics The pulmonary lymphatics form an extensive network that reaches from the corner vessels to the interlobular septa and bronchovascular bundles. In alveolar septa, lymphatics are nonexistent. These channels are normally collapsed and inconspicuous but are more prominent if there is pulmonary edema or they contain tumor. They can be highlighted with monoclonal antibody D2–40, which recognizes podoplanin, an O-linked sialoglycoprotein expressed by lymphatic endothelium.

Vascular remodeling during development and aging At birth, the first breaths expand the thoracic volume and expose the lung to high oxygen levels. The resultant abrupt and marked drop in pulmonary arterial resistance causes a significant rise in pulmonary blood flow, while shunting via the foramen ovale and the ductus arteriosus is abolished. Some

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Chapter 18: Pulmonary vascular pathology

(a)

(b)

Figure 3. Normal pulmonary vasculature in neonate. (a) Elastic artery and a supernumerary artery branching from it. The elastic artery features discontinuously layered elastic lamellae and a prominent adventitia (EvG stain). (b) Muscular artery with the media sandwiched between the internal and external elastic lamina. In the neonate, the adventitia is still prominent (EvG stain).

of these profound hemodynamic changes are caused by the response of pulmonary arteries, especially the muscular arteries. Before birth these arteries are constricted, so the media is thick in comparison to the vascular diameter. After birth, muscular pulmonary arteries rapidly relax, and a more gradual medial thinning follows during the first 3 weeks of life. The adult medial thickness of up to 5–7% of the external vascular diameter is reached at the end of the first year. The number of axial and especially supernumerary arteries increases substantially during the first 2 years of life. These vessels expand the total pulmonary vascular bed and thus contribute to a further decrease in the pulmonary vascular resistance.24 Pulmonary arterial adventitial changes have received little attention. This compartment is more than a simple connective tissue sheath. The adventitia thins in the transition from the low-oxygen fetal circulation to the high-oxygen newborn and infant pulmonary circulation (Figure 3). Experimental evidence also indicates the adventitia is actively involved in vascular remodeling in hypoxia.25 After the age of 40 to 50 years, elastic arteries start to show some intimal fibrosis, most pronounced in the apices of the upper lobes.26 The medial collagen content also gradually increases with advancing age.27

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Radiological findings in pulmonary hypertension Pulmonary hypertension produces distinct radiological abnormalities. There is enlargement of the central pulmonary arteries. On chest X-ray (CXR) the right interlobar artery is considered enlarged when over 16 mm in diameter. The main pulmonary artery is dilated when it measures greater than 29 mm in diameter, on computed tomography (CT) or magnetic resonance imaging (MRI) (Figure 4). In pulmonary hypertension, pulmonary arteries show a rapid decrease in caliber from the center to the lung periphery. This is referred to as “pruning”, since the vacular tree resembles a pruned tree. Evaluation of these features is not very reproducible and correlates poorly with the severity of pulmonary hypertension. In addition, these features provide no clue to the etiology of the disease. The main values of CT and MRI do not lie in diagnosing pulmonary hypertension per se, but rather in the exclusion of interstitial lung disease, chronic obstructive pulmonary disease (COPD) and congestion, and the evaluation of possible thromboembolic disease. In thromboembolic disease, high resolution CT scans may reveal occlusion of central pulmonary arteries with

Chapter 18: Pulmonary vascular pathology

characteristic dilatation upstream of the blockage. There is an abrupt decrease in caliber downstream of the occlusion (so-called Westermark sign) (Figure 5a). Occlusion of smaller pulmonary arteries typically features a mosaic pattern of high- and low-density areas, representing uneven distribution of blood flow (Figure 5b). In congestive vasculopathy and pulmonary veno-occlusive disease (PVOD), there may be widening of interlobular septa, which is best identified near the pleura.28 In PVOD, these patchy changes are best

appreciated in areas where the hydrostatic pressure is low, i.e., in the ventral and upper areas on a supine CT scan. Ground-glass opacification, due to alveolar edema and/or accumulation of iron-laden alveolar macrophages, may be seen in both entities, the distribution being more patchy in PVOD (Figure 6) (see below).29,30 Cardiac ultrasound studies are helpful as they allow diagnosis of congenital heart disease, as well as tricuspid valve insufficiency resulting from right ventricular decompensation.

Figure 4. CT scan shows a markedly distended pulmonary artery in a case of longstanding pulmonary arterial hypertension.

Figure 6. High-resolution CT scan of a limited cutaneous systemic sclerosis patient with pulmonary hypertension, and a PVOD pattern at autopsy. The CT shows diffusely distributed centrilobular fine-nodular ground-glass opacities, some septal lines (so-called Kerley B lines), not only dorsally but also on the ventro-lateral side of the upper lobes.

(a)

(b)

Figure 5. (a) High-resolution CT scans showing an embolus occluding the right pulmonary artery, with characteristic dilatation upstream of the occlusion. There is an abrupt decrease in caliber downstream of the occlusion (so-called Westermark sign). (b) High-resolution CT scan showing a mosaic pattern of high- and low-density areas, representing an uneven distribution of blood flow in a case of pulmonary thrombo-emboli. Note the larger caliber of the pulmonary arteries in the areas of increased perfusion, compared to areas with low perfusion.

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Chapter 18: Pulmonary vascular pathology

Ultrasound also provides some estimate of pulmonary arterial pressure. An estimate of a highly elevated pulmonary artery pressure can safely be assumed to be genuinely above the normal range. However, a mildly/moderately elevated pulmonary artery pressure, as estimated by ultrasound, warrants verification by right-heart catheterization.

Genetics of pulmonary hypertension Pulmonary hypertension may be familial. Most such pulmonary arterial hypertensive cases are linked to the BMPR2 gene (see below).31,32 Uniallelic inactivating germline mutations in this gene lead to haploinsufficiency, which may or may not suffice to cause clinical disease. An autosomal dominant pattern of inheritance with incomplete penetrance results.31–33 Germline BMPR2 gene mutations are also overrepresented among sporadic iPAH and PVOD patients,28,34 as well as in those developing pulmonary hypertension after appetite suppressant use.35,36 In these apparently isolated cases, BMPR2 haploinsufficiency is associated with a younger age and more severe hemodynamic compromise at presentation, as well as a more rapid disease progression.37 BMPR2 haploinsufficiency has not been noted in chronic thromboembolic pulmonary hypertension (CTEPH) (reviewed by Lang38). Germline mutations in other genes involved in the BMPR-2 transduction pathway, especially ALK-1 (activin receptor-like kinase type I) and endoglin, also pose a risk for pulmonary hypertension (reviewed by Machado et al.39). Accordingly, the “familial” PAH of the previous classification10 has now been subspecified as BMPR-2, ALK-1 or endoglin mutation associated PAH (Table 2).11

Pathogenesis of pulmonary hypertension Introduction The wide variety of causes and risk factors of pulmonary hypertension suggest different pathogenetic pathways. Once established, however, arterial hypertension itself comes to act as a stress factor on the vasculature, causing shear stress. In addition the hypoxia that results from a low diffusion capacity and/or low mixed venous oxygen pressure possibly contributes to vascular changes. Thus, in addition to pathogenetic pathways related to its cause, pulmonary hypertension shares pathogenic features; a notion supported by the partly overlapping, partly distinctive patterns of vasculopathy in human pulmonary hypertension. The pathogenesis of pulmonary hypertension has been studied in a variety of animal models such as rats, calves, pigs and mice (including transgenic mice), and by means of different stress factors. These include pulmonary hypoxia (alone, or in combination with other manipulations, e.g. VEGF blocking), increased flow (e.g. due to intracardiac shunting, pulmonary artery banding or pneumonectomy), local arterial obstruction, left-sided cardiac failure and toxic endothelial damage with monocrotalin. Elucidated cellular and molecular mechanisms depend on the cellular composition of vessels at

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particular sites along the pulmonary vasculature, and microenvironmental factors. Each of the vascular cells (endothelium, smooth muscle cell, adventitial fibroblast) may undergo siteand time-dependent alterations in proliferation, matrix protein production, expression of growth factors, cytokines, and receptors (Figure 7).40 Each resident cell type, and/or possibly circulating precursors, plays a role in the remodeling process. Many of these pathways are closely interconnected, and the relative contribution of specific pathways in pulmonary hypertension of different etiologies is still largely obscure. Our understanding as to how these mechanisms translate to the histopathological features of vasculopathy in pulmonary hypertension in humans is far from complete. We have limited our discussion to the main players in the pathogenesis of pulmonary hypertension, and refer to the various histopathological alterations seen in pulmonary hypertension where possible.2,3,39–42

Bone morphogenic protein receptor-2 (BMPR2) The bone morphogenic protein receptor-2 (BMPR2) signal transduction pathway is central to the pathogenesis of pulmonary hypertension, notably in plexogenic arteriopathy and PVOD. BMPR2, located at 2q31–32, is a member of the transforming growth factor beta (TGFb) superfamily. It is widely expressed in pulmonary arterial endothelium and BMPR signaling controls angiogenesis by regulating apoptosis of vascular smooth muscle cells.43 It also opposes TGFb-induced AKT activation.33,44 BMPR2þ/45 or BMPR2/ conditional knockout mice have increased susceptibility to apoptosis and pulmonary hypertension.46 The combination of increased endothelial apoptosis and failure of growth suppression in pulmonary artery smooth muscle cells is probably of direct relevance in the pathogenesis of PAH (Figure 7).47 Exonic mutations in the BMPR2 gene and several of its signal transduction pathway members have been found in familial pulmonary hypertension.28,34 The transduction pathway members, endoglin and SMAD4, are seen in hemorrhagic hereditary telangiectasia (HHT/Rendu-Osler-Weber disease) associated with pulmonary hypertension.48 Mutations in the TGFb type I receptor gene and activin receptor-like kinase type I (ALK-1) gene49 are encountered in HHT patients but occasionally also in pulmonary hypertension unassociated with HHT. Since familial PAH is strongly linked to chromosome 2q33, it is possible that familial cases without exonic mutations have alterations in non-coding regions of the BMPR2 gene or in regulatory genes.50 Increased frequency of germline BMPR2 mutations are found in pulmonary hypertension patients who have used appetite suppressants35 and in patients with congenital heart defects.51 In scleroderma, a connective tissue disease with a high risk of pulmonary hypertension, BMPR2 mutations are infrequent52 but a polymorphism in endoglin, which is part of a functional complex involving BMPR2, has been reported in some cases (see Chapter 21).53

Chapter 18: Pulmonary vascular pathology

Figure 7. Diagram depicting potential mechanisms involved in the development of pulmonary arterial hypertension. In this figure a viral infection propagates a myriad of cellular events. Ang, angiopoeitin; AVD, apoptotic volume decrease; BMP, bone morphogenetic protein; BMPR, bone morphogenetic protein receptor; CaM, calmodulin; CREB, cAMP-response element binding protein; DAG, diacylglycerol; Em, membrane potential; EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ET, endothelin; GAP, GTPase activating protein; GF, growth factor; GPCR, G protein-coupled receptor; HHV, human herpes virus; HT, hydroxytryptamine (serotonin); HTT, hydroxytryptamine (serotonin) transporter; IP3, inositol 1,4,5-trisphosphate; Kv, voltage-gated Kþ; MAPK, mitogen-activated protein kinase; MLC, myosin light chain; MLCK, myosin light chain kinase; NA(D)PH, nicotinamide adenine dinucleotide phosphate; NO, nitric oxide; PASMC, pulmonary artery smooth muscle cell; PDGF, platelet-derived growth factor; PGI2, prostacyclin; PKC, protein kinase C; PLC, phospholipase C; ROC, receptor-operated Ca2þ channels; ROS, reactive oxygen species; RTK, receptor tyrosine kinase; SR, sarcoplasmic reticulum; SRF, serum response factor; TCF, T cell factor; TIE, endothelial-specific tyrosine kinase; VDCC, voltage-dependent calcium channel. (Reproduced from Morrell et al. Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 2009;54(1), Suppl S, with permission from Elsevier.)

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Germline mutations and other rearrangements in the BMPR2 gene lead to haploinsufficiency, a major risk factor for pulmonary hypertension.31–33,35,51 However, haploinsufficiency is neither necessary nor sufficient to cause pulmonary hypertension. The penetrance of clinical disease in BMPR2 mutation carriers is approximately 20%, and varies among families.33 In pulmonary hypertension patients, BMPR2 expression is reduced in mutation carriers and non-mutation carriers alike. In the former, there is a greater reduction in BMPR2 expression than can be explained by haploinsufficiency alone. This points to a contribution of other, genetic or environmental, factors associated with BMPR2 dysfunction.33,47 Interestingly, human immunodeficiency virus (HIV)-1 tat protein inhibits BMPR2 expression. This may explain the increased prevalence of PAH in HIV-infected patients.4 Conversely, overexpression of BMPR2, for example by enhancing BMPR2 promoter activity by simvastatin,54 prevents or attenuates pulmonary hypertension in BMPR2 wild-type rats.55–57 Once pulmonary hypertension is established in BMPR2 haploinsufficient, monocrotalin-treated rats, overexpression of BMPR2 by adenoviral transfection is no longer effective in reversing the disease.58

Serotonin (5-hydroxytryptamine, 5HT) and appetite suppressants Serotonin (5-HT) is a vascular smooth muscle constrictor, and, in concert with its transporter protein (5-HTT or SERT), acts as a smooth muscle mitogen.20,21 Serotonin regulates BMPR-SMAD signaling.45 BMP-4 and BMP-6, ligand to the BMPR-2 signal transduction pathway, which in turn, inhibit 5-HT transporter (5-HTT) expression.45 BMPR2 haploinsufficient mice show increased sensitivity for serotonin-induced pulmonary hypertension.45 The vascular smooth muscle growth-enhancing effect of serotonin is already potentiated in pulmonary hypertensives.59 Such patients overexpress 5-HTT in their lungs and platelets20,21 and underexpress BMPR2.33,47 These mechanisms are further potentiated by hypoxia.60 As in pulmonary hypertension,21 an increased frequency of the long promoter polymorphism in the 5-HTT gene is found in COPD patients and is associated with more severe pulmonary hypertension in these subjects.61 Increased plasma levels of 5-HT in pulmonary hypertension patients are also attributed to decreased platelet serotonin storage.62 In summary, these data imply a pulmonary hypertension-promoting effect of serotonin, which is enhanced both by functional insufficiency of BMPR2 and by hypoxia. Several appetite suppressants have been identified as risk factors for pulmonary hypertension.5 All the implicated drugs, including aminorex, fenfluramine, D-fenfluramine and fenfluramine-phentermine, inhibit serotonin reuptake.60 The group of serotonin re-uptake inhibitors contains not only appetite suppressants, but also several widely used

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antidepressants. Some evidence has emerged that antidepressants may increase the risk of pulmonary hypertension in genetically predisposed individuals.63 Other drugs known to increase circulating serotonin levels, such as amphetamines, have similarly been incriminated as risk factors for pulmonary hypertension.64,65

Inflammation There is an incompletely understood association between chronic inflammatory disorders, such as systemic lupus erythematosus (SLE), and various immune deficiencies including acquired immunodeficiency syndrome (AIDS) and BMPR2 function. This association involves both humoral and T-cell responses. For example, IL-6, a pleiotropic acute phase cytokine, negatively regulates several BMPs.66 Conversely, BMPR2 haploinsufficiency leads to increased levels of IL-6, which may augment humoral immune responses, notably immunoglobulin production. Circulating B cells in pulmonary hypertension patients are activated,67 and are found in vascular lesions.68,69 Interestingly, the POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes), characterized by high levels of circulating IL-6 and other cytokines, is associated with pulmonary hypertension.70 In connective tissue diseases, the humoral response may be directed at the lung endothelium, since anti-endothelial antibodies are identified in SLE and systemic sclerosis.71–73 In the latter disease, antibodies against anti-platelet-derived growth factor receptor (PDGFR) antibodies have been demonstrated.74,75 PDGFR activation mediates collagen gene expression, fibroblast-to-myofibroblast differentiation, and smooth muscle proliferation. PDGFR is expressed at higher levels in lung endothelium of pulmonary hypertension patients.76,77 Whether these antibodies have activating properties is controversial.74,75,78 Imatinib (Gleevec ), a tyrosine kinase inhibitor of the PDGF receptor, may prove to be an effective treatment for at least some cases of PH.79–83 In mice, T cells appear to protect against the development of pulmonary hypertension. When treated with a vascular endothelial growth factor (VEGF) blocker, athymic mice develop pulmonary hypertension.69 This effect is abolished by supplying the mice with T cells from euthymic mice.69 These findings are consistent with the fact that depletion of T helper cells in HIV infection predisposes to pulmonary hypertension.4 Intriguingly, Th2 type skewing of CD4þ cells promote both vascular remodeling and sensitivity to hypoxia.84 Perhaps the role of T cells in the development of pulmonary hypertension depends in part on the tolerogenic capacity of the immune system, in which dendritic cells are pivotal. The tolerogenic state of dendritic cells and subsequent T regulatory cell function is maintained in the presence of the neuropeptide vasointestinal peptide (VIP), promoting Th2 skewing.85,86 A role for VIP in pulmonary hypertension is supported by observations that VIP knockout mice develop pulmonary hypertension spontaneously.87 In addition, exogenous VIP

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Chapter 18: Pulmonary vascular pathology

appeared to improve physiological parameters of pulmonary hypertension in an uncontrolled open label study in humans.88

Endothelium and thrombosis The endothelium is a central player in the regulation of pulmonary vascular tone and the clotting cascade.89,90 These cells produce many vasoactive pro-inflammatory and mitogenic mediators, including endothelin-1, nitric oxide (NO), serotonin, prostacyclins and thromboxane. Endothelins, which are potent vasoconstrictors, bind to the endothelin A (ET-A) receptor of vascular smooth muscle cells, which increases their intracellular free Ca2þ. This function is counteracted by cyclooxygenase 2.91,92 Chronic hypoxia enhances the sensitization to Ca2þ through radical oxygen species-dependent activation of RhoA/Rho kinase signaling.93 Endothelin-1 also has a vasodilator effect, via its binding to endothelin-B (ET-B) receptor on endothelial cells; this vasodilator effect is mediated by endothelial NO release.94,95 In addition, endothelin-1 acts as a mitogen of pulmonary vascular smooth muscle cells and induces extracellular matrix formation.96 Platelet activating factor (PAF), another endotheliumderived vasoactive peptide, is a potent phospholipid activator and mediator of platelet aggregation and inflammation. It is produced by various leukocytes and is present in platelets. It increases pulmonary vascular resistance, and may be involved in hypoxia-induced remodeling.97 It is apparently not involved in hypoxic vasoconstriction.98 Pressure, strain and shear stress cause endothelial dysfunction.99 Increased flow, due to shunts and pulmonary arterial hypertension, alters the flow pattern in pulmonary arteries and increases shear stress. Shear stress occurs in the areas of most intense turbulence, downstream of bifurcations and probably also near the origins of supernumerary arteries. Besides endothelial activation caused by pulmonary hypertension, the endothelium may be directly targeted by other insults, such as a thromboemboli, foreign-body material, schistosoma eggs and auto-antibodies. The effects of endothelial dysfunction are wide-ranging and include vasoconstriction, vascular leakage, inflammation, thrombosis and, ultimately, vascular remodeling. Damage or activation of endothelial cells is implicated in local activation of the clotting cascade and intimal fibrosis. The notion of locally impaired fibrinolysis in pulmonary hypertension5 is supported by evidence from the following factors; an imbalance between thromboxane A2 and prostacyclin,100 local production of plasminogen activator inhibitor (PAI), increased levels of von Willebrand factor,101 and the release of endothelium-derived procoagulant microparticles containing CD105 (endoglin) in pulmonary arterial hypertension (PAH) patients.102 These microparticle levels correlate with the pulmonary artery pressure. In pulmonary hypertension associated with connective tissue diseases, various auto-antibodies, including antiendothelial antibodies, can be detected.71–73,103 These may induce upregulation of proinflammatory and immuno-active

molecules, such as intercellular adhesion molecule-1 (ICAM-1), endothelial leukocycte adhesion molecule-1 (ELAM-1) and major histocompatibility complex class II.104 Autoantibodies are also probable contributors to local inflammation, which further promotes thrombosis. Anticoagulant therapy is a mainstay in pulmonary hypertension treatment.2,3,5 Pulmonary arterial hypertension patients have elevated endothelial levels of the chemokines RANTES105 and fractalkine.106 The latter also acts as a cell adhesion molecule in its membrane-anchored form. These patients also have increased levels of circulating endothelium-derived soluble vascular cellular adhesion molecule-1 (sVCAM-1).102 In addition, they have circulating proinflammatory markers, such as monocyte chemoattractant protein-1 (MCP-1) and C-reactive protein,102 reflecting a pro-inflammatory state. It is currently unknown whether this is a cause or a consequence of pulmonary hypertension and/or vascular remodeling. Taken together, the endothelial proinflammatory, prothrombotic and profibrotic state may be caused by various direct insults, as well as by shear stress resulting from the pulmonary hypertension.

Hypoxia

Hypoxia and pulmonary vascular tone Pulmonary vascular recruitment and vascular tone are the main regulators of pulmonary arterial pressure. Local oxygen tension is considered the key factor in the regulation of pulmonary vascular tone (reviewed in Marshall et al.107). In contrast to its effect in the systemic circulation, low oxygen tension elicits a vasoconstrictive response of pulmonary blood vessels, especially pulmonary arteries. This is probably beneficial in the case of uneven ventilation of the lungs, as it redistributes blood flow away from underventilated areas. However, generalized pulmonary hypoxia results in increased pulmonary vascular tone and pulmonary hypertension. Hypoxic pulmonary hypertension has been extensively studied in laboratory animals. Histologically, hypoxic pulmonary hypertension, as observed in humans (e.g. people resident at high altitude) and in animals, differs markedly from plexogenic arteriopathy, thrombotic arteriopathy, congestive vasculopathy and pulmonary veno-occlusive disease (see below). It is unknown whether there is pathogenic overlap between hypoxic pulmonary hypertension and these other forms of pulmonary hypertensive disease. The regulation of the pulmonary vascular tone is the net result of the action of vasodilator and vasoconstrictor mediators, including endothelium-derived and neurohumoral factors.89,90 In addition, many other vasoactive factors, such as adenosine, bradykinin, calcitonin gene-related peptide, histamine, serotonin, neuropeptide Y, reactive oxygen species, substance P, vasopressin and vasoactive intestinal peptide (VIP), all influence pulmonary vascular tone. BMPR2 haploinsufficiency and the altered interplay of BMPR2 and serotonin in pulmonary hypertension may sensitize the pulmonary vessels to hypoxia (see above).

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In chronic hypoxia, acute re-exposure to normal or even high levels of inhaled oxygen becomes progressively less effective in immediately reducing the pulmonary arterial pressure. This can be attributed to both loss of responsiveness to oxygen and structural vascular changes. Both these factors increase pulmonary vascular resistance (PVR) (reviewed by Moudgil et al.108). The loss of responsiveness to oxygen increases PVR and does not contribute to ventilation-perfusion matching and optimization of gas exchange. The loss of responsiveness to oxygen involves the downregulation of expression of several potassium channels including voltage-gated channel Kv1.5.109,110 Sustained hypoxic vasoconstriction appears to be Rho-kinase-dependent,111 which involves endothelin-1mediated, ROS-dependent activation of the RhoA/ROK signaling pathway. This leads to increased smooth muscle myofilament Ca2þ sensitization.93 Endothelin-1 receptor antagonists are variably effective in pulmonary hypertension.5 Rho kinase inhibitors are under investigation.112

Hypoxia and vascular remodeling Hypoxia-induced vascular remodeling involves arterial medial smooth muscle hyperplasia, endothelial remodeling and adventitial fibrosis. Chronic hypoxic vasoconstriction initiates a proproliferative and anti-apoptotic state in pulmonary vascular smooth muscle cells, resulting in medial hyperplasia.110 These mechanisms involve mitochondrial hyperpolarization, upregulation of the anti-apoptotic molecule BCL-2,113,114 and increased levels of intracellular free Ca2.113 In pulmonary vascular smooth muscle, the latter is regulated, at least in part, by the nuclear factor of activated T cells (NFAT).113 This transcription factor is expressed to higher levels in leukocytes of pulmonary hypertension patients.113 This suggests a link between inflammation and hypoxia-driven vascular remodeling. In mice, some arterial intimal cells respond to hypoxia by differentiating into myofibroblasts.115 This finding fits with the observed subendothelial bundles of longitudinally oriented smooth muscle enveloped by elastin fibers seen in hypoxic arteriopathy.116 This feature may not be specific to hypoxia, as it is also occasionally observed in bronchial arteries.13 The intimal remodeling process is TGFb-dependent and linked to a BMPR2-related pathogenesis.47 Medial hyperplasia is neither necessary nor sufficient to cause pulmonary hypertension.117–119 Generalized marked medial hyperplasia, as induced by hypoxia111 or repeated bronchial challenge with allergen,84 does not lead to increased luminal narrowing111 or pulmonary hypertension under normoxic conditions. It does, however, enhance sensitivity to hypoxia and subsequent pulmonary hypertension.84,111 Conversely, the prevention of medial hyperplasia does not limit right ventricular hypertrophy in a rat model of hypoxia-induced pulmonary hypertension.120 Medial hyperplasia may still be present in the absence of vasoconstriction in rats recovering from hypoxia.118 Adventitial remodeling occurs in both large and small pulmonary arteries. Adventitial cell proliferation,

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production/deposition of extracellular matrix proteins, especially collagens, and increased numbers of myofibroblasts (a-smooth muscle actin-expressing fibroblasts) are responsible. In addition, hypoxia induces neovascularization of the adventitia.121–123 Fibroblasts increase in number, not only by local proliferation, but also by recruitment of circulating bone marrow-derived precursor cells that enter via the vasa vasorum. These fibroblasts differentiate into fibrocytes122,123 and/or smooth muscle cells.121,124,125 Adventitial thickening is especially prominent in congestive vasculopathy (see below).126 It can, however, be seen in many forms of chronic pulmonary hypertension, including persistent pulmonary hypertension in infants with congenital diaphragmatic hernia.127 In this condition, less adventitial thickening is found in those infants effectively treated with extracorporeal membrane oxygenation (ECMO). In view of these findings, one could postulate that hypoxia drives pulmonary arterial adventitial thickening in humans.

Histopathological patterns of hypertensive pulmonary vascular disease Introduction The role of biopsy pathology in the diagnosis and clinical management of pulmonary hypertension is limited to those cases where clinical presentation and hemodynamic parameters do not provide an unequivocal diagnosis. Only in those cases does the benefit of a histopathological diagnosis for further clinical management outweigh the risk of a surgical biopsy. Importantly, the histopathological identification of an underlying disease may shift therapeutic options from pulmonary hypertension therapy alone to combined treatment of pulmonary hypertension and an underlying disease, potentially improving prognosis.5,128 Histopathological findings should always be evaluated in conjunction with clinical, immunological and radiological findings. Although histopathological assessment of vascular lesions in explants and at autopsy may not benefit those individuals, such evaluations allow for correlation with the clinical diagnosis, hemodynamics, the effects of therapy, as well as the detection of undiagnosed or misdiagnosed disease. These investigations are also indispensable for furthering knowledge in this field. For example, little is known about development of histopathological patterns over time, or pharmacological effects on disease. As our insight into the genetics of pulmonary hypertension expands, a tissue diagnosis and, indeed, the DNA from tissue obtained at post-mortem may contain valuable information for other family members. The following sections describe the recognized histological patterns of pulmonary hypertensive vascular disease. These are plexogenic arteriopathy, thrombotic arteriopathy, hypoxic arteriopathy, congestive arteriopathy and pulmonary veno-occlusive disease/capillary hemangiomatosis (Table 3A).13 Each pattern of vasculopathy is described, with reference to the specific risk factors, underlying diseases and pathogenic pathways.

Chapter 18: Pulmonary vascular pathology Table 3A: Basic patterns of pulmonary vascular disease in pulmonary hypertension13

Table 3B: Dana Point clinical classification of pulmonary hypertension (2008) versus histopathological patterns of pulmonary hypertensive disease

Plexogenic arteriopathy

Dana Point clinical classification 2008

Thrombotic arteriopathy

Histopathological pattern

1.

Pulmonary arterial hypertension (PAH)

1.1.

Idiopathic PAH

1.2.

Hereditary

1.2.1.

BMPR2

PPA PVOD

1.2.2.

ALK1, endoglin

PPA

1.2.3.

Unknown

PPA PVOD

1.3.

Drug- and toxin-induced

PPAa

1.4.

Associated with

1.4.1.

Connective tissue diseases

PPA PVOD SSc Congestive

1.4.2.

HIV infection

PPA

1.4.3.

Portal hypertension

PPA

1.4.4.

Congenital heart diseases

PPA

Plexogenic arteriopathy

1.4.5.

Schistosomiasis

PPA Thrombotic

Epidemiology and risk factors

1.4.6.

Chronic hemolytic anemia

PPA Thromboticb

Plexogenic arteriopathy constitutes the most common histopathological pattern of pulmonary vascular disease in the clinical group of pulmonary arterial hypertension (PAH, group 1; see Table 2). It affects women two to three times more often than men.7 Plexogenic arteriopathy may be familial, or associated with a variety of conditions (Table 4). Congenital heart disease with a post-tricuspid left-to-right shunt is a major risk factor. This condition requires early surgical correction to prevent irreversible remodeling of the pulmonary vasculature and persistent pulmonary hypertension, eventually progressing to Eisenmenger syndrome (see below).129,130 Other risk factors include portal hypertension of any cause, various germline mutations (e.g. in BMPR2), hemorrhagic hereditary telangiectasia (HHT, Rendu-Osler disease), SLE and HIV infection.11 In the late 1960s, an epidemic of pulmonary hypertension was linked to the use of aminorex (Menocil ), a serotonin re-uptake inhibitor with appetite-suppressant activity.14 Since this initial description, other agents including fenfluramine/ponderal , Dfenfluramine/Adifax /Redux and fenfluramine-phentermine have been found to increase the risk of pulmonary hypertension (Table 1) (see Chapter 16).5,11,18 Globally, chronic Schistosoma infection, particularly S. mansoni, is a major risk factor for plexogenic pulmonary hypertension. Schistosomiasis-associated plexogenic pulmonary arteriopathy is largely limited to areas where schistosomiasis is endemic, such as northwestern South America (Brazil, Venezuela and Surinam), parts of the Caribbean (lower Antilles, Puerto Rico and the Dominican Republic) and northeast Africa.8 Thromboembolic lesions caused by embolizing schistosoma eggs were the predominant pulmonary lesions

1.5

Persistent pulmonary hypertension of the newborn

Various, depending on type. See also alveolar capillary dysplasia/ misalignment of the vessels (see Chapter 3)

10

Pulmonary veno-occlusive disease (PVOD) and/or pulmonary capillary hemangiomatosis (PCH)

PVOD

2.

Pulmonary hypertension owing to left heart disease

2.1.

Systolic dysfunction

Congestive vasculopathy

2.2.

Diastolic dysfunction

Congestive vasculopathy

2.3.

Valvular disease

Congestive vasculopathy

3.

Pulmonary hypertension owing to lung diseases and/ or hypoxia

3.1.

Chronic obstructive pulmonary disease

Hypoxic arteriopathy

3.2.

Interstitial lung disease

Hypoxic thromboticc

3.3.

Other pulmonary diseases with mixed restrictive and obstructive pattern

Hypoxic thrombotic

3.4.

Sleep-disordered breathing

Hypoxic arteriopathy

3.5.

Alveolar hypoventilation disorders

Hypoxic arteriopathy

Hypoxic arteriopathy Congestive vasculopathy Pulmonary veno-occlusive disease/capillary hemangiomatosis

In the clinical classification of pulmonary hypertension (Table 2)11 the various forms of pulmonary hypertension are grouped into five categories, mainly on the basis of clinical parameters, including major causes, risk factors and response to treatment. These five clinical categories correspond only partially to the histopathological classification. An overview of the clinical classification and corresponding histopathological patterns of vasculopathy is given in Table 3B. Since histopathology provides the most direct assessment of the type, distribution and severity of tissue lesions, our discussion of pulmonary vascular hypertensive disease is based on microscopy.

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PPA

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Chapter 18: Pulmonary vascular pathology Table 3B: (cont.)

Table 4 Conditions associated with plexogenic arteriopathy

Dana Point clinical classification 2008

Histopathological pattern

Congenital malformations of heart or great vessels, with left-to-right shunting

3.6.

Chronic exposure to high altitude

Hypoxic arteriopathy

Hereditary (BMPR2, ALK1, endoglin (with or without hereditary hemorrhagic telangiectasia))

3.7.

Developmental abnormalities

Hypoxic arteriopathy (see also 1.5)

Drugs and toxins (including appetite suppressants)

4.

Chronic thromboembolic pulmonary hypertension (CTEPH)

Thrombotic arteriopathy

HIV infection

5.

Hematological disorders: myeloproliferative disorders, splenectomy

5.2.

Systemic disorders:

5.3.

Shistosoma infection Unknown (idiopathic PAH) Thrombotic arteriopathy vascular occlusion by leukemic cells

Sarcoidosis

Thrombotic hypoxic congestive

Langerhans cell histiocytosis, lymphangioleiomyomatosis, neurofibromatosis

Thrombotic

Vasculitis

Thrombotic

Arterial medial hyperplasia Cellular intimal proliferation Plexiform lesions Focal fibrinoid necrosis Dilatation lesions, including angiomatoid lesions Concentric laminar intimal fibrosis Necrotizing arteritis (occasionally in severe, advanced disease).

PPAd

Others: tumoral obstruction

Thrombotic

fibrosing mediastinitis

Congestive

chronic renal failure on dialysis

PVODd

In drugs causing rise in serotonin levels, such as appetite suppressants, amphetamines, antidepressants. b In sickle cell disease: thrombotic arteriopathy. c Post-thrombotic remodeling or endarteritis obliterans. d Own anecdotal observation. PPA, plexogenic pulmonary arteriopathy; PVOD, pulmonary veno-occlusive disease; SSc, systemic sclerosis; ALK1, activin receptor-like kinase type 1; BMPR2, bone morphogenetic protein receptor type 2; HIV, human immunodeficiency virus. a

originally described by Chaves in the 1960s,131–133 hence its former classification in group 4 (thrombotic/embolic disease).10 However, the clinical presentation, histopathological features and course of the disease are mostly those of plexogenic pulmonary arteriopathy, rather than pulmonary thrombotic/embolic disease.132,134 In recognition of these newer data, this disease has now been placed in group 1 of the updated Dana Point classification (see Table 2) (see Chapter 8).11 The conditions mentioned above, except for intracardiac post-tricuspid shunts, are by themselves neither necessary nor sufficient to cause pulmonary hypertension, but they

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Table 5 Pulmonary vascular lesions in plexogenic arteriopathy12,13

Tortuosity of pulmonary arteries

Metabolic disorders: glycogen storage disease, thyroid disorders Gaucher disease

5.4.

Portal hypertension

Pulmonary hypertension with unclear multifactorial mechanisms

5.1.

Connective tissue diseases: SLE

substantially increase the risk of this disease. For example, penetrance of clinical disease in BMPR2 mutation carriers (see below) is approximately 20% and varies among families, as does the age at presentation.33 The use of appetite suppressants increases the risk of developing pulmonary hypertension 30-fold,35,36 but many users remain free of disease. Apparently, this form of pulmonary hypertension requires at least one additional stress factor to initiate the cascade of events that leads to clinically manifest disease.33,50

Histopathology of plexogenic arteriopathy (Table 5) In the earliest stages of the disease, medial hyperplasia of elastic and muscular arteries is a constant finding and often the only abnormality (Figure 8).130,135–137 Medial thickening results from both hypertrophy and hyperplasia of medial smooth muscle.135 The media is thickened when it exceeds 7% of the arterial diameter as measured at the outer elastic lamina. In addition to true medial hyperplasia, subendothelial intimal cells may show myofibroblastic differentiation,135,138 which probably contributes to vessel wall functional alterations.139,140 In adults with plexogenic arteriopathy, the degree of medial hyperplasia is roughly proportional to pulmonary artery pressure and vascular resistance.141–143 Some arterial intimal fibrosis is common in plexogenic arteriopathy.144 It is usually eccentric in the larger arteries, but sometimes concentric in smaller ones. Such intimal

Chapter 18: Pulmonary vascular pathology

fibrosis can be widespread and may be the most conspicuous finding, but it is not specific for plexogenic arteriopathy. There is, however, a type of intimal fibrosis, concentric laminar intimal fibrosis, which is at least highly suggestive of plexogenic arteriopathy.130 Concentric layering of collagen and myofibroblasts within small arteries is evident on H&E-stained sections, and has been likened to the appearance of onion skins, hence the alternative term “onion skin fibrosis” (Figure 9). For unknown reasons, concentric laminar intimal fibrosis is especially prominent in systemic sclerosis-associated pulmonary hypertension (see Chapter 21).145 Since the typical histological picture of concentric laminar intimal fibrosis

distinguishes it from post-thrombotic fibrosis, it seems probable that its pathogenesis also has distinctive elements. Concentric laminar intimal fibrosis should not be confused with the development of a new muscular media around a residual lumen in a case with marked nonspecific intimal fibrotic thickening. Such a newly formed muscular media consists of smooth muscle cells hugging the residual lumen, sometimes with newly formed elastin on either side, thus mimicking the media of a muscular pulmonary artery. This double media is another characteristic feature of systemic sclerosis-associated PAH (Figure 10) (see below).

Figure 8. Axial muscular pulmonary artery showing marked media hyperplasia. Arrows indicate the inner (lower arrow) and outer (upper arrow) elastic laminae (EvG stain).

Figure 10. Newly formed muscular media in a small pulmonary artery. There may be associated newly formed elastin mimicking the inner and/or outer elastic laminae of a muscular pulmonary artery. Fibrosis is not concentrically layered, so the new muscle layer stands out from this deeper intimal fibrosis.

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Figure 9. Concentric laminar intimal fibrosis. (a) The intima shows concentric layering of collagen and myofibroblasts within small arteries, nearly or completely occluding the vascular lumen. (b) EvG stain shows the outer and inner elastic laminae that delineate the media, and some fine, discontinuously layered elastic fibers (neo-elastica) within the intima.

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Figure 11. Plexogenic arteriopathy. (a) Distal pulmonary arteries in hypertensive pulmonary vascular disease. A computer-aided reconstruction shows plexiform lesions (white) occur in small side branches (supernumerary arteries) arising from larger parent arteries. Intimal lesions (yellow) are located in peripheral dichotomous branches. A few patent vessels remain (arrows). (Reproduced with permission from Yaginuma et al. Distribution of arterial lesions and collateral pathways in the pulmonary hypertension of congenital heart disease: a computer aided reconstruction study.Thorax 1990;45:586–90, with permission from BMJ Publishing Group Ltd.) (b) The plexiform lesion is noted at a pulmonary artery branching point. Note vessel distension. (c) The major artery can feature intimal fibrosis. (EvG stain.)

The plexiform lesion is a small (generally < 300 mm) but highly characteristic lesion of this pattern of hypertensive pulmonary vascular disease. It usually involves a supernumerary artery close to its origin from the axial parent vessel (Figure 11).18,146,147 The lesion consists of a plexus of slit-like or wider channels lined by small, flat or slightly plump endothelial cells and subjacent myofibroblasts with small and dark nuclei. One or several markedly dilated vein-like arterial branches are noted distally (Figure 12).139,148,149 Distal to the plexus the arterial branch is usually markedly dilated. Interestingly, these “dilatation lesions” may be solitary or may cluster together as angiomatoid lesions.150 They also occur without the associated plexus that characterizes the plexiform lesion (Figure 13). The parent axial artery commonly exhibits focal intimal fibrosis near the origin of the supernumerary artery. The affected supernumerary artery may display a small area of fibrinoid necrosis, most commonly between the origin and the

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plexus of vessels (Figure 14).148 Affected arterial segments show intense eosinophilia and apparent loss of nuclei in H&E-stained sections. There may be associated thrombus within the lumen of the affected branch. Fibrin and/or thrombus are commonly present, either within the parent vessel or as small clumps in the plexus (Figure 12b).144 Small areas of partial or circumferential fibrinoid necrosis are occasionally seen, especially at or immediately distal to the origins of supernumerary arteries. Focal arteritis is rare, and manifests as a transmural roundcell inflammatory infiltrate, most commonly affecting an axial muscular pulmonary artery. It may or may not be accompanied by fibrinoid necrosis. Necrotizing arteritis (Figure 15) may be seen in severe advanced plexogenic arteriopathy, but is a consequence rather than a cause of the hypertension. Plexogenic arteriopathy can be diagnosed in the absence of plexiform lesions, when there is distinct concentric laminar

Chapter 18: Pulmonary vascular pathology

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(c)

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Figure 12. Plexogenic arteriopathy. (a) This plexiform lesion features adjacent vascular dilatation and interstitial fibromyxoid tissue reaction. Note the medial thickening in the adjacent artery section. (b) Prominent distended thin-walled vessels are considered “vein-like” branches. Scattered vascular channels contain fibrin thrombi. (c) This lesion vaguely resembles a renal glomerulus. Stromal tissue is scant. (d) Endothelial cells are spindled to cuboidal and often hyperchromatic.

fibrosis, vein-like branches or angiomatoid lesions, fibrinoid necrosis in proximal segments of supernumerary arteries or, preferably, a combination of these findings. Expression of both vascular endothelial growth factor-1 (VEGF-1) and its receptor are markedly increased in the lesion.146,151–154 In experimental animal models pulmonary hypertension with complex lesions, closely resembling plexiform lesions, can be demonstrated after VEGF-receptor blockade in combination with hypoxia or shear stress. In this prototype, the endothelial lining of the plexus consists of a proliferating apoptosis-resistant population.155,156 In humans, the angioproliferation has been attributed by some to a high prevalence of angiotropic human herpes virus-8 (HHV-8) in plexiform lesions.157,158 This finding has not been confirmed by all.159–164

The cause of the “dilatation lesions” is unknown. It has been suggested that the dilatation represents a decompensation of the vascular wall caused by increased intraluminal pressure.

Plexogenic arteriopathy: differential diagnosis Plexiform lesions are not a feature of severe hypoxic pulmonary hypertension, congestive vasculopathy or PVOD. Despite claims to the contrary, the plexiform lesion can and should be distinguished from the commoner organizing thrombus or thromboembolus.144 The histopathological patterns differ. Since occasional post-thrombotic lesions may be encountered in all types of pulmonary hypertensive vascular disease,

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Chapter 18: Pulmonary vascular pathology

(a)

(b)

Figure 13. Plexogenic arteriopathy: dilatation and angiomatoid lesions. (a) Dilated arterial branches distal to the plexus without the associated plexiform lesion are easy to overlook. The parent vessel shows intimal fibrosis (EvG stain). (b) Vein-like branches may cluster together to form an angiomatoid lesion. These small lesions often surround the small parent pulmonary artery (EvG stain).

Figure 15. Arteritis in plexogenic arteriopathy. A muscular pulmonary artery features transmural mixed lymphocytic and granulocytic infiltrates, fibrinoid changes of the vessel wall and a thrombus in the vessel lumen.

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Figure 14. Plexogenic arteriopathy. Fibrinoid necrosis can be seen.

Effects of therapy

including plexogenic arteriopathy, the finding of an obvious post-thrombotic lesion does not indicate thrombotic arteriopathy. Neither does their presence, in addition to the “tell-tale” lesions of plexogenic pulmonary arteriopathy or any other type of hypertensive vascular disease, point to some vague and allencompassing “spectrum of hypertensive vascular disease”. Such reasoning effaces essential distinctions in our understanding of pulmonary hypertension.

Much of our knowledge of pulmonary vascular histopathology was gathered in a time when, apart from cardiac surgery in cases of cardiac malformations or valvular disease, treatment options were virtually non-existent. In 1984 the effectiveness of continuous infusion with prostacyclin (PGI2, epoprostanol) for the treatment of primary pulmonary hypertension (now referred to as idiopathic pulmonary arterial hypertension, group 1) was first demonstrated.165 Only in 1998 did the United States Food and Drug Administration (FDA) approve continuous infusion with PGI2 for the treatment of iPAH. At

Chapter 18: Pulmonary vascular pathology

present, oral therapy (endothelin receptor antagonists, e.g. bosentan, phosphodiesterase 5 inhibitors, e.g. sildenafil) is the first line of medical treatment. Continuous intravenous administration of prostanoids is reserved for those whose response to oral treatment is insufficient. The effects of treatment on vascular remodeling and the resultant histological phenotype of end-stage disease have been studied.166 The vascular morphology in explanted lungs of prostacyclin-treated and that in non-treated patients with either idiopathic pulmonary hypertension or Eisenmenger syndrome were compared. Treated cases showed more frequent and extensive perivascular and peribronchiolar inflammation and alveolar edema. Treated and non-treated control groups did not differ with respect to medial, intimal or adventitial thickness, or number of plexiform lesions.

Thrombotic arteriopathy and chronic thromboembolic pulmonary hypertension (CTEPH) Epidemiology, risk factors

Thrombotic arteriopathy may result from thromboembolism or, more rarely, from primary pulmonary thrombosis. Injury to endothelium, alterations in normal blood flow and alterations in the blood (hypercoagulability) predispose to thrombosis. Specific conditions are listed in Table 6. CTEPH is clinically defined as pulmonary hypertension after acute pulmonary thromboembolism, arising immediately or insidiously within the first 2 years after the initial thromboembolic event. About 30% of CTEPH patients develop pulmonary hypertension without documented acute thromboembolic episodes;167 this situation is known as “silent recurrent pulmonary thromboembolism”. Evidence of an acute embolic event is lacking in these cases. Some may be due to local thrombosis, or deficient clearing of small thromboemboli that may normally occur over time, rather than due to acute and massive thromboembolism.38 CTEPH is classified in group 4 of the Dana Point classification 2008.11 After a symptomatic thromboembolic event, further embolic episodes are not uncommon, and are noted in up to 8% of patients in the ensuing year, despite adequate anticoagulation.168–170 The cumulative risk of CTEPH after acute pulmonary emboli without evidence of deep-vein thrombosis is no greater than 4% at 2 years.168,171 After this period, further cases of CTEPH are rare, even though this asymptomatic socalled honeymoon period may very sporadically extend for up to 40 years.172 Risk factors for CTEPH after pulmonary embolism are: younger age, a history of previous pulmonary embolism, pulmonary emboli from an unknown source and pulmonary emboli associated with a detectable perfusion defect.168 Anti-phospolipid antibodies, known for their prothrombotic propensity, inflammatory bowel disease and osteomyelitis are also associated with an increased risk of CTEPH.167 Hampered thrombolysis, caused by infection, inflammation,

Table 6 Conditions associated with thrombotic pulmonary arteriopathy38,167

Previous pulmonary embolism Idiopathic pulmonary embolism Young age at first embolic event Persisting perfusion defect after acute pulmonary embolism High hematocrit Diminished pulmonary flow Temporary risk factors of pulmonary thromboembolism: Recent trauma Fracture Surgical intervention Hospitalization Immobilization along with pressure on the calves Pregnancy Use of oral contraceptives or thyroid hormone-replacement therapy Ventriculo-atrial shunt Long-term risk factors of pulmonary thromboembolism: Deficiency of antithrombin, protein C, protein S, Factor V Leiden, prothrombin G20210A mutation Two or more first-degree relatives with venous thromboembolism Lupus anticoagulant, anticardiolipin/antiphospholipid antibodies Malignancy, especially adenocarcinomas of stomach, pancreas and ovary Immobilization from chronic disease Sickle cell disease b-Thalassemia Asplenic state Blood group, non-O

autoimmunity and malignancy (various solid tumors including mucinous adenocarcinomas of the pancreas, lung and gastrointestinal tract173,174) is a further risk factor (see Chapter 24).38 Infected thrombi may cause a localized pulmonary arteritis.175 For example, staphylococcal infection delays thrombus resolution and promotes fibrotic vascular remodeling.176 Various additional factors associated with a hypercoagulable state and an increased risk of CTEPH or thrombotic pulmonary arteriopathy include sickle cell disease,177,178 inherited thrombophilias such as Factor V Leiden deficiency and the prothrombin G20210A mutation,179,180 asplenia,181 particularly in b-thalassemia patients,182 as well as thrombocythemia in myeloproliferative disorders10 (see also review by Esmon183). The commonest genetic contributor to venous thromboembolism is the Factor V Leiden mutation (FVL).183 This mutation decreases the ability of activated protein C to inactivate activated Factor V. The incidence of heterozygosity ranges from 2–8% in Caucasians to less than 0.1% in Asians.184 Heterozygous carriers of FV Leiden have an approximately 5-fold increased risk of venous thromboemboli, whereas homozygous carriers have a 20–80-fold increased risk179,185

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Hence, the mutation is likely to predispose to CTEPH, but it has not been shown to be overrepresented among PAH patients.184 Sickle cell disease (SCD) patients have a particularly high risk of developing pulmonary hypertension and additional comments are warranted (reviewed by Kato186). Pulmonary hypertension develops in up to 40% of patients,187–189 with a mortality rate of almost 50%.177,187,189 This autosomal recessive hereditary hemoglobinopathy is caused by a codon 6 mutation in the hemoglobin B chain, resulting in a valine substituting a glutamine. SCD was the first human disease to be defined at the molecular level. Persons with homozygous mutations, i.e. inherited from both parents, HbSS, develop SCD. Heterozygous individuals (HbS) rarely suffer serious consequences but HbS when combined with other inherited gene defects, such as B-thalassemia, also results in SCD.190,191 The gene is distributed across west, central and east Africa, parts of north Africa and in some parts of Asia. Thus, with historical slave-related transportation and other migrations, SCD has a significant prevalence in Africa, the Caribbean, North America and Europe. In the USA, 8% of AfricanAmericans are heterogyzgous for the sickle mutation, while less than 0.25% are homozygous.192 With greatly improved management over the past 20 years, mortality rates have declined and patients live into adulthood. Thus, disease prevalence continues to rise. There is great variation in the clinical presentation of SCD, depending on the amount of protective fetal HbF in the blood, other inherited genetic variations and treatment. However, increased susceptibility to infection is certainly due to repeated episodes of splenic infarction, leading to autospenectomy.193,194 Lung involvement, aside from pneumonia, manifests as either acute chest syndrome or chronic lung disease. Acute chest syndrome or acute vasculopathy is a potentially fatal sickle crisis with a clinicoradiological definition. Patients present with fever, wheeze, cough, tachypnea, chest pain, bone pain, hypoxemia and new pulmonary infiltrates on chest imaging (Figure 16a).192,195 Precipitating factors promoting sickling include bacterial pneumonia and sepsis, bone sickle cell crisis and necrotic marrow embolism (Figure 16b), pulmonary fat embolism, and general anesthesia.196 Infection or other inflammatory stimuli cause pulmonary hypoxia and increased expression of endothelial adhesion molecules, including a4b1 and vascular cell-adhesion molecule-1 (VCAM-1). This precipitates HbS polymerization and vaso-occlusion, causing further hypoxia and inflammation. Vaso-occlusion causes the release of free plasma hemoglobin, which reduces NO availability, altering VCAM-1 expression. Vaso-occlusion and bone marrow infarction can cause fat embolism, further damaging the pulmonary circulation. Secretory phospholipase A2 concentrations, which increase in response to inflammation and are known to be very high in acute chest syndrome, further increase expression of adhesion molecules in the pulmonary vasculature, causing more vasoocclusion.196

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Morphologically, the lungs are heavy and congested. Arterioles, capillaries and venules are dilated and engorged with sickled erthyrocytes (Figure 16c,d). Alveolar septal edema, necrosis and hemorrhage are common. Pleural-based infarcts are rare.197 In some cases, microvascular thrombosis is also identified. Pulmonary edema and even diffuse alveolar damage may be seen in fatal crises. One should distinguish the iatrogenic effects of cardiopulmonary resuscitation (CPR) from acute chest syndrome at autopsy, as CPR can cause fat embolism to the lung and, after death, sickling of previously nonsickled red blood cells occurs. Chronic lung disease manifests in SCD patients with chronic dyspnea. Chronic lung disease may develop independently of the acute chest syndrome.198 Chronic hypoxia, along with restrictive lung disease due to generalized pulmonary fibrosis, leads to pulmonary hypertension and cor pulmonale. It is probable that pulmonary infarction and endothelial injury contribute to the hypertension. In addition, hemolysis may detrimentally affect the availability of NO and increase cellular exposure to oxidants.199,200 Finally, left heart failure and hepatic cirrhosis with portal hypertension may be independent contributing factors. Accurate descriptions of pulmonary vasculopathy in well-characterized patients are lacking. Thus, the pathogenesis of pulmonary hypertension in sickle cell disease is multifactorial, rather than purely thrombotic.201

Histopathology of thrombotic arteriopathy (Table 7) Thrombotic occlusion of a pulmonary artery may result from thromboembolism, primary thrombosis or a combination of both. A thrombus formed locally cannot be distinguished reliably from a thromboembolus by histology alone (Figure 17a). The clinical features may not be discriminatory either, as the risk factors, signs and symptoms overlap. The term “thrombotic arteriopathy” conveniently covers both entities, avoiding the problematic distinction.13 An increased caliber of the pulmonary arteries proximal to the obstruction, is characteristically found in chronic thromboembolic lung disease.202–204 The lesional distribution and histopathology of thrombotic pulmonary arteriopathy are distinctive.148,205 If a patient survives the acute thromboembolic episode, a recent thrombus is rarely encountered, since thrombi are quickly lysed or organized (Figure 17b). The timeline of various stages of thrombus organization are said to be discernible histologically.206 However, experimental data correlating the various stages of organization with the age of the thrombus are largely lacking, and histological dating of thrombi should therefore be regarded as somewhat conjectural. In most instances one can simply discern acute from organizing or chronic thrombi. Organization of a thrombus is generally accompanied by ingrowth of endothelium, which leads to the formation of channels traversing the organizing thrombus (Figure 17c,d). Such recanalization results in the formation of one or more “neolumina”, which freely anastomose and commonly succeed

Chapter 18: Pulmonary vascular pathology Table 7 Pulmonary vascular lesions in thrombotic arteriopathy13

in re-establishing the blood flow through the affected artery (Figure 18a). Eccentric intimal fibrosis may be seen (Figure 18b). So-called “colander” lesions may widen so that ultimately, they are separated only by thin fibrotic septa (Figure 18c). Thin septa and “bands and webs” may be detected macroscopically at autopsy, when large arteries are involved (Figure 18d).

Eccentric, irregular intimal fibrosis Intravascular fibrous septa (webs and bands), colander lesions Recanalizing thrombi (uncommon) Recent thrombi (usually rare or absent) Medial hyperplasia of muscular pulmonary arteries: mild or absent

(a)

(b)

(c)

(d)

Figure 16. Sickle cell disease. (a) Chest X-ray in a sickle cell disease patient with the acute chest syndrome features bilateral pulmonary infiltrates. (Image courtesy of Professor S. Lucas, London, UK.) (b) Sickle cell disease patient with acute chest syndrome. Pulmonary artery is partially occluded with a necrotic bone marrow embolus. (Image courtesy of Professor S. Lucas, London, UK.) (c) Dilated small lung vessel clogged with sickled red blood cells. (Image courtesy of Professor S. Lucas, London, UK.) (d) Electron microscopic image of a sickled red blood cell in an alveolar capillary. (Courtesy of Professor P. Hasleton, Manchester, UK. Reproduced from P. Hasleton et al. Evolution of acute chest syndrome in sickle cell trait: an ultrastructural and light microscopic study.Thorax 1989;44:1057–8, with permission from the BMJ Publishing Group Ltd.)

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Chapter 18: Pulmonary vascular pathology

(a)

(b)

(c)

(d)

Figure 17. Thromboemboli. (a) Acute thromboemboli fill pulmonary arteries (arrows). (b) Recent thromboembolus. Alternating layers of platelets mixed with fibrin and darker layer containing red blood cells (Lines of Zahn) attest to the premortem nature of the blood clot. (c) Endothelial cell ingrowth into the thrombus leads to either resolution or recanalization. (d) Thrombi may be entirely removed by mesenchymal cells.

The media and adventitia of a thrombosed artery are usually unremarkable. There may be slight medial hypertrophy or atrophy, but the changes are usually mild. There are anecdotal reports of epithelioid granulomas within thrombi, not only in the vessel wall as in sarcoidosis, and of a diffuse myofibroblastic proliferation resembling

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inflammatory myofibroblastic tumor in a thrombus mass.175 Such combinations probably indicate secondary thrombus formation in a vascular inflammatory lesion, rather than inflammatory change in a thrombus. While the first phase of recognizable thrombus is a short one, the fibrotic remnants are long-lived. At any time in the

Chapter 18: Pulmonary vascular pathology

(a)

(c)

(b)

(d)

Figure 18. Post-thrombotic remnants. (a) Endothelial-lined channels traverse the thrombus and result in the formation of neolumina. These freely anastomosing spaces re-establish blood flow through the affected artery. (b) Post-thromotic pads of intimal fibrosis (EvG stain). (c) Fibrous webs or bands may be the only signs of prior thromboembolic events (EvG stain). (d) Fibrous bands may be seen macroscopically (arrows).

course of the disease, fibrotic remnants therefore greatly outnumber recent thrombi, creating a spurious impression that disease activity has abated. As the lesions are focal, many arteries may appear normal or near normal, even when they are severely stenosed at some point along their length. Documented severe pulmonary hypertension with a normal or near-normal appearance of pulmonary blood vessels in a biopsy is therefore suggestive of thrombotic pulmonary arteriopathy, and diagnostic lesions should be sought in step sections. Remodeling of small vessels, similar to other types of vasculopathy, has been described in thrombotic arteriopathy.207 This is likely to be the result of a chronic hyperdynamic flow state in the still patent pulmonary arteries after an acute thromboembolic event,38 or multiple thrombotic or thromboembolic occlusions of pulmonary arteries. One might expect this condition to result in a pattern of vasculopathy found in pulmonary hypertension cases associated with a hyperflow

state. Such a condition causes vascular damage, as in portopulmonary hypertension and left-to-right shunting, and thus may be a cause of plexogenic arteriopathy. In view of this, claims as to the presence of genuine plexiform lesions in some CTEPH patients are of interest.207 However, images of plexiform lesions in CTEPH have not convinced us.

Pulmonary infarcts Since the local pO2 in ventilated lung tissue is not low, and there is a dual lung blood supply, pulmonary thrombotic arterial occlusion alone is usually insufficient to cause parenchymal infarction. In fact, pulmonary infarcts are noted in less than 20% of autopsy lungs from patients with deep vein thrombi.208 However, when blood flow stagnates due to an outflow impediment, conditions for pulmonary infarction are met. Since congestive pulmonary vasculopathy, as in mitral valve insufficiency, is often complicated by arterial thrombosis, pulmonary infarcts may arise in that condition. Chest

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Chapter 18: Pulmonary vascular pathology

radiographs demonstrate a peripheral infiltrate while CT scan may feature a wedge-shaped subpleural consolidation with a rim of ground-glass opacity (Figure 19a). Incipient infarcts demonstrate capillary congestion with alveolar hemorrhage and edema but tissue necrosis is not seen (Figure 19b).209 If the collateral circulation is inadequate, tissue necrosis occurs (Figure 19c,d). Pleural-based wedge-shaped hemorrhagic lung with an apex pointing toward the hilum is composed of neutrophils and erythrocytes. Epithelial and endothelial cell nuclei disappear while elastic fragments remain, raising the impression of elastosis (Figure 19e,f). Organization starts at the periphery. Granulation tissue and hemosiderinladen macrophages replace the necrotic tissue and fill up alveolar spaces. Overlying pleura is thickened and inflamed. Squamous metaplasia and endarteritis obliterans are also noted. This tissue is ripe for infection or abscess formation. Infective thrombophlebitis, catheters or right heart valve endocarditis may be sources for such an infection.210,211 Cavitation may ensue. Completely organized infarcts shrink and often form thin scars. Overlying pleural adhesions may develop.

Differential diagnosis Some recanalized post-thrombotic lesions, especially when situated in a small vessel, may resemble a plexiform lesion.148 Post-thrombotic lesions are found in all types of pulmonary hypertensive vascular disease, so no special significance should be attributed to their occasional presence in plexogenic arteriopathy.144 However, plexiform lesions are distinct, rather than a stage in organization and canalization of a thrombus.153 Morphologically, the distinction is generally not too difficult. Arteries containing organized thrombi are generally larger than those incurring most damage in plexogenic arteriopathy.148 Post-thrombotic lesions rarely affect the proximal part of supernumerary arteries, which is the preferential site for plexiform lesions. Post-thrombotic lesions are less cellular, the endothelium and myofibroblasts have paler nuclei, and postlesional dilatation is not seen.

Non-thrombotic causes of pulmonary artery embolism There are several causes of embolic pulmonary hypertension, other than pulmonary thromboembolism. These were formerly classified in group 4,10 but seem to have lost their place in the updated classification. Some have been moved to group 5, presumably due to their rarity.11 Parasites, notably schistosoma eggs, can embolize to the pulmonary circulation, causing thrombosis and granulomatous inflammation (Figure 20). Chronic hepatosplenic schistosomiasis accounts for about a third of all pulmonary hypertension cases in endemic areas, such as Brazil.8 Data on incidence in other endemic areas for Schistosoma mansoni, such as the Nile (Sudan, Egypt) and sub-Saharan Africa, are scarce but unlikely to be more favorable.212 Once pulmonary hypertension has been established in schistosomiasis patients, antiparasitic therapy (e.g. praziquantel) has no effect on disease progression. Clinically, the presentation and course of the

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disease are said to resemble pulmonary arterial hypertension more than thrombotic/embolic disease.8 In Schistosoma mansoni infection, cercaria penetrate the skin and enter the circulation, homing to the mesenteric vascular bed, via the lungs. After this acute stage, the cercaria mature into adult helminths, which produce the highly antigenic eggs. In the early chronic stage of the disease, most of the eggs terminate in the liver, via the portal vein. At this site they evoke an intravascular granulomatous reaction, localized vasculitis with thrombosis and post-thrombotic changes, pipe stem fibrosis (Symmers’ fibrosis), and even cirrhosis. Increased portal pressure, and shunting and bypass of the liver via the collateral circulation, may develop. By this route, eggs can directly reach the pulmonary vascular bed, again causing vascular damage. This includes vascular occlusion by granulomas or post-thrombotic remodeling.131–133 Plexogenic arteriopathy appears to be the prevalent pattern of schistosoma-induced pulmonary hypertension. Portal hypertension presumably is an important contributing factor,13 since only a few granulomas are present.134 In most recent series, only low-grade or “burnt out” infections are seen. Thus, schistosomia-induced PH can be placed in classification groups 111 and/or 410 depending on severity and duration of their infection. Intravenously injected substances, such as used by drug addicts, may cause physical blockade or damage of pulmonary arterial branches, resulting in thrombosis and inflammation (Figure 21). Some of these substances can be recognized histologically, e.g. talc-containing materials are birefingent under polarized light. Material introduced by intervention radiologists attempting to block bleeding bronchial arteries in patients with bronchiectasis, vascular dysplasia and vascular malformations may also be encountered (Figure 22) (see Chapter 16). Finally, malignant tumors, particularly carcinomas, can embolize to the lungs and elicit local thrombosis and subsequent intimal fibrosis (Figure 23). These tumor cells may be so rare and inconspicuous that they are easily overlooked. In other instances, the converse is true and tumor cells occlude the vessels but a thrombotic reaction is absent. This is especially common when tumor clumps are covered by endothelium, as is the case with endocrine carcinomas (adrenal, thyroid), renal cell, and hepatocellular carcinomas (Figure 24). In rare cases, adenocarcinoma may grow in a lepidic fashion along the endothelial basement membrane of the pulmonary arteries (A.G. Nicholson, personal communication). Tumor emboli within blood vessels are generally easily distinguished from lymphangitic tumor spread. If there is doubt, a D2–40 immunohistochemical stain allows easy distinction, since lymphatic but not blood vessel endothelium reacts (Figure 24c). Pulmonary hypertension due to carcinoma emboli tends to run a rapidly progressive fatal course. Acute leukemia may cause acute pulmonary hypertension. While thrombosis is a recognized etiology in these patients, peripheral leukocyte counts exceeding 200 000/ml can lead to blockage in the absence of fibrin thrombi.213 Rarely,

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Figure 19. Lung infarct. (a) Computed tomogram demonstrating a right lower lobe wedge-shaped recent infarct. Note surrounding ground-glass opacity. (b) Wedge-shaped hemorrhage infarct with subpleural necrosis. An arterial thrombus is seen (arrow). (Courtesy of Keith Kerr FRCPath, FRCPEd, Aberdeen, UK.) (c) Subpleural hemorrhagic wedge-shaped infarct with thrombosed artery (arrow). The proximal artery is dilated. (d) Subpleural infarcted parenchyma features cell and stromal outlines. (e) Infarcts shrink and become elastotic. (f) While the infarct scars, the feeding artery undergoes recanalization (EvG stain).

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Figure 20. Pulmonary vascular disease associated with Schistosoma infection. (a) Pulmonary artery with granulomatous inflammation and many eosinophils around a Schistosoma egg. The adjacent pulmonary artery is occluded by intimal fibrosis and a swollen endothelium (Black 6 mouse, experimental Schistosoma mansoni infection) (EvG stain). (b) Remnant of a Schistosoma mansoni egg in a pulmonary artery with extensive intimal fibrosis.

Figure 21. Foreign body pulmonary emboli. Foreign body giant cell reaction to birefringent matter of unknown origin in a distended vessel, localized in the media of a pulmonary artery. This location suggests that the embolus is lodged in a supernumerary arterial branch.

Figure 22. Therapeutic bronchial artery embolization in a patient with pulmonary hemorrhage. “Blue-ish”, refractile, but not birefringent, foreign body in the wall of an elastic pulmonary artery.

Hypoxic arteriopathy pulmonary hypertension ensues from chronic myeloproliferative disorders, including myeloid metaplasia with myelofibrosis (agnogenic myeloid metaplasia) (see Chapter 34), essential thrombocythemia, polycythemia vera, myelodysplastic syndrome, and chronic myeloid leukemia214 (Group 5 Dana Point classification 200811). The etiology is unknown but the fact that symptomatic improvement may be seen after a single low dose of thoracic radiotherapy suggests that microvascular blockage by hematopoietic elements, such as megakaryocytes, may also play a role in this situation.215

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Epidemiology and causal factors As discussed above, the pulmonary circulation responds to a low oxygen tension with vasoconstriction. This response is beneficial in cases of local pulmonary hypoventilation, since it shifts blood flow away from areas where gas exchange will be incomplete. However, when hypoxia affects the entire lung, this essentially protective vasoconstrictive response becomes pathogenic, causing pulmonary hypertension. Hypoxic arteriopathy (Dana Point classification 2008 group 311) in its purest form is found in people living at high

Chapter 18: Pulmonary vascular pathology

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Figure 23. Thrombotic arteriopathy associated with carcinomatous pulmonary emboli. (a) An axial pulmonary artery is occluded by a partly organized thrombus mass with small clumps of tumor. (b) Immunohistochemical staining for CD31 (PECAM-1) illustrates endothelial ingrowth into the thrombus. Note the distended vasa vasorum in the adventitia. (c) Immunohistochemical staining for cytokeratin 7 confirms the presence of carcinoma within the thrombus mass. This patient presented with rapidly progressive pulmonary hypertension during pregnancy secondary to metastatic breast carcinoma.

altitudes, as in the high Andes and Himalayas.116,216 In some of these high-altitude dwellers it leads to chronic mountain sickness (Monge disease), i.e. polycythemia and hyperviscosity. This hampers perfusion and causes ventilation/perfusion (V/Q) mismatching. This, in turn, tips the balance towards

deeper hypoxemia rather than increased oxygen-carrying capacity. Hypoxemia causes further pulmonary vasoconstriction, pulmonary hypertension and, eventually, right heart failure.217 These symptoms and the neurological manifestations of Monge disease (headache, dizziness, depression, irritability

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Figure 24. Non-thrombotic tumor emboli. (a) A corner vessel is occluded by hepatocellular carcinoma. Note the cytoplasmic bile pigment. (b) Immunohistochemical staining for CD31 (PECAM-1) highlights tumor cells surrounded by endothelial cells. Note the absence of a host reaction. (c) Immunohistochemical staining for podoplanin (D2–40) shows the lymphatic vessels without tumor.

and coma) may be directly life-threatening. On descent to sea level, the clinical condition improves substantially; the pulmonary hypertension usually abates and may reverse to normal within 2 weeks. At lower altitudes, hypoxic arteriopathy commonly complicates chronic lung disease with a reduced diffusion capacity. This is particularly seen in chronic obstructive pulmonary disease (COPD) and fibrotic lung diseases.13,218 Alveolar

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hypoventilation, e.g., sleep apnea syndrome and morbid obesity, is also a well-known risk factor for hypoxic arteriopathy (Table 8).11,219 Pulmonary vascular hypoxia is not only caused by a low alveolar oxygen pressure. It can also be a consequence of a low mixed venous oxygen pressure. The determinants of central mixed venous O2 pressure (MVPO2) are central arterial oxygen saturation, cardiac output and peripheral oxygen

Chapter 18: Pulmonary vascular pathology Table 8 Conditions associated with hypoxic arteriopathy

Table 9 Pulmonary vascular lesions in hypoxic arteriopathy

Chronic obstructive lung disease

Medial hyperplasia, especially of small muscular pulmonary arteries

Pulmonary fibrosis Alveolar hypoventilation due to CNS dysfunction: i.e. Pickwickian syndrome, sleep apnea syndrome Neuromuscular disorders Thoracic skeletal disorders Upper airways obstruction Low ambient oxygen; high altitude dwellers

Table 10 Conditions associated with congestive vasculopathy

Left ventricular failure Mitral valve dysfunction Aortic valve dysfunction Congenital stenosis of major pulmonary veins or left atrial orifice Anomalous pulmonary venous connections or obstruction in left atrium (myxoma, cor triatriatum persistans) Mediastinal disease impeding pulmonary venous outflow (fibrosis, lymphadenopathy)

extraction. Peripheral oxygen extraction increases with exercise. This may result in a low MVPO2, if the cardiac output cannot keep pace with the increased oxygen demand, as is often the case in pulmonary hypertension. Thus, low MVPO2 and hypoxic pulmonary vasoconstriction often complicate established pulmonary hypertension of other etiologies.107

Histopathology of hypoxic arteriopathy (Table 9) Increased muscle content of small arterial branches is the most striking histological feature of hypoxic pulmonary vasculopathy. Small muscular pulmonary arteries show marked medial hyperplasia while arterioles normally devoid of a muscular coat develop a distinct muscular media sandwiched between inner and outer elastic laminae (so-called “muscularization of arterioles”) (Figure 25a,b).116,218 Large pulmonary arteries are affected to a much lesser extent, if at all. Pulmonary veins and venules also show a slight increase in smooth muscle content but not artertialization, as identified in congestive vasculopathy (see below).13,216,220 The arterial and arteriolar intima may feature some intimal fibrosis, in which smooth muscle differentiation may be detected (Figure 25c).218,220 Longitudinally oriented smooth muscle cells, lying as solitary cells or as small bundles along the inner side of the internal elastic lamina of pulmonary arteries and muscularized arterioles, are a common feature of hypoxic pulmonary vasculopathy. Such bundles are typically lined with newly formed elastic laminae. This finding is suggestive, but not pathognomonic, of hypoxic arteriopathy.

Muscularization of arterioles Intimal longitudinal smooth muscle bundles in small arteries and in arterioles Arterial adventitial thickening Mild increase in venous smooth muscle Intimal fibroelastosis

It may be encountered in other conditions, and may affect larger pulmonary, bronchial and systemic arteries, particularly when there is extensive vascular smooth muscle hyperplasia.13,221 Finally, some adventitial thickening of the pulmonary arteries may be observed in hypoxic pulmonary vasculopathy (Figure 25a). The adventitia is markedly thicker in the normal fetus and newborn, gradually decreasing to its adult thickness during the first years of life.13 Thus, in adults, the arterial adventitia is normally inconspicuous. Hypoxia-driven adventitial remodeling has been described in animals (reviewed by Stenmark et al.25). Reference values for adventitial thickness in humans are not available, so this feature has limited diagnostic utility. At best, a subjective division into “unremarkable” versus “thickened” adventitia can be made.

Congestive vasculopathy Epidemiology, causal factors

Chronic elevation of the pulmonary venous blood pressure results in a distinct set of adaptive pulmonary vascular changes, termed congestive pulmonary vasculopathy.150,222 The outflow obstruction is reflected by an elevated wedge pressure (> 15 mmHg) at right heart catheterization. This finding excludes pulmonary arterial hypertension as a single diagnosis. Most cases result from left ventricular failure or mitral valve dysfunction (Dana Point classification 2008 group 211) (Table 10). Fewer cases are due to venous obstruction at the level of the large pulmonary veins. Hilar, mediastinal or left atrial tumor, and fibrosing mediastinitis are examples (Dana Point classification 2008 group 511). Congestive pulmonary vasculopathy is one of the major causes of non-PAH PH (formerly known as secondary PH) and may occasionally be a component of mixed pattern vasculopathy, particularly in the elderly.

Histopathology of congestive vasculopathy (Table 11) Arterialization of veins is the most distinctive vascular alteration in congestive pulmonary vasculopathy.126 Arterialized veins resemble muscular pulmonary arteries as they acquire distinct inner and outer elastic laminae sandwiching a compact layer of medial smooth muscle. Veins, particularly

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Figure 25. Hypoxic arteriopathy. (a) Medial hyperplasia and cellular intimal proliferation of a small muscular pulmonary artery. Note the prominent adventitia. (b) This corner vessel has a recognizable media sandwiched between inner and outer elastic laminae. (c) There is an almost complete formation of a neo-elastica.

when arterialized, may be difficult or impossible to distinguish from pulmonary arteries by morphology alone (Figure 26a–c). Their identity is revealed by their localization in the interlobular septa. Pulmonary arteries show medial hyperplasia, which may be prominent and out of proportion compared to the venous changes (Figure 26b). The marked arterial medial hyperplasia is paralleled by the well-known observation that the rise in pulmonary artery pressure usually exceeds that of the venous pressure.91 Characteristically, there is substantial thickening of the arterial adventitia. This is a feature shared by hypoxic arteriopathy, but it tends to be more marked in congestive vasculopathy.126,223 Secondary findings of chronic congestion include intraalveolar edema, dilated lymphatics, interstitial edema, and relatively diffuse interstitial fibrosis (Figure 26d). Increased

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numbers of mast cells are seen. Intra-alveolar calcification and/or focal ossification may arise (Figure 26e). Interstitial fibrosis is often accompanied by siderophages, probably resulting from minor recurrent extravasation of blood at the capillary level (Figure 26f). Focal encrustation of venous elastin fibers by extracellular iron salts is often seen (Figure 26g).224 When subtle, these important diagnostic clues can be easily overlooked, unless an iron stain is performed. At the other end of the spectrum, marked interstitial fibrosis and iron pigmentation results in so-called “brown induration of the lung”. This term is derived from the macroscopic appearance seen in autopsies of patients with longstanding left ventricular failure or diseased aortic and/or mitral valves. Pulmonary infarction is not a feature of uncomplicated congestive vasculopathy. If present, it points to a mixed

Chapter 18: Pulmonary vascular pathology Table 11 Pulmonary vascular lesions in congestive arteriopathy

Pulmonary arteries: Prominent medial hyperplasia of muscular pulmonary arteries and muscularization of arterioles Eccentric, non-laminar, non-obstructive intimal fibrosis extending over long stretches Marked adventitial thickening Pulmonary veins: Medial hypertrophy and arterialization Mild to moderate intimal fibrosis Lymphatics: Dilatation Lung tissue: Interstitial edema Interstitial fibrosis Hemosiderosis Microlithiasis, osseous nodules (rare)

pattern of congestion, in combination with thrombotic occlusion of the pulmonary artery, as in CTEPH. As mentioned above, mitral valve insufficiency in particular has been identified as a risk factor for pulmonary infarcts, probably due to venous stasis.13 Congestive pulmonary vasculopathy often regresses to a significant extent when its cause is removed, i.e., by valvular surgery. It lacks the self-perpetuation and progressive deterioration characteristic of all but the earliest stages of plexogenic arteriopathy.

Pulmonary veno-occlusive disease and pulmonary capillary hemangiomatosis Introduction

Pulmonary veno-occlusive disease (PVOD) and pulmonary capillary hemangiomatosis (PCH) are rare but severe and often rapidly progressive pulmonary hypertensive diseases characterized by a decreased diffusion capacity out of proportion to a mild elevation of pulmonary arterial pressure.225 Patients may have (occult) alveolar hemorrhage. Although PVOD/PCH has been placed in group 1 of the Dana Point classification 2008,11 the histopathological pattern of vascular disease is very different from plexogenic arteriopathy seen in most other entities of group 1. PVOD-like disease was first described in 1938.226 The term PVOD was proposed by Heath et al. in 1966.227 In 1978, a case of extensive and destructive capillary congestion with venous and venular obstructive intimal fibrosis was described by Wagenvoort et al.228 Despite its resemblance to PVOD, the capillary congestion was deemed so prominent, and in excess of what could be explained by congestion, that Wagenvoort proposed the term PCH. Since then, the terms PVOD and PCH have sometimes been used interchangeably, and the term

PCH has even occasionally been used for intense capillary congestion outside the context of pulmonary hypertension.229,230 Evidence that PCH is a capillary proliferative disease has, so far, not been substantiated.231 When both PVOD and PCH are considered within the context of pulmonary hypertension, PCH overlaps considerably with PVOD clinically, radiologically, histologically, and in their adverse response to prostanoids.28,29,228,232–234 It seems likely that PCH and PVOD are closely related or may even represent manifestations of the same disease entity.233,234

Epidemiology and clinical features PVOD/PCH is not easily diagnosed by clinical and radiological parameters and a surgical lung biopsy may be required.225 As a consequence, its true incidence is difficult to ascertain; it has been estimated to account for about 5–10% of all PAH initially diagnosed as idiopathic.235 Most patients present in adulthood, but some pediatric cases have been reported.234 In the pediatric population, both sexes are equally affected. In the adult population, PVOD/PCH affects men more frequently than women in a 2:1 ratio.28,225,235,236 PVOD, or a pattern of vascular lesions closely resembling it, complicates a number of connective tissue diseases, particularly SSc and occasionally SLE. An association has also been found with autoimmune thyroid disease. Interestingly, the Raynaud phenomenon often precedes the development of PVOD and anti-endothelial antibodies have been described in connective tissue diseases that may feature a PVOD-like pattern.71–73 PVOD may develop as a late complication of radiotherapy, especially for Hodgkin lymphoma.237 Some chemotherapeutic agents, including BCNU, bleomycin and mitomycin,238 have also been incriminated (see Chapter 16). Radiotherapy and drug toxicity directed at endothelial cells is the hypothesized mechanism. Additional cases have been associated with previous bone marrow or stem cell transplantation,239–241 and heart and/or lung transplantation. It is postulated that the pathology is secondary to obliterative bronchiolitis along with arterial and venous intimal fibrosis.242,243 This rare vascular disease also develops in individuals with granulomatous angiitis.244 Finally, PVOD has been reported to occur as a familial disease.245 BMPR2 mutations have been identified in several cases (Table 12).28,34,246 PVOD/PCH tends to run an inexorable downhill course and most patients die within two years of diagnosis.246 Importantly, vasodilator drugs, such as prostacyclin, are usually less effective than in other types of PAH. They should be used with caution as they may precipitate an attack of acute pulmonary edema.232,246 At present, the only effective treatment is lung transplantation, but preliminary evidence suggests a possible beneficial effect of imatinib (STI571), a PDGFR tyrosine kinase inhibitor.79–83,247 Case reports indicate the possibility of occurrence229 or recurrence248 after bilateral lung transplant.

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Chapter 18: Pulmonary vascular pathology Table 12 Conditions associated with PVOD/PCH

Radiological features of PVOD/PCH

Hyper- and hypothyroidism

High-resolution chest CT is useful in the diagnosis of PVOD/ PCH. In addition to revealing general features of pulmonary hypertension and a possible underlying disease, it may demonstrate several characteristic features not seen in other forms of PAH. These include diffusely distributed centrilobular groundglass opacities and/or mosaic lung attenuation.30,249,250 In addition, subpleural septal lines, so called Kerley B lines, are often noted and represent widened interlobular septa (Figure 6).246 The latter feature is predominantly subpleural. This feature is particularly telling when observed in the upper lung fields, where general perfusion is low compared to the bases. The caliber of pulmonary veins is normal, while mediastinal lymph node enlargement is seen in some patients. Pleural and/or

Late complication of radiotherapy, notably after Hodgkin lymphoma Chemotherapeutic agents: BCNU, bleomycin, mitomycin Previous bone marrow or stem cell transplantation Collagen vascular diseases, particularly systemic sclerosis/CREST syndrome, occasionally in SLE (often mixed pattern, including PVOD-like changes) Granulomatous angiitis Familial

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Figure 26. Congestive vasculopathy. (a) Distended and arterialized vein with marked thickening of the media situated in the interlobular septa. Note the markedly dilated lymphatic (EvG stain). (b) Distended vein with intimal fibrosis (left), and a bronchovascular bundle (right). The muscular artery features medial hyperplasia and prominent adventitia (EvG stain). (c) Venous intimal fibrosis is striking (EvG stain). (d) Diffuse alveolar septal fibrosis with collections of intra-alveolar hemosiderin-laden macrophages are seen. (e) Intra-alveolar bone formation in long-standing congestion. (f) Hemosiderin-laden macrophages are prominent on a Perls’ iron stain. (g) Interstitial iron salt deposition around a small vein (Perls’ iron stain).

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pericardial effusions do not discriminate between PVOD/PCH and other types of PAH or CTEPH. The radiologist must also ensure the heart and cardiac valves are normal since causes of left atrial hypertension can mimic PVOD/PCH.

Histopathology (Table 13) PVOD/PCH is characterized by focal progressive fibrotic narrowing and obliteration of venules and small veins. Patchy subpleural parenchymal congestion and often infarction are seen (Figure 27a,b). This fibrotic venular obliteration is characteristically loosely textured and associated with a mild lymphocytic infiltrate in approximately half of cases (Figure 27c–e).234 Webs and colander type lesions may be found in veins and venules (Figure 27f,g). Significantly, larger (interlobular) veins are normal or near normal, and there is no arterialization. The characteristic resultant patchy congestion227,228,234,251–253 contrasts with the more diffuse congestion of congestive vasculopathy; however, diffuse congestion can be observed. Bleeding from these engorged capillaries probably explains the hemoptysis some patients experience. Hemosiderin deposition is frequently seen in the periphery of lobules beneath the visceral pleura and interlobular septa. Calcium and iron salt encrustation of venular and alveolar wall elastic fibers may cause a giant cell response,254 sometimes referred to as pseudo-pneumoconiosis or endogenous pneumoconiosis (Figure 27h,i). Such ferruginous encrustation can be seen on H&E sections as black staining of elastin fibers, resembling the elastin fibers in an Elastic van Gieson stain. Their nature is confirmed with an iron stain (Figure 27i). This feature is not entirely specific, as it can occasionally be seen also in congestive vasculopathy, though usually to a lesser extent.224 Occasionally small arteries and arterioles also display intimal fibrosis, but to a far lesser degree than seen in veins. Thrombosis and recanalization are common (Figure 27j). In fact, the term vaso-occlusive rather than veno-occlusive disease is preferred by some.236 As in congestive vasculopathy, pulmonary arteries show medial hyperplasia, which on occasion may be prominent, drawing attention away from the subtle but diagnostically more telling focal venular pathology (Figure 27k). Lymphatics are often dilated and may resemble lymphangiomatosis lesions. The term PCH was originally applied to PVOD-like cases with focal areas consisting of a pronounced increase in patent capillaries within alveolar walls and bronchovascular bundle interstitium (Figure 28a–c).228,252,255 The process is now characterized by patchy and sometimes lobular interstitial proliferations of small blood-filled capillary-like vascular channels. Endothelial cells are cytologically bland (Figure 28d,e). Venous infiltration leads to intimal fibrosis and veno-occlusive changes (Figure 28f). Arteries are not spared and demonstrate medial and intimal changes (Figure 28g). The cause of this striking increase in capillaries is unclear; its histology with gradual transitions in the severity of the proliferation and absence of cellular atypia suggests a reactive

rather than a neoplastic process.231 As PCH appears to be otherwise similar to PVOD, PCH probably represents the severe end of a spectrum of capillary congestion and perhaps a proliferative capillary response, resulting from local venular obstruction. The variations in descriptions of PVOD/PCH may relate in part to the fact that PVOD/PCH is associated with various conditions, including SLE and SSc.145,256 Plexiform lesions are not part of the PVOD/PCH pattern,227,234,236,253 despite occasional claims to the contrary.10 However, both plexogenic arteriopathy and the PVOD pattern of pulmonary vascular disease may occur in the context of various connective tissue diseases, such as SLE. Although our knowledge of connective tissue diseases and vascular pathology is still limited, it is clear that an underlying connective tissue disease should be excluded in cases with PVOD/PCH histology.5 Histological evidence favoring a connective tissue disease-associated process includes the presence of diffuse interstitial pneumonitis and (lymphocytic) vasculitis. Interstitial fibrosis should be distinguished from that associated with longstanding congestion.

Differential diagnosis Lesser degrees of focal congestive changes, reminiscent of PVOD/PCH, may occasionally be observed in various situations associated with pulmonary parenchymal congestion, such as (congestive) heart disease (see below),255,257,258 with vasodilative prostacyclin treatment (authors’ observation), or rarely as an incidental finding.229,230 There is histological overlap between congestive vasculopathy and PVOD/PCH. Clinical information, hemodynamic parameters (notably wedge pressure) and HR-CT scan findings28–30,249 must be considered. Usually, congestion in congestive vasculopathy tends to be diffuse rather than patchy, as in PVOD, but exceptions occur. The distinction is best appreciated in areas that have the lowest hydrostatic pressure, i.e. the ventral-upper areas, where the patchy aspect of PVOD/PCH stands out more clearly. Veins tend to be more severely affected than venules in congestive vasculopathy, whereas small venous and venular intimal fibrosis are more prominent in PVOD/PCH. Pronounced capillary congestion from any cause, including left heart disease, may mimic capillary hemangiomatosis.229,230,255,257,258

Congenital heart disease with hypo- or hyperperfusion Cardiac malformations with pulmonary hypoflow

A minority of cardiac malformations result in pulmonary hypoperfusion (Table 14). Fallot’s tetralogy is the best known example. Congenital pulmonary stenosis, valvular or infundibular, or within the pulmonary trunk or a peripheral artery may rarely occur as an isolated anomaly. In the latter case, the vascular changes are limited to the vessels

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(k) Figure 27. Pulmonary veno-occlusive disease (PVOD)/Pulmonary capillary hemangiomatosis (PCH). (a) Macroscopic section demonstrating prominent interlobular septa and pulmonary infarction. A venous thrombus (arrow) is also noted. (b) Subpleural congestion with an edematous interlobular septum and scattered prominent alveolar wall proliferations are typically seen in PVOD/PCH. Dilated lymphatics resemble lymphangiomatosis lesions. (c and d). This pulmonary vein features edematous intimal fibrosis. Congested alveolar capillaries and intra-alveolar hemosiderin-laden macrophages are also noted (panel c: Movat pentachrome stain). (e) The vein may be practically obliterated. (f and g) Thrombosed veins may recanalize (panel f: Movat pentachrome stain). (h and i) Iron encrustation of a small venule with giant cell reaction is a common finding. (j) Arterial thrombosis and recanalization is commonly seen. (k) Medial arterial hypertrophy is often seen.

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Figure 28. Pulmonary veno-occlusive disease (PVOD)/Pulmonary capillary hemangiomatosis (PCH). (a) Capillary congestion in a lung from a patient with PVOD extends into the bronchiolar mucosa (EvG). (b) Scattered nodular lesions are noted at low magnification. (c) The pulmonary interstitium is expanded by capillary-sized vascular channels. (d) Alveolar septa are not spared. (e) Flat to cubodial endothelial cells are cytologically bland. (f) A pulmonary vein adjacent to a capillary lesion features intimal proliferation. (g) Pulmonary arteries also feature intimal and medial changes.

distal to the obstruction. In tetralogy of Fallot, the flow in the pulmonary vascular bed depends on the size of the ventricular septal defect in relation to the pulmonary stenosis. When a right-to-left shunt is present, the lungs are perfused via the ductus arteriosus, in which case pressure may be normal, although flow is greatly diminished.259–261 This situation is further complicated by polycythemia and a tendency to thrombosis, as a consequence of hypoxemia and low flow.

The histopathological features of cardiac malformations with pulmonary hypoflow are listed in Table 15. Hypoflow causes medial atrophy of muscular pulmonary arteries.13,262 This may not be easily appreciated, as the normal pulmonary arterial media is already thin. In some cases the vessel wall is exceedingly thin, so the internal and external elastic laminae can barely be seen as separate layers. This medial atrophy may be explained by the greatly diminished pulse pressure, even when a normal mean arterial pressure is maintained.13

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Chapter 18: Pulmonary vascular pathology Table 13 Pulmonary vascular lesions in PVOD/PCH

Table 14 Congenital cardiac defects associated with pulmonary hypoperfusion

Pulmonary veins and venules Focal obstructive intimal fibrosis, initially of loose texture, within venules and small, pre-septal veins Mild medial hypertrophy and arterialization of larger veins (occasionally) Pulmonary arteries Medial hyperplasia Intimal changes similar to those in venules, but generally to a far lesser extent (variable) Recent thrombi (variable) Lung tissue Patchy congestion, evolving to focal interstitial fibrosis Prominent focal hemosiderosis Iron salt encrustation of vascular elastin fibers (“pseudopneumoconiosis”)

Isolated pulmonary artery stenosis or atresia Tricuspid valve atresia Tetralogy of Fallot Peripheral obstruction of the pulmonary arteries Unilateral stenosis or absence of the main pulmonary arterya Distal to an acquired occlusion, e.g. an organized thrombus (uncommon) a

This condition may be found in persistent ductus arteriosus. As a result, there will be hyperflow in the contralateral lung, which may be severe enough to cause plexogenic arteriopathy.

Table 16 Congenital heart defects associated with left-to-right (systemicto-pulmonary) shunts

Atrial septal defect (ASD) Table 15 Vascular lesions in pulmonary hypoperfusion

Ventricular septal defect (VSD)

Pulmonary arteries Dilatation Medial atrophy Post-thrombotic intimal fibrosis Intra-arterial thin fibrous septa and colander lesions

Atrioventricular septal defect (AVSD)

Pulmonary veins Dilatation Intimal fibrosis and septa (occasionally)

Acquired (surgical) left-to-right shunt for tetralogy of Fallot

Especially in tetralogy of Fallot, there is marked dilatation of arteries, which can be appreciated when they are compared to the accompanying bronchiole. Pulmonary veins are also dilated. In tetralogy of Fallot, post-thrombotic lesions develop over time, and are usually present from adolescence onwards. These typically present as thin, fibrotic septa and colander lesions within widened, thin-walled arteries. These post-thrombotic lesions are uncommon in isolated pulmonary stenosis. Extensive collaterals between the bronchial and pulmonary arteries provide the lungs with additional blood flow.

Cardiac malformations with pulmonary hyperflow Congenital cardiac malformations, resulting in systemic-topulmonary (left-to-right) shunting, lead to pulmonary hyperflow and eventually to pulmonary hypertension (group 1 of the Dana Point classification 200811) (Table 16). Intracardiac shunts can be either pre-tricuspid, i.e. atrial-septal defects, or post-tricuspid, e.g. ventricular-septal defects. Both types of shunting lead to pulmonary hyperflow, but post-tricuspid shunts are generally more damaging to the pulmonary arterial tree, since they also transmit the high pressure of the left ventricle and aorta to the pulmonary trunk. Accordingly, post-tricuspid shunts lead to pulmonary

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Transposition of the great arteries with VSD Truncus arteriosus persistens Patent ductus arteriosus

hypertension early in life, though symptoms rarely emerge before the age of 2 years. The chance of advanced and irreversible plexogenic pulmonary arteriopathy is substantial in the case of an uncorrected large post-tricuspid shunt, but minimal in a pre-tricuspid one. Pulmonary hypertension caused by pre-tricuspid shunts rarely manifests clinically before adulthood.13 The histopathological consequences of cardiac defects associated with hyperflow are similar to those described in detail under general features and plexogenic arteriopathy. Briefly, early and histologically nonspecific changes consist of medial hypertrophy and cellular intimal fibrosis of muscular pulmonary arteries. More advanced disease is identified by proliferative responses and dilatation of small branches, leading to focal fibrinoid necrosis of supernumerary arteries, concentric laminar intimal fibrosis, plexiform lesions, veinlike branches and angiomatoid lesions. In the presence of advanced lesions, the pulmonary hypertension has progressed to an irreversible stage and even progresses, despite surgical correction of the causative cardiac defect.130,259 Ultimately, the progressive pulmonary hypertension leads to diminution of the shunting, and may even reverse the direction of flow. The resultant right-to-left shunt, which leads to severe systemic hypoxemia, is known as Eisenmenger syndrome, and always denotes irreversibility of the pulmonary hypertension.130,136 Although the prognosis is ultimately dismal, it is

Chapter 18: Pulmonary vascular pathology

not uncommon for patients with this condition to survive for several years.263 In the past, open lung biopsies were undertaken to assess the reversibility of pulmonary vascular changes.130,141,264,265 Improved diagnostic testing and pediatric surgical techniques generally lead to repairs at a young age, before irreversible pulmonary artery damage occurs. For this reason, lung biopsies are infrequently performed. To categorize the severity of plexogenic arteriopathy due to a cardiac defect, Heath and Edwards proposed a grading system in 1958.150 The system has fallen into disuse on account of several shortcomings. Their higher-grade lesions (grades 4–6) do not reflect increasingly severe disease and the system does not take into consideration the severity and extensiveness of the intimal fibrosis. Subsequent studies have shown that patients with mild intimal fibrosis do better than those with severe concentric laminar intimal fibrosis.130 The grading system has also been inappropriately used by others outside the context of congenital heart disease and in histological patterns of pulmonary vascular disease other than plexogenic arteriopathy. Wagenvoort was correct in concluding over 20 years ago that the Heath-Edwards classification was obsolete and should be discarded.266

Pulmonary vasculopathy in pulmonary hypertension associated with connective tissue diseases Introduction Pulmonary hypertension is a serious complication of several connective tissue diseases (see Chapter 21). It is most frequent in SSc and SLE. The other diseases that may cause pulmonary hypertension, albeit less frequently, include mixed connective tissue disease, rheumatoid arthritis, dermatomyositis and Sjögren disease.128,267 Fibrosis, vascular inflammation, in situ thrombosis and pulmonary thromboembolism (due to underlying hypercoagulabilty states) all contribute to the development of pulmonary hypertension in these patients.268–271 Anti-endothelial antibodies are commoner when connective tissue diseases are associated with pulmonary hypertension.103 Antibody-mediated endothelial injury may be the basis of vascular remodeling and the fibrotic, reparative response.269 In addition to intrapulmonary causes, the pulmonary hypertension may be due to passive elevation in the pulmonary artery pressure caused by right ventricular diastolic dysfunction.272 Cardiac failure, mainly due to pericarditis and/or pericardial effusion, ischemic heart disease, myocarditis, congestive heart failure and valvular heart disease may substantially contribute to the pulmonary hypertension in this group of patients.273 It is imperative to determine the cause(s) of pulmonary hypertension, as the treatments are very different. It is in these complex cases of pulmonary hypertension associated with (suspected) connective tissue

disease that the value of the information obtained from a surgical lung biopsy not uncommonly outweighs the risk of the biopsy procedure.

Systemic sclerosis Systemic sclerosis (previously known as scleroderma) and the CREST syndrome (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactyly and telangiectasia) are expressions of an autoimmune disease (see Chapter 21). Deposition of excessive amounts of extracellular matrix components, endothelial dysfunction and altered immune tolerance result in thickening and tightening of the skin, and ulcers on the fingers, cutaneous hyperpigmentation and subcutaneous calcification. Pulmonary hypertension commonly develops as a consequence of extensive pulmonary fibrosis.274–276 Even without fibrosis, patients are at risk of developing pulmonary hypertension, especially in the limited cutaneous form of systemic sclerosis. Other predisposing factors include female gender and an advanced age at onset of the disease.277,278 The estimated prevalence of pulmonary hypertension is 8 to 12%.274,279,280 Characteristically, in cases of pulmonary hypertension lacking pulmonary fibrosis (referred to as SScPAH), there is a disproportionate reduction in the diffusion capacity, while the pulmonary arterial pressure is only moderately elevated.278,281 The short-term prognosis in SScPAH compares unfavorably to idiopathic pulmonary arterial hypertension (iPAH), with 3-year survival rates of 50% for SScPAH vs. 84% for iPAH.282,283 Those with extensive pulmonary fibrosis have an even poorer prognosis.274,276 A favorable response to various therapies (epoprostanol, sildenafil, calcium channel blockers) has been reported.278,284,285 This response is less noticeable in SScPAH compared to iPAH.286,287 Age, gender, mixed venous oxygen saturation and WHO functional class are independent predictors of survival in isolated SScPAH.288 This disease is in group 1 of the Dana Point classification 2008.11 Pulmonary hypertensive vasculopathy associated with SSc is characterized by extensive intimal fibrosis of pulmonary arteries and arterioles of all sizes (Table 17).13,256,289,290 The prominence and widespread nature of these changes, particularly in small vessels without plexiform lesions, distinguishes SScPAH from plexogenic arteriopathy.145 Concentric-laminar intimal fibrosis, with an onion skin-like layering of cells and matrix in a greatly thickened intima, may be seen (Figure 9a,b).13,145,256 In small vessels, the intimal fibrosis is usually concentric but not necessarily concentric-laminar. It ranges from cellular or paucicellular loose fibrosis, to compact collagen-rich and pauci-cellular fibrosis (Figure 29a–c). Some affected small vessels may show a densely collagenous or muscular layer of tissue beneath the endothelium, separated from the elastic lamina (a single elastic lamina at this level) by a zone of loose connective tissue.13,145 On H&E stains, this picture may

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Chapter 18: Pulmonary vascular pathology Table 17 Pulmonary vascular lesions in systemic sclerosis-associated vasculopathy

Pulmonary arteries Medial hyperplasia Generalized intimal fibrosis, extending into parenchymal arterioles (common) Smooth muscle or densely collagenous subintimal fibrotic rim in corner vessels Concentric laminar intimal fibrosis (occasional) Venules PVOD-like changes (occasionally): focal venular intimal fibrosis associated with patchy congestion Lung tissue Interstitial fibrosis (nonspecific interstitial pneumonia pattern) (variable)

easily be mistaken for medial hyperplasia, as in hypoxic arteriopathy. The position of this layer, in relation to the elastic lamina, is readily recognized by the EvG stain (Figure 10). In larger arteries this phenomenon may raise the impression of a double media.13 In large vessels, as in any type of pulmonary hypertension, intimal fibrosis is more commonly eccentric. Intravascular accumulation of lymphocytes and transmural lymphocytic infiltrates, indicative of lymphocytic vasculitis, can occasionally be seen (Figure 29d).13,145 However, arteritis and fibrinoid necrosis, as seen in severe plexogenic pulmonary arteriopathy, are uncommon in SScPAH.13,145 In a subset of patients, patchy congestion is reminiscent of PVOD. Also, hemosiderosis and encrustation of iron on vascular elastin fibers (pseudo-pneumoconiosis) are occasionally seen.145,256,291 Such indicates that widespread smallvessel intimal fibrosis involves at least some of the venules and correlates with anecdotal reports of pulmonary edema developing in PVOD patients treated with NO or prostanoids.28,232,292,293 Plexiform lesions have been reported in one study,251 but we and others have not encountered unequivocal examples.145,256,275,289,290

Systemic lupus erythematosus (SLE) Pulmonary hypertension complicating SLE has been reported in up to 14% of published cases, corresponding to approximately 25% of patients with pulmonary symptoms.273,294,295 Pulmonary hypertension may develop as a consequence of interstitial fibrosis, vasculitis, or thrombotic vasculopathy. Antiphospholipid, anticardiolipin and/or antilupus antibodies probably promote this process.167,270,271 In addition, pulmonary hypertension may develop due to cardiac failure, mainly as a result of pericarditis and/or pericardial effusion, systemic arterial hypertension, ischemic heart disease, myocarditis, congestive heart failure or valvular disease.273 Raynaud’s phenomenon is frequently present

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in SLE.296,297 It is associated with a higher pulmonary artery systolic pressure, implicating vascular spasm as a contributor to the raised pulmonary vascular resistance.297,298 Apart from therapy directed specifically at pulmonary hypertension, treatment of the SLE is beneficial.5,128,299 SLE-associated PAH is placed in group 1 of the Dana Point classification 2008.11 The lesions encountered in SLE-associated pulmonary hypertension vary according to the causes, and are usually heterogeneous, even within a single patient. In heart failure, congestive vasculopathy is seen. In the absence of cardiac failure and an elevated wedge pressure, the patterns of vasculopathy may vary from medial hyperplasia of the pulmonary arteries300,301 to, most commonly, full-blown plexogenic arteriopathy.302–305 Capillary hemangiomatosis has been reported in SLE, together with intimal fibrosis, thrombotic lesions256,301,306 and hemorrhagic pulmonary edema.256 The latter might be attributed to prostacyclin therapy. Vasculitis, including fibrinoid necrosis, has been found,301,303 even after steroid therapy, in a case of refractory pulmonary hypertension.307 Thrombotic disease may be more prominent than in other forms of pulmonary hypertension,302,308 particularly in the presence of antiphospholipid, anticardiolipin antibodies and/or lupus anticoagulant.167,270 Aneurysmal dilatation of vessels is a recognized complication.301 Vasoconstrictive mechanisms, similar to those operative in Raynaud’s phenomenon, may contribute to increased pulmonary vascular resistance, but are impossible to evaluate histologically.13,298

Pulmonary hypertension in interstitial lung diseases Any interstitial inflammatory and/or fibrosing lung disease may eventually be complicated by pulmonary hypertension.309,310 Connective tissue diseases are categorized in group 1, fibrosing lung diseases are in group 3, and systemic and metabolic diseases are in group 5 in the Dana Point classification 2008.11 About 30–40% of cases with end-stage fibrosing interstitial lung diseases develop pulmonary hypertension, most commonly in idiopathic pulmonary fibrosis (up to 84%) and sarcoidosis (6%) (see Chapters 10 and 13).311 Metabolic disorders, including Gaucher disease, can also cause pulmonary hypertension (Figure 30) (see Chapter 11). Pulmonary hypertension often requires lung transplantation.309,312 In view of the differing causes, the patterns of vasculopathy are likely to be diverse and heterogeneous. In general, pulmonary hypertension is thought to result from loss of vascular bed due to vascular remodeling and vascular destruction associated with progressive parenchymal fibrosis, vasculitis, perivascular fibrosis, thrombotic angiopathy, hypoxia, fluid overload, or a combination of these factors.13,218,313 Any fibrotic and/or inflammatory change of lung parenchyma is associated with intimal

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(a)

(b)

(c)

(d)

Figure 29. Vascular disease in systemic sclerosis. (a) Concentric laminar intimal fibrosis. (b) Loose textured intimal fibrosis (EvG stain). (c) Collagenous pauci-cellular intimal fibrosis (EvG stain). (d) A thin-walled vessel features transmural lymphoid infiltrates. Necrosis is not seen.

fibrosis, medial hyperplasia and adventitial thickening of arteries and veins.13 Extensive post-thrombotic type intimal fibrosis and vasculitis are often seen in cases with organizing pneumonia or diffuse alveolar damage. This phenomenon is referred to as endarteritis obliterans. Obliteration of small vessels may be less striking histologically, but is probably significant hemodynamically. When a focal lung lesion is involved, these changes may be beneficial, since they probably divert the blood flow to normal or less affected parts of the lung. In diffuse interstitial lung disease, the resultant increase in vascular resistance causes pulmonary hypertension. Symptoms of pulmonary hypertension in interstitial lung disease are nonspecific, but a disproportionate reduction in

diffusion capacity, severe hypoxemia or oxygen desaturation upon exercise may indicate the need for further diagnostic procedures, including right heart catheterization.128,309 Besides treatment of the underlying disease and specific treatment for pulmonary hypertension, a diagnosis of pulmonary hypertension complicating interstitial lung disease warrants listing for lung transplantation, if age and (co-)morbidity permit.309 There are several possible causes of pulmonary hypertension in sarcoidosis.309 Within the lungs, extensive fibrosis in longstanding disease is common.311 In addition, mediastinal lymphadenopathy or significant left ventricular myocardial involvement may compromise pulmonary venous outflow, causing congestive vasculopathy. Granulomatous

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(a)

(b)

Figure 30. Gaucher disease. (a) Alveolar septal and airspace accumulation of Gaucher cells (EvG stain). (b) Gaucher cells percolate through the layers of a small vessel. Plexogenic arteriopathy may result.

Figure 31. Sarcoidosis. Granulomatous venulitis destroys the elastic lamina and partially occludes the lumen (EvG stain).

vasculitis, affecting mostly the pulmonary veins, is not uncommon in sarcoidosis (Figure 31).314–316 Rarely, liver cirrhosis with portal hypertension may account for the pulmonary hypertension.

Pulmonary hypertension in vasculitis Vasculitis can lead to pulmonary hypertension (group 5 in the Dana Point classification 200811) (see Chapter 19). Focal, low-grade lymphocytic vasculitis may be a feature of a number of autoimmune diseases, including but not limited to SSc, rheumatoid arthritis and SLE. Vasculitis is a hallmark

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of Behçet disease,317 and “Wegener’s-like vasculitis” has been reported in ulcerative colitis.318,319 Vasculitis usually affects only short segments of vessels at one point in time. Therefore, multiple tissue levels should be studied when one’s clinical suspicion is high. As the endothelium is activated and/or damaged in vasculitis, thrombosis and its sequelae may ensue. This creates the spurious impression that thrombosis is the primary cause of the disease. Pulmonary vasculitis may be detected by chance in the absence of any histological or clinical etiological clues.13 To establish the correct diagnosis, the vasculitic lesions should be actively sought in non-thrombosed vessels. Fragmentation of the elastic laminae suggests previous vasculitis. Necrotizing arteritis is occasionally seen in severe, advanced plexogenic arteriopathy and is considered a consequence, rather than a cause of the hypertension. This type of arteritis morphologically resembles polyarteritis nodosa (PAN). PAN is more likely to involve the bronchial arteries, while pulmonary arteries are usually spared. PAN is therefore an unlikely cause of pulmonary hypertension.13 A few lymphocytes may be found within vessel walls at the site of any organizing thrombus, and should not be interpreted as vasculitis. Infectious thromboemboli should be included in the differential diagnosis. Extensive post-thrombotic intimal fibrosis may be seen with vasculitis in cases of organizing pneumonia. This endarteritis obliterans is considered a bystander effect, rather than a clue as to the cause of the organizing pneumonia.

Acknowledgements The authors gratefully acknowledge Professor Anton VonkNoordegraaf, MD, PhD, pulmonary physician at VU University Medical Center Amsterdam, for critical review

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of this chapter, and his contribution on clinical and radiological diagnosis of pulmonary hypertension, Dr. Rutger J. Lely, MD, radiologist at VU University Medical Center

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antibody-mediated microvascular injury in the evolution of pulmonary fibrosis in the setting of collagen vascular disease. Am J Clin Pathol 2007;127(2):237–47. 270. Ostrowski RA, Robinson JA. Antiphospholipid antibody syndrome and autoimmune diseases. Hematol Oncol Clin North Am 2008;22(1):53–65, vi. 271. Tanaseanu C, Tudor S, Tamsulea I, et al. Vascular endothelial growth factor, lipoporotein-associated phospholipase A2, sP-selectin and antiphospholipid antibodies, biological markers with prognostic value in pulmonary hypertension associated with chronic obstructive pulmonary disease and systemic lupus erithematosus. Eur J Med Res 2007;12(4):145–51. 272. Overbeek MJ, Lankhaar JW, Westerhof N, et al. Right ventricular contractility in systemic sclerosis-associated and idiopathic pulmonary arterial hypertension. Eur Respir J 2008;31(6):1160–6. 273. Badui E, Garcia-Rubi D, Robles E, et al. Cardiovascular manifestations in systemic lupus erythematosus. Prospective study of 100 patients. Angiology 1985;36(7):431–41. 274. Mathai SC, Hummers LK, Champion HC, et al. Survival in pulmonary hypertension associated with the scleroderma spectrum of diseases: Impact of interstitial lung disease. Arthritis Rheum 2009;60(2): 569–77. 275. Young RH, Mark GJ. Pulmonary vascular changes in scleroderma. Am J Med 1978;64(6):998–1004. 276. Mathai SC, Hummers LK, Champion HC, et al. Survival in pulmonary hypertension associated with the scleroderma spectrum of diseases: Impact of interstitial lung disease. Arthritis Rheum 2009;60(2):569–77. 277. Schachna L, Wigley FM, Chang B, et al. Age and risk of pulmonary arterial hypertension in scleroderma. Chest 2003;124(6):2098–104.

arterial hypertension in systemic sclerosis: a French nationwide prospective multicenter study. Arthritis Rheum 2005;52(12):3792–800. 280. Mukerjee D, St George D, Knight C, et al. Echocardiography and pulmonary function as screening tests for pulmonary arterial hypertension in systemic sclerosis. Rheumatology (Oxford) 2004;43(4):461–6. 281. Sun XG, Hansen JE, Oudiz RJ, Wasserman K. Pulmonary function in primary pulmonary hypertension. J Am Coll Cardiol 2003;41(6):1028–35. 282. Fisher MR, Mathai SC, Champion HC, et al. Clinical differences between idiopathic and scleroderma-related pulmonary hypertension. Arthritis Rheum 2006;54(9):3043–50. 283. Kawut SM, Taichman DB, ArcherChicko CL, Palevsky HI, Kimmel SE. Hemodynamics and survival in patients with pulmonary arterial hypertension related to systemic sclerosis. Chest 2003;123(2):344–50. 284. Badesch DB, Tapson VF, McGoon MD, et al. Continuous intravenous epoprostenol for pulmonary hypertension due to the scleroderma spectrum of disease. A randomized, controlled trial. Ann Intern Med 2000;132(6):425–34. 285. Badesch DB, Hill NS, Burgess G, et al. Sildenafil for pulmonary arterial hypertension associated with connective tissue disease. J Rheumatol 2007;34(12):2417–22. 286. Girgis RE, Mathai SC, Krishnan JA, Wigley FM, Hassoun PM. Long-term outcome of bosentan treatment in idiopathic pulmonary arterial hypertension and pulmonary arterial hypertension associated with the scleroderma spectrum of diseases. J Heart Lung Transplant 2005;24(10):1626–31. 287. Mathai SC, Girgis RE, Fisher MR, et al. Addition of sildenafil to bosentan monotherapy in pulmonary arterial hypertension. Eur Respir J 2007;29(3):469–75.

278. Steen V, Medsger TA Jr. Predictors of isolated pulmonary hypertension in patients with systemic sclerosis and limited cutaneous involvement. Arthritis Rheum 2003;48(2):516–22.

288. Condliffe R, Kiely DG, Peacock AJ, et al. Connective tissue disease associated pulmonary arterial hypertension in the modern treatment era. Am J Respir Crit Care Med 2009;179:151–7.

279. Hachulla E, Gressin V, Guillevin L, et al. Early detection of pulmonary

289. al Sabbagh MR, Steen VD, Zee BC, et al. Pulmonary arterial histology and

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morphometry in systemic sclerosis: a case-control autopsy study. J Rheumatol 1989;16(8):1038–42. 290. Yousem SA. The pulmonary pathologic manifestations of the CREST syndrome. Hum Pathol 1990;21(5):467–74. 291. Morassut PA, Walley VM, Smith CD. Pulmonary veno-occlusive disease and the CREST variant of scleroderma. Can J Cardiol 1992;8(10):1055–8. 292. Humbert M, Sanchez O, Fartoukh M, et al. Short-term and long-term epoprostenol (prostacyclin) therapy in pulmonary hypertension secondary to connective tissue diseases: results of a pilot study. Eur Respir J 1999;13(6):1351–6. 293. Preston IR, Klinger JR, Houtchens J, et al. Pulmonary edema caused by inhaled nitric oxide therapy in two patients with pulmonary hypertension associated with the CREST syndrome. Chest 2002;121(2):656–9. 294. Bertoli AM, Vila LM, Apte M, et al. Systemic lupus erythematosus in a multiethnic US Cohort LUMINA XLVIII: factors predictive of pulmonary damage. Lupus 2007;16(6):410–7. 295. Simonson JS, Schiller NB, Petri M, Hellmann DB. Pulmonary hypertension in systemic lupus erythematosus. J Rheumatol 1989;16(7):918–25. 296. Hodson P, Klemp P, Meyers OL. Pulmonary hypertension in systemic lupus erythematosus: a report of four cases. Clin Exp Rheumatol 1983;1(3):241–5. 297. Kasparian A, Floros A, Gialafos E, et al. Raynaud’s phenomenon is correlated with elevated systolic pulmonary arterial pressure in patients with systemic lupus erythematosus. Lupus 2007;16(7):505–8. 298. Callejas-Rubio JL, Lopez-Perez L, Moreno-Escobar E, Ortego-Centeno N. Raynaud’s phenomenon and pulmonary arterial hypertension. Lupus 2008;17(4):355. 299. Hennigan S, Channick RN, Silverman GJ. Rituximab treatment of pulmonary arterial hypertension associated with

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systemic lupus erythematosus: a case report. Lupus 2008;17(8):754–6. 300. Wilson L, Tomita T, Braniecki M. Fatal pulmonary hypertension in identical twins with systemic lupus erythematosus. Hum Pathol 1991;22(3):295–7. 301. Fayemi AO. Pulmonary vascular disease in systemic lupus erythematosus. Am J Clin Pathol 1976;65(3):284–90. 302. Yokoi T, Tomita Y, Fukaya M, et al. Pulmonary hypertension associated with systemic lupus erythematosus: predominantly thrombotic arteriopathy accompanied by plexiform lesions. Arch Pathol Lab Med 1998;122(5):467–70. 303. Roncoroni AJ, Alvarez C, Molinas F. Plexogenic arteriopathy associated with pulmonary vasculitis in systemic lupus erythematosus. Respiration 1992;59(1):52–6. 304. Kanemoto N, Sato M, Moriuchi J, et al. An autopsied case of systemic lupus erythematosus with pulmonary hypertension – a case report. Angiology 1988;39(2):187–92. 305. Samet P, Bernstein WH. Loss of reactivity of the pulmonary vascular bed in primary pulmonary hypertension. Am Heart J 1963; 66:197–9. 306. Fernandez-Alonso J, Zulueta T, ReyesRamirez JR, Castillo-Palma MJ, Sanchez-Roman J. Pulmonary capillary hemangiomatosis as cause of pulmonary hypertension in a young woman with systemic lupus erythematosus. J Rheumatol 1999;26(1):231–3. 307. Srock K, Kerr LD, Poon M, Fallon JT. Refractory pulmonary hypertension in a lupus patient with occult pulmonary vasculitis. J Clin Rheumatol 2003;9(4):263–6. 308. Yutani C, Imakita M, Ishibashi-Ueda H, et al. Pulmonary thromboembolic hypertension in systemic lupus erythematosus with lupus anticoagulant: histopathological analysis of localization and distribution of thromboemboli in pulmonary vasculature. Intern Med 1995;34(10):1030–4.

309. Ryu JH, Krowka MJ, Pellikka PA, Swanson KL, McGoon MD. Pulmonary hypertension in patients with interstitial lung diseases. Mayo Clin Proc 2007;82(3):342–50. 310. Behr J, Ryu JH. Pulmonary hypertension in interstitial lung disease. Eur Respir J 2008; 31(6):1357–67. 311. Handa T, Nagai S, Miki S, et al. Incidence of pulmonary hypertension and its clinical relevance in patients with sarcoidosis. Chest 2006;129(5):1246–52. 312. Shigemitsu H, Nagai S, Sharma OP. Pulmonary hypertension and granulomatous vasculitis in sarcoidosis. Curr Opin Pulm Med 2007;13(5):434–8. 313. Hopkins N, McLoughlin P. The structural basis of pulmonary hypertension in chronic lung disease: remodelling, rarefaction or angiogenesis? J Anat 2002; 201(4):335–48. 314. Nunes H, Humbert M, Capron F, et al. Pulmonary hypertension associated with sarcoidosis: mechanisms, haemodynamics and prognosis. Thorax 2006;61(1):68–74. 315. Smith LJ, Lawrence JB, Katzenstein AA. Vascular sarcoidosis: a rare cause of pulmonary hypertension. Am J Med Sci 1983;285(1):38–44. 316. Judd PA, Finnegan P, Curran RC. Pulmonary sarcoidosis: a clinicopathological study. J Pathol 1975;115(4):191–8. 317. Uzun O, Erkan L, Akpolat I, et al. Pulmonary involvement in Behçet’s disease. Respiration 2008;75(3):310–21. 318. Kasuga A, Mandai Y, Katsuno T, et al. Pulmonary complications resembling Wegener’s granulomatosis in ulcerative colitis with elevated proteinase-3 antineutrophil cytoplasmic antibody. Intern Med 2008;47(13):1211–4. 319. Gal AA, Velasquez A. Antineutrophil cytoplasmic autoantibody in the absence of Wegener’s granulomatosis or microscopic polyangiitis: implications for the surgical pathologist. Mod Pathol 2002;15(3):197–204.

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19

Pulmonary vasculitis and pulmonary hemorrhage syndromes Eugene J. Mark, Rex Neal Smith, John H. Stone, Douglas B. Flieder, Amita Sharma and Osamu Matsubara

Introduction Vasculitis is defined generically as inflammation of a vessel. Hemorrhage is defined generically as the escape of blood from vessels. Were pulmonary vasculitis and pulmonary hemorrhage so directly connected that inflammation of blood vessels led to rupture and then hemorrhage, the classification of the vasculitic and hemorrhagic syndromes would be considerably easier. However, inflamed vessels may leak or not leak; inflammation in vessels may have great or little clinical or pathophysiological significance; and hemorrhage may be due to trauma, vascular anomalies, pulmonary hypertension, or a bleeding diathesis in the absence of inflammation or vascular necrosis. There are many schemes for classifying vasculitis.1,2–4 One can classify vasculitis by organ involvement, etiology, histology, size of blood vessel, immunological parameters, cellular mediation, or associated systemic disease. Various categorizations are given in Table 1. In this chapter we separate the large and complex topic into three major groups: antineutrophil cytoplasmic antibody (ANCA)-associated diseases; pulmonary hemorrhage syndromes; and idiopathic vasculitides that rarely affect the lung. Many other entities, including but not limited to pulmonary infections, interstitial lung diseases such as sarcoidosis/necrotizing sarcoid granulomatosis and Langerhans cell histiocytosis, pulmonary hypertension, drugs, transplantation, radiation, systemic processes including connective tissue disease and inflammatory bowel disease, and malignancies, especially lymphoproliferative disorders such as lymphomatoid granulomatosis, cause pulmonary vasculitis and/or hemorrhage. These entities are discussed elsewhere in this text.

ANCA-associated diseases In 1982 antibodies directed against neutrophil cytoplasmic antigens were first described in patients with pauci-immune glomerulonephritis, and thought to be associated with Ross River virus infections.5 Within several years, though, autoantibodies were linked to Wegener granulomatosis (WG), ChurgStrauss syndrome (CSS) and microscopic polyangiitis (MPA)

as well as idiopathic crescentic glomerulonephritis.6,7 These findings confirmed the mid-1950 belief of Godman and Churg that the entities shared a pathogenic mechanism.8 ANCA interactions with neutrophils are likely responsible. In vitro ANCA can activate neutrophils to produce reactive oxygen species and the release of lytic enzymes.9 Animal models also support a pathogenic role for some of the diseases.10 ANCA is pathogenic in humans. A 2005 report documenting placental transmission of maternal P-ANCA (anti-myeloperoxidase; MPO) antibodies leading to pulmonary-renal syndrome in the newborn serves as a definite human model (see below).11 When the sera of patients with ANCA-associated vasculitis are incubated with ethanol-fixed human neutrophils, two major immunofluorescence staining patterns are recognized. Patients with antibodies directed against proteinase-3 (PR3), a 29-kDa serine protease found in lysosomal granules and on plasma membranes of neutrophils and monocytes, demonstrate diffuse cytoplasmic staining of neutrophils.12,13 This pattern is termed cytoplasmic ANCA (C-ANCA) (Figure 1). Ninety-five percent of C-ANCAs have specificity for PR3. Patients with antibodies directed against myeloperoxidase (MPO) demonstrate a perinuclear staining pattern.6 This pattern is termed peri-nuclear ANCA (P-ANCA) (Figure 2). While MPO is also a cytoplasmic protein, ethanol fixation causes the positively charged granules to rearrange around the negatively charged nuclear membrane. A P-ANCA staining pattern is also caused by antibodies directed against lactoferrin, elastase, catalase, to name a few.14 Approximately 90% of P-ANCAs have specificity for MPO. While disease associations with ANCAs will be discussed below, Table 2 lists generally accepted sensitivity values of C-ANCA and P-ANCA for WG, CSS and MPA. Two ANCA test types are available. Indirect immunofluorescence assay and enzyme-linked immunosorbent assay (ELISA). The former uses alcohol-fixed buffy coat leukocytes, and the latter uses purified specific antigens. The immunofluorescence assay is more sensitive while the ELISA is more specific. Patients should be screened with immunofluorescence assays, and positive results confirmed with ELISA.15,16

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Table 1 Vasculitis: manners of categorization

Etiology Infectious Bacterial Mycoplasmal Viral Fungal

Scleroderma Dermatomyositis Mixed connective tissue disease Inflammatory bowel disease Ulcerative colitis Crohn disease

Immunological Cell-mediated Transplant rejection Graft versus host ANCA-related Hypertensive Cryoglobulinemia Toxin Drug Radiation Histological reaction pattern Normal Fibrinoid necrosis Leukocytoclastic Eosinophilic Granulomatous

Figure 1. Cytoplasmic ANCA immunofluorescence pattern. Most cases with this complete cytoplasmic staining pattern have antibodies directed against proteinase-3.

Giant cell Lymphocytic Plasmacytic Obliterative Size and type of vessels Large artery Polyarteritis nodosa Giant cell arteritis Behçet syndrome Wegener granulomatosis Mid to small artery Wegener granulomatosis Churg-Strauss syndrome Microscopic polyarteritis Capillary Connective tissue disease, especially systemic lupus erythematosus Wegener granulomatosis Henoch-Schönlein purpura Vein Wegener granulomatosis Inflamed veno-occlusive disease Associated systemic disease Sarcoid Connective tissue disease Rheumatoid

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Figure 2. Perinuclear ANCA immunofluorescence pattern. Most cases with this perinuclear staining pattern have antibodies directed against myeloperoxidase.

However, several caveats regarding ANCA results should be noted. ANCA testing is not standardized and lacks reference values for normal ranges. Immunofluorescence assay interpretation is quite subjective and the results are not highly specific. C-ANCA has a greater specificity than P-ANCA for vasculitis, but positive C-ANCA immunofluorescence results may only be associated with a vasculitis in 50% of patients.17 ANCA positivity has been reported in patients with virtually all nonvasculitic rheumatic disorders. A P-ANCA pattern is

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Table 2 C-ANCA and P-ANCA disease associations

Disease

PR3-ANCA (%)

MPO-ANCA (%)

Wegener granulomatosis

66

24

Microscopic polyangiitis

26

58

Churg-Strauss syndrome

1500 cells/mm3 Evidence of vasculitis involving at least two organs 1990 American College of Rheumatology based on clinical information with or without pathological material (four or more features must be present) Asthma

Histopathology

Small vessel vasculitis?

Tissue infiltration by eosinophils?

Pathogenesis

ANCA-related

Toxic products from eosinophils

Modified from213.

Eosinophilia > 10% Mononeuropathy (including multiplex) or polyneuropathy Migratory or transient pulmonary opacities detected radiographically Paranasal sinus abnormality Biopsy with extravascular eosinophils 1994 Chapel Hill Consensus Conference based on clinical information and pathological material Eosinophil-rich and granulomatous inflammation involving the respiratory tract Necrotizing vasculitis affecting small to medium sized vessels Asthma and eosinophilia 181,210,211

From

.

states that CSS features eosinophil-rich and granulomatous inflammation involving the respiratory tract, and necrotizing vasculitis affecting small to medium-sized vessels associated with asthma and eosinophilia. It is satisfying for pathologists to note that the European League Against Rheumatism recommends that histology should be obtained in all cases to assist in diagnosis.212 Recent medical advances and research further complicate attempts to neatly classify this vasculitic process. Since no more than 50% of patients with CSS demonstrate ANCA positivity, it is uncertain whether the ANCA-positive group represents a different disease entity from the ANCA-negative patients (see Table 9).213,214 It is postulated that the ANCAassociated process has small-vessel vasculitis-driven findings while the ANCA-negative patients have pathology secondary to eosinophilic infiltration of organs. The former may be associated with renal involvement, peripheral neuropathy and biopsy-proven vasculitis, while ANCA-negative status might be associated with heart disease and fever.215

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Epidemiology CSS is extremely rare, with an incidence between 1.3 and 6.8 cases per one million patients per year and a prevalence of 10 to 14 per million patients.216–218 The incidence is higher in asthmatics, ranging from 34 to 64 cases per one million patients per year.219,220 A slightly higher prevalence is reported in northern Europe as compared to southern Europe, and in urban rather than rural areas. Caucasian ethnicity is most common.216 A sex preponderance is not reported.218 Most patients are in their fifth or sixth decade of life.214,215,221 Children may be afflicted.222

Genetics Rare case reports document CSS amongst relatives of WG patients.223 Several genetic factors may be risk factors. Susceptibility foci include HLA-DRB1*04 and HLADRB1*07.216,224,225 It is intriguing to note that the HLA-DRB4 gene, present in patients carrying either of the above-mentioned alleles, is far more frequent in CSS patients than in controls. Furthermore, its frequency correlates with vasculitic symptoms rather than those secondary to eosinophilic tissue infiltration.224 HLA-DRB3 is reportedly associated with protection against the disease.224 IL-10, a pleomorphic cytokine involved in immune modulation, has also been studied. The IL10.2 haplotype, associated with increased IL10 expression, is significantly associated with ANCA-negative CSS.226 This finding suggests a real difference between the ANCA-positive and ANCA-negative disease forms.

Clinical manifestations CSS usually develops through three successive phases. However, not all patients have clear-cut stepwise progression as up

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Table 10 Churg-Strauss syndrome: main clinical features

Asthma Eosinophilia > 10% Sinusitis Lung involvement Peripheral neuropathy Skin involvement Heart involvement Gastrointestinal tract involvement Renal involvement Central nervous system involvement

to 20% may have overlapping manifestations from the different phases.218,227 The prodromal phase features asthma and allergic manifestations including allergic rhinitis and nasal polyposis. Patients are usually in their 30s and the phase can last from 3 to 30 years, although most patients progress within 8 to 10 years.211,228 The eosinophilic phase follows and manifests with peripheral blood eosinophilia and eosinophilic infiltration of organs. The upper and lower respiratory tracts, heart and gastrointestinal tract are favored sites. Such infiltrates may manifest as Löffler syndrome or chronic eosinophilic pneumonia, eosinophilic gastroenteritis or myocarditis. The third and final phase heralds the appearance of a life-threatening small and medium-sized vasculitis. Peripheral nerves, skin and kidneys are often affected. This phase also features signs and symptoms from the prodromal and eosinophilic phases. One can only diagnose CSS during the vasculitic phase.211 Almost all patients present with fatigue, malaise, fever and weight loss. The most frequently involved organs are the lungs, peripheral nerves, skin, gastrointestinal tract and heart.211 Recent clinical studies found that only 50% of patients have pulmonary disease aside from asthma (see Table 10).214,215,221 Radiographic infiltrates and pleural effusions may contribute to shortness of breath. Rhinorrhea and nasal polyps are the most common upper respiratory tract manifestations. Peripheral neuropathies are the most common extrapulmonary manifestations. Mononeuritis multiplex and asymmetric or symmetric sensory or sensorimotor polyneuropathies are more frequent than Guillain-Barré-like syndromes. The common peroneal nerve is most frequently involved, and those with mononeuritis multiplex suffer with motor palsy and sensory deficit including painful hyperesthesia and muscular atrophy.229 Central nervous system manifestations including cranial nerve involvement or optic neuritis are rare.230 Palpable purpura on the legs and feet, cutaneous nodules or papules and sometimes migratory urticarial rashes are seen in up to 75% of patients. Gastrointestinal symptoms include diarrhea, bleeding, ascites and pain. Eosinophilic enteritis involves the small intestine more often than the stomach or colon.231,232 Small bowel

Figure 42. Pulmonary Churg-Strauss syndrome. Axial chest computed tomogram demonstrates bilateral mainly peripheral ground glass opacities.

obstruction or perforation is a rare event. Pancreatitis and cholecystitis are also reported.233 Cardiac involvement is noted in up to 50% of cases and is the leading cause of death. Myocarditis, coronary artery vasculitis, valvular abnormalities, congestive heart failure and pericarditis are reported.214 Renal abnormalities, present in approximately 25% of patients, include ANCA-associated necrotizing crescentic glomerulonephritis, eosinophilic tubulointerstitial nephritis and mesangial proliferative glomerulonephritis. Obstructive uropathy secondary to ureter or prostate involvement are also rare occurrences.234

Radiographic findings Parenchymal and airway abnormalities are detected on chest X-ray or CT.235–238 Parenchymal opacification, either consolidation or ground-glass opacity, is reported in 60% of cases238 and corresponds to eosinophilic pneumonia, organizing pneumonia or granulomatous vasculitis. It may be transient, is usually bilateral and symmetrical, and has a peripheral predilection (Figure 42).235,238,239 Centrilobular nodules may be seen in areas of ground-glass opacity and are secondary to small vessel vasculitis or eosinophilic bronchiolitis.235 Larger nodules, measuring up to 3 cm in diameter, have been reported but are less common than in WG. Ground-glass halos surround many of the nodules secondary to eosinophilic and giant cell infiltration (Figure 43).235 Septal thickening is a common finding secondary to edema and eosinophilic infiltration.239 Airway involvement secondary to muscle wall hypertrophy and eosinophil wall infiltration occurs in more than one third of cases.235,237,238 It presents on CT as bronchial wall thickening with or without dilatation and lobular

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Figure 44. Pulmonary Churg-Strauss syndrome. Eosinophils infiltrate a small pulmonary artery and surrounding alveoli. Airspaces also contain fibrin.

Figure 43. Pulmonary Churg-Strauss syndrome. Axial chest computed tomogram features multiple lung nodules with ground glass halos (arrows).

hyperinflation. Parenchymal abnormalities in treated patients resolve more completely than the airway manifestations.240 Pleural effusions are reported in 30% of cases.241 Pericardial effusions and mediastinal lymphadenopathy are rare.237,242 The radiological differential diagnosis includes chronic eosinophilic pneumonia and WG. Homogeneous peripheral consolidation favors a diagnosis of chronic eosinophilic pneumonia, while larger nodules favor WG.

Macroscopic pathology Pulmonary hemorrhage or nodules are noted in a small percentage of samples.

Histopathology There are three major pulmonary findings in CSS. Diagnostic cases feature necrotizing eosinophilic vasculitis, tissue infiltration with eosinophils, and extravascular granulomas. However, no more than 20% of patient samples contain all three findings.243 Only rarely will a transbronchial biopsy suffice. Of note, detailed studies of ANCA-positive versus ANCAnegative morphologies have not been reported. Vasculitis usually involves small to medium-sized arteries and/or veins (Figures 44 and 45). Eosinophils along with lymphocytes, neutrophils, histocytes and even multinucleated giant cells infiltrate small vessel walls. Fibrinoid necrosis is common. Cicatricial vascular fibrosis indicates inactive lesions. While the entire pulmonary lobule may be involved, vasculitis can be confined to a single compartment, such as the interlobular septa or bronchovascular bundle. Capillary involvement manifests with capillaritis and pulmonary hemorrhage. Tissue infiltration with eosinophils includes typical airway findings seen in asthma, and/or eosinophilic pneumonia.

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Figure 45. Pulmonary Churg-Strauss syndrome. The arterial media is filled with eosinophils while the intima is starting to collapse.

Bronchial walls are thickened with prominent basement membranes, hypertrophy of submucosal glands and smooth muscle, edema and eosinophils. Intra-alveolar accumulations of eosinophils along with macrophages and fibrin are also seen (Figure 46). Extravascular granulomas, also referred to as “allergic granulomas”, are distinctive parenchymal lesions. These serpiginous or geographic zones of eosinophilic necrosis are lined by palisading histiocytes and multinucleated giant cells (Figure 47). Allergic granulomas are often more prominent that the vasculitic component. Non-pulmonary organ involvement usually manifests with vasculitis and/or eosinophilic infiltrates. Eosinophilic infiltrates in the upper respiratory tract are seen more often than vasculitis. Eosinophilic myocarditis leads to fibrosis. Coronary artery vasculitis causes thrombosis and myocardial infarction. Acute fibrinous pericarditis leads to restrictive pericarditis. Vasculitis of the vaso nervorum leads to peripheral nerve dysfunction.

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

Figure 46. Pulmonary Churg-Strauss syndrome. Parenchymal infiltration with eosinophils, epithelioid histiocytes and multinucleated giant cells (arrow) may lead to hyaline membrane formation.

Cytology Bronchoalveolar lavage samples contain many eosinophils, occasional Charcot-Leyden crystals, and perhaps blood. Pleural effusions are usually exudative and eosinophil-rich.241

Laboratory findings

Figure 47. Pulmonary Churg-Strauss syndrome. Allergic granulomas, not unlike Wegener granulomatosis, feature geographic necrosis.

Blood hypereosinophilia, high IgE titers and P-ANCA positivity are the main laboratory abnormalities seen in CSS patients. Serum eosinophil levels fluctuate during the disease and disappear with corticosteroid therapy. While an eosinophil count exceeding 1500/mm3 is one of the diagnostic criteria for disease, mean values range from 4400 to almost 8200.244 Nonspecific findings include elevated ESR and C-reactive protein, leukocytosis, hypergammaglobulinemia and elevated complement (C3, C4, CH50) levels. IgE is also elevated at diagnosis in 75% of patients but is nonspecific and usually not seen in those taking steroids for asthma.229 No more than 50% of CSS patients are P-ANCA-positive with specificity for MPO. Less than 5% demonstrate C-ANCA positivity. Interestingly, positive rheumatoid factor is reported in more than half of studied pateints.211

IL-13.249 These cytokines are involved in eosinophil differentiation, activation, proliferation and adhesion to endothelium, and tissue infiltration.250 Granuloma formation is associated with heightened T-helper type-1 cytokines, including tumor necrosis factor-a, interferon-g and IL-2.250–252 It has also been reported that patients at disease onset or relapse have fewer CD4þCD25þ T-cells producing IL-10, and fewer CD4þCD25- T-cells producing IL-2 than seen in those in remission, and those with chronic eosinophilic pneumonia or asthma.253 Increased numbers of T-helper type-17 are also reported.254 Decreased eosinophil apoptosis may also contribute to disease development.251 Altered humoral immunity probably plays a role. Elevated IgE levels, rheumatoid factor and the presence of ANCA in some cases represent indirect evidence.255

Pathogenesis

Differential diagnosis

Our understanding of this multiphase disease is extremely limited. It is not known whether ANCA plays a pathogenic role or is merely associated with vasculitic manifestations. Proposed disease triggers include desensitization treatment, inhaled antigens, free-base cocaine and the use of leukotriene receptor antagonists (LTRA).245 Whether LTRA cause CSS or unmask an underlying disease by allowing for steroid reduction in asthmatics is not certain.221 Associations between CSS development and other asthma medications, including beta-agonists and oral corticosteroids, are also noted.246–248 An imbalance of major effector T-cell subpopulations may play a large pathogenetic role. Allergic features suggest a major role for T-helper type-2 cytokines, such as IL-4, IL-5, IL-10 and

The light microscopic differential diagnosis for CSS is broad and includes WG, eosinophilic pneumonia, fungal and parasitic diseases, as well as drug-induced vasculitis. Morphological features of CSS and WG are identical in lung samples except that CSS always contains many eosinophils. Distinguishing the two may be impossible in the 5% of WG cases with striking eosinophilia.122 Hence, one must rely on clinical features such as serum eosinophilia, peripheral neuropathy and cardiac involvement in CSS, as opposed to destructive upper airway disease and severe renal disease in WG. ANCA subtyping is not useful since overlap occurs. If clinical features of CSS are lacking, then one should diagnose WG with prominent eosinophils.

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

Eosinophilic pneumonia and allergic bronchopulmonary fungal disease differ morphologically from CSS in that they lack necrotizing vasculitis. Non-necrotizing vasculitis, however, may be seen (see Chapter 15). Parasitic infections, most notably Strongyloides stercoralis and Toxocara canis, present with asthma and systemic illness.256 Lung morphology is similar to CSS findings. Excluding parasitic infections is an integral part of the clinical workup. Stool and sputum samples should be examined and one should be aware that finding organisms in tissue sections is exceedingly rare. Serological and molecular tests may be useful (see Chapter 8). Many drugs can cause eosinophilic pneumonia and vasculitis. Carbamazine and estrogen may lead to necrotizing granulomatous vasculitis (see Chapter 16).257,258

Prognosis and natural history Although fatal before the advent of corticosteroid therapy, overall 5-year survival for CSS patients is greater than 70% and in some series approaches 97%.221,259 However, prognosis depends on the initial disease extent and organs involved. A five-factor score is used to guide therapy. Patients are assessed for cardiac involvement, gastrointestinal disease, renal insufficiency, proteinuria and central nervous system involvement. In addition, 5-year mortality rates correlate with the score. The reported 5-year mortality rate for patients with none of the factors is 12%, 26% when one factor is present and 46% when three or more are present.260 Vasculitis is the main cause of death, while cardiac disease, namely cardiomyopathy and myocardial infarction, gastrointestinal complications including bleeding and mesenteric infarction, and status asthmaticus are also implicated.229 Patients are usually treated with steroids, while refractory or severe disease also requires cyclophosphamide. Plasma exchange is not beneficial.261 Following remission induction, steroids are tapered and either azathioprine or methotrexate replaces cyclophosphamide. Treatment-associated side-effects are usually not severe and are reversible. However, infectionrelated deaths do occur.259 While approximately 90% of patients enter remission, 25% relapse. Rates approach 75% for those treated with only steroids.259 Most relapses occur during the first year of follow-up and, apart from a rise in serum eosinophilia, are difficult to predict. Thus, maintenance therapy, corticosteroid-sparing therapies and eosinophil monitoring are very important.

Goodpasture syndrome Introduction Goodpasture syndrome (GPS) or anti-basement membrane antibody disease receives its eponym from Ernest Goodpasture. While on leave from Harvard Medical School to study the influenza pandemic after World War I, he described systemic vasculitis in an 18-year-old male who died of

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pulmonary hemorrhage.262 Although the disease that Goodpasture described was different from the entity that now carries his name,263 GPS is a well recognized cause of pulmonary hemorrhage and/or acute renal failure. The eponym describes the triad of diffuse pulmonary hemorrhage, glomerulonephritis and circulating anti-basement membrane (anti-BM) antibodies. Unlike other pulmonary-renal syndromes, many of which are part of a systemic illness, GPS manifestations are limited almost exclusively to the lungs and kidneys.264–268

Epidemiology The incidence of GPS is unknown but is considered to be less than 1 case/million population/year.269 It represents no more than 2% of all cases of rapidly progressive glomerulonephritis. Both sexes are affected, with a male:female ratio ranging from 2:1 to 9:1. Patient ages range from 10 years to the elderly, with a bimodal distribution featuring incidence peaks at 20 to 30 years and 60 to 70 years.270 A Caucasian ethnicity is also the predominant ethnic background.271 Lung and renal disease are more likely to afflict young white males, while exclusive renal disease can be seen in elderly women.272 A strong correlation with smoking exists, while hydrocarbon exposure is an uncertain association.271,273–275 De novo GPS also occurs in patients with Alport disease who have received a kidney transplant.276,277

Genetics Disease rates are notably higher in siblings, cousins and identical twins.272 There are strong positive and negative associations between class II genes and GPS, suggesting that immune response genes are predisposing factors in antibody and disease development.278,279 HLA DRB1–15, DRB1–4, DRW2, DQB1 and DQA1 are strongly associated with disease. In fact, DRB1–15 alleles have been found in 70–90% of patients of western European descent compared with no more than 30% in the general population. However, since 30% of the nonaffected population have this allele, other factors must exist. Conversely, HLA DRB1–7, DRB1–1 and DPB1–4 are dominantly protective, the latter in Chinese patients.279–285

Clinical manifestations Manifestations vary greatly. Between 60% and 80% of patients have clinical manifestations of both pulmonary and renal disease, between 20% and 40% have only renal disease and less than 10% have disease limited to the lungs.272 Hemoptysis is the presenting symptom in most patients with lung involvement and almost all patients experience hemoptysis during the disease.286 Hemoptysis has been reported up to 12 years before clinical diagnosis.272 Blood loss is initially small but massive hemoptysis may occur. This presentation leads to rapid respiratory failure and is the most common cause of death.272 In addition, patients often present

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Figure 48. Pulmonary Goodpasture syndrome. Axial chest computed tomogram demonstrates diffuse centrilobular ground-glass nodules secondary to pulmonary hemorrhage.

with a flu-like prodrome of weakness, lethargy and sometimes cough. Individuals with circulating anti-BM antibodies and ANCA may have significant constitutional symptoms.287 Crackles can be heard on chest auscultation. Azotemia, a slightly elevated ESR, and an active urine sediment featuring proteinuria, either gross or microscopic hematuria and red blood cell casts are noted. Iron deficiency anemia is present.271 Pulmonary function testing is rarely helpful in the clinical evaluation of these patients; however, elevated diffusing capacity for carbon monoxide (DLCO) may be noted. This is secondary to the binding of inhaled carbon dioxide to intraalveolar hemoglobin. This test is useful when working up a patient for recurrent disease. In fact, recurrent pulmonary hemorrhage may be diagnosed when a patient presents with two of the following three criteria: fresh alveolar opacities on chest radiograph; a 30% rise in DLCO; and/or an otherwise unexpected fall in hemoglobin concentration of 2 g/dl in 24 hours.272

Radiographic findings The hallmark of GPS is diffuse pulmonary hemorrhage and is visible as airspace opacities on chest radiography and CT. Findings range from centrilobular ground-glass opacities to dense consolidation (Figure 48).288,289 Involvement has a predilection for the perihilar regions in the mid and lower zones. Less common manifestations include unilateral or focal opacity and centrilobular nodules. After a few days, interlobular septal thickening is visible on CT secondary to accumulation of hemosiderin-laden macrophages in the interstitium. Hemorrhage clears within 10–14 days but with repeated episodes chronic changes of mild fibrosis may occur. In such cases, CT will show traction bronchiectasis, mild honeycombing and reticular opacities.

Macro- and histopathology Pathological findings do not differ greatly from those seen in other causes of diffuse pulmonary hemorrhage (see below). Grossly, lungs are heavy and red to brown. Histologically, diffuse alveolar hemorrhage with hemosiderin-laden macrophages is usually accompanied by slight septal widening and occasional neutrophils. Type II pneumocytes may be hyperplastic. Neutrophilic capillaritis, i.e. neutrophilic infiltration of alveolar septal walls, may be patchy but may not be seen. Capillaries may be thrombosed or demonstrate fibrinoid necrosis. Karyorrhectic debris and scattered eosinophils can be seen (Figures 49 and 50). Microscopic foci of organizing pneumonia or even hyaline membranes may be noted but convincing vasculitis or phlebitis in areas without hemorrhage should not be seen.27,290 Diagnosis of diffuse pulmonary hemorrhage as GPS requires immunofluorescence and Western blotting studies. By immunofluorescence there is strong linear staining for IgG, which exceeds albumin (Figure 51). Confirmation requires a positive Western blot, which is the most sensitive assay. Diagnostic bands are noted at 28 and 48–50 kilodaltons (Figure 52).291

Cytology Bronchoalveolar lavage samples may demonstrate blood and numerous hemosiderin-laden macrophages.292

Ultrastructural findings Ultrastructural study may identify mononuclear cells in alveolar walls, and endothelial basement membrane breaks. Alveolar spaces contain surfactant and erythrocytes.293,294

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

Laboratory findings Patients with GPS have low hemoglobin while serum creatinine is usually elevated. The urinary sediment should be active with red cell casts. ESR is only mildly elevated. Complement proteins C3 and C4 are usually normal. Circulating anti-BM antibodies may be detected by ELISA or Western blotting. Different ELISA assays and multiplex-based detection systems are commercially available although all have similar degrees of sensitivity (> 95%), but variable specificity ranging from

Figure 49. Pulmonary Goodpasture syndrome. Pulmonary hemorrhage is due to leaky capillary walls. Intra-alveolar erythrocytes and fibrin are obvious.

91 to 100%.295,296 The antibodies can be detected in situ by direct immunostaining of kidney or lung biopsies. While the autoantibodies are not identified in healthy individuals, human immunodeficiency virus or hepatitis C patients may be serum anti-BM-positive without clinical evidence of disease. In addition, rare cases of HIV-negative individuals with Pneumocystis jirovecii pneumonia may have detectable circulating anti-BM antibodies.297 Alveolar injury or

Figure 50. Pulmonary Goodpasture syndrome. Capillary walls often contain fibromyxoid connective tissue indicative of prior injury, but neutrophilic capillaritis may not be seen. Figure 51. Pulmonary Goodpasture syndrome: immunofluorescence findings. Strong linear IgG staining can be seen in pulmonary as well as renal samples.

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Figure 52. Pulmonary Goodpasture syndrome: Western blot. A patient’s serum (lane 1) demonstrates bands at 28 and 48–50 kilodaltons. These correspond to the epitopes of alpha-3 (IV) non-collagen (lane 2 is a negative control serum).

host response to the organism must affect the basal membrane antigen or a similar antigen. Conversely, case reports of patients with histopathological proof of GPS without detectable circulating anti-BM antibodies suggest that circulating antibodies can be absorbed by diseased tissue.298 As alluded to in the clinical section, up to one-third of GPS patients also have circulating ANCA at some point during their illness.299–301 Although uncertain, it appears that ANCA with a MPO specificity (P-ANCA) is more commonly seen than the proteinase-3 (C-ANCA) pattern.287,302

Pathogenesis GPS is an autoimmune disorder. Tissue injury is caused by antibody binding to reactive epitopes in basement membranes. This is a classic Type II hypersensitivity reaction. The autoantibodies are IgG, predominantly IgG1303 and complement

fixing.304 Antibody binding and complement activation initiates an inflammatory pathway leading to disruption of the glomerular and alveolar basement membranes. In addition, autoreactive T cells play a role in disease pathogenesis.280,305 It is also noted that de novo anti-BM disease in transplanted Alport disease patients is caused by alloantibodies to native (non-cryptic) epitopes.276,277 For not entirely understood reasons, anti-BM antibodies bind to the glomerular basement membrane and less commonly to the alveolar basement membrane. The antibody is directed against the noncollagenous domain of the alpha-3 chain of type IV collagen and in some cases the alpha-5 chain.306 Preferential binding to glomerular and alveolar basement membranes is in part due to the fact that there is greater expression of the alpha-3 (IV) non-collagen chains in glomerular basement membrane and alveolar basement membrane than in other basement membranes. At these sites the chains are integrated into the membrane in such a way that the lining epitopes are more accessible to circulating antibodies.307 Additional structural differences probably explain why not all GPS patients develop pulmonary hemorrhage. Alveolar capillaries lack fenestrations within the basement membrane. Thus, the alveolar endothelium is normally a barrier to the anti-BM antibody. Increased capillary hydrostatic pressure, high concentrations of inspired oxygen, bacteremia and/or endotoxemia increase alveolar capillary permeability, and all have been observed to precipitate recurrent pulmonary hemorrhage in GPS patients.272,308 Upper respiratory tract infections, tobacco use and perhaps exposures to volatile hydrocarbons may also alter pulmonary capillary permeability. The mere presence of anti-BM antibodies does not necessarily lead to disease. In spite of the normal clonal selection against B cells with reactivity to alpha-3 (IV) non-collagen chain epitopes,309 some HIV patients, possibly due to B cell dysregulation, have anti-basement membrane antibodies without GPS.297,310 Alpha-3 (IV) non-collagen chain antibodies are found in normal sera311 with much lower titer and avidity as compared to sera from GPS patients. In addition the subclass distribution is different, with IgG1 present in sera of patients with disease compared to IgG2 in normal sera, suggesting the importance of complement. T cell tolerance may be broken by T cell priming to bacterial antigens, which cross-react on alpha-3 (IV) non-collagen chain epitopes (epitope mimicry).312 Variation in host natural killer T cells, TGFb, Fc receptor and kallikrein esterase activity may exacerbate or mitigate disease activity.313–315 Overlap with WG is not understood.287,291,316–319 A detailed serological analysis suggested that double positive patients had fewer antibodies to the two pathogenic epitopes of alpha-3 (IV) non-collagen chain than patients with only antiBM antibodies. This suggests that some double positive patients should have a disease pattern more closely resembling so-called ANCA disease. However, some double positive patients have fulminant disease, more closely resembling

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

GPS. It is possible that the ANCA disease in anti-BM susceptible patients causes sufficient basement membrane injury to unmask the cryptic alpha-3 (IV) non-collagen chain epitopes and allows T cell epitope spreading.320–322

Differential diagnosis The histological differential diagnosis for GPS includes ANCA-associated pulmonary vasculitis, immune-complexmediated diseases such as cryoglobulinemia and systemic lupus erythematosus, anti-phospholipid antibody syndrome and toxic exposures including cocaine. Since pulmonary hemorrhage is a nonspecific morphological finding, one needs to correlate pathology with clinical and laboratory tests to arrive at the correct diagnosis (see below). Of note, GPS samples differ from many of the other diagnostic considerations in that it is not primarily a vasculitis. Thus, pulmonary hemorrhage in the setting of hypocomplementemia and a markedly elevated ESR should raise the possibility of an alterative diagnosis.

Prognosis and natural history The mortality rate for untreated GPS exceeds 90%. Early diagnosis is an important determinant of response to therapy and long-term prognosis.323,324 Current therapies have reduced mortality to less than 20%.325 Successful treatment requires rapid removal of autoantibodies by plasma exchange, and inductive immunosuppression with prednisone and cyclophosphamide. Maintenance immunosuppression is required, which may include rituximab and/or mycophenolate mofetil.267,326,327 Pulmonary hemorrhage responds well to plasma exchange. Following anti-alpha-3 chain of type IV collagen titers guides therapy and can identify early relapse.328 Relapse rates are less than 5% but are higher in smokers.329,330 The outcome in patients with recurrent disease is better than in the initial presentation since treatment is instituted immediately.331 Anti-BM and ANCA double positive patients may have a slightly better renal outcome, but survival rates in this cohort appear similar. Relapse rates may be slightly higher, but only when due to the vasculitis and not the GPS.332 Those with higher titers of ANCA and lower titers of anti-BM seem to have a better response to therapy and better short-term prognosis.

Diffuse pulmonary hemorrhage syndromes and capillaritis Diffuse pulmonary hemorrhage is not always associated with vasculitis syndromes. However, those associated with vasculitides almost always feature capillaritis (see above). Capillaritis in the lung has been a controversial phenomenon and is still not accepted by all pathologists. Recognition of capillaritis in other organs is facilitated by the leaking blood and nuclear fragments of neutrophils, which are retained around the blood vessel by encircling collagen. Since pulmonary capillaries lie in interalveolar septa that contain only isolated fibers of

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Figure 53. Pulmonary hemorrhage syndrome. This autopsy lung from a patient with systemic lupus erythematosus is overexpanded with blood.

collagen, erythrocytes and nuclear dust quickly transit the interstitial space and come to lie principally in the alveolar airspaces. Fibrinoid necrosis of pulmonary capillaries is not usually detectable in light microscopic sections. If larger blood vessels are not also necrotic, the blood might be incorrectly assumed secondary to a bleeding diathesis, or the neutrophils might be incorrectly assumed to be due to an infectious process. Pulmonary capillaritis is a potentially fatal process requiring rapid diagnosis and proper treatment. While different etiologies may require different treatments, morphology is usually similar in all cases (Figure 53).27,111 Patients present with rapid or several days complaint of cough, dyspnea, fever, and hemoptysis. However, up to onethird of patients do not present with hemoptysis.333 A small percentage present with acute respiratory distress syndrome requiring intubation and mechanical ventilation. Patients have varying degrees of hypoxia. A sequential increase in diffusing capacity for carbon dioxide (DLCO) secondary to increased amounts of intra-alveolar hemoglobin may be seen. Chest radiographs and CT demonstrate nonspecific patchy or diffuse alveolar opacities with perihilar and mid/lower lung

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

Figure 54. Pulmonary hemorrhage syndrome due to primary antiphospholipid syndrome. The entire pulmonary lobule is filled with blood.

zone predominance.288,334 Patients with recurrent disease may have interstitial opacities and scarring. At bronchoscopy blood is seen in all airways. Bronchoalveolar lavage demonstrates increasingly hemorrhagic samples with serial lavages. If the process is at least 3 days old, hemosiderin-laden macrophages are also noted.335 However, these macrophages may be seen in non-vasculitic processes including left ventricular cardiac failure with chronic pulmonary edema. Lavage samples must be sent for culture as hemorrhage may be seen in a variety of infections. Although capillaritis can be diagnosed on a transbronchial biopsy, a surgical lung biopsy is the diagnostic gold standard.336 Suspicion of pulmonary capillaritis arises when the alveolar lattice is obscured by blood at low magnification but extra neutrophils hug the alveolar walls (Figure 54) or spill into blood-filled alveolar airspace. Neutrophilic infiltration and necrosis of capillary walls with nuclear dust (Figures 55 and 56) and hemosiderin in the interstitium or clotted excrescences of fibrin lying upon walls (Figure 57) permit a diagnosis. Periodic acid Schiff stain is often more useful than stains for elastic tissue, reticulin or immunopathological markers for endothelial cells. In cases with impressive hemosiderosis, iron encrustation of arteriolar and venular walls with giant cells may be mistaken for granulomatous vasculitis (see Chapter 2). Many causes are recognized, and correct diagnosis requires a thorough history including drug use and pathology work-up (Table 11). In addition to usual blood studies, ANCA, anti-BM antibodies, rheumatological serologies, antiphospholipid antibodies, blood cultures and drug screening are suggested in each case. Snap-frozen tissue samples should be studied for linear IgG deposition (consistent with GPS) (Figure 51),

Figure 55. Pulmonary hemorrhage syndrome due to primary antiphospholipid syndrome. Neutrophilic capillaritis along with intra-alveolar fibrin and scattered histiocytes are noted. Type II pneumocytes are reactive.

Figure 56. Pulmonary hemorrhage syndrome due to primary antiphospholipid syndrome. The alveolar wall including the capillary is destroyed (arrow) (elastic van Gieson stain).

granular immune complex deposition (indicative of a connective tissue disorder) or IgA deposition (suggesting HenöchSchonlein purpura or IgA nephropathy).333,337 Isolated pulmonary capillaritis lacks evidence of an underlying systemic process.338

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Table 11 Causes of diffuse pulmonary hemorrhage

Pulmonary hemorrhage with capillaritis Wegener granulomatosis Microscopic polyangiitis Goodpasture syndrome Connective tissue diseases Primary antiphospholipid syndrome Behçet’s sydnorme Henöch-Schonlein purpura Drug-induced Immune-complex-associated glomerulonephritis Pauci-immune glomerulonephritis Mixed cryoglobulinemia Isolated pulmonary capillaritis Acute lung allograft rejection Figure 57. Pulmonary hemorrhage syndrome due to primary antiphospholipid syndrome. Fibrin balls with hemosiderin are likely to be incorporated into the alveolar wall.

In WG capillaritis is usually associated with arteritis or phlebitis as well as with focal pathergic necrosis, palisading histiocytes, and diffuse granulomatous tissue,26,27,111,339 However, the latter findings represent subacute or chronic disease, and occasionally the capillaritis is seen in acute disease without the other histological features. In systemic lupus erythematosus, capillaritis may be associated with fibrinous pleuritis, arteriolar hypertension and diffuse interstitial fibrosis (see Chapter 21). However, as with WG, these latter findings represent subacute and chronic changes, and the capillaritis and hemorrhage may be the only acute finding in the lung. Pulmonary hemorrhage occurs in a variety of pulmonaryrenal syndromes. Some are well characterized, such as GPS and IgA nephropathy. Others are poorly characterized and idiopathic. A leukocytoclastic capillaritis is not apparent in the lung in most cases of GPS, even when there is extensive hemorrhage. Very high P-ANCA titers are associated with drug-induced diffuse hemorrhage.18 Implicated agents include hydralazine, propylthiouracil, carbimazole, methimazole, penicillamine, allopurinol and all-trans-retinoic acid.18,340,341 Other conditions which may display pulmonary capillaritis include MPA,187 acute rheumatoid disease, Henoch-Schönlein purpura, hepatitis B,342 transfusion reaction, idiopathic glomerulonephritis, polyarteritis nodosa and plexogenic hypertension, including patients with acquired immunodeficiency syndrome.343 Localized pulmonary hemorrhage secondary to capillary or venular thrombosis without capillaritis is seen in anti-cardiolipin antibody syndrome.344–346 Some cases are idiopathic. Idiopathic pulmonary hemosiderosis is a diagnosis which leaves important unanswered

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Pulmonary hemorrhage without capillaritis Systemic lupus erythematosus Goodpasture syndrome Diffuse alveolar damage Drug-induced Idiopathic pulmonary hemosiderosis Left ventricular dysfunction Mitral stenosis Coagulation disorders Pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis Human immunodeficiency virus infection Neoplasms

questions for the clinician. Although the histological features of interstitial fibrosis and extensive hemosiderin are easy to recognize, the pathological diagnosis does not have a consistent clinical presentation or natural history (Figures 58 and 59). Most often seen in children and associated with celiac disease, juvenile rheumatoid arthritis, cardiomyopathy and ANCA serologies, adult patients do not share the associations.347,348 Recurrent episodes of hemorrhage, often presenting with hemoptysis, eventually lead to pulmonary fibrosis. Anemia also ensues. There are probably several causes, including previous capillaritis and hemorrhage. Idiopathic pulmonary hemosiderosis in these cases is the chronic sequel to the acute bleed. Diagnoses of WG and GPS have been established at our hospital during later episodes of acute pulmonary hemorrhage in patients with a prior diagnosis of idiopathic pulmonary hemosiderosis. The etiological differential diagnosis for diffuse pulmonary hemorrhage includes acute bacterial or viral pneumonia without capillaritis. Filling of the alveoli and bronchioles with

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

Figure 58. Idiopathic pulmonary hemosiderosis. This rare entity features diffuse alveolar septal fibrosis.

neutrophils, much in excess of those in interstitium, suggests infection. Hyaline membranes suggest viral infection among many other possibilities. A bleeding diathesis leaves no telltale histological finding in the lung, unless one finds an intravascular leukemia which might be causing it. Arteriovenous malformations are generally difficult to identify unless an angiogram has localized the lesion (see Chapter 33). Even then, pulmonary arteriovenous malformations may destroy themselves when they rupture. There may be no way to prove that blood in an open lung biopsy has been aspirated from a proximal source; however, aspirated blood may fill one lobule entirely and the adjacent lobule not at all. Lastly, one must exclude the possibility that the blood is artifactual. Bronchoscopic and surgical manipulations can mimic hemorrhage syndromes. Procedure-related hemorrhage often lacks fibrin or hemosiderin and features normal alveolar walls with marginated neutrophils. Treatment and survival rates for patients with diffuse pulmonary hemorrhage depend on the underlying etiology. Early mortality rates for those with systemic lupus erythematosus, WG, MPA or GPS range from 30% to 50%.343 While steroids are the mainstay of treatment, cyclophosphamide, azathioprine or plasma exchange may be utilized. Complications of disease include pulmonary fibrosis and obstructive lung disease with emphysema.202

Polyarteritis nodosa Introduction Polyarteritis nodosa (PAN) is a necrotizing, focal segmental vasculitis that affects predominantly medium-sized arteries in many different organ systems. The lungs are rarely involved.181 Polyarteritis was first described by Rokitansky in 1852,349 followed by Virchow, who described endoarteritis deformans in 1863.350 Kussmaul and Maier described an autopsy case of a 27-year-old male who had proteinuria, myalgia, neuritis and

Figure 59. Idiopathic pulmonary hemosiderosis. Airspaces are lined by reactive type II pneumocytes and filled with hemosiderin-laden macrophages. Septal thickening is seen.

abdominal pain.351 The autopsy showed the blood vessels throughout the body had been transformed into thickened cords with nodule formation. Ferrari used the term polyarteritis, recognizing both the multiplicity of vessel involvement and the inflammatory process involving the entire wall thickness.352 With the widespread use of light microscopy it became clear that polyarteritis nodosa of the microscopic type was frequent.353 With the recognition of other forms of systemic vasculitis and widespread availability of ANCA testing, the incidence of PAN has dropped. Many previous cases are now diagnosed as microscopic polyangiitis (MPA). Diagnostic criteria are offered by the American College of Rheumatology and the Chapel Hill Consensus Conference, but one should note that the diagnosis is one of exclusion (Table 12).4,354 PAN is defined as “necrotizing inflammation of medium-sized or small arteries without glomerulonephritis or vasculitis in arterioles, capillaries, or venules”.181 When the ACR criteria are properly applied, sensitivity and specificity for the diagnosis approach 82 and 87%, respectively. The kidneys, gastrointestinal tract, skin, nerves, joints and muscles are commonly affected.355 While Kussmaul and Maier noted that pulmonary involvement was unusual, Rose and Spencer in 1957 reported that approximately half of PAN cases involved the lungs.356 It is now evident that these so-called pulmonary cases represented either WG or CSS. On the rare occasions when PAN involves the lungs, it is generally limited to the bronchial arteries.

Epidemiology Because MPA has been recently separated from PAN and the diagnostic standard is not uniform, the true incidence of PAN remains obscure.357 The overall incidence is estimated as 0.2 to 0.7 per 100 000 persons per year. It may be more common in men than in women. The diagnosis is most frequently made in middle-aged or older individuals. It is associated with hepatitis

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Table 12 Polyarteritis nodosa: diagnostic criteria

A diagnosis of PAN can be rendered in a patient with documented vasculitis who has at least three of the following criteria: Otherwise unexplained weight loss more than 4 kg Livedo reticularis Testicular tenderness or pain Myalgias (excluding shoulder and hip girdle), muscle weakness, leg muscle tenderness or polyneuropathy Mononeuropathy or polyneuropathy New-onset diastolic blood pressure greater than 90 mmHg Elevated serum blood urea nitrogen (> 40 mg/dl) or creatinine (> 1.5 mg/dl) Hepatitis B virus infection confirmed with serum antibody or antigen serology Characteristic arteriographic abnormalities not resulting from noninflammatory disease processes A biopsy of small or medium-sized artery containing neutrophils Source354.

B virus (HBV) in approximately 7% of cases.358,359 Other infections such as hepatitis C virus, HIV and parvovirus B 19 are rarely associated.194 Patients with connective tissue diseases or hairy cell leukemia may develop PAN.

Clinical manifestations Renal disease, polyneuritis, polymyositis and abdominal pain represent the most common tetrad of patient symptoms.360 Various clinical manifestations depend on the distribution of disease. Fever, body weight loss, myalgia, abdominal pain, hypertension, muscle weakness, arthralgia, swelling of the joints, ischemic heart diseases, thromboembolism, pericarditis, heart tamponade, brain hemorrhage, cerebral infarction, polyneuritis, gastrointestinal hemorrhage, perforation and necrosis, peritonitis and peritoneal hemorrhage are reported.361 Hepatitis B- related PAN has more frequent peripheral neuropathy, abdominal pain, cardiomegaly, orchitis and hypertension than non-virus-related disease.361 Dyspnea and cough along with constitutional symptoms are possible pulmonary signs.362

Four overlapping morphological stages are described.360 The first stage is characterized by edema and a fibrinous exudate, followed by fibrinoid necrosis. Each arterial lesion is no more than a millimeter long and may involve the entire circumference of the arterial wall, or only a part of it. In an area of fibrinoid necrosis the medial muscle and adjacent tissues are fused into a structureless eosinophilic mass. Neutrophils, lymphocytes, plasma cells and macrophages are present in varying proportions, and eosinophils may be conspicuous. In the second stage, the fibrinoid necrosis is more widespread, and involves the entire arterial wall. The internal elastic lamina is attenuated and fragmented. Intimal fibroblastic proliferation occurs along with secondary thrombosis and organ infarction (Figure 60). With the destruction of elastic fibers an aneurysm develops (Figure 61). Rupture results in massive hemorrhage. In the third or reparative stage, the fibrinoid necrosis is replaced by granulation tissue with marked intimal fibrous proliferation and partial or total luminal occlusion. In the final healed stage, the destroyed arterial wall is replaced by fibrous scar tissue with luminal obliteration. Bronchial artery lesions demonstrate these findings.362–365

Immunohistochemistry Although immune complexes are suggested as a possible pathogenic mechanism, immunofluorescence microscopy does not reveal evidence of any immune complex deposition in the arterial lesions.

Electron microscopy Arteries show endothelial cell swelling, degeneration and desquamation associated with fibrin deposition. Electron-dense deposits are not seen.

Laboratory findings Specific laboratory findings are not reported; however, ANCA, antinuclear antibodies and rheumatoid factor should be negative when considering a diagnosis of primary PAN.

Pathogenesis

Radiographic findings

Multiple poorly understood mechanisms may contribute to the pathogenesis.366 PAN associated with hepatitis B virus infection is probably an immune complex process. However, it remains unknown why arterioles, capillaries and venules are spared.

Arterial aneurysms may be detected by angiography. The finding is not disease-specific since any necrotizing arteritis as well as other entities can produce aneurysms.

Differential diagnosis

Macroscopic pathology The kidneys, gastrointestinal tract and heart demonstrate hemorrhage, infarction, thrombosis and infarctions. The lungs do not show gross abnormalities.

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Histopathology

The differential diagnosis includes MPA, WG and CSS. In fact, many previously reported cases of PAN probably represent these ANCA-positive differential entities. While PAN only involves medium-sized arteries, MPA involves small and medium sized arteries. MPA also features glomerulonephritis and may present with massive pulmonary hemorrhage.33,187

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

(a)

(b)

Figure 60. Pulmonary involvement with polyarteritis nodosa. (a) The bronchial artery demonstrates intimal proliferation with near occlusion of the lumen, medial granulation tissue, and adventitial fibrosis. (b) The internal elastic lamina is partially destroyed while intimal proliferation is striking (Masson-elastica stain).

disease.372 Renal and GI signs seem to be the most serious prognostic factors.260 Relapses are more likely if diagnosis and treatment are delayed.307,373 The prognosis is not well established for PAN confined to the lung. However, the disease is generally not fatal.

Giant cell arteritis Introduction

Figure 61. Pulmonary involvement with polyarteritis nodosa. This high magnification image shows inflammatory cell destruction of an elastic layer (arrow).

WG features extravascular granulomatosis inflammation while CSS also demonstrates tissue eosinophilia.109 In addition, clinical asthma and eosinophilia further support a diagnosis of CSS.

Prognosis and natural history PAN is usually fatal without treatment. One and 5-year survival rates are reported as 50% and 13%, respectively.367,368 Anti-inflammatory and immunosuppressive therapy lead to remissions or cures in most patients, with 5-year survival rates approaching 80%.209 Plasma exchange is also utilized for those with secondary PAN.369 Relapses are very rare, unlike the more frequent relapses in MPA.194,370 In the patients with disease related to HBV, anti-viral therapy suppresses the etiological agent of the vasculitis.371 This patient group survival is slightly lower, 73% at 5 years, than those with primary

Giant cell arteritis (GCA) was first reported in 1937 by Horton et al. and was named arteritis of the temporal vessels by Jennings374,375 GCA is also known as temporal arteritis, cranial arteritis, or granulomatous arteritis. GCA is a systemic necrotizing vasculitis that mainly affects the intracranial branches of the carotid arteries. Extracranial branches of the aortic arch are reportedly involved in 10–15% of cases. Temporal artery biopsy is the gold standard for diagnosis.

Epidemiology GCA predominantly affects Caucasians and is infrequently found in Asians and Africans.376 The annual incidence in the population above 50 years old can be as high as 76 per 100 000 in Denmark and more than 30 per 100 000 in Olmsted County, Minnesota, USA, but only 1.6 per 100 000 in Tennessee.377,378–380 One Japanese nationwide survey also showed that the prevalence of GCA in Japan is extremely low (1.47 per 100 000).381 In one study only two of 1435 patients with biopsy-proven GCA were younger than 50.382 Women develop the disease two to six times more often than men.383 There is a suggestion that the frequency of this disease may be on the rise.384 Tobacco use and atheromatous disease increase the risk of disease in women, but not in men.385

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

(a)

(b)

Figure 62. Pulmonary involvement with giant cell arteritis. (a) The pulmonary artery features nodular expansion (arrows). (b) Granulomatous inflammation widens and destroys the media. Note the fragmented elastic fibers (elastic van Gieson stain).

Genetics HLA-DR4 and a sequence polymorphism within the secondary hypervariable region of the HLA-DRB1 gene are correlated with GCA. The HLA-DRB1 gene maps to the antigen-binding cleft of the HLA-DR molecules. Interestingly, patients with polymyalgia rheumatica (PMR) share this sequence polymorphism (see below). Gene polymorphisms for intercellular adhesion molecule-1 (ICAM-1) enhance the risk of GCA and PMR.386

Clinical manifestations Headache, scalp tenderness, jaw claudication and visual symptoms are the most typical features of the disease.376 Systemic manifestations such as fever, anorexia and weight loss are reported. Less common manifestations are related to the central or peripheral nervous systems, the respiratory tract and extra-cranial large-vessel involvement. Permanent vision loss is the most serious complication of GCA.387 Although the relationship is not well understood, PMR occurs in 40% to 50% of patients.388

Histopathology GCA demonstrates granulomatous inflammation with necrosis, multinucleated giant cells, macrophages, lymphocytes and fibroblasts.389,390 The arterial media is most frequently affected. Arteritis is most commonly found in the arteries arising from the aorta, particularly the superficial temporal, ophthalmic, posterior ciliary and vertebral arteries. GCA of large- and medium-sized peripheral arteries typically leads to long tapering and occlusion of the arterial lumen due to concentric intimal thickening, sometimes accompanied by spontaneous dissection. Depending on the extent of the arterial obliteration and on the anatomy

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of the involved arterial segment, this may result in severe ischemia of the limbs during the acute phase of the disease. GCA of the aorta usually remains asymptomatic for many years, and leads to a markedly increased risk of aneurysms and dissections. The thoracic aorta is particularly susceptible. Few cases of GCA complicated by pulmonary artery disease are reported. The first case was reported by Wagenaar et al.,391 in which a 33-year-old male had giant cell arteritis limited to large elastic pulmonary arteries (Figure 62). Additional reports127,392,393 showed isolated granulomatous pulmonary giant cell arteritis without systemic vascular involvement. Cases of GCA involving the pulmonary artery are difficult to differentiate from isolated pulmonary Takayasu arteritis (see below).394,395

Laboratory findings While elevated erythrocyte sedimentation rates (ESR) are often 100 mm/h or greater, less than 10% of patients have an ESR of less than 50 mm/h and 4% of patients have normal values.382 Elevated C-reactive protein (CRP) in conjunction with elevated ESR greater than 50 mm/h has 97% specificity in diagnosing GCA.396

Pathogenesis Peak disease rates occurring every 5 to 7 years suggests a possible infectious cause or trigger.386 Parvovirus B19, human parainfluenza virus type 1 as well as Chlamydia pneumoniae are proposed agents.397 Persistent infection or persistent antigen may lead to interferon-g-dependent chronic vessel inflammation.398 Humoral and cellular immune systems are implicated in the pathogenesis, but details are lacking beyond generalizations that the process is antigen-driven and probably T-cell dependent.398,399

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Table 13 Giant cell arteritis and Takayasu arteritis: comparative features

Giant cell arteritis

Takayasu arteritis

Female:male ratio

3:2

7:1

Age at onset

> 50 years

< 40 years

Ethnicity

European

Asian

HLA association

HLA-DR4

HLA-Bw52

Primary vessels involved

External carotid artery branches

Aorta and branches

Renovascular hypertension

Rare

Common

Histopathology

Granulomatous inflammation

Granulomatous inflammation

Natural history

Self-limited

Chronic

Response to steroids

Excellent

Excellent

Surgical intervention

Rare

Common

Data from499.

Differential diagnosis Pulmonary involvement is much more common in Takayasu arteritis than in GCA. Because pulmonary arteries affected are usually large elastic-type arteries, it is reasonable to regard isolated pulmonary arterial disease as a manifestation of Takayasu arteritis (see Table 13). Disseminated visceral giant cell arteritis is very rare but is reported to involve the pulmonary arteries.400,401 WG involving the temporal artery can mimic GCA clinically by virtue of a painful artery, visual disturbances, elevated ESR and intracranial involvement of vessels.402,403 However, WG also features parenchymal necrosis and usually ANCA positivity.

Natural history and prognosis If untreated, GCA can cause permanent visual loss in one or both eyes in up to 50% of patients.387 Early recognition and treatment of this condition is thus essential. Although never studied in a placebo-controlled manner, systemic steroids are the standard therapy for patients with GCA. In a high percentage of patients the treatment may extend for more than one year.404 Disease flares are associated with steroid tapering.

Takayasu arteritis Introduction Takayasu arteritis is a primary arteritis of unknown cause that commonly affects the thoracic and abdominal aortas and their major branches, including the pulmonary artery. In 1905 Takayasu, Professor of Ophthalmology at Kanazawa University, Japan, presented a case of a 21-year-old woman who

showed characteristic fundal arteriovenous anastomoses.405–408 At the discussion time of the same meeting, Onishi and Kagoshima each mentioned individually similar cases associated with absent radial pulses.409 In 1920, the first post-mortem case of a 25-year-old woman demonstrated panarteritis and suggested that the fundal appearances resulted from retinal ischemia.410 In 1951, Shimizu and Sano summarized the clinical features of this “pulseless disease”.411 The American College of Rheumatology suggests six criteria for diagnosis: (1) onset at age less than 40 years; (2) claudication of an extremity; (3) decreased branchial artery pulse; (4) more than 10 mmHg difference in systolic blood pressure between arms; (5) a bruit over the aorta or subclavian arteries; and (6) arteriographic evidence of narrowing or occlusion of the entire aorta, its primary branches, or large arteries in the proximal upper or lower extremities.412 The presence of three of these criteria reportedly has a sensitivity of 91% and specificity of 98%.

Epidemiology Takayasu arteritis is relatively rare, but most commonly seen in Japan, Korea, China, South East Asia, India and Mexico. In 1990, it was included in the list of intractable diseases maintained by the Japanese government,413 and to date more than 5000 patients have been registered. Variable disease presentation between different populations is well illustrated.414 Nearly 100% of Japanese patients are female while almost 50% of Indian patients are male. Although it has been estimated that 150 new cases are diagnosed in Japan yearly, the incidence in Europe and the United States is no more than three new cases per year per million population per year.415 The disease commonly presents in the second or third decade of life.

Genetics Takayasu arteritis is associated with different human leukocyte antigen (HLA) alleles in different populations.416,417 In Japan and Korea there is a clear association with the extended haplotype HLA B*52, DRB1*1502, DRB5*0102, DQA1*0103, DQB1*0601, DPA1*02-DPB1*0901.416 Sequence analysis has shown some of the alleles share specific epitopes. It may be that the epitopes are more important as a disease susceptibility factor than the allele in which they are found. However, specific autoantigens have not yet been identified. Pulmonary involvement is more common in individuals with HLABw52/Dw12.418

Clinical manifestations TA is notoriously difficult to diagnose. From the onset of symptoms, diagnosis may be delayed for months to years. Manifestations range from non-palpable pulses and vascular bruits to catastrophic neurological disorders. Nonspecific clinical features include fever, night sweats, malaise, weight

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

loss, arthralgia and myalgia.415 As the disease progresses and arterial stenoses develop, the more characteristic features become apparent, influenced by the development of collateral circulation. Stenotic lesions predominate and tend to be bilateral.419,420 Nearly all patients with aneurysms also have stenoses. Most have extensive vascular lesions. Characteristic signs and symptoms include diminished or absent pulses associated with limb claudication and blood pressure discrepancies between the arms.419,421 Vascular bruits affect the carotids, subclavian and abdominal vessels. Hypertension is usually caused by renal artery stenosis. Takayasu retinopathy may be seen.415,422 Aortic regurgitation can result from dilatation of the ascending aorta, sometimes leading to congestive heart failure. Neurological features are also described, but the frequency with which Takayasu arteritis involves the brain parenchyma is not clear. Pulmonary artery involvement is reported in 14–100% of patients, depending on the method used to examine the pulmonary artery. Pulmonary artery disease shows little correlation with the systemic pattern of arterial involvement,406,422,423and is rarely the presenting manifestation.424 Pulmonary symptoms include dyspnea and chest wall pain. Pulmonary hypertension has also been reported.425 Of note, renal, cardiac, skin and gastrointestinal tract involvement is also reported.

Radiographic findings Since histological diagnosis is usually impractical and pathological assessment is limited to those cases undergoing lung biopsy or cardiovascular surgery, angiography is the gold standard for diagnosis Examination of the pulmonary arteries by angiography is not universally recommended, but reserved for patients with symptoms of pulmonary hypertension. Typical angiographic findings include stenosis, occlusion and aneurysm formation.406 CT can demonstrate irregular thickening of the vascular wall in patients with active disease.407 CT also demonstrates inhomogeneous lung attenuation owing to localized areas of decreased perfusion. This finding is mirrored in ventilation perfusion scans where multiple perfusion defects may be noted.426

Macroscopic pathology In the chronic phase, the aorta is thickened secondary to fibrosis of all three vessel layers. If disease progression is slow, the affected arterial wall loses the medial layer, fibrosis progresses and the lumen usually becomes narrow in a patchy distribution. If disease progression is rapid, fibrosis can be incomplete with subsequent dilatation and aneurysm formation. The intima may be ridged, with a “tree bark” appearance, a feature common to many aortitides.406,413,427

Histopathology The vasculitis may be divided into an acute florid inflammatory phase, a granulomatous phase, and a healed fibrotic phase. In

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the acute phase, microabscesses are the initial finding while inflammation in the adventitial vasa vasorum follows. In the granulomatous phase, the media is infiltrated by lymphocytes and occasional giant cells with neovascularization and loss of elastic fibers. Inflammation is most prominent in the adventitia, with possibly nodular collections of B- and T-lymphocytes and dendritic cells.428 Mucopolysaccharides, smooth muscle cells and fibroblasts thicken the intima. In the chronic phase there is fibrosis with destruction of remaining elastic tissue.

Laboratory findings Anemia may be severe, and thrombocytosis is often striking. Elevated ESR and C-reactive protein (CRP) are a good reflection of the underlying inflammatory process. Serum concentrations of the pro-inflammatory and chemotactic cytokines interleukin 1b, IL-6 and RANTES are elevated during active disease.429 ANCA and antiphospholipid antibodies are not found in this disease.430

Pathogenesis The cause of the disease is unknown. Cell-mediated mechanisms are postulated. A great proportion of infiltrating mononuclear cells are gamma delta T lymphocytes.431 High levels of antiendothelial antibodies have also been observed.430 Infection is also considered.432 Tuberculosis has been particularly implicated in view of the high prevalence of infection, past or present, in affected patients, largely from endemic areas.421,433 More recently, viral infections have been investigated as potential disease triggers.434

Differential diagnosis The differential diagnosis includes inflammatory aortitis and developmental abnormalities. The former category contains syphilis, tuberculosis, systemic lupus erythematosus, rheumatoid arthritis, spondyloarthropathies, Behçet syndrome, Kawasaki disease, GCA and IgG4-related systemic disease. Coarctation of the aorta and Marfan syndrome are also considerations (see Chapter 11). Although GCA and Takayasu arteritis are dissimilar in many respects, GCA involves the aorta in 10–15% of patients.406,427,429 Since tissue samples are rarely obtained, one must differentiate the diseases on clinical grounds. In a study of 280 patients identified through the American College of Rheumatology Vasculitis Criteria Databank – 217 of whom had giant cell arteritis and 63 of whom had Takayasu arteritis – an age of disease onset before the age of 40 was the single most helpful factor in identifying Takayasu arteritis (see Table 13).181

Prognosis and natural history Takayasu arteritis is a chronic disease with exacerbations and remissions over time. Vascular involvement is usually progressive, although the disease may burn out. Complications such as

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

retinopathy, hypertension or aortic regurgitation and progression suggest a 66% 15 year-survival as compared with those well controlled with medical therapy, who have over 90% 15-year survival.435 The management of patients can be problematic. There may be uncertainty with regard to the onset and course of the disease, a poor correlation between clinical assessment and disease activity, poor disease activity markers in peripheral blood, and a lack of useful treatment in up to 25% of patients with progressive disease. The risk of increased morbidity and mortality means most patients who present will ultimately receive immunosuppression in addition to corticosteroids. Early treatment of those with complicated, progressive disease may lead to a better prognosis for this group since steroids are only palliative, with a high incidence of recurrences. Percutaneous transluminal angioplasty or bypass grafts may be indicated in attempts to reverse vascular obstruction and ischemic symptoms. While vascular stenosis is common, aneurysm rupture, with all its morbidity and mortality, is rare. As inflammation is a risk factor for atherosclerosis, significant atherosclerotic complications are likely in the long term.410 Approximately 50% of patients have chronic active disease that resists steroid-induced remissions. Methotrexate or azathioprine may be useful.436,437

Behçet syndrome Introduction Behçet syndrome (BS) is a chronic inflammatory disorder of unknown etiology characterized by the triple symptom complex of recurrent oral and genital ulcers and relapsing uveitis. BS, unlike other systemic vasculitides, involves both arterial and venous vessels of all sizes. The first patient with the syndrome was reported by Hippocrates. Dr. Hulusi Behçet, a Turkish dermatologist, wrote the first scientific description of this disease in 1937.438 BS does not have pathognomonic symptoms or laboratory findings. Thus, the diagnosis is made on the basis of criteria proposed by the International Study Group in 1990 (see Table 14).439 Recurrent oral ulcer must be present along with at least two of the following: recurrent genital ulcer, eye lesions, skin lesions or a positive pathergy test (development of a papule or pustule following a needle prick to the skin). Interestingly, pathergy is not common in Northern Europeans or North Americans.

Epidemiology Although BS has a worldwide distribution, most cases are seen in Turkey, Japan and other Asian countries. Thus, cases of the disease cluster along the ancient Silk Road, which extends from far eastern Asia to the Mediterranean basin (see Table 15).440–447 The highest prevalence rate is reported in Turkey with 80–370 cases per 100 000 persons. The prevalence rate ranges from 2 to 30 cases per 100 000 persons in other Asian countries, but is very low in Europe and North America.

Table 14 Behçet syndrome: International Study Group diagnostic criteria

In the absence of other clinical explanations patients must have: (1) Recurrent oral ulceration (aphthous or herpetiform) observed by the physician or patient recurring at least three times in one 12 month period AND two of the following: (2) Recurrent genital ulceration (3) Eye lesions: anterior uveitis posterior uveitis (cells in the vitreous observed by slit lamp examination); or retinal vasculitis observed by an ophthalmologist (4) Skin lesions: erythema nodosum pseudofolliculitis papulopustular lesions or acneiform nodules in post-adolescent patients not on corticosteroids (5) Positive skin pathergy test read by a physician at 48 h – that is, a 2 mm erythematous papule or pustule at the prick site 48 h after the application of a sterile hypodermic 20–22 gauge needle which obliquely penetrated avascular antecubital skin to a depth of 5 mm Source439.

Table 15 Behçet syndrome: epidemiological data

Prevalence rate (/100 000)

Reference

Turkey

80–370

456

Japan

7–8.5

440

Other Asian countries

2–30

457

UK

0–64

458

Germany German natives Turkish immigrants

0.42–0.55 21

459 459

The age of disease onset is usually in the second or third decade. The male to female ratio is approximately equal in regions where the disease is more common, but women are affected more often in the United States and northern Europe. Men are more likely to have lung involvement and hemoptysis.

Genetics Although the majority of patients with BS have no family history, familial clustering is reported. Recent studies have shown that the risk of developing BS by siblings of index patients is increased compared with the general population.448 The association of HLA-B51 with BS is the strongest finding

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes Table 16 Behçet syndrome: clinical manifestations

Lesion

Frequency (%)

Oral ulcers

96–100

Skin lesions Folliculitis Erythema nodosum Positive pathergy test

40–50 25–80 10–50

Genital ulcers

65–90

Eye lesions

35–70

Arthritis

30–80

Neurological involvement

10–50

Gastrointestinal involvement

5–60

Vascular involvement

5–30

Pulmonary involvement

1–18

440,452–460

Sources

.

supporting the contribution of genetics to disease pathogenesis.449,450 Associations between non-HLA genes may also play a role in determining disease susceptibility. Polymorphisms of the ICAM-1 gene, tumor necrosis factor genes and endothelial nitric oxide synthase gene are implicated.451

Clinical manifestations Clinical manifestations include involvement of the skin, central nervous system, gastrointestinal and genitourinary tracts and the lung. Subcutaneous thrombophlebitis, deep vein thrombosis, epididymitis, arterial occlusion, aneurysms, arthralgia, arthritis and renal disorders are also observed (see Table 16).440,452–460 Pulmonary artery aneurysms, pulmonary emboli, pulmonary infarctions and involvement of small-sized vessels are common in BS. Pulmonary symptoms include hemoptysis, dyspnea, cough and chest pain along with general fatigue. Massive hemoptysis is also reported.461,462 Three types of pulmonary disorders associated with BS are recognized: (1) pulmonary artery aneurysm; (2) pulmonary parenchymal disorders; and (3) other thoracic disorders including pulmonary artery occlusion, pleural effusion, and obstructive airway disease.463 A cumulative analysis of published cases reveals that pulmonary problems affect from < 1 to 18% of patients.464–467 In a large review of Japanese autopsy cases, 75% had pulmonary lesions. Pneumonia and pulmonary edema were the most common.455 Pleuritis, pleural effusion, lung abscess, thrombosis and infarction were also reported.

Radiographic findings Nonspecific radiographic abnormalities include hilar enlargement or mass lesions, alveolar infiltrates, wedge-shaped peripheral opacities, hyperlucent lung, diaphragmatic elevation, pleurisy and pleural effusions.468 Although angiography is

750

Figure 63. Behçet syndrome. Small pulmonary arteries may be infiltrated with mostly mononuclear cells.

generally the gold standard diagnostic method in vascular diseases, selective pulmonary arteriography and catheterization may be harmful.469

Macroscopic pathology Pulmonary arterial aneurysms are saccular or fusiform dilatations. This lesion most frequently involves the right lower lobe lobar arteries, followed by the right and left main pulmonary arteries.470 The aneurysm diameter ranges from 1 to 7 cm. Multiple aneurysms occur. Bronchial obstruction is a dangerous complication. Pulmonary parenchyma disorders include atelectasis, pulmonary hemorrhage and/or infarcts. Other thoracic disorders include occlusion of the superior vena cava and thrombosis of the innominate and subclavian veins.471 Pseudoaneurysms of the aortic arch as well as the subclavian and coronary arteries have been described.472,473

Histopathology The basic histopathology of BS is a necrotizing lymphocytic vasculitis.455,456 The vasculitis involves arterial and venous large, medium and small vessels as well as capillaries. Inflammatory cell infiltrates are usually composed of lymphocytes with fewer numbers of neutrophils and macrophages (Figure 63). Thrombi are also seen. Pulmonary arterial aneurysms show cellular perivascular infiltrates around the vasa vasorum, marked intimal thickening, decrease in amount or complete loss of the elastic lamina, fresh and organized thrombi, thrombotic occlusion and recanalized blood vessels (Figure 64).474,475 Perivascular fibrosis

Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

(a)

(b)

Figure 64. Behçet syndrome. (a) A pulmonary artery aneurysm often contains adherent thrombus. (b) The intima is thickened and the elastic lamina is absent from part of the arterial wall (elastic van Gieson stain).

Pathogenesis

Figure 65. Behçet syndrome. Pleural involvement features a proliferation of small collateral blood vessels within the wall of the destroyed artery (not shown).

and newly formed collateral vessels with thick muscular walls and intimal fibrosis lacking elastic lamellae may be striking (Figure 65). Pulmonary infarcts, eosinophilic pneumonia, diffuse alveolar hemorrhage, diffuse alveolar damage and fibrinous pleuritis have been reported.475–478

Laboratory findings There are no disease-specific laboratory findings. Elevated ESR and C-reactive protein levels are nonspecific.

The etiology of BS is unknown. Clinical observations and laboratory investigations support the concept that immunological mechanisms induced by microbial pathogens occur in genetically susceptible individuals.479,480 Activation of neutrophils with increased chemotaxis and superoxide generation has long been suggested as the main pathogenic mechanism in BS. Increased production of some cytokines, such as IL-8, TNF alpha and IL-1, from lymphocytes, macrophages, neutrophils and/or endothelial cells may have a regulatory role in neutrophil function and might be responsible for this primed state.481–487 Nonspecific hyperreactivity seems to be an important feature of BS. The example is the skin pathergy reaction in which a papule or pustule occurs following a simple needle prick to the skin, similar to those appearing spontaneously in the disease.488 This hyperresponsiveness to the minor trauma is not unique to the skin, and the phenomenon can be observed at other sites.489 Several microbial agents, including herpes simplex virus and certain strains of streptococci, are suspected pathogenic participants. A hypothesis suggests that certain epitopes of microbial heat shock proteins (HSP) are putative antigens that trigger a specific immune response and produce a crossreacting inflammatory reaction. An increased T cell response against four peptides derived from 65-kDa mycobacterial HSP and their human homologs in British, Japanese and Turkish patients has been reported.482,490,491

Differential diagnosis The academic differential diagnosis includes other systemic vasculitides such as GCA; however, pulmonary artery aneurysm evaluation requires clinical context. Hughes-Stovin

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Chapter 19: Pulmonary vasculitis and pulmonary hemorrhage syndromes

syndrome (young men with idiopathic pulmonary artery aneurysms and recurrent venous thrombosis) may be related to BS (see Chapter 21).236,492,493

Prognosis and natural history The natural disease history features a series of exacerbations and remissions. Clinical symptoms may lessen after 20 years. Male sex and younger age of onset are markers of severe disease courses. Pulmonary arterial aneurysm appears to be associated with a particularly poor prognosis. Twenty percent of patients with this complication die within 2 years.474,494,495

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Chapter

20

The pathology of lung transplantation Gerald J. Berry and Helen M. Doran

Introduction Lung transplantation has evolved over the last 30 years from an experimental procedure to a proven therapeutic intervention for a wide variety of end-stage parenchymal and vascular disorders in infants, children and adults. Guthrie and Carrel first demonstrated the technical feasibility of the method over a century ago in a heterotopic procedure in which the heart and lungs of a kitten were implanted in the neck of a cat.1 Early experimental work is credited to the Russian physiologist, Petrovich Demikhov, although much of his work remained in obscurity until recently.2 The first human lung transplant was performed by Hardy and colleagues at the University of Mississippi in 1963 but the recipient survived only 18 days before succumbing to multiorgan failure.3 Cooley et al. performed the first combined heart-lung transplant in a 2-monthold infant with complex congenital heart disease. The child survived 14 hours and died of respiratory failure.4 Between 1963 and 1980, 38 more combined transplant procedures were performed but few patients survived more than a few days or weeks.5 Reitz and colleagues at Stanford University developed the first successful combined heart and lung transplant program in the early 1980s.6,7 Advanced pulmonary vascular disease including idiopathic pulmonary arterial hypertension and complex congenital heart disease with Eisenmenger’s physiology were indications for this procedure. Four of the five patients survived beyond a 6-month period of follow-up. Single lung and double lung (including bilateral sequential single lung procedures) transplant programs became established later in that decade.8 Lung transplantation in the pediatric population was introduced in 1986 and the first procedure in an infant was reported in 2000. The living-related lobar transplant program was developed by Starnes and colleagues in the mid-1990s and has more recently been expanded to cadaveric lobar lung transplantation.9–11 In the 2010 report of the Registry of the International Society for Heart and Lung Transplantation (ISHLT) 1400 lung transplant procedures and 559 combined heart-lung transplants were performed in pediatric patients from 1986 through June 2009.12 The accompanying adult registry recorded 3546 heart-

lung and 32 652 lung transplants since 1984 from centers around the world.13 Approximately 2800 lung procedures are performed annually in adults and a little over 100 procedures in children. The unadjusted 1-year, 5-year and 10-year survival figures for adults are 79%, 52% and 29%, respectively. The results are similar in the pediatric age group. These results have improved incrementally over the years and reflect improvements in surgical techniques, patient selection, perioperative and post-transplant patient management, immunosuppressive regimes, and the detection and treatment of infectious organisms. In the perioperative and postoperative period up to 30 days, primary graft failure, infection, technical and other complications and acute rejection account for the majority of deaths. In the period from 1 month to 1 year, infection and rejection are the most common causes of death. After 1 year chronic airway rejection is the commonest cause of death and remains an indication for retransplantation in some centers. Less common causes include malignancy and infection. The ISHLT registries also reported the indications for transplantation in adult and pediatric populations along with the types of surgical procedure. The primary indications for lung transplantation in adults between 1995 and 2009 were chronic obstructive lung disease (COPD), idiopathic pulmonary fibrosis (IPF), cystic fibrosis (CF), alpha-1-antitrypsin deficiency (A1AT) and idiopathic pulmonary arterial hypertension (IPAH). Less frequent indications include sarcoidosis, bronchiectasis, lymphangioleiomyomatosis (LAM) and retransplant for chronic airway rejection.13 Interestingly, there has been a gradual shift in the preferred surgical procedure from single lung to bilateral lung transplants and this now accounts for the majority of procedures. The decision on the type of operation (single lung transplant (SLT) versus bilateral/sequential lung transplant (B/SSLT)) remains controversial and largely appears to depend on center preferences, recipient age and donor organ availability. In the United States, the lung allocation score (LAS) was introduced in 2005 in an attempt to prioritize patients on the wait-list for lung transplants based on the urgency of the patient’s condition and the likelihood of survival after transplant.14,15

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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The LAS represents a paradigm shift from the traditional “time on the wait-list” approach to the implementation of the mathematical model that targets those most likely to benefit and those most in need of transplantation. The program has resulted in a significant decline in the wait-list times for patients from years to months and a drop in the number of deaths on the wait-list. It has also led to an increase in the proportion of patients transplanted for IPF and decline in those transplanted for COPD.16–18 There have been a number of recent studies that have shown that the overall survival time has not changed, reflecting the problem that sicker patients are now being transplanted. Not surprisingly, patients with high LAS scores have increased morbidity and mortality after transplant.19 Guidelines for patient evaluation have been carefully enumerated by the ISHLT to optimize both the timing and benefit of the procedure.20,21 The indications for lung transplantation in the pediatric population vary, depending on the age of the recipient in the pediatric group, and differ from adults. In the infant group the most common indications are IPAH or other vascular disorders, congenital heart disease and surfactant deficiencies. In the childhood group of 1 to 5 years, IPAH and pulmonary fibrosis account for the majority of cases. In children older than 6 years, CF remains the most common indication.12,22–25 In this population size-matching of the graft, donor shortages and technical alterations due to anatomic developmental anomalies introduce additional challenges.26

Table 1 Role of the pathologist in lung transplantation

1. Effective member of multidisciplinary team 2. Establish the primary native lung diagnosis on transbronchial or open lung/video-assisted thoracoscopic specimen prior to transplant 3. Thoroughly evaluate explanted lung(s) to confirm primary pathological diagnosis and identify additional lung pathologies, such as infection or occult malignancy 4. In post-transplant biopsies, identify etiology of early graft dysfunction such as primary graft dysfunction/ ischemia-reperfusion injury, hyperacute rejection, infection, anastomotic complications 5. Diagnosis and grade acute cellular rejection using ISHLT criteria and exclude morphological mimics. Determine efficacy of anti-rejection or anti-infection therapy in followup biopsy specimens 6. Diagnose other causes of graft dysfunction such as aspiration pneumonia, drug toxicity, infection, acute antibodymediated rejection 7. Identify the presence of obliterative bronchiolitis in late biopsy specimens 8. Diagnose and classify post-transplant lymphoproliferative disorder 9. Establish recurrence of primary parenchymal diseases in the allograft such as sarcoidosis, lymphangioleiomyomatosis 10. Preserve tissue or bronchoalveolar lavage specimens for research protocols

The pathologist’s role in lung transplantation The pathologist plays a critical role in the management of transplant recipients and is a key member of the multidisciplinary team (Table 1). Many patients, particularly those with interstitial lung disease (ILD), will have had prior transbronchial or video-assisted thoracoscopic (VATS) biopsies as part of their diagnostic work-up. The establishment of the correct pattern of ILD will have important therapeutic and prognostic implications such as the type of immunosuppressive agent (e.g. steroids versus mycophenolate mofetil). The explanted lung specimen should be carefully evaluated to establish the primary diagnosis and exclude secondary complications such as infections or occult malignancies.27 In the setting of pulmonary vascular disease the distinction of IPAH from chronic recurrent thromboembolic disease (CRPE) is important, as patients with CRPE are at risk of recurrent injury to the allograft. Patients with COPD can harbor occult malignancies such as bronchogenic carcinomas.28 We thoroughly examine the distal cartilaginous bronchi and small airways and routinely perform histochemical stains for fungal organisms to evaluate the possibility of fungal colonization of the airways as part of the primary disease process or as a complication of immunosuppressive therapy. In the perioperative and early postoperative period primary graft failure must be distinguished from hyperacute rejection,

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severe acute cellular rejection, and infection, as these will be treated differently. In the later post-transplant setting the pathologist is responsible for the diagnosis and grading of acute allograft rejection. As we will discuss in more detail, acute cellular rejection is a diagnosis of exclusion and a variety of morphological mimics must be considered. Following the institution of augmented immunosuppressive therapy for the treatment of allograft rejection, follow-up biopsies are usually performed to determine treatment effect. More recently the entity of acute antibody-mediated rejection (AMR) of the lung allograft has emerged and a multidisciplinary approach for diagnosis and treatment is necessary. The diagnosis of chronic airway rejection by transbronchial biopsy can be problematic on account of sampling issues. In the setting of declining pulmonary function 6 or more months following transplant, careful evaluation of the small airways is essential to identify obliterative bronchiolitis (OB). Less common findings include post-transplant lymphoproliferative disease (PTLD) and recurrence of the primary graft disease. Finally, the important role of the post-mortem examination for its clinical and educational functions must be emphasized. We attempt to obtain permission for autopsy evaluation of every thoracic transplant recipient and discuss in detail the clinical and pathological findings in the setting of a

Chapter 20: The pathology of lung transplantation

multidisciplinary conference. It is not uncommon to find clinically significant missed diagnoses in post-mortem studies. In a recent study 21% of autopsy cases found a discrepancy between the autopsy findings and clinically suspected cause of death. Hyperacute rejection, myocardial infarction, pulmonary embolism, high-grade acute cellular rejection and disseminated fungal infection were the most frequently missed diagnoses.29

Methods for monitoring the pulmonary allograft The post-transplant assessment and monitoring of recipients requires a complex and multidisciplinary approach. Not surprisingly these patients are challenging and often have multiple interrelated clinical problems. Frequent clinic visits, serial radiological imaging with roentgenograms and CT scans, daily spirometry measurements, interval pulmonary function studies, protocol bronchoscopy with bronchoalveolar lavage (BAL) and transbronchial biopsy, microbiological assessments, nutritional evaluations, measurement of immunosuppressive drug levels and psychosocial assessments are all part of the postoperative care to detect alterations in graft function and to support patient compliance and other critical issues. The use of noninvasive techniques to monitor the immunological status of cardiac transplant recipients has been established but remains an elusive strategy in lung transplantation.30–35

Transbronchial biopsy In many centers the surveillance of patients after transplantation includes protocol assessment by bronchoscopy with BAL and transbronchial biopsy.36–42 In some centers asymptomatic patients are followed without protocol transbronchial biopsy sampling and they reserve biopsy for clinical indications, such as new onset of symptoms or decline in pulmonary function.43,44 The goal of surveillance biopsy is the identification of treatable processes such as infection or acute rejection before allograft dysfunction develops, to identify potential risk factors for chronic airway rejection and ultimately to delay or prevent OB. This stems in large part from the poor sensitivity and specificity of clinical signs and symptoms, radiological techniques and functional techniques in distinguishing acute rejection, infection and airway anastomotic complications. As a result the transbronchial biopsy is widely regarded as the “gold standard” for the evaluation of the pulmonary allograft. It should be recognized, however, that there are limitations to this technique that include both technical and interpretative issues. The criteria for the diagnosis and reporting of acute cellular rejection (ACR) and other forms of allograft rejection have been established by the ISHLT and have undergone a series of modifications and revisions by the Lung Rejection Study Group since 1990.45–47 In a recent study of interobserver variability for the grading of ACR, a central panel of transplant pathologists reviewed over 1500 biopsies from 845 patients performed at 20 transplant centers. The kappa value for

interobserver agreement was 0.183.48 Cases were upgraded from no rejection to ACR in 9% of cases, downgraded from treatable rejection categories to no rejection or low-grade rejection in 35% of cases, and cases of low-grade rejection were downgraded to no rejection in 36% of cases and upgraded to treatable rejection in 19% (95% CI 0.147–0.220). In many cases the biopsies were deemed “ungradeable” using the ISHLT criteria. In another interobserver study of 59 biopsies for ACR, only moderate agreement was shown between pathologists (kappa 0.470). There was less robust agreement for the diagnosis of either airway inflammation or obliterative bronchiolitis (OB). Excellent intraobserver agreement was, however, found (kappa 0.795).49 In addition to the interpretative challenges, there are a number of technical problems related to the transbronchial biopsy. Tissue atelectasis and artifactual distortion are found to varying degrees with every sample. Gentle swirling agitation of the biopsy pieces in formalin fixative can reduce the amount of atelectasis. The liberal use of leveled sections and connective tissue stains can resolve crush artifact and render a biopsy fragment interpretable in some cases. The Lung Rejection Study Group of the ISHLT made recommendations for tissue handling and processing in the original grading scheme and these have been reiterated in the subsequent revisions. Specifically, a minimum of five pieces of alveolated tissue that are not completely collapsed should be obtained and immediately fixed in a standard fixative such as 10% neutral buffered formalin. In the 1990 and 1996 versions it was recommended that each piece should contain bronchioles and greater than 100 air sacs but this specific requirement was omitted from the 2007 document.45–47 Additional pieces may be necessary if focal processes, such as parenchymal nodules or obliterative bronchiolitis, are primary diagnostic considerations. Further, centers that utilize immunofluorescence antibody staining for the diagnosis of acute antibodymediated rejection (AMR) will require one or more additional tissue pieces in saline or another appropriate fixative before snap freezing. Electron microscopy has no role in routine transplant biopsy evaluation but may be part of research protocols. It is important to emphasize that frequently five pieces alone will not be sufficient as one or more piece is composed of airway wall, strips of airway mucosa, blood vessel or thrombus and so generous sampling is encouraged. The histological assessment of transplant pathology requires optimum handling and processing. Overnight processing in an automated processor is optimal but a variety of rapid processing programs are available for handling emergency biopsies or clinically indicated biopsies that yield slides in 3–4 hours. Following embedding in paraffin wax a minimum of three “leveled” sections each with multiple ribbons prepared at 4–5 mm thickness and routinely stained with hematoxylin-eosin (H&E). A connective tissue stain such as Masson’s trichrome and/or an elastic stain is helpful for assessing airway and vascular integrity. We still routinely perform silver stains such as Gomori-methenamine-silver (GMS) for

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fungal organisms but some centers prefer other microbiological, serological or molecular methods. Additional histochemical stains, immunohistochemistry and molecular techniques are advocated on a case-by-case basis, e.g. viral infections such as cytomegalovirus or for the diagnosis and classification of PTLD.50 Some centers alter the method of slide preparation and obtain seven levels, then stain levels 1, 4 and 7 with H&E, one with elastic trichrome (level 3), one with GMS (level 6) and two are left unstained for additional stains if necessary.51

Bronchoalveolar lavage In many centers bronchoalveolar lavage is used in conjunction with transbronchial biopsy. It can be used for the rapid assessment of infection in a patient with clinical deterioration. The specific methodologies differ among institutions but generally small aliquots of normal saline are put into the airways and then aspirated by manual or mechanical suction. Fractions of the fluid can be sent for microbiological culture, for cytopathological evaluation, a cell count and differential quantitation. The exclusion of bacterial or fungal infection is important in the early postoperative period and in patients with chronic airway rejection. Viral organisms can be seen on routine Papanicolaou or May-Grünwald-Giemsa stained preparations and fungal organisms can be visualized with fungal stains such as GMS. Some centers analyze the functional characteristics of the cells retrieved from the lavage as an adjunct test for infection, as well as acute and chronic rejection.52 On occasion acute cellular rejection can present with increased levels of eosinophils but this can also occur in drug reactions and some viral infections.53 In some centers long-term surveillance of BAL characteristics such as the predominance of lymphocytes or neutrophils, the CD4:CD8 ratio, the mean percentage of neutrophils and lavage levels of myeloperoxidase have been used to predict patient outcomes and in patients with bronchiolitis obliterans syndrome (BOS).54,55

Open lung biopsy (OLB) or video-assisted lung biopsy (VATS) The role of OLB and VATS biopsy in the management of transplant recipients has been controversial. Historically, concern for persistent air-leaks or bronchopleural fistulas limited the indication to gravely ill patients who had failed antirejection or anti-infection therapy or who suffered from multiple concurrent processes such as infection and PTLD. A limited number of studies published in the pediatric and adult transplant populations have shown similar findings.56–59 The majority of patients underwent OLB by way of a minithoracotomy incision and the biopsies were performed either in the early post-transplant or late transplant periods. The most common indications were unexplained deteriorating pulmonary dysfunction after thorough clinical, serological and bronchoscopic evaluation, or the onset of new or persistent

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pulmonary infiltrates or nodules. In 30–70% of cases a new diagnosis requiring therapeutic intervention or confirmation of the clinically suspected diagnosis was achieved, including acute rejection, OB, organizing pneumonia pattern, infection and malignancy. The procedure was found to be most helpful in the early postoperative period for identifying acute rejection or infection. In this setting there is a role for frozen section examination, as it can provide an initial impression of infection versus rejection versus PTLD and can direct the handling of tissue for special studies such as cytogenetics and molecular analysis. In every case a piece of tissue should be sent for microbiological analysis. Complications arose in 5–25% of reported cases and ranged from minor issues such as wound infection, postoperative pain and prolonged air-leaks to more serious problems such as respiratory failure and intrathoracic bleeding requiring surgical re-exploration. Importantly, resolution of air-leaks occurred in all patients although in some cases the course was protracted. The results of this small number of studies and our own experience indicate that there are specific clinical indications that warrant consideration for this invasive procedure.

A temporal approach to lung transplant pathology Lung transplant pathologists recognize that many of the clinical and pathological issues in the lung allograft occur within a reasonably narrow temporal framework. For example, primary graft failure and its morphological correlate of diffuse alveolar damage occur in the early postoperative period. Acute rejection is uncommon during this early period but is usually identified later in the first year. Obliterative bronchiolitis is uncommon in the first 6 months and typically presents after 1 year. We arbitrarily separate the pathological changes along various time points but recognize that some disorders such as acute rejection, infection or PTLD can occur anytime following transplantation (Table 2).

Perioperative and early post-transplant period (up to 1 month) Complications in the immediate perioperative and early postoperative period may present with similar clinical features and include primary graft dysfunction, hyperacute rejection, pulmonary venous obstruction, acute left ventricular dysfunction and overwhelming pulmonary infection with sepsis. The etiology, treatment and prognosis differ for each and their prompt recognition is important. With the exception of primary graft dysfunction, the other disorders in this group are rare.

Primary graft dysfunction / ischemic-reperfusion injury Primary graft dysfunction (PGD) represents an acute lung injury pattern that develops in the first 72 hours after

Chapter 20: The pathology of lung transplantation Table 2 Temporal paradigm for lung transplant pathology

1. Perioperative and early post-transplant period (up to 1 month) Primary graft dysfunction/failure Hyperacute rejection Anastomic complications Infections 2. Intermediate complications (1 month – 1 year) Acute cellular rejection Airway inflammation Acute antibody mediated rejection Infections Post-transplant lymphoproliferative disorder Drug toxicity Aspiration changes 3. Late complications (after 1 year) Obliterative bronchiolitis Chronic vascular rejection Post-transplant lymphoproliferative disorder and other EBV-related disorders Recurrence of primary pulmonary disease

transplant and affects 5–25% of lung allografts.60 It has previously been termed ischemia-reperfusion injury, reimplantation response or edema, early graft dysfunction and primary graft failure. It exhibits both a clinical and a morphological spectrum which in its severe form is characterized by acute hypoxemic respiratory failure, diffuse pulmonary infiltrates on radiographs and diffuse alveolar damage in lung biopsy specimens. The ISHLT recently proposed a clinical grading scheme (grades 0–3) for PGD severity based on the partial pressure of oxygen to the fraction of inspired oxygen ratio (PaO2:FiO2) and the presence or absence of radiological infiltrates at varying time points (0–6 h, 24 h, 48 h and 72 h) after transplant.60 Numerous studies have shown mortality in the first 30 days is significantly higher for patients with severe PGD and that patients who survive this injury are at increased risk of long-term functional impairment and the development of chronic allograft rejection in the form of pathological OB or the clinical bronchiolitis obliterans syndrome (BOS).61–63 Currently there is no specific clinical marker for PGD but a number of methodologies are under clinical investigation.64,65 A variety of donor, procurement, recipient and operative variables promote risk factors for PGD. Donor risk factors include older or very young donors, African-American race, female gender, history of smoking, prolonged mechanical ventilation, aspiration episodes, lung trauma and hemodynamic instability after brain death.66 The list of recipient risk factors includes patients with pulmonary arterial hypertension, elevated pulmonary artery pressures at transplant and patients with diffuse parenchymal lung disease.67 Prolonged ischemic time, the need for cardiopulmonary bypass and blood product administration have been found in some studies to be operative risk factors but some remain controversial. PGD probably involves a

multifactorial pathogenesis with numerous contributing factors. The mechanisms at the cellular and molecular level are now beginning to be elucidated towards the goal of limiting or eliminating its onset and development.67–70 The clinical differential diagnosis includes hyperacute rejection, cardiogenic pulmonary edema, venous anastomotic complications/ obstruction and infectious pneumonia, especially viropathic and bacterial. A morphological spectrum of changes of PGD can be found in transbronchial and open lung biopsies. As described above, diffuse alveolar damage (DAD) represents the morphological pattern in the most severe form of PGD. At the other end of the spectrum patterns of acute lung injury include neutrophilic infiltration or sequestration of the alveolar septa, patchy alveolar fibrinous aggregates and increased pulmonary macrophages (Figure 1). Severe PGD usually shows the proliferative or organizing phase of DAD with fibroblastic foci, scattered residual hyaline membranes, sparse interstitial inflammation but widened, edematous septa with fibroblastic aggregates. Similar to the clinical differential considerations, other morphological causes of acute alveolar injury must be considered including hyperacute rejection, vascular anastomotic problems with intravascular thrombi, infection and severe acute cellular rejection. Immunohistochemical staining for viropathic organisms and acute antibody-mediated rejection is recommended. Appropriate histochemical and immunohistochemical staining, microbiological cultures and serologies (viral and donor specific antibody) are required. The management of PGD is primarily supportive care with careful attention to fluid balance and infection prevention.

Hyperacute rejection Hyperacute rejection (HAR) is an uncommon but devastating complication that presents within minutes to hours after revascularization of the allograft. The presence of preformed circulating anti-HLA Type I or II or anti-ABO antibody against donor antigen triggers the complement system resulting in acute clinical dysfunction and morphological alterations. Currently only a few case reports have been published detailing the pathological changes.71–76 Clinically, patients present with abrupt onset of respiratory failure with a sharp increase in mean pulmonary artery and airway pressures, release of copious amounts of bloody, frothy fluid into the airways, dramatic decline in pulmonary compliance and a drop in both systemic blood pressure and arterial oxygenation. Radiological changes rapidly progress to “white-out” of the transplanted lung.77 Of the six cases detailed in the literature, five occurred in patients with emphysema, four had negative pretransplant studies for panel reactive antibodies (PRA) and two patients had elevated PRA. Death occurred in five patients despite aggressive interventions with plasmaphoresis and potent immunosuppressive agents. Currently, prevention of HAR by identifying patients at risk, e.g. prior pregnancies, transfusions

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(a)

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Figure 1. Histopathological patterns of primary graft dysfunction. (a) Interstitial accumulations of neutrophils and minimal alteration of the alveolar lining cells are observed. (b) Edematous interlobular septa with scattered inflammatory cells. (c) Exudative phase of DAD with numerous hyaline membranes, sloughed alveolar lining cells and rare inflammatory cells. (d) Proliferative or organizing phase of DAD with edematous septa, fibroblastic foci and reactive lining cells.

or transplantation, or underlying connective tissue disorders, and depletion of PRA by plasmaphoresis or intravenous immunoglobulin (IVIg) prior to transplant are routinely performed. The current techniques, sensitivities and specificities for cross matches are detailed elsewhere.78–80 The allograft in HAR is heavy, firm in consistency and purplish-red in color. Microscopically, the changes range from florid alveolar edema with fibrinous aggregates in the airspaces, to conspicuous neutrophilic infiltrates within the septal walls and/or flooding of the airspaces with blood and neutrophils to classic DAD with hyaline membrane formation and endothelial and epithelial injury (Figure 2). In the reported cases platelet-fibrin thrombi in the capillaries and small vessels were inconspicuous or absent. Deposits of immunoglobulin or complement in the vessels, alveolar septal structures and airspaces have been reported.

Vascular and airway anastomotic complications The transplanted allograft is at risk of vascular and/or airway complications on account of a number of donor-related, surgical and immunosuppressive factors. Colonization of the

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donor airways by bacteria or fungi, mismatch of donor and recipient airway or vascular luminal measurements, the use of positive-pressure mechanical ventilation and the tension placed on the anastomosis, devascularization of the proximal airways with bronchial artery disruption and the delay in graft healing due to immunosuppression all play roles in the initiation and development of anastomotic problems.81 While vascular anastomotic complications are less common than airway disruptions, the clinical implications are usually more serious. In particular, partial or complete venous obstruction remains a serious cause of early morbidity and mortality if not recognized and treated.82 The majority of venous difficulties occur early after transplant and present with signs and symptoms of persistent pulmonary edema, pleural effusions, hemodynamic instability, high pulmonary capillary pressure and parenchymal consolidation.83 Systemic embolization and stroke are also potential complications of pulmonary vein thrombosis. Complete venous obstruction causes hemorrhagic infarction of the lung if surgical revision of the anastomosis is not performed promptly. Transesophageal echocardiography is an effective modality for assessing the anastomosis and for detecting obstruction.84–85 Other

Chapter 20: The pathology of lung transplantation

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Figure 2. Hyperacute rejection. (a) Scanning magnification showing minimal alteration. (b) Alveolar edema and platelet-fibrin thrombi within the interstitial capillaries. Figure 3. Post-ischemic scarring with luminal narrowing of the airway. Other airways required stent placement.

techniques for diagnosis of these problems include CT or MRI imaging and CT angiography.86 Late venous complications have also been reported.87 Airway complications including infarction, dehiscence, overgrowth by bacteria or fungi, luminal narrowing by granulation tissue and scar tissue are commoner complications. The bronchial arteries provide the primary blood supply to the distal trachea and initial 4–5 cm of the bronchi. Ligation of the bronchial arteries places the anastomosis at risk of ischemic injury for the first 2–4 weeks after transplantation. In the early transplant experience, complete dehiscence occurred. Currently luminal narrowing by granulation tissue and scar formation is more common and in some cases requires laser excision and stent placement or retransplantation (Figure 3). Devitalized airways are also sites for infectious colonization.88

We have observed fungal tracheobronchitis and bacterial bronchitis in infarcted airway cartilage.

Infection in the early post-transplant period As with anastomotic complications, a variety of donor, operative and recipient factors place the allograft at risk of infection and sepsis.89 Prolonged hospitalization and mechanical ventilation promote nosocomial infection. The transplant procedure results in disruption of lymphatic circulation, ciliary motility, mucous clearance and neural connections and ischemia in the proximal airways. Following transplantation extended ventilatory requirements, line placements, nutritional problems and diminished ambulation contribute to the development of infections. Likewise recipient factors such as

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Chapter 20: The pathology of lung transplantation Table 3 2007 International Society for Heart and Lung Transplantation grading for allograft rejection

1. Acute cellular rejection (Grade A) No evidence of acute rejection (Grade 0) Minimal acute rejection (Grade A1) Mild acute rejection (Grade A2) Moderate acute rejection (Grade A3) Severe acute rejection (Grade A4) 2. Airway inflammation without scarring/ lymphocytic bronchiolitis (Grade B) * Ungradeable biopsy (Grade BX) No lymphocytic bronchiolitis (Grade B0) Low-grade lymphocytic bronchiolitis (Grade B1R) High-grade lymphocytic bronchiolitis (Grade B2R) * Designates revised grade Figure 4. Acute bronchopneumonia characterized by dense acute inflammatory exudates within alveolar spaces.

paranasal sinus and airway colonization in patients with cystic fibrosis, smoking history, nutritional and functional deconditioning and the intense level of immunosuppression after transplant are significant risk factors.90 As a result bacterial infections and, specifically, nosocomial infections account for the majority of infections.91,92 Gramnegative organisms such as Pseudomonas and Burkholderia and Gram-positive Staphylococcus are the most common organisms during this period. Nosocomial fungal infections also occur, with candida species constituting most cases. Viral, mycobacterial and parasitic infections are uncommon in this period. The incidence of overwhelming infection/sepsis has greatly diminished over the last two decades. Careful patient (both donor and recipient) selection, judicious use of antimicrobial prophylaxis, and refined post-transplant management have led to a significant decrease from previous rates, which were as high as 35%.93 Fiberoptic bronchoscopy with bronchoalveolar lavage and culture are generally used to establish the diagnosis. The typical pattern of bronchopneumonia is seen in tissue specimens (Figure 4). Because of the overlap of features with HAR, careful clinical correlation and the use of histochemical and immunohistochemical stains may be warranted.

Intermediate period after transplantation (1 month – 1 year) The common complications during this interval are acute cellular rejection (ACR), airway inflammation, antibodymediated rejection (AMR), infections and post-transplant lymphoproliferative disorder (PTLD). There is morphological overlap for many of these disorders.

Acute cellular rejection Episodes of ACR are common after lung transplant and develop in single and double lung and combined heart-lung

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3. Chronic airway rejection/obliterative bronchiolitis (OB) (Grade C) Absent (Grade C0) Present (Grade C1) 4. Chronic vascular rejection/transplant-associated vasculopathy (Grade D) Modified from reference 47

recipients. Both adult and pediatric patients are affected and the incidence is as high as 90%.22,23,37–40,94,95 Most episodes occur in the first 3–6 months. The Lung Rejection Study Group first presented the classification for grading and reporting ACR in 1990 and issued revisions in 1996 and 2007 (Table 3). Parenthetically, the grading of ACR has changed little since the 1990 scheme. Most patients are asymptomatic, reflecting the low-grade nature of most rejection episodes. However, on occasion even patients with moderate rejection have few or no complaints. Signs and symptoms are nonspecific and include low-grade fever, cough, drop in arterial oxygenation or expiratory flow rates, or new onset and/or increase in pulmonary infiltrates or pleural effusions.1 On account of the overlap of clinical and radiological findings with infection, imaging techniques are not sensitive and PFTs are useful for patient surveillance but not as diagnostic tools.96,97 The transbronchial biopsy has been used as the “gold standard” for the diagnosis and classification of ACR. The yield is higher in procedures performed for clinical indications, compared to those done as part of surveillance protocols. The cardinal features are the presence of mononuclear inflammatory cell infiltrates in the perivascular tissue spaces with extension along adjacent alveolar interstitial structures and into airspaces with increasing severity of ACR (Table 3). The immunophenotype of the inflammatory cells includes CD4þ and CD8þ T-cells, CD68þ macrophages and CD21þ dendritic cells. With increasing grades of rejection the cellular infiltrates may become more polymorphous with eosinophils and scattered neutrophils and can cause endothelialitis. Further, the

Chapter 20: The pathology of lung transplantation

airways including cartilaginous bronchi and bronchioles can have concurrent lymphocytic infiltrates in their walls and epithelial layers (lymphocytic bronchitis/bronchiolitis). The A grade designates the grade of acute rejection and the B grade designates the airway inflammation.47

The cells are typically small and transformed lymphocytes with occasional plasmacytoid cells form rings 2–3 cell layers thick (Figure 6). These are generally inconspicuous at lowpower magnification but can be detected at scanning magnification.

Grade AX (ungradeable specimen)

Grade A2 (mild acute rejection)

A numeric grade cannot be rendered because of insufficient (< 5 adequate) pieces of tissue, crush artifactual distortion, etc. (Figure 5).

Perivenular and occasionally periarteriolar infiltrates are readily observed at low and scanning magnifications and are composed of more than three cell layers in thickness. The composition includes small and transformed lymphocytes, macrophages and, not infrequently, eosinophils. The infiltrates are restricted to the perivascular spaces and do not extend along the alveolar septa (Figure 7). Besides the extent of the infiltrates and the presence of eosinophils in Grade A2, another feature that distinguishes it from Grade A1 is the presence of subendothelial mononuclear cells with a resultant “lifting” or expansion of the endothelial cells termed endothelialitis, or intimitis. In our experience, co-existing airway inflammation is more common in mild rejection than minimal rejection. The liberal use of leveled sections is often helpful in borderline cases. In most centers this is the threshold for instituting augmented immunosuppression on account of concern for immune-mediated damage of the allograft and the risk of developing chronic airway rejection.98

Grade A0 (no evidence of rejection / NER) No perivascular infiltrates are seen in an adequate biopsy sample.

Grade A1 (minimal acute rejection) Limited, infrequent circumferential cuffs of mononuclear inflammatory cells are seen around one or a few venules.

Grade A3 (moderate acute rejection)

Figure 5. Ungradeable biopsy (grade AX). Marked crush artifactual distortion and collapse of alveolar spaces preclude the confirmation of perivascular mononuclear inflammatory cells for the diagnosis of ACR.

(a)

The hallmarks of moderate rejection include the frequency and density of the perivascular cuffs and the extension of the inflammatory infiltrates along peribronchiolar alveolar septa and into alveolar spaces (Figure 8). The latter is often characterized by macrophage and lymphocyte collections within alveolar spaces and type II changes of the alveolar epithelium. Endothelialitis is often present and concurrent airway inflammation may also be found.

(b)

Figure 6. Minimal acute rejection (grade A1). Sparse perivascular mononuclear cells cuff the vessels.

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Figure 7. Mild acute rejection (grade A2). Distinct perivascular collections are observed at scanning magnification (panels a and c) and confirmed at higher magnification. The infiltrates are restricted to the perivascular tissue spaces. Note the presence of eosinophils in addition to mononuclear cells in panel d.

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Figure 8. Moderate acute rejection (grade A3). Extension of the inflammatory infiltrates from the perivascular tissue spaces along the surrounding alveolar septa (a) and into the alveolar spaces (b).

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Grade A4 (severe acute rejection) In our experience this stage of ACR is now uncommon. Most patients are symptomatic with marked dyspnea or acute respiratory failure. In addition to the perivascular, interstitial and airspace mononuclear infiltrates and endothelialitis this pattern of ACR is characterized by parenchymal damage in the form of alveolar damage, hyaline membranes, necrotic cellular debris, alveolar hemorrhage and often a conspicuous neutrophilic component (Figure 9). Vasculitis, thrombosis and tissue infarction are seen in the late stages.

Morphological mimics and pitfalls in the diagnosis and grading of acute rejection There are a number of morphological mimics of ACR (Table 4). Firstly, perivascular inflammatory infiltrates are not specific for ACR. A variety of viral, mycobacterial and fungal infections and PTLD can have perivascular infiltrates. In particular CMV and Pneumocystis jirovecii pneumonia can both have perivascular and/or interstitial components (Figure 10). For this reason the diagnosis of ACR is defined as a diagnosis of exclusion and should be evaluated together with the clinical, serological and microbiological information. Occasionally, the question of concurrent non-bacterial infection and rejection arises. For example, classic CMV pneumonitis is found in one biopsy piece and perivascular mononuclear infiltrates without associated viral inclusions are present in another piece. Stewart and others have observed that CMV pneumonitis usually shows a predominance of interstitial and septal changes over the perivascular cuffing and frequently also exhibits prominent perivascular edema, small neutrophilic microabscesses, cytological atypia of the alveolar lining cells and concurrent acute airway inflammation.90 Appropriate viral serologies and cultures are recommended along with immunohistochemical and/or molecular tissue studies. In cases that cannot be resolved the Lung (a)

Rejection Study Group has recommended that the pathologist indicate which process is favored and that a repeat biopsy should be performed after an appropriate period of antimicrobial therapy to exclude ACR.46 Bronchus-associated lymphoid tissue (BALT) is localized to the wall of small airways at branching points. In most cases its nodular configuration and airway position confirms the diagnosis. Small vessels in the walls of the airways can display a circumferential inflammatory cell cuff as part of BALT and should not be confused with ACR (Figure 11). In some cases the use of additional leveled sections is helpful in demonstrating the airway locale. This finding accounted for some of the discrepancy in the ACR readings between local and central pathologists in the study by Arcasoy and colleagues.48 Collections of mononuclear and other inflammatory cells can be seen in biopsy site changes. Although uncommon, the repetition of transbronchial biopsy can result in the sampling of a healing or healed site of prior biopsy. The presence of granulation tissue, accumulations of fibrin, blood and thrombus are useful histological clues (Figure 12). Perivascular mononuclear cell infiltrates in the walls of conducting airways or within interlobular septa likewise Table 4 Morphological mimics of acute cellular rejection

Infection (Viral (CMV) and fungal (Pneumocystis) Bronchus-associated lymphoid tissue Biopsy site changes Post-transplant lymphoproliferative disorder Primary graft dysfunction Acute antibody-mediated rejection Drug toxicity

(b)

Figure 9. Severe acute rejection (grade A4). In addition to dense interstitial and intra-alveolar infiltrates there are alveolar fibrinous exudates and hyaline membranes. Endothelialitis is shown in panel A (H&E  100). At high magnification consolidation of inflammatory cells within the alveolar structures is found.

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Figure 10. Infectious mimics of ACR. (a) CMV pneumonitis with numerous inclusion bodies. There is conspicuous perivascular edema along with the mixed inflammatory cell infiltrates and prominent interstitial infiltrates. (b) Pneumocystis jirovecii pneumonia (PJP) with cysts found adjacent to small vessels and a sparse mononuclear cell infiltrate (H&E and GMS stains).

Issues related to the diagnosis and classification of acute rejection

Figure 11. Bronchus-associated lymphoid tissue (BALT). A nodular, circumscribed collection of predominantly small lymphocytes is noted within the wall of the conducting airway.

should not be classified as ACR. In some of these cases the inflammatory cells are admixed with anthracotic pigment. PTLD is defined as a proliferation of lymphoid and/or plasmacytoid cells and is usually EBV-associated. In most cases these present as solitary or multiple lung masses. An interstitial component with or without perivascular involvement can be seen around the edge of the lesions. A transbronchial biopsy specimen may yield the periphery of the lesion rather than the diagnostic component. Communication between the clinical team and the pathologist is essential for proper specimen handling and selection of appropriate immunophenotypic and molecular panels. Interestingly, we have observed both ACR and PTLD in a VATS biopsy and used immunohistochemistry to delineate the lesions.

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A number of technical and interpretative issues related to the ISHLT grading scheme warrant further clarification. Firstly, it is not uncommon to find different patterns of severity of ACR in a transbronchial or VATS biopsy. For example, one piece of tissue may display a pattern of minimal rejection and another show mild or moderate ACR. The designated grade is based on the most advanced grade of rejection and not the predominant pattern. Secondly, some samples may exhibit ACR but with fewer than the recommended five pieces of alveolated tissue. We advocate a descriptive diagnosis such as “Mild Acute Rejection in a Suboptimal Sample” and refrain from a numeric grade to alert the clinician to the ACR. The concern for a higher grade of ACR in the lung must be considered in this setting. Thirdly, the diagnosis of ACR requires that the inflammatory infiltrate is completely circumferential around the vessel. The arrangement can vary from compact, “tight” collections to more loosely arranged cuffs. The problem of a partial rather than complete cuff is not uncommon. Additional leveled sectioning of the paraffin block resolves many of these problematic cases. In equivocal cases we report the findings and recommend careful clinical and microbiological correlation to exclude an infectious etiology. Fourthly, it is not uncommon to find a number of different pathologies in a transbronchial biopsy sample.99 For example, we have observed concurrent cases of ACR and OB in patients transplanted more than 1 year. All significant findings should be documented in the pathology report irrespective of the overall adequacy of the sample. The clinical significance of Grade A1 or minimal ACR is controversial. Episodes of higher grades of ACR (mild or greater) have been shown to be independent risk factors for the development of chronic rejection. Recent studies have

Chapter 20: The pathology of lung transplantation

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Figure 12. Biopsy site changes. (a) Low-power magnification showing a well-delineated round focus of scar with central hemosiderin-laden macrophages. (b) Higher magnification showing granulation tissue admixed with early fibrous scar, blood and hemosiderin-laden macrophages.

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Figure 13. Mimics of ACR. (a and b) Collections of mononuclear cells in the wall of the airway, including around mural blood vessels. (c) Nodular collection of mononuclear inflammatory cells within an interlobular septum. (d) Note the absence of a perivascular distribution at high magnification.

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Figure 14. Low-grade lymphocytic bronchiolitis (grade B1R) is characterized by submucosal and peribronchiolar collections of mononuclear inflammatory cells with only rare intramucosal lymphocytes.

raised concern that even asymptomatic minimal rejection (Grade A1), including patients with only a single episode, increases the risk for BOS.100–101 Unfortunately, the reports do not provide photomicrographs of cases interpreted as minimal rejection. As the current threshold for initiating treatment is mild rejection, it is important that the pathologist strictly adheres to the ISHLT diagnostic criteria. In addition, patients with minimal ACR warrant close follow-up and repeat biopsy after a designated interval. The concept of AMR in lung transplantation has recently received much deserved attention. As will be discussed in detail later, the diagnostic criteria remain elusive. Nonetheless, the morphological findings include neutrophilic interstitial infiltrates and DAD with hyaline membranes. The overlap with severe ACR is considerable and therefore all cases of severe ACR should be studied for AMR with tissue immunophenotyping and serological donor specific antibody studies.

Airway inflammation without scarring As previously described, the large and small airways of the pulmonary allograft are the targets of a variety of inflammatory and infectious lesions. In some cases of ACR the accompanying airways display submucosal mononuclear inflammatory cell infiltrates. If other etiologies can be excluded, the infiltrates are thought to be a manifestation of airway rejection. In the original 1990 ISHLT scheme, each grade of ACR was further divided into subgroups a–d depending on the presence or absence of bronchiolar inflammation (a, b), large airway inflammation (c) or absence of bronchioles (d). In the absence of ACR, active airway inflammation without associated scarring was classified as either lymphocytic bronchitis or bronchiolitis Grade B1 or B2. In the 1996 revised ISHLT scheme the four subtypes in ACR (subgroups a–d) were eliminated as they were

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considered cumbersome and confounding. However the concept of airway inflammation was further expanded into six groups to reflect the concept of airway inflammation as a risk factor or harbinger of chronic airway rejection (Table 4). The grades of Minimal (B1), Mild (B2), Moderate (B3) and Severe (B4) represented airway alterations along a morphological spectrum from scant mononuclear cells in the bronchial or bronchiolar submucosa to dense band-like mural infiltrates with epithelial sloughing and ulceration. Grade 0 and ungradeable or Grade BX were retained from the original form. The Lung Rejection Study Group simplified the classification in the 2007 ISHLT grading scheme (Table 3). Firstly, the evaluation of airway inflammation is now restricted to bronchioles and the term “lymphocytic bronchiolitis” (LB) is applied. Secondly, the minimal and mild grades (B1 and B2) were reduced to low-grade small airway inflammation and the numerical grade B1R was created to emphasize that this grade represents the revised grade. Low grade LB is characterized by mononuclear inflammatory cells in the submucosal layer of the bronchioles. The patterns range from patchy or scattered infiltrates to circumferential bands of mononuclear cells (Figure 14). Eosinophils are found occasionally but in small numbers and, by definition, mononuclear cells are not present amongst epithelial cells. Currently, the term high-grade small airway disease and its numeric counterpart B2R replace the moderate and severe grades (B3 and B4) of the 1996 scheme. It is defined by the presence of dense mononuclear cells in the submucosa, including small and transformed lymphocytes admixed with other inflammatory cells such as eosinophils, neutrophils and plasma cells in association with intra-epithelial infiltrates and epithelial injury. The damage ranges from respiratory cell necrosis and metaplasia to epithelial sloughing, ulceration and cellular and fibrinous exudates (Figure 15).

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Figure 15. High-grade lymphocytic bronchiolitis (grade B2R) displays dense intraepithelial, submucosal and mural infiltrates composed of lymphocytes, eosinophils and scattered neutrophils. The mucosal layer is sloughed in panel B.

Grade B0 is defined as the absence of airway inflammation and the numeric grade, Grade BX is employed for ungradeable biopsy specimens, e.g. absence of airways, tangential sectioning, artifactual distortion of airways or infection. There are a number of diagnostic issues that need to be further elaborated. Firstly, the 2007 classification acknowledged the poor reproducibility of the earlier schemes and attempted to alleviate the confusion. Secondly, it reiterated that the etiology of airway inflammation is multifactorial and that airway infection is an important consideration. A variety of bacterial infections including Pseudomonas, Mycoplasma and chlamydial organisms, as well as viruses and fungi, can produce a similar histological pattern and should be excluded by culture and/or serological testing. If eosinophils or neutrophils are the predominant cell type in the airways, an infectious or aspiration etiology must be excluded. Another common cause of airway inflammation is aspiration injury. Oropharyngeal dysphagia is common after lung transplantation and aspiration of gastric contents, including bile acids, has been shown to be a risk factor for the development of BOS.103,104 Other causes of airway inflammation including smokingrelated injury should also be considered. The diagnosis and classification of LB is often challenging for technical and interpretative reasons. Tangential or incomplete cross sections of airways are not uncommon in transbronchial specimens. In one study of 2981 samples, 46% were ungradeable (Grade BX).48 The interobserver agreement was even poorer than that for ACR, with a kappa value of 0.035. Some of the reasons for discrepancy have been previously discussed, such as BALT and biopsy site changes. BALT displays rounded aggregates of small lymphocytes, often with small germinal centers containing B-cells and high endothelial venule-like vessels. The epithelium overlying the lymphoid aggregates exhibits diminished numbers of goblet cells, CD4þ T-cells and more epithelial cells containing microvilli.

PTLD can occasionally present either centered on the airway or as a peripheral parenchymal lesion with an airway component but usually shows cytological atypia or necrosis. The classification of LB in the setting of ACR warrants brief mention. It is not uncommon, particularly in the higher grades of ACR, to find concurrent LB.105 We grade and report the patterns of ACR and LB separately in the pathology report, e.g. mild acute cellular rejection (Grade A2) and low-grade lymphocytic bronchiolitis (Grade B1R). The prognostic significance of LB and, in particular, its severity has been clearly documented. Glanville et al. reported that the severity of LB is linked to both the subsequent risk of BOS and mortality.106 The pathogenesis of LB is the subject of current investigation in animal models and transplant patients. The mechanism remains unknown but inadequate suppression of peripheral blood T-cell granzyme B, interferon-g and tumor necrosis factor-a is postulated.107

Antibody-mediated rejection Antibody-mediated rejection is firmly established as a cause of morbidity and mortality in renal and heart transplant recipients. The histopathological and immunophenotypic criteria were recently established for the cardiac allograft but are elusive in the lung transplant setting.108–109 Currently, pulmonary AMR has been reported in the literature as single case reports and small series.110–112 The 2007 ISHLT Working Formulation reached no consensus but recommendations for terminology, antibody staining and future studies were proposed. Specifically, the term “acute capillary injury” was favored over terms such as “acute capillaritis” and “septal capillary necrosis” by the majority of transplant pathologists.113–115 The patterns of injury in pulmonary AMR include acute alveolar injury with fibrinous alveolar exudates, alveolar hemorrhage, septal neutrophilic

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Figure 16. Acute antibody-mediated rejection. (a) Transbronchial biopsy sections showing fibrinous intra-alveolar collections and reactive-appearing alveolar lining cells. (b) Linear continuous C4d staining alveolar capillary walls. This represents staining of subendothelial structures.

congestion and margination, and hyaline membrane formation (Figure 16). These features are nonspecific and can be seen in primary graft dysfunction, HAR, non-immunological causes of DAD, severe ACR and infection. More importantly, these findings should warrant a thorough histochemical, immunohistochemical, molecular, serological and microbiological work-up for other possible causes. The immunohistochemical criteria for AMR are also unsettled. In some studies positive C4d staining was restricted to linear, continuous endothelial/subendothelial deposition in septal capillaries, while others have included arterioles and venules for the diagnosis.110–112 Staining of elastic membranes of bronchial walls, hyaline membranes in DAD and vessels in the walls of airways is nonspecific and is not considered in the evaluation. The association of AMR and ACR is also controversial and conflicting results have been reported.110,111 A recent study by Westall and colleagues found C4d and C3d deposition in 33% and 60% of biopsies, respectively, in primary graft failure and early post-transplant period (1–3 months) infections.116 Moreover, they did not demonstrate an association with ACR, chronic airway rejection or with the morphological features of AMR. There are a number of unresolved issues.117 There is currently no consensus on the histopathological or immunophenotypic criteria for the diagnosis of AMR. As with cardiac AMR there is probably a morphological spectrum of changes that progress to a diffuse alveolar injury pattern in the allograft. Currently we perform immunostaining on formalinfixed paraffin-embedded transbronchial biopsy specimens when an acute alveolar injury pattern is observed. We utilize a panel approach of antibodies that includes C4d and CD31. We require continuous linear staining of interstitial capillaries and use venules and arterioles as internal controls for C4d.

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Analogous to cardiac AMR we also carefully evaluate the small vasculature for the presence of intravascular macrophages (Figure 16). Careful clinical and microbiological correlation is essential in light of the previous discussion of the nonspecificity of histological and immunophenotypic staining. We routinely recommend serological donor-specific antibody (DSA) studies in this setting. Our group thinks that the diagnosis of AMR should be made as a diagnosis of exclusion, and only in the setting of clinical dysfunction, circulating anti-HLA antibodies (positive DSA) and positive histological and immunophenotypic results. Additional multicenter efforts will be required to further evaluate the histological and immunological components of AMR. In particular, frequent serial assessment of patients by transbronchial biopsy with routine immunohistochemical stains is needed along with clinical and serological correlation. In some centers 10–15% of transplant recipients are presensitized prior to transplant and are at risk of developing both early and late complications.118–121 Further, patients can develop donor-specific and non-donor specific anti-HLA antibodies following transplantation and are likewise at risk for the development of AMR, persistent ACR and BOS with diminished overall survival. The incidence ranges from 10% to 56% and it occurs within the first 3 months in the majority of patients.122–123 In some centers treatment protocols using rituximab and intravenous globulin (IVIG) or IVIG alone are used. Multicenter trials are under way to further evaluate the efficacy of therapeutic intervention for the prevention of chronic allograft dysfunction such as BOS in patients who develop anti-HLA antibodies after transplant. In addition to anti-HLA antibodies, some patients develop non-HLA antibodies after transplantation that may also contribute to chronic injury. In particular, autoantibodies against Type V collagen and K-alpha-1-tubulin have been shown to have a deleterious effect.124–126 These studies suggest the

Chapter 20: The pathology of lung transplantation

importance of serological monitoring of patients as part of routine surveillance.

Infection As mentioned previously the lung allograft is at risk from a variety of infectious complications on account of immunological, mechanical and functional issues related to the donor, the recipient, the procedure and post-transplant management. Among the solid organ transplant groups, lung recipients are at the highest risk of infection. Both adult and pediatric groups are vulnerable, with respiratory viral infections reported in more than half of pediatric recipients.127 Fishman and Rubin classify the types of infectious etiologies according to a temporal paradigm.89 In the period 1 to 6 months following transplantation opportunistic infections occur as patients are maximally immunosuppressed to prevent acute allograft rejection. In addition to the immunomodulating viruses of the herpes group (CMV, EBV and others), fungal infections such as aspergillosis and Pneumocystis pneumonia, bacterial infections such as nocardiosis and mycobacterial infections and, rarely, parasitic infections arise.128 After 6 months most patients have reached their lowest maintenance levels of immunosuppression and are at risk primarily from community-acquired infections. Community-acquired respiratory viruses can trigger acute rejection episodes or promote the development of chronic rejection.129 In the small subset of patients who receive augmented immunosuppression for recurrent acute rejection or in an attempt to stabilize chronic rejection, recrudescence of opportunistic infections can arise (Table 5). With the widespread use of antimicrobial prophylaxis for viral and fungal organisms new infectious complications have now emerged.130 We will discuss the common infections in lung recipients that occur in this time period.

Viral infections including cytomegalovirus (CMV) Viral infection and CMV in particular is the most common opportunistic infection in this population as the lung is the principal reservoir for latent CMV virus. The two mechanistic scenarios are primary allograft infection through viral transmission or reactivation of latent virus.90 Seronegative recipients who receive grafts from seropositive donors (Dþ/R) and the first transplant for a seropositive recipient (Dþ/Rþ) are high-risk groups. ISHLT registry data have shown that these patients are at risk of diminished long-term survival.131 There remains a great deal of confusion in the literature regarding CMV terminology and our approach is based on the following definitions:132,133 CMV infection: evidence of CMV replication regardless of symptoms (differs from latent CMV). CMV disease: evidence of CMV infection with attributable symptoms. It can be further classified as CMV syndrome and tissue-invasive disease.

Table 5 Timeline of infections in lung allografts

1. Perioperative to 1 month i. Nosocomial infections ii. Infection in devitalized airways following ischemia 2. One month to 6 months i. Opportunistic infections such as aspergillosis, CMV pneumonitis ii. Relapse or residual nosocomial infection from early postoperative period 3. After 6 months i. Opportunistic infection following therapeutic interventions for acute or chronic rejection ii. Community-acquired infections such as respiratory viruses iii. Endemic infections restricted to geographical region Modified from references 89, 90, 150

CMV viral syndrome: presence of fever (> 38 C for at least 2 days within a 4-day period), malaise, leukopenia and thrombocytopenia and the detection of CMV in blood. Tissue-invasive disease: presence of signs and/or symptoms of pulmonary disease combined with demonstration of CMV in BAL fluid or lung tissue specimens by virus isolation, histopathology and immunohistochemical or in situ hybridization staining. Prior to the era of CMV prophylaxis, transplant patients typically developed clinical CMV disease (fever, leukopenia and end-organ involvement) or CMV infection between 1 and 3 months. Currently most programs have implemented prophylaxis regimes using anti-viral agents such as oral valganciclovir or oral/intravenous ganciclovir although the treatment duration varies considerably in centers from 1 to 12 months.134–137 Rather than eliminating the disease, studies have shown that these programs delay the onset of disease and the problem of “late-onset CMV disease” has emerged in some patients.138 Other problems with long-term prophylactic regimens include drug toxicity and neutropenia and the development of drug resistance in 10–15% of patients.139,140 There are a variety of diagnostic approaches to monitoring patients and for the diagnosis of active CMV infection/disease. These include surveillance bronchoscopy with BAL lavage and transbronchial biopsy, rapid shell viral culture, determination of viral load from peripheral blood of BAL lavage with pp65 antigenemia assay and PCR techniques. Each has documented sensitivities, specificities and positive and negative predictive values.134 The ultimate goal of surveillance programs is to prevent ventilator-dependent CMV pneumonitis and secondarily to prevent the triggering of acute rejection, other infections and BOS. In up to 15% of biopsies demonstrating CMV pneumonitis patients are asymptomatic.90 The diagnosis requires the identification of the classic amphophilic, large nuclear inclusion separated from the nuclear membrane by a translucent rim or halo with or without cytoplasmic inclusions amidst a variable inflammatory response (Figure 17). CMV or “owl’s eye” inclusions can be found in pneumocytes, endothelial cells, dendritic cells, macrophages and smooth muscle cells.

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The inflammatory response can range from a sparse interstitial pneumonitis of mixed inflammatory cells to an alveolar damage pattern with fibrinous exudates, hyaline membranes and microabscesses. Immunohistochemical or molecular techniques are useful for biopsies demonstrating equivocal findings. Following anti-viral therapy, cells infected by CMV can contain smudgy, inhomogeneous inclusions with irregular outlines and lose the characteristic perinuclear halos (Figure 18). As discussed earlier, the problem of distinguishing ACR from CMV pneumonitis can be problematic. Perivascular infiltrates are reported in up to 45% of biopsies with CMV pneumonitis and endothelialitis can also be found.39 In this setting we recommend anti-viral therapy with an early followup biopsy to evaluate acute rejection. Subtle clues that can favor CMV pneumonitis and other viral infections over ACR

include perivascular edema and mixed inflammatory infiltrates that contain neutrophils, more loosely arranged perivascular cuffs and interstitial and septal infiltrates that predominate over the perivascular component (Figure 10).141 Other viruses from the beta-herpes group are encountered in lung recipients. We will discuss Epstein-Barr virus (EBV) in detail in the discussion of PTLD. The role of human herpesvirus 6 (HHV-6) and human herpesvirus 7 in graft dysfunction and other complications is the subject of recent investigation.142–144 Adenovirus and herpes simplex virus are other opportunistic viral infections that can be encountered in transbronchial biopsies. In addition to acute morbidity and mortality, some patients with adenovirus pneumonia progress to chronic rejection. Adenoviral pneumonia is characterized by necrotizing, hemorrhagic bronchopneumonia with irregular ground-glass intranuclear inclusions or smudge cells (Figure 19).145 Patterns

Figure 17. CMV pneumonitis is characterized by the presence of classic “owl eyes” intranuclear inclusions in epithelial, endothelial, smooth muscle and other cells.

Figure 18. Following treatment with ganciclovir the CMV-infected cells lose their classic nuclear halos and appear more smudgy and inhomogeneous.

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Figure 19. (a) Adenovirus pneumonia in a lung transplant recipient. The epithelial cells display small smudgy intranuclear inclusions. (b) Herpes pneumonia showing a necrotizing hemorrhagic pneumonia with abundant karyorrhectic debris and numerous type A and type B inclusions. Inset: immunostaining for HSV.

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of HSV pneumonia include ulcerative or necrotizing tracheobronchitis and bronchiolitis, miliary nodules and DAD patterns. Single or multinucleated giant cell inclusions are found adjacent to or within the necrotic foci (see Chapter 5).

Common community-acquired viral infections Community-acquired respiratory viral illnesses in immunocompetent patients are usually limited to upper respiratory tract symptoms. However, in lung transplant recipients lower respiratory tract involvement is more common and is associated with increased morbidity and mortality. Both RNA and DNA viruses have been identified and the most common agents are respiratory syncytial virus (RSV), influenza A/B, parainfluenza (serotype 3), metapneumovirus and rhinovirus. The incidence ranges from 2% to 15% of recipients in some reports.146 The histological findings are usually nonspecific and the diagnosis is confirmed by viral culture, fluorescent antibody or PCR tests. Unfortunately, vaccine for the paramyxoviruses is not currently available. In addition to the risk of life-threatening viral pneumonia and superimposed bacterial infections, these viruses (except for rhinovirus) have been associated with both acute and chronic rejection. Antiviral agents have been used in cases of severe infection but the results have been quite variable.147–149

Fungal infections Fungal infections remain a serious concern following transplantation and a significant cause of mortality. The patterns and frequency of specific fungal infections have changed over the last two decades, reflecting in large part greater clinical awareness and management and the implementation of antifungal prophylaxis. The problem has been discussed in detail in a number of recent publications and the current discussion will be restricted to histopathological patterns.90,150–154 The temporal paradigm outlined by Kubak provides a useful framework.150 In the first month after transplantation infections are donor-derived or related to surgical complications such as airway ischemia. In the period of 1–6 months fungal infections are usually opportunistic in origin but either relapsed or residual infection are considerations. In this setting, predisposing factors include transplant indications such as COPD, ILD and CF, augmented immunosuppression for acute rejection, concurrent or recent bacterial or viral infections and other causes of graft dysfunction. Vadnerkar and colleagues emphasize the importance of detailed histopathological examination of the explanted lung(s) at the time of transplantation and reported mold infections in 5% of explanted lungs.155 The patterns included chronic necrotizing pneumonia, mycetoma and invasive fungal pneumonia and the diagnosis was not suspected in over half of the patients. In the period after 6 months fungal infections usually arise after therapeutic interventions for chronic rejection. Aspergillus, Candida, Scedosporium and Cryptococcus infections are responsible for the majority of clinical

infections. Of these, Aspergillus infections are the most common. A range of lesions have been documented, including airway dehiscence, vascular anastomotic disruption, tracheobronchitis including ulcerative and pseudomembranous forms, angioinvasive/disseminated parenchymal disease, empyema, aspergilloma, endobronchial stent obstruction, mucoid bronchial impaction and other patterns of allergic bronchopulmonary aspergillosis (see Chapter 15).150 Tracheobronchitis involves foci of ischemic airway including the anastomotic site and is characterized by necrotic cartilage containing hyphal forms (Figure 20). At the other end of the spectrum, invasive aspergillosis demonstrates foci of ischemic necrosis with intravascular plugs of fungi that spill out into the adjacent necrotic parenchyma (Figure 20). Widely disseminated aspergillosis is less common now but involvement of the heart, central nervous system, thyroid, adrenal glands and other sites is often found at autopsy. Subtle clues for the possibility of airway fungal colonization/infection include the presence of numerous mural eosinophils. A number of noninvasive techniques are currently being evaluated for the detection of invasive disease patterns, but prevention or early detection are the optimal modalities for the prevention of invasive aspergillosis.155–157 Serious Candida infections are less common today than in the early transplant experience, where it accounted for the majority of infections in the first month.158 Identification of Candida mold in BAL fluid by culture or cytopathology is not uncommon and most represent upper airway colonization. Scedosporium species (formally Pseudoallescheria) are indistinguishable from Aspergillus on morphology and by radiological techniques. They cause a similar spectrum of disease but, importantly, are resistant to amphotericin-B and other polyene antifungal drugs but do respond to azole compounds such as voriconazole. Cryptococcus infection is rare in pediatric lung recipients and usually reflects reactivation of latent disease in adults. In immunocompetent hosts the disease usually manifests as nodular lesions but in immunosuppressed patients alveolar or interstitial patterns are seen.159 A detailed discussion of uncommon and emerging fungal pathogens was recently reported by Kubak and colleagues.153 Pneumocystis jirovecii pneumonia (PJP) (previously known as Pneumocystis carinii or PCP) is now uncommon with routine prophylaxis. Poor compliance and the development of drug resistance currently account for most of the cases and lifetime prophylaxis is required. The classic radiological appearance is bilateral ground-glass opacities. The histological patterns include sparse interstitial lymphoplasmacytic infiltrates, the classic frothy alveolar exudates, granulomatous lesions, and DAD (see Chapter 7). Importantly, in lung transplant recipients the alveolar exudates may be diminished. Perivascular mononuclear inflammatory cell infiltrates mimicking ACR are reported, so we continue to routinely perform silver stains (Figure 20).

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Figure 20. Patterns of Aspergillus infection in lung transplant recipients. (a and b) Fungal tracheobronchitis in necrotic cartilage and supporting soft tissues of the cartilaginous airway. Note the presence of fungal hyphae within and around the necrotic cartilage. (c and d) Tissue invasive aspergillosis with numerous fungi seen within necrotic parenchyma (b and d GMS stains).

Post-transplant lymphoproliferative disorder and other EBV-related disorders In the most recent edition of the World Health Organization (WHO) Classification of Tumours of Hematopoietic and Lymphoid Tissues PTLD is defined as “lymphoid or plasmacytic proliferations that develop as a consequence of immunosuppression in a recipient of a solid organ, bone marrow (BM) or stem cell allograft”.160 Lung transplant recipients continue to have the highest incidence among all the solid organ groups. A recent report by Wudhikarn and colleagues from the University of Minnesota reported an incidence of 5% in heart-lung and lung recipients over a 20-year period but older series have reported rates closer to 10%.161,162 Over the last decade the median interval to PTLD has increased from 6 months (80% within the first year) to approximately 3 years.160 This increase is due in part to the increase in EBV-negative PTLD cases. Previously 80–90% of PTLD were EBV-associated but currently up to 40% of cases are EBV-negative.

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Risk factors for developing PTLD include sero-negative allograft recipients (which includes a significant proportion of children), intensity and duration of immunosuppression, CMV disease, and the degree of donor/recipient HLA mismatch.163–165 Treatment protocols have changed although reduction in immunosuppression remains the central component. The introduction of the anti-CD20 antibody, rituximab, has expanded therapeutic options and systemic chemotherapy is generally reserved for cases that fail to respond to these two options or for patients with central nervous system involvement. Conventional antiviral therapy alone with acyclovir and ganciclovir has not been shown to be of therapeutic benefit. However, clinical trials are currently evaluating the efficacy of antiviral drugs in combination with arginine butyrate, a drug that induces EBV thymidine kinase transcription.166,167 Other novel modalities include infusion of EBV-specific cytotoxic T-cells to help reestablish immunological control of EBVinfected B-cell activation and proliferation.168

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Figure 21. The work-up for PTLD includes careful evaluation for light chain restriction and in situ hybridization for EBER-1 genome. (a) This monomorphic lymphoid proliferation appears malignant. (b) CD20 staining confirms the B-cell nature of the process. (c) Lambda light chain restriction is apparent. (d) Strong EBER-1 nuclear staining is noted by ISH.

There is tremendous heterogeneity in both the clinical presentation and histopathological appearance of PTLD. Diagnostic modalities include image-guided needle core biopsy, transbronchial biopsy and open biopsy. Flow cytometry along with immunohistochemical and molecular studies are required to determine clonality and other diagnostic, potential therapeutic and prognostic information (Figure 21). Currently, the WHO grading scheme is used by most centers and is divided into four patterns (Table 6).

Table 6 Classification of post-transplant lymphoproliferative disease (PTLD)

1. Early lesions (plasmacytic hyperplasia and infectious mononucleosis-like PTLD) 2. Polymorphous PTLD 3. Monomorphic PTLD (including diffuse large B cell, Burkitt-like, plasma cell lesions and NK/T-cell lymphoma) 4. Classic Hodgkin-lymphoma-like PTLD 5. Other (including cutaneous and primary effusion lymphomas)

1. Early lesions including plasmacytic hyperplasia (PH) and infectious-mononucleosis (IM)-like PTLD

Modified from reference 160.

These patterns are characterized by the formation of mass lesions with lymphoid or lymphoplasmacytic proliferation but preservation of nodal or extranodal architecture. These typically occur in EBV-seronegative recipients (children and occasionally adults) and represent primary EBV infections. In tissue sections PH exhibits numerous plasma cells and small lymphocytes with occasional immunoblasts in a background of follicular hyperplasia while IM-like lesions display paracortical expansion, and immunoblasts admixed with plasma cells and

T-cells (Figure 22). Immunostaining reveals polytypic B-cells and plasma cells with variable detection of EBV by EBER in situ hybridization.

2. Polymorphous PTLD This pattern is characterized by architectural effacement of nodal or extranodal structures by a mixture of cell types, including small and transformed lymphocytes, immunoblasts and plasma cells (Figure 22). In some cases cells resembling

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Figure 22. Patterns of PTLD. (a) Reactive lymphoid hyperplasia showing small and transformed lymphocytes without necrosis or atypia. (b) Polymorphous PTLD demonstrating an admixture of small and large lymphocytes and plasma cells. This pattern can be found in either polytypic or monotypic PTLD. (c) Monomorphic PTLD showing a uniform population of atypical large lymphocytes resembling diffuse aggressive large B-cell lymphoma. (d) Hodgkin-like PTLD with an admixture of cell types including a central Reed-Sternberg cell.

Reed-Sternberg (RS) cells are noted and necrosis and mitoses can be variable. It is the most common pattern reported in pediatric PTLD and usually represents primary EBV infection rather than reactivation. A range of clonality results have been found including polyclonal, oligoclonal and monoclonal cases. The RS-like cells are usually CD30þ, CD20þ but CD15-negative and generally EBV-positive by ISH.

3. Monomorphic PTLD Monomorphic PTLD resembles the classic forms of B-cell and NK/T-cell lymphomas encountered in immunocompetent patients. The B-cells lesions are usually diffuse large B-cell lymphoma but Burkitt-like lymphomas and plasma cell neoplasms are also included in this group (Figure 22). Both nodal and extranodal lesions are reported and the lung is a common site of involvement presenting as endobronchial or parenchymal nodular masses. The B-cells lesions are monoclonal and cytogenetic abnormalities are common but the presence of EBV is less consistent than in polymorphous PTLD.

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Monomorphic NK/T-cell PTLD is less common than the B-cell type and is frequently extranodal. Numerous patterns have been described including peripheral T-cell lymphoma, hepatosplenic T-cell lymphoma, T-cell large granular lymphocytic leukemia, adult T-cell leukemia/lymphoma, extranodal NK/T-cell lymphoma, nasal type, and the spectrum of T-cell cutaneous lymphoid neoplasms.160 Up to a third are EBVpositive.

4. Classic Hodgkin lymphoma-type PTLD This is the least common pattern of PTLD and the most diagnostically challenging. It must be distinguished from the polymorphous PTLD group and requires both the morphology and immunophenotype of classical HD such as CD30þ and CD15þ immunostaining. In the setting of diffuse alveolar consolidative rather than nodular distribution of PTLD or with small biopsy specimens, the distinction of PTLD from ACR can be challenging for the transplant pathologist. The liberal use of leveled tissue sections, immunohistochemistry and in situ hybridization along with

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EBV Figure 23. EBV-associated endobronchial leiomyomatous polyp. (a) CT scan showing intraluminal nodular projection in proximal right bronchus. (b) At bronchoscopy a smooth, nodular mass was partially occluding the airway. (c) Low magnification demonstrating expansion of the submucosa and wall of the airway by a spindled cell proliferation. (d) At high magnification the spindled cells resemble mature smooth muscle cells and show strong EBER-1 nuclear staining by ISH.

careful attention to the morphological findings can usually resolve the problems. Findings that favor PTLD include monomorphic and atypical lymphoid cells, necrosis, and numerous mitotic figures.169

Other EBV-associated neoplasms The association of EBV and smooth muscle neoplasms was first reported in 1995 in solid organ recipients and has now also been described in a variety of immunocompromised patients, including congenital/primary and acquired immunodeficiency states including human immunodeficiency virus (HIV) and therapy-induced immunosuppression. To date 127 cases have been reported, with half arising in solid organ transplant recipients.170 Two-thirds of these cases involve the liver or lungs. Of the 40 cases in the lung, seven were classified as leiomyosarcoma, seven as benign leiomyomas and the

remainder as smooth muscle tumors of uncertain malignant potential. We have observed two cases in lung allografts presenting as multiple polypoid endobronchial lesions with the morphology of benign leiomyoma (Figure 23). The smooth muscle cells express desmin and smooth muscle actin and ISH for EBER shows strong nuclear staining. Small T cell lymphocytes are often scattered throughout the lesion. Excision by bronchoscopic snaring can be curative. Interestingly, there are reports of metachronous or synchronous development of PTLD in some patients.171

Other pulmonary complications in lung transplant recipients

The 2007 ISHLT report identified a number of “non-rejection” findings that can be seen in transbronchial or VATS biopsy

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Figure 24. Patterns of aspiration pneumonia. (a) Foreign material engulfed by foreign body-type giant cells and abundant inflammatory exudates. (b) Exogenous lipoid pneumonia with numerous alveolar macrophages containing lipid in macrovesicular and microvesicular patterns.

specimens. These may be of either donor or recipient origin and could warrant further investigation or treatment in some cases. As discussed previously, oropharyngeal dysfunction with aspiration of food and other materials can elicit a foreignbody-type reaction along with bronchiolitis and organizing pneumonia patterns. Aspiration of exogenous lipoid materials leaves intra-alveolar macrophages vacuolated (Figure 24). Smoking-related lesions such as respiratory bronchiolitis or pulmonary Langerhans’ cell histiocytosis could be from a donor with heavy tobacco abuse or may indicate that the recipient is actively smoking (Figure 25) (see Chapter 10). The presence of intravascular talc granulomas or numerous intra-alveolar macrophages with anthracotic pigment are indicators of illicit drug use (see Chapter 16). Drug toxicity remains an under-recognized problem in these patients. The histopathological findings are often nonspecific and careful clinical-pathological correlation is needed. Sirolimus pulmonary toxicity was first reported in renal transplant recipients. The histopathological findings are nonspecific and include an organizing pneumonia pattern, interstitial pneumonitis, pulmonary alveolar proteinosis, patchy fibrosis and alveolar hemorrhage (Figure 26).172–175 Other drugs used in the transplant setting with reported pulmonary complications are everolimus and rituximab.176–177 The pulmonary toxicity of antibiotics, cardiovascular agents and other medications is discussed in detail in Chapter 16.

Late period after transplantation (> 12 months) Like other solid organ recipients, lung transplant patients can sustain complications to numerous organ systems such as renal, gastrointestinal including hepatobiliary, hematological, ocular and central nervous system, cardiovascular and musculoskeletal.178–185 However, chronic allograft rejection with its morphological manifestation as obliterative bronchiolitis (OB)

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and its clinical counterpart, BOS remains the major impediment to achieving long-term survival in lung transplantation.

Obliterative bronchiolitis and bronchiolitis obliterans syndrome In the current era BOS develops in 30%, 49% and 75% of adult recipients by 2.5, 5 and 10 years, respectively, following transplantation.13 Like the adult group, OB remains the most common cause of death or retransplant in pediatric patients who survive at least 1 year.12 Further, the type of operation does not influence these results. As discussed above, alloimmune and non-alloimmune mechanisms, such as ACR, AMR, lymphocytic bronchiolitis, increased number of HLA mismatches between donor and recipient, primary graft dysfunction, GER with aspiration, development of new HLA antibodies after transplant and respiratory infections, have been invoked as risk factors. These have been reviewed in detail in recent publications.186–193 Recently, the role of small airway microvascular injury as a mechanism for OB has been proposed.194,195 The clinical component of chronic allograft dysfunction or BOS is defined as a sustained decline in the baseline forced expiratory volume in 1 second (FEV1) or mid-expiratory flow rate (FEF25–75) over a specified period of time.196,197 In some patients the onset and progression are abrupt while others manifest a more gradual, linear decline in PFTs. Many patients are asymptomatic until the late stages, when dyspnea on exertion and cough are common symptoms.193 Other causes of airway obstruction must first be excluded, such as anastomotic narrowing, infection, acute rejection or recurrent/progressive COPD. The numeric grades of BOS (Grades 0–3) reflect increasing severity of the obstruction. Radiological findings observed on expiratory-phase high-resolution computed tomography (HRCT) imaging include foci of air

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Figure 25. Smoking related changes found in post-transplant biopsies. (a and b) Nodular collections of anthracotic pigment. (c and d) Low and high magnification of recurrent pulmonary Langerhans cell histiocytosis.

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Figure 26. Sirolimus-associated pulmonary toxicity. (a) Scanning power magnification showing nonspecific organizing pneumonia with interstitial inflammation and fibroblastic foci. (b) EVG stain highlighting the loose fibromyxoid plugs within the lumens of small distal airways.

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Figure 27. Obliterative bronchiolitis. The arrows indicate discrete nodular foci of scar tissue centered on the bronchovascular bundles.

trapping or hyperlucency, mosaic patterns of parenchymal attenuation, septal thickening, bronchiectasis and tree-in-bud alterations of small airways.198–200 The histopathological equivalent of these patterns is constrictive obliterative bronchiolitis. In the 1990 ISHLT Working Formulation different patterns of airway luminal alteration were recognized and the presence or absence of an inflammatory component was recorded. The lesions were classified as total or subtotal narrowing and active if intra- and/or peribronchiolar mononuclear cell infiltrates were present.45 In the 1996 ISHLT revision the patterns of “total” and “subtotal” were eliminated but inflammatory activity was retained.46 In the 2007 ISHLT Grading Scheme the distinction between active and inactive OB was removed and the designation was simplified to the absence (Grade C0) or presence (Grade C1) of OB (Table 3).47 The simplification of the grading reflects, in part, the accumulated transplant experience that the level of disease activity does not predict response to augmented immunosuppression, the problem of tangential sectioning in classifying OB lesions and the overall poor sensitivity of the transbronchial biopsy for demonstrating OB in the setting of BOS. The macroscopic features of OB can be subtle and obscured by adjacent parenchymal infection or scarring. Nodular fibrous thickening of bronchovascular bundles can be discerned by inspection or palpation (Figure 27). The histopathological hallmarks of OB are fibro-inflammatory lesions centered on membranous and respiratory bronchioles, and eosinophilic collagenous submucosal fibrosis producing partial or complete luminal obliteration (Figure 28). These hyalinized plaques can produce concentric or eccentric lesions. If present, mononuclear inflammatory cells are distributed within the submucosal fibrosis and/or in the peribronchiolar tissue spaces. In early lesions abundant inflammation and fibromyxoid aggregates may be observed but the submucosal scarring lies beneath these lesions (Figure 29).

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At scanning magnification complete luminal obstruction can be easily overlooked on H&E stains and careful attention to the parenchyma adjacent to the muscular pulmonary arteries is important. Connective tissue stains, such as elastin-van Gieson (EVG), Masson’s trichrome or combined Masson’s trichrome/elastic Verhoeff van Gieson stains, are helpful in identifying the small airways. In the late cicatricial stages of OB lumina are filled with mature collagenous tissue. Disruption of the elastic membrane and atrophy of the muscular layer of the airway are also seen (Figure 28). The scar tissue can extend distally into the alveolar ducts and sacs and septal scarring is often present. Mucostasis and/or intraluminal foam cells in the distal airways are markers of airway obstruction. Bronchiectasis and bronchiolectasis are usually found at postmortem or at the time of retransplant in cases of advanced OB but are not discernible in transbronchial biopsy specimens.158 Bronchiectasis/bronchiolectasis is characterized by mucous plugging, goblet cell hyperplasia, squamous metaplasia of the epithelial lining and variable mural inflammation (Figure 30). The pathogenesis is probably multifactorial and involves recurrent bacterial or fungal infections, mucous plugging, denervation of the allograft and distal airway obstruction (see Chapter 17). The differential diagnosis of OB is limited. Prior biopsy site lesions can produce mural scarring and variable inflammation. Fibromyxoid plugs of granulation tissue in the small airways can be found in a variety of organizing airway injury patterns including resolving rejection, organizing infection, ischemicreperfusion injury and drug toxicity. It should not be confused with constrictive OB. There are limited treatment options available for patients with established OB or BOS. With the exception of retransplantation, treatments only produce temporary “stabilization” in the rate of decline of measured pulmonary function.193 Current therapies include augmentation of immunosuppression, cytolytic therapy with lymphocyte-depleting agents, antibody therapy with alemtuzumab, antibiotics such as azithromycin, and photophoresis therapy.201–209 Despite the poor long-term outcome, most patients survive for a number of years after the diagnosis is established.210

Chronic vascular rejection The arteries and veins of the lung allograft can develop transplant-associated vasculopathy (TAV). The lesions are similar to those seen in other solid organ transplants and are designated by Grade D in the 2007 ISHLT Working Formulation. Combined heart-lung recipients can develop concurrent TAV of the coronary circulation.211 In our experience these lesions are rarely seen in transbronchial biopsy specimens but are readily found at the time of post-mortem or retransplant. They usually accompany OB and recent studies have documented hemodynamic evidence of pulmonary hypertension in these patients.212 Concentric intimal thickening, composed of smooth muscle cells and myofibroblasts, variable lymphocytes

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Figure 28. Patterns of obliterative bronchiolitis. (a and b) H&E and EVG sections showing eccentric, raised luminal plugs of scar tissue. (c) Complete luminal occlusion by collagenous scar tissue with intraluminal and peribronchiolar collections of mononuclear cells. Note the preservation of the muscular layer of the bronchiole. (d) Dense cicatricial scarring of the airway without inflammatory cells. (e) The EVG stain highlights the fragmented elastica and loss of smooth muscle cells in the airway. (f) The EVG stain confirms that the small aggregate of scar tissue represents a bronchiole replaced by scar. This can be easily overlooked on routine sections.

and macrophages including foam cells, is found (Figure 31). On occasion superimposed thrombosis complicates the lesions. Intimal sclerosis of veins and venules is commonly found in transbronchial biopsy specimens and should not be designated as chronic rejection in isolation.

Recurrent disease in the allograft The list of disorders that have recurred in the transplant allograft expands annually and is published as case reports and small series (Table 7). In our experience sarcoidosis

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Figure 29. H&E- and EVG-stained sections showing “early” OB with submucosal scarring and loose aggregates of mononuclear cells, fibrin and cellular debris.

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Figure 30. Bronchiectasis in the setting of OB. (a) The distal airway is ectatic and inflamed. (b) The epithelial lining ranges from squamous metaplasia (not shown) to respiratory-type mucosa with intraepithelial mononuclear cells and epithelial injury. Table 7 Recurrent disease in the transplanted lung

Sarcoidosis Lymphangioleiomyomatosis Bronchioloalveolar carcinoma Desquamative interstitial pneumonia Giant cell interstitial pneumonia Pulmonary alveolar proteinosis Idiopathic pulmonary hemosiderosis Diffuse panbronchiolitis Pulmonary Langerhans’ cell histiocytosis Pulmonary veno-occlusive disease Figure 31. Chronic vascular rejection. The EVG-stained section shows concentric intimal proliferation in a muscular artery. Note the adjacent bronchiole showing OB.

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and LAM are the most common lesions and are usually detected as incidental findings on surveillance transbronchial biopsy.213–238 The histopathological changes are similar to the native disease. Knowledge of the patient’s primary disease and careful scrutiny of the biopsy specimens after transplantation is required. Infection should be excluded in all cases of granulomatous inflammation after transplantation.

Future directions Great strides have been made in patient selection and management after transplant. Survival rates have continued to improve for patients in their first year. However, a number of serious issues remain and constitute impediments to lung transplantation. Firstly, the pool of lungs available for transplant has remained stagnant over the last two decades despite the increasing number of potential candidates for transplant in North America and Europe. Less than a fifth of lungs are now considered suitable or available for donation and a third or more patients die on the wait-list. A number of techniques under experimental and clinical investigation might expand the donor pool by reconditioning marginal lungs into acceptable grafts. Ex vivo lung perfusion (EVLP) is a novel approach to both extend the period of evaluation of the lung and/or institute directed therapy to promote lung recovery prior to transplantation. The technique has moved into the clinical arena and small series have been published promoting its success.239–244 Experimental gene therapy work is under way using adenovirus vectors encoding interleukin-10 to repair injured lungs.245

References 1. Woo MS. Overview of lung transplantation. Clinic Rev Allerg Immunol 2008;35:154–63. 2. Shoja M, Tubbs RS, Ardalan MR, et al. A testimony to the history of heart and lung transplantation: English translation of Demikhov’s paper, “Transplantation of the heart, lungs and other organs”. Int J Cardiol 2010;143:230–4. 3. Hardy JD, Webb WR, Dalton ML Jr, Walker GR Jr. Lung homotransplantations in man: report of the initial case. JAMA 1963;186:1065–74. 4. Cooley DA, Bloodwell RD, Hallman GI, et al. Organ transplantation for advanced cardiopulmonary disease. Ann Thorac Surg 1969;8:30–42. 5. Pearson FG. Lung transplantation. Arch Surg 1989;124:535–8. 6. Reitz BA, Wallwork JL, Hunt SA, et al. Heart-lung transplantation: successful therapy for patients with pulmonary

Another direction of active investigation is the development of artificial lung technology to serve as a bridge to transplant and eventually as destination therapy. Extracorporeal life support systems have evolved from extracorporeal membrane oxygenation (ECMO) to more sophisticated devices that use a polymethylpentene membrane oxygenator, a centrifugal pump and heparin-coated tubing circuits that can be configured for specific clinical situations. A number of small series have been published showing some success as a bridging technique.246 The paracorporeal artificial lung was developed with the goal of creating an ambulatory device that can be used for extended periods of time for patients awaiting transplant. To date the technique has been successfully used in children and adults.247–249 The conceptual and mechanical aspects of these devices are the subject of recent reviews.250,251 The goal of tissue engineering is still experimental but holds promise for pulmonary parenchymal reconstruction and regeneration.252–254 Currently the primary obstacle to long-term survival after transplant remains chronic airway rejection. The overall incidence has not changed significantly although the clinical onset is delayed. The pathogenesis of OB is multifactorial and numerous risk factors have been elicited. As molecular mechanisms are clarified, prevention rather than treatment becomes the goal. To date retransplantation is the only definitive therapy but there are a number of technical, ethical and management issues to be considered.255 The future brings many challenges and the pathologist will continue to be an integral member of the multidisciplinary transplant team.

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12. Aurora P, Edwards LH, Kucheryavaya AY, et al. The registry of the International Society for Heart and Lung Transplantation: thirteenth official pediatric lung and heart-lung transplantation report- 2010. J Heart Lung Transplant 2010;29:1129–41. 13. Christie JD, Edwards LB, Kucheryavaya AY, et al. The registry of the International Society for Heart and Lung Transplantation: twenty-seventh official adult lung and heart-lung transplant report- 2010. J Heart Lung Transplant 2010;29:1104–18. 14. Levine GN, McCullough KP, Rodgers AM, et al. Analytical methods and database design: implications for transplant researchers, 2005. Am J Transplant 2006; 6(Part 2): 1228–42. 15. Egan TM, Murray S, Bustami RT., et al. Development of the new lung allocation system in the United States. Am J Transplant 2006; 6(5 Pt 2): 1212–27.

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16. Takahashi SM, Garrity ER. The impact of the lung allocation score. Semin Respir Crit Care Med 2010;31:108–14. 17. O’Beirne S, Counihan IP, Keane MP. Interstitial lung disease and lung transplantation. Semin Respir Crit Care Med 2010;31:139–46. 18. Diamond J, Kotloff RM. Lung transplantation for chronic obstructive pulmonary disease: special considerations. Semin Respir Crit Care Med 2010;31:115–22. 19. Liu V, Zamora MR, Dhillon GS, Weill D. Increasing lung allocation score predict worsened survival among lung transplant recipients. Am J Transplant 2010;10:915–20. 20. Orens JB, Estenne M, Arcasoy S, et al. Pulmonary Scientific Council of the International Society for Heart and Lung Transplantation. International guidelines for the selection of lung transplant candidates: 2006 update – a consensus report from the Pulmonary Scientific Council of the International Society for Heart and Lung Transplantation. J Heart Lung Transplant 2006;25:745–55. 21. Merlo CA, Orens JB. Candidate selection, overall results and choosing the right operation. Semin Respir Crit Care Med 2010;31:99–107. 22. Dishop MK, Mallory GB, White FV. Pediatric lung transplantation: perspectives for the pathologist. Ped Develop Pathol 2008;11:85–105. 23. Solomon M, Grasemann H, Keshavjee S. Pediatric lung transplantation. Pediatr Clin N Am 2010;57:375–91. 24. Huddleston CB. Pediatric lung transplantation. Curr Treat Opt Cardiovasc Med 2011;13:68–78. 25. Zafar F, Heinle JS, Schecter MG, et al. Two decades of pediatric lung transplant in the United States: have we improved? J Thorac Cardiovasc Surg 2011;141:828–32.

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unexpected explant carcinoma: a management dilemma. J Heart Lung Transplant 2007;26:1206–8. 29. Akindipe OA, Fernandez-Bussy S, Staples ED, Baz MA. Discrepancies between clinical and autopsy diagnoses in lung transplant recipients. Clin Transplant 2010;24:610–14. 30. Deng MC, Eisen HJ, Mehra MR, et al. Noninvasive discrimination of rejection in cardiac allograft recipients using gene expression profiling. Am J Transplant 2006;6:150–60. 31. Morgun A, Shulzhenko N, Perez-Diez A, et al. Molecular profiling improves diagnoses of rejection and infection in transplanted organs. Cir Res 2006;98: e74–83.

the management of lung transplant recipients. J Heart Lung Transplant 1993;12:308–24. 40. Chakinala MM, Ritter J, Gage BF, et al. Yield of surveillance bronchoscopy for acute rejection and lymphocytic bronchitis/bronchiolitis after lung transplantation. J Heart Lung Transplant 2004;23:1396–1404. 41. McWilliams TJ, Williams TJ, Whitford HM, Snell GI. Surveillance bronchoscopy in lung transplant recipients: risk versus benefit. J Heart Lung Transplant 2008;27:1203–9. 42. Glanville AR. Bronchoscopic monitoring after lung transplantation. Semin Respir Crit Care Med 2010;31:208–21.

32. Bhorade SM, Janata K, Vigneswaran WT, Alex CG, Garrity ER. Cylex ImmuKnow assay levels are lower in lung transplant recipients with infection. J Heart Lung Transplant 2008;27:990–4.

43. Valentine VG, Taylor DE, Dhillon GS, et al. Success of lung transplantation without surveillance bronchoscopy. J Heart Lung Transplant 2002;21:319–26.

33. Husain S, Raza K, Pilewski JM, et al. Experience with monitoring in lung transplant recipients: correlation of low immune function with infection. Transplantation 2009;87:1852–7.

44. Valentine VG, Gupta MR, Weill D, et al. Single-institution study evaluating the utility of surveillance bronchoscopy after lung transplantation. J Heart Lung Transplant 2009;28:14–20.

34. Pham MX, Teuteberg JJ, Kfoury AG, et al. Gene-expression profiling for rejection surveillance after cardiac transplantation. N Eng J Med 2010;362:1890–900.

45. Yousem SA, Berry GJ, Brunt EM, et al. A working formulation for the standardization of nomenclature in the diagnosis of heart and lung rejection: Lung Rejection Study Group. The International Society for Heart Transplantation. J Heart Transplant 1990;9:593–601.

35. Snyder TM, Khush KK, Valantine HA, Quake SR. Universal noninvasive detection of solid organ transplant rejection. Proc Natl Acad Sci USA 2011;108:6229–34. 36. Starnes VA, Theodore J, Oyer PE, et al. Evaluation of heart-lung transplant recipient with prospective, serial transbronchial biopsies and pulmonary function studies. J Thorac Cardiovasc Surg 1989;98:945–50.

26. Benden C, Inci I, Weder W, Boehler A. Size-reduced lung transplantation in children – an option worth to consider! Pediatr Transplantation 2010;14:529–33.

37. Scott JP, Fradet G, Smyth RL. Prospective study of transbronchial biopsies in the management of heart-lung and single lung transplant patients. J Heart Lung Transplant 1991;10:626–36.

27. Stewart S, McNeil K, Nashef SA, et al. Audit of referral and explant diagnoses in lung transplantation: a pathologic study of lungs removed for parenchymal disease. J Heart Lung Transplant 1995;14:1173–86.

38. Trulock EP, Ettinger NA, Brunt EM, et al. The role of transbronchial lung biopsy in the treatment of lung transplant recipients. An analysis of 200 consecutive procedures. Chest 1992;102:1049–54.

28. Richie AJ, Mussa S, Sivasothy P, Stewart S. Single-lung transplant complicated by

39. Sibley RK, Berry GJ, Tazelaar HD, et al. The role of transbronchial biopsies in

46. Yousem SA, Berry GJ, Cagle PT, et al. Revision of the 1990 working formulation for the classification of pulmonary allograft rejection: Lung Rejection Study Group. J Heart Lung Transplant 1996;15:1–15. 47. Stewart S, Fishbein MC, Snell GI, et al. Revision of the 1996 working formulation for the standardization of nomenclature in the diagnosis of lung rejection. J Heart Lung Transplant 2007;26:1229–42. 48. Arcasoy SM, Berry G, Marboe CC, et al. Pathologic interpretation of transbronchial biopsy for acute rejection of lung allograft is highly variable. Am J Transplant 2011;11:320–8. 49. Stephenson A, Flint J, English J, et al. Interpretation of transbronchial biopsies from lung transplant recipients: interand intraobserver agreement. Can Respir J 2005;12:75–7.

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227. Gómez-Román JJ, Del Valle CE, Zarrabeitia MT, et al. Recurrence of bronchioloalveolar carcinoma in donor lung after lung transplantation: microsatellite analysis demonstrates a recipient origin. Pathol Int 2005; 55(9):580–4. 228. Toyooka S, Waki N, Okazaki M, et al. Recurrent lung cancer in the mediastinum noticed after a livingdonor lobar lung transplantation. Ann Thorac Cardiovasc Surg 2009; 15:119–22. 229. Chen F, Hasegawa S, Bando T, et al. Recurrence of bilateral diffuse bronchiectasis after bilateral lung transplantation. Respirology 2006;11:666–8. 230. King MB, Jessurun J, Hertz MI. Recurrence of desquamative interstitial pneumonia after lung transplantation. Am J Respir Crit Care Med 1997;156:2003–5. 231. Verleden GM, Sels F, Van Raemdonck D, et al. Possible recurrence of desquamative interstitial pneumonitis in a single lung transplant recipient. Eur Respir J 1998;11:971–4. 232. Frost AE, Keller CA, Brown RW, et al. Giant cell interstitial pneumonitis. Disease recurrence in the transplanted lung. Am Rev Respir Dis 1993;148:1401–4. 233. Calabrese F, Giacometti C, Rea F, et al. Recurrence of idiopathic pulmonary hemosiderosis in a young adult patient after bilateral single-lung transplantation. Transplantation 2002;74:1643–5. 234. Baz MA, Kussin PS, Van Trigt P, et al. Recurrence of diffuse panbronchiolitis after lung transplantation. Am J Respir Crit Care Med 1995;151(3 Pt. 1): 895–8. 235. Gabbay E, Dark JH, Ashcroft T, et al. Recurrence of Langerhans’ cell granulomatosis following lung transplantation. Thorax 1998;53:326–7.

238. Izbicki G, Shitrit D, Schechtman I, et al. Recurrence of pulmonary venoocclusive disease after heart-lung transplantation. J Heart Lung Transplant 2005;24:635–7. 239. Steen S, Ingemansson R, Eriksson L, et al. First human transplantation of a nonacceptable donor lung after reconditioning ex vivo. Ann Thorac Surg 2007;83:2191–4. 240. Cypel M, Yeung JC, Hirayama S, et al. Technique for prolonged normothermic ex vivo lung perfusion. J Heart Lung Transplant 2008;27:1319–25. 241. Cypel M, Rubacha M, Yeung J, et al. Normothermic ex vivo perfusion prevents lung injury compared to extended cold preservation for transplantation. Am J Transplant 2009;9:2262–9. 242. Ingemansson R, Eyjolfsson A, Mared L, et al. Clinical transplantation of initially rejected donor lungs after reconditioning ex vivo. Ann Thorac Surg 2009;87:255–60. 243. Zych B, Marczin N, Carby M, et al. Ex vivo perfusion- a way to salvage lungs for transplantation? Br J Transplantation 2009;4:10–16. 244. Lindstedt S, Hlebowicz J, Koul B, et al. Comparative outcome of double lung transplantation using conventional donor lungs and non-acceptable donor lungs reconditioned ex vivo. Interact Cardiovasc Thorac Surg 2011;12:162–5. 245. Cypel M, Liu M, Rubacha M, et al. Functional repair of human donor lungs by IL-10 gene therapy. Sci Transl Med 2009;1(4):4ra9. 246. Cypel M, Keshavjee S. Extracorporeal life support as a bridge to lung transplantation. Clin Chest Med 2011;32:245–51. 247. Fischer S, Simon AR, Welte T, et al. Bridge to lung transplantation with the novel pumpless interventional lung assist device NovaLung. J Thorac Cardiovasc Surg 2006;131:719–23.

Chapter 20: The pathology of lung transplantation

248. Camboni D, Philipp A, Arlt M, et al. First experience with a paracorporeal artificial lung in humans. ASAIO J 2009;55:304–6. 249. Gazit AZ, Sweet SC, Grady RM, Huddleston CB. First experience with a paracorporeal artificial lung in a small child with pulmonary hypertension. J Thorac Cardiovasc Surg 2011;141:e48–50. 250. Ha RR, Wang D, Zwischenberger JB, Clark JW Jr. Hemodynamic analysis

and design of a paracorporeal artificial lung device. Cardiovasc Eng 2006;6: 10–29. 251. Nolan H, Wang D, Zwischenberger JB. Artificial lung basics: fundamental challenges, alternative designs and future innovations. Organogenesis 2011;7:23–7. 252. Wagner WR, Griffith BP. Reconstructing the lung. Science 2010;329:520–2.

253. Petersen TH, Calle EA, Zhao L, et al. Tissue-engineered lungs for in vivo implantation. Science 2010;329: 538–41. 254. Ott HC, Clippinger B, Conrad C, et al. Regeneration and orthotopic transplantation of a bioartificial lung. Nat Med 2010;16:927–33. 255. Kawut SM. Lung retransplantation. Clin Chest Med 2011;32: 367–77.

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Chapter

21

The lungs in connective tissue disease Donald G. Guinee Jr. and William D. Travis

Introduction to connective tissue diseases Pleuropulmonary involvement by connective tissue diseases (CTD) is varied and includes pleural, interstitial, bronchial/ bronchiolar and vascular manifestations. Common patterns of involvement overlap with those of idiopathic lung disease. They include patterns of nonspecific interstitial pneumonia (NSIP), lymphocytic interstitial pneumonitis (LIP), organizing pneumonia (OP), usual interstitial pneumonia (UIP), diffuse alveolar damage (DAD), chronic bronchiolitis, pulmonary capillaritis with alveolar hemorrhage and primary pulmonary hypertension among others (see Table 1).1 Some patterns (e.g. OP and NSIP) can occur in all CTD although their incidences, as pulmonary manifestations, differ greatly between individual disorders.2 This chapter reviews common and rare pulmonary manifestations of CTDs. Radiological and histological features of individual patterns of interstitial lung disease (e.g. NSIP pattern) are similar to those of their idiopathic counterparts (see Chapter 10). Accordingly this chapter will refer to such patterns but will not repeat detailed descriptions of the pathology. The status of Behçet syndrome, inflammatory bowel disease, as well as some dermatological conditions (e.g. Sweet syndrome, pyoderma gangrenosum) as components of CTD is less clear. They may have pulmonary and other systemic manifestations and are also briefly discussed. Prior to discussing specific pulmonary manifestations of individual CTDs, it is important to consider several issues which affect lung biopsy interpretation in this setting. These include the occurrence of secondary pulmonary complications, acute exacerbation of CTD, and the distinction of idiopathic interstitial pneumonias from interstitial lung disease occurring as a presenting feature in CTD patients.

Secondary complications in connective tissue disease Prior to diagnosing primary lung involvement in a patient with underlying CTD, it is important to consider whether the

findings may, in fact, reflect a nonspecific complication of therapy or the indirect effects of the CTD on other organ systems. For example, patients with underlying CTD frequently receive immunosuppressive treatment and are at risk of secondary infections. Likewise drug reactions can be an important cause of pulmonary morbidity and mortality.3–8 Pulmonary amyloidosis may occur in many CTDs, including rheumatoid disease, Sjögren syndrome and systemic lupus erythematosus.9–18 Esophageal dysmotility may lead to aspiration in scleroderma and mixed connective tissue disease. Finally, there appears to be an increased risk of malignancy, (small cell and non-small cell lung carcinoma and lymphoproliferative disorders) which varies depending upon the type of disease. For example, polymyositis/dermatomyositis is sometimes associated with underlying lung cancer, where it probably represents a paraneoplastic syndrome (see Chapter 24).19–22 Sjögren syndrome is associated with extranodal marginal zone B-cell lymphoma of bronchus-associated lymphoid tissue (BALT) (see Chapter 34).23–26 An increased incidence of lung cancer has also been noted in rheumatoid disease, scleroderma and systemic lupus erythematosus.27–33

Acute exacerbation of connective tissue diseases Acute exacerbation of CTD is a recently recognized complication reported in systemic lupus erythematosus, rheumatoid arthritis, mixed connective tissue disease, Sjögren syndrome and systemic sclerosis.9,34,35 This complication presents with rapidly progressive respiratory failure. Biopsies show DAD. The condition may occur in patients with pre-existing interstitial lung disease or de novo without prior pulmonary involvement.34 Occasional cases have also been noted in association with etanercept and infliximab – agents which modulate the activity of tumor necrosis factor or its receptor.4,36 Patients with this complication have a poor prognosis with mortality rates exceeding 50%.35

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 21: The lungs in connective tissue disease Table 1 Patterns of lung disease in connective tissue and systemic disease

RA Pleural disease Pleuritis þ/ effusion Pleural fibrosis Sterile or septic empyema Spontaneous pneumothorax Interstitial/parenchymal Usual interstitial pneumonia Diffuse alveolar damage Nonspecific interstitial pneumonia Organizing pneumonia Lymphocytic interstitial pneumonia Granulomatous interstitial pneumonia Apical fibrobullous disease Aspiration pneumonia Necrobiotic nodule Sterile abscess/neutrophilic infiltrates Interstitial neutrophilic infiltrates Amyloid deposits Alveolar lesions Eosinophilic pneumonia Alveolar proteinosis Vascular lesions Vasculitis Alveolar hemorrhage þ/ capillaritis Thromboembolism Pulmonary artery aneurysms Pulmonary hypertension Plexogenic arteriopathy Chronic thrombotic arteriopathy Pulmonary veno-occlusive disease Pulmonary capillary hemangiomatosis Airway lesions Bronchiolitis þ/– fibrosis Follicular bronchiolitis Constrictive bronchiolitis Bronchiectasis Xerotrachea Bronchocentric granulomatosis Diffuse panbronchiolitis Tracheobronchial stenosis

SLE

SCL

SS

PM/DM MCTD

111 111 111 111 1

11 11

1 1

1 1

111 1

11

11 11 111 11 11 1

11 1

1

1

APS

UCTD AS

111 111

11

11

1

1 1 111 111 111

1 11

111

1

1 1

1 11

11

11

BD

11 11 1 1

1

1

PC

PG

SW 1

11

1

1

1

1

1 1

111

1

11

IBD

11 1

1 11

11 1 1

111 11

1

111 11

1

1

1 1

1 1

1 11

1

11

111

11 11 1

11

1

11

1 1

1

1

1

1 1

1 1

1 1

1

1 1

1

111 1 11 111

111 1 111a 1

1 1

1

111 111 1

1

11 11 11 11

1 1

1

1

1

11

111 1 11 11

1 1 1 1

1

1

1

11 1 1 11 1

111

1 11

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Chapter 21: The lungs in connective tissue disease Table 1 (cont.)

RA Indirect respiratory effects Diaphragmatic or respiratory muscle dysfunction Thoracic cage immobility Atelectasis (shrinking lung syndrome) Neoplasms Lung cancer Lymphoma Kaposi’s sarcoma

SLE

APS

SCL

SS

1 1

111

1

1 1 1

PM/DM MCTD

1

11

11

PC

BD

PG

SW

IBD

11 11 1

1

11

UCTD AS

1 1

1

Modified from reference 1, Table 6–1, p. 292, with permission. RA, rheumatoid arthritis; SLE, systemic lupus erythematosus; APS, antiphospholipid antibody syndrome; SCL, scleroderma; SS, Sjögren syndrome; PM/DM, polymyositis/dermatomyositis; MCTD, mixed connective tissue disease; UCTD, undifferentiated connective tissue disease; AS, ankylosing spondylitis; PC, polychondritis; BD, Behçet disease; PG, pyoderma gangrenosum; SW, Sweet syndrome; IBD, inflammatory bowel disease. a Scleroderma shows marked mucoid intimal thickening as part of a spectrum of plexogenic arteriopathy

Idiopathic interstitial pneumonias as a presenting manifestation of connective tissue disease Pulmonary involvement by CTD may mimic idiopathic interstitial pneumonias.37 Of patients presenting with chronic interstitial lung disease, approximately 15–20% either have or subsequently develop an associated connective tissue disease.37,38 The most frequent patterns of interstitial lung disease occurring as manifestations of an underlying connective tissue disease include NSIP, UIP, LIP, OP and DAD.37 While the histological and radiological features of idiopathic and CTD-associated interstitial lung disease are similar, their distinction is nonetheless important. Patients with interstitial lung disease occurring in the context of a CTD have a significantly better prognosis than patients with idiopathic interstitial lung disease. This difference appears to stem from improved survival of CTD patients with a UIP pattern compared to patients with idiopathic pulmonary fibrosis (IPF).37,39 One recent study suggests that areas with a cellular NSIP pattern in biopsies otherwise showing UIP histology, while not specific, is commoner in patients with underlying connective tissue disease. This may be a helpful clue in suggesting this differential diagnosis.40

Genetics of connective tissue diseases The genetic associations of CTD are complex and multifactorial. Recent gene mapping studies have enhanced our understanding of these disorders, including the role of the TNF and NFKappab signaling pathways, which are important to many of the CTDs such as rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus and

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others. The interested reader is referred to several excellent recent reviews of this subject.41–45

Rheumatoid arthritis Pulmonary involvement by rheumatoid arthritis is present in up to 40% of these patients and accounts for approximately 10–20% of patient mortality.46–48 Pulmonary involvement often occurs after the diagnosis of rheumatoid arthritis, but may be the presenting manifestation in 10–20% of patients.48 Similar to patients with SLE, pleuropulmonary manifestations of rheumatoid arthritis are varied and affect the pleura, interstitium, airways or vessels. Primary and secondary patterns of pleuropulmonary involvement are listed in Table 2.

Pleuritis At autopsy, approximately 40–70% of patients with rheumatoid arthritis show a pleuritis.49 Clinically symptomatic pleural effusion and/or pleuritis are present less commonly in 5% to 25% of patients.48–50 Effusions may be a reflection of rheumatoid arthritis but may also be a secondary complication of congestive heart failure, infection and chylothorax, among others.48 Biopsies show nonspecific changes, including patchy fibrosis, chronic inflammation and occasional fibrin (Figure 1).51 Rheumatoid nodules may occasionally occur within the visceral pleura.48,49 In some cases there is palisading of histiocytes in the pleura, which must be distinguished from other causes of granulomatous disease. Lymphoid follicles may be present as part of the spectrum of lymphoid hyperplasia (see below).52

Chapter 21: The lungs in connective tissue disease Table 2 Pleuropulmonary pathology of rheumatoid arthritis

Pleuritis Rheumatoid nodules Interstitial lung disease UIP pattern NSIP pattern (cellular, fibrosing and cellular or fibrosing) Organizing pneumonia pattern Lymphocytic Interstitial pneumonitis Diffuse alveolar damage Airway Follicular bronchiolitis/lymphoid hyperplasia Obliterative (constrictive) bronchiolitis Chronic bronchiolitis, not otherwise specified Bronchiectasis Vascular Pulmonary hypertension

Figure 1. Rheumatoid arthritis. Chronic pleuritis. The pleura is expanded by a patchy chronic inflammatory infiltrate and granulation tissue.

Pulmonary capillaritis/vasculitis with alveolar hemorrhage Other rare manifestations Apical fibrobullous disease with pneumothorax Eosinophilic pneumonia Secondary complications Infection Pulmonary edema from congestive heart failure Drug reactions Increased risk of malignancy (lung cancer, lymphoproliferative disorders) Metabolic disturbances (amyloidosis)

Rheumatoid nodules Rheumatoid nodules are common, occurring in over 30% of open lung biopsies from patients with rheumatoid arthritis. They may occur either singly or multiply and are distributed preferentially in a subpleural location or along interlobular septa.53 Rarely, endobronchial nodules may occur.49 Grossly, nodules are well circumscribed with a necrotic center (Figure 2). Histological features are identical to those of rheumatoid nodules in subcutaneous tissue, consisting of a central area of necrosis surrounded by a rim of epithelioid histiocytes, often in a palisaded array (Figures 3 and 4). Special stains for acid-fast bacilli and fungi should always be performed to exclude infection.49 When rheumatoid nodules occur singly, they may be biopsied to exclude lung cancer.54 Numerous multiple nodules may rarely occur and have been termed “rheumatoid nodulosis”.55 For unknown reasons, treatment with anti-TNF-a therapy precipitates rheumatoid nodulosis in some patients.56

Figure 2. Rheumatoid nodules. Well circumscribed nodules with tan necrotic centers are seen. (Image courtesy of Dr D. Flieder, Philadelphia, PA, USA.)

Rare cases of pulmonary rheumatoid nodules have been associated with the secondary development of adenocarcinoma.57 Khazeni et al. reported massive “cavitary” rheumatoid nodules occurring in a patient with rheumatoid arthritis and human immunodefiency virus (HIV). They speculated that HIV infection may alter the phenotypic expression of this disease.58 Rheumatoid nodules may rarely be colonized by aspergillus, sometimes with an associated adjacent eosinophilic pneumonia (see Chapter 15).59,60

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Chapter 21: The lungs in connective tissue disease

Figure 3. Rheumatoid arthritis. Rheumatoid nodules. Randomly located necrobiotic nodules mimic an infectious process.

Figure 4. Rheumatoid arthritis. Rheumatoid nodule. The histological features are identical to those of rheumatoid nodules in subcutaneous tissue, consisting of a central area of necrosis surrounded by a rim of epithelioid histiocytes often in a palisaded array.

before, during or after the onset of arthritis. New nodules may appear after intervals of a few months or they may remain stable.66 In Caplan’s original series, approximately 25% of nodules cavitated.61 Histologically, the nodules are similar to rheumatoid nodules and show central necrosis with a fibrotic border, containing chronic inflammation and occasional multinucleated giant cells. In contrast to rheumatoid nodules, there is variable accumulation of dust present in a concentric ring at the periphery of the nodules (Figure 5).67,68

Interstitial lung disease Figure 5. Rheumatoid arthritis. Caplan syndrome. Necrotic nodules with a fibrotic border contain pigmented macrophages. Inset shows higher-power view of dust within macrophages.

The finding of rheumatoid nodules without other features of pulmonary disease is generally associated with a favorable prognosis. Nodules are usually asymptomatic, although they may rarely cavitate.49 Significant pulmonary disease rarely develops and death has not been reported.53 Caplan syndrome (rheumatoid pneumoconiosis) refers to the development of multiple bilateral pulmonary nodules in patients with rheumatoid arthritis and an underlying pneumoconiosis (see Chapter 14). The syndrome was initially described in a series of coalminers in 1953.61 It has since been recognized in association with other environmental exposure histories, including asbestosis, foundry work, marble work and others.47,62–65 The nodules may occur

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Interstitial lung disease has long been recognized as an important pulmonary manifestation of rheumatoid arthritis. Historical reports described a variety of patterns, including UIP, bronchiolitis obliterans with organizing pneumonia, cellular interstitial pneumonitis and lymphoid hyperplasia. More recent studies have refined and added to these observations in accordance with the 2002 American Thoracic Society/European Respiratory Society (ATS/ERS) consensus classification.2,69,70 While the various histological patterns all reflect pulmonary manifestations of rheumatoid arthritis, diagnosis of the pattern of involvement is important in determining prognosis and treatment options. Clinical features of interstitial lung disease in patients with rheumatoid arthritis are similar to those occurring in patients with idiopathic disease and include cough and exertional dyspnea. In up to one-third of patients, the interstitial lung disease either pre-dates or occurs concurrently with the diagnosis of rheumatoid arthritis. The majority of patients show a restrictive defect on pulmonary function tests variably accompanied by a low diffusion capacity for carbon monoxide (DLCO).70

Chapter 21: The lungs in connective tissue disease

High-resolution CT (HRCT) scan studies show that most cases of rheumatoid arthritis-associated interstitial lung disease are associated with four major patterns including UIP, NSIP, OP and bronchiolitis.71 HRCT features are similar to patients with idiopathic interstitial lung disease. Basically, the UIP pattern consists of bilateral reticulation and honeycombing with a subpleural lower lobe predilection. These findings are variably accompanied by traction bronchiectasis. The NSIP pattern mostly affects the lower lobes and consists predominantly of ground-glass opacities with or without reticulation and may show traction bronchiectasis. The organizing pneumonia pattern shows bilateral airspace consolidation with air bronchograms and ground-glass opacities with a subpleural distribution. These findings are variably accompanied by centrilobular micronodules and reticulation. The inflammatory airway disease pattern (bronchiectasis, bronchiolitis) consists of centrilobular micronodules and bronchiectasis, sometimes accompanied by nodules without a zonal predisposition.70,71 In constrictive bronchiolitis, routine chest radiographs and HRCT may appear either normal or show hyperinflation. Air trapping or mosaic attenuation is characteristic and is best seen on expiration. Histopathological features generally correlate well with CT. In a study of open lung biopsies from 18 patients with rheumatoid arthritis and interstitial lung disease, a UIP histology was most common (56%), followed by an NSIP pattern (33.3% including mixed cellular and fibrotic (22.2%) and fibrotic (11.1%)) and inflammatory airway disease with OP (11.1%). The pattern of inflammatory airway disease with an organizing pneumonia was sometimes associated with follicular bronchiolitis or chronic nonspecific bronchiolitis.70

UIP pattern in rheumatoid arthritis Rheumatoid arthritis differs from other connective tissue diseases in that UIP is the commonest pattern seen in interstitial lung disease (Figure 6), whereas NSIP is most frequent in the majority of other disorders.70–73 A UIP pattern correlates strongly with male gender and prior history of smoking, whereas NSIP histology correlates with female gender and non-smoking status.70 As in the idiopathic setting, the UIP pattern is associated with a worse prognosis than NSIP. In the largest study, no deaths were observed in the six patients with an NSIP pattern, whereas five of 10 patients with UIP histology died after a median follow-up of 4.2 years. Nonetheless, the prognosis of patients with a UIP pattern in this context appears better than in patients with idiopathic UIP/ IPF.2,70,72 Although half of patients5 with a UIP pattern in the abovementioned study died, pulmonary function in the other half 5 remained stable or slightly improved.70 Flaherty et al. found a correlation with fewer fibroblastic foci in the UIP pattern in CTD patients compared to idiopathic cases. They suggested that this finding explained the more favorable survival.74

NSIP pattern in rheumatoid arthritis NSIP histology is the second most common type of interstitial lung disease in rheumatoid arthritis. Fibrosing NSIP is most common, although cellular or mixed cellular and fibrosing patterns may also occur (Figure 7).2,70,73 The radiological and histological features are similar to patients with idiopathic NSIP. As the radiological findings in patients with this pattern are often diverse, surgical lung biopsy is often helpful for diagnosis. As noted above, the prognosis of rheumatoid arthritis patients with an NSIP pattern is better than that for those with a UIP histology. In Lee et al.’s study, all six patients with an NSIP pattern were alive at follow-up.70

Organizing pneumonia pattern in rheumatoid arthritis An organizing pneumonia histology (bronchiolitis obliterans organizing pneumonia/BOOP) is another common type of pulmonary involvement by rheumatoid arthritis. In one large series, it was the second most common histological pattern (after rheumatoid nodules).53 Clinical symptoms include the subacute onset (weeks to months) of cough, dyspnea, weight loss and fever. A restrictive defect is often present when assessed by pulmonary function tests. Histological features are identical to organizing pneumonia in patients without CTD. Patchy bronchiolocentric involvement of alveolar ducts and sacs with polypoid intraluminal plugs of loose fibromyxoid connective tissue (“Masson bodies”) is seen and occasionally extends into respiratory and terminal bronchioles (Figure 8). In involved areas, there is often an associated mild chronic interstitial inflammatory infiltrate. The underlying alveolar septal framework is intact without significant fibrosis or honeycombing. The organizing pneumonia pattern is not specific and may arise from a number of etiologies. It may represent a minor histological component in the context of other more prominent pathological changes. For example, organizing pneumonia may be a manifestation of underlying infection, drug, toxin or fume exposure, aspiration as well as a nonspecific reaction around a mass lesion. It may also be a focal pattern in an otherwise typical UIP histology, DAD or hypersensitivity pneumonitis. For this reason, careful clinical correlation to exclude these and other etiologies is necessary in any biopsy showing this pattern in a patient with underlying CTD. Special stains for microorganisms (acid-fast bacilli and fungi) should be performed in all cases with a note of any drugs that could be associated with this pattern.69,75 Most patients with an organizing pneumonia pattern respond readily to steroids. Yet compared to patients with idiopathic cryptogenic organizing pneumonia, some rheumatological patients are steroid-resistant. Based on these observations, some have postulated that an organizing pneumonia pattern has a worse prognosis in patients with underlying CTD.47,76 Steroid-resistant patients will often respond to additional immunosuppressant or cytotoxic therapy, such as cyclophosphamide or cyclosporine.47,77

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Chapter 21: The lungs in connective tissue disease

(a)

(b)

(c)

Figure 6. Rheumatoid arthritis. UIP pattern. (a) Note the temporally heterogeneous fibrosis. (b) Occasional fibroblast foci (arrows) are present. (c) Areas of honeycomb lung were present in other areas of the biopsy.

Figure 7. Rheumatoid arthritis. Cellular NSIP pattern. Chronic interstitial inflammatory infiltrate expand alveolar septa.

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Figure 8. Rheumatoid arthritis. Organizing pneumonia pattern. There are intraluminal plugs of loose fibromyxoid connective tissue (“Masson bodies”) within alveoli and alveolar ducts.

Chapter 21: The lungs in connective tissue disease

(a)

(b)

Figure 9. Rheumatoid arthritis. Lymphocytic interstitial pneumonia pattern. (a) Diffuse expansion and distortion of alveolar septa by a polymorphous mononuclear cell infiltrate is seen. (b) The infiltrate consists of a mixture of lymphocytes, histiocytes and plasma cells.

Lymphocytic interstitial pneumonia pattern in rheumatoid arthritis A LIP pattern may occasionally occur as a pulmonary manifestation of rheumatoid arthritis (Figure 9).78,79 LIP is reported a histological pattern of pulmonary involvement by rheumatoid arthritis in the 2002 ATS/ERS classification of idiopathic interstitial pneumonias.69 In one review of 15 patients with LIP, three cases were associated with rheumatoid arthritis.80

Diffuse alveolar damage pattern in rheumatoid arthritis DAD may occur in patients with rheumatoid arthritis, either alone or as an acute exacerbation of pre-existing interstitial lung disease (Figure 10). 34,81 Acute exacerbations occur in almost 20% of rheumatoid arthritis patients with pre-existing UIP. Park et al. estimated an 11.1% one-year frequency of an acute exacerbation in rheumatoid arthritis patients with a UIP histology.81 DAD may rarely be the presenting manifestation of rheumatoid arthritis, as well as other CTD.34 The prognosis of this complication is poor.

Airway disease

Obliterative bronchiolitis pattern (constrictive bronchiolitis) in rheumatoid arthritis Obliterative bronchiolitis (constrictive bronchiolitis) is a rare complication of rheumatoid arthritis. Despite its name, it represents a completely different pattern from organizing pneumonia (BOOP), with which it should not be confused. Clinical symptoms are nonspecific, consisting of cough, dyspnea and irreversible airflow obstruction, which progresses rapidly over weeks to months. The pattern of “mosaic attenuation” with air-trapping on HRCT expiration studies is a characteristic radiographic feature.75,82–84

Figure 10. Rheumatoid arthritis. Diffuse alveolar damage. Pink hyaline membranes line edematous alveolar septa. This histological pattern may occur either alone or as an acute exacerbation of pre-existing interstitial lung disease. In some cases it may be the presenting manifestation of rheumatoid arthritis.

Histological features of obliterative bronchiolitis consist of submucosal and peribronchiolar fibrosis, leading to partial or complete narrowing and obliteration of bronchiolar lumina (Figure 11a,b).83–86 These changes are associated, in some cases, with a cellular bronchiolitis. Necrosis of bronchiolar epithelium may be present. Secondary changes include mucostasis and bronchiolectasis. In some cases biopsies may show only a few affected bronchioles and the changes may be inconspicuous, even in patients with marked symptomatology. Thus, biopsies of multiple lobes are usually necessary to make the diagnosis.82,85 Elastic tissue stains may help highlight the extent of the submucosal fibrosis (Figure 11c).

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Chapter 21: The lungs in connective tissue disease

(a)

(b)

(c)

Figure 11. Rheumatoid arthritis. Obliterative bronchiolitis. (a) Luminal bronchiolar narrowing is seen (arrows). Normally, the lumen should be approximately equal or only slightly smaller than the accompanying pulmonary artery. (b) Marked peribronchiolar submucosal fibrosis results in luminal compression. The bronchiolar epithelium should normally be juxtaposed to the bronchiolar smooth muscle. (c) Pentachrome stain highlights the marked submucosal fibrosis which extends eccentrically from the original elastic lamina of the bronchiole.

Like other patterns, obliterative bronchiolitis is not specific to rheumatoid arthritis. It most commonly occurs as a sequel of chronic rejection in lung transplant patients (see Chapter 20).87 Other etiologies include post-infectious, fume or toxin exposure (e.g. herbal tea from Sauropus androgynus, World Trade Center lung), drugs, inflammatory bowel disease, as a manifestation of graft versus host disease in bone marrow transplantation and in association with diffuse idiopathic neuroendocrine cell hyperplasia (see Chapter 17).75,82–84 The prognosis of obliterative bronchiolitis is generally poor and often fatal. Treatment with corticosteroids or immunosuppressants, such as cyclophosphamide, may benefit occasional patients.47

Follicular bronchiolitis / lymphoid hyperplasia in rheumatoid arthritis Follicular bronchiolitis represents hyperplasia of the bronchusassociated lymphoid tissue (BALT) (see Chapter 34).88 While often associated with rheumatoid arthritis, follicular bronchiolitis may also occur in association with other connective tissue diseases (e.g. Sjögren syndrome) and as an idiopathic

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process. It is also a secondary finding in patients with bronchiectasis, chronic bronchitis, asthma, chronic infections or cystic fibrosis.89–92 Follicular bronchiolitis may rarely precede the development of rheumatoid arthritis.93 Clinical symptoms are nonspecific and include cough and dyspnea. Chest X-ray shows bilateral reticular or reticulonodular infiltrates. High-resolution CT scan shows small 1–3 mm centrilobular and sometimes peribronchial nodules.94 Histologically, follicular bronchiolitis consists of lymphoid follicles, usually with abundant germinal centers in a peribronchial or peribronchiolar location (Figure 12). Lymphoid follicles may also have a lymphangitic distribution, along the interlobular septa and beneath the pleura, a pattern sometimes referred to as lymphoid hyperplasia. Follicular bronchiolitis and lymphoid hyperplasia probably represent BALT hyperplasia.89–92 Studies of patients with follicular bronchiolitis with prognostic data are limited. In Hayakawa’s series, eight patients with follicular bronchiolitis in the setting of rheumatoid arthritis showed a chronic but relatively favorable course. This

Chapter 21: The lungs in connective tissue disease

Figure 12. Rheumatoid arthritis. Follicular bronchiolitis. Reactive lymphoid follicles with secondary germinal centers cluster around a terminal bronchiole. Lymphoid hyperplasia features similar findings along with the additional presence of lymphoid follicles along the septa and pleura in a lymphangitic distribution.

Figure 13. Rheumatoid arthritis. Chronic bronchiolitis. A chronic inflammatory infiltrate permeates the wall and mucosa of this bronchiole.

Bronchiectasis Bronchiectasis is sometimes noted in rheumatoid arthritis, unassociated with other patterns of interstitial lung disease (Figure 14).70,98 Patients are asymptomatic or may present with features that clinically mimic interstitial lung disease.98 Clinical symptoms, when present, include cough, purulent sputum and repeated bouts of pneumonia.82 HRCT scan shows dilatation of airways with ectasia and mucous plugging.82,99 Prognosis is poor, with many patients ultimately succumbing to infectious complications.82,100

Vascular disease

Primary pulmonary hypertension in rheumatoid arthritis

Figure 14. Rheumatoid arthritis. Bronchioloectasis. A dilated bronchiole contains inspissated mucus. The associated pulmonary artery should generally be of comparable size but is much smaller. An adjacent terminal bronchiole also shows a pattern of follicular bronchiolitis.

result is mirrored in anecdotal case reports, as well as in small series of patients with follicular bronchiolitis outside the setting of rheumatoid arthritis.92 Treatments with steroids and erythromycin have variable success. Rarely patients develop a low-grade BALT lymphoma.89,91,92,95,96

Chronic bronchiolitis In addition to follicular bronchiolitis, occasional biopsies from patients with rheumatoid arthritis may show a nonspecific chronic bronchiolitis (Figure 13). This finding is noted in fewer than 10% of patients.70 Rare patients with a clinical and histological pattern of diffuse panbronchiolitis have also been reported (see Chapter 17).97

In contrast to some other CTD, primary pulmonary hypertension is rare as a primary pulmonary manifestation of rheumatoid arthritis. Small series and case reports with limited pathology descriptions of pulmonary hypertension and plexogenic arteriopathy have been reported (see Chapter 18).101–103

Diffuse alveolar hemorrhage/capillaritis in rheumatoid arthritis Diffuse alveolar hemorrhage is rare but may occur as a pulmonary manifestation of rheumatoid arthritis. In reported cases limited to the lung, there is often an associated capillaritis, accompanied by variable deposition of IgG and C3 in alveolar walls by immunofluorescence.104 Occasional cases have an accompanying vasculitis, often necrotizing, involving medium-sized arteries and veins (Figure 15).104–106 In separate reports, alveolar hemorrhage has occurred either in association with a necrotizing and crescentic glomerulonephritis and/or with serological evidence of anti-neutrophil cytoplasmic antibody (ANCA).107,108 Rarely rheumatoid arthritis can present with lung involvement resembling idiopathic pulmonary hemosiderosis.109 Pulmonary vasculitis in the absence of hemorrhage may occur (see Chapter 19).

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Chapter 21: The lungs in connective tissue disease

Figure 15. Rheumatoid arthritis. Vasculitis involving a small pulmonary artery. There is fibrinoid necrosis of the vessel wall with marked intimal edema and a mixed mural and subintimal inflammatory infiltrate.

Figure 17. Rheumatoid arthritis. Eosinophilic pneumonia. Eosinophilic pneumonia may rarely be associated with rheumatoid arthritis, sometimes as a presenting manifestation. Typical features of eosinophilic pneumonia are present in this section with aggregates of eosinophils and histiocytes within alveolar spaces.

Other rare pulmonary manifestations of rheumatoid arthritis Other rare pulmonary manifestations of rheumatoid arthritis are reported. Amyloid deposits have been described, usually of the AA type, occurring in both the nodular and alveolar septal forms (Figure 16).11,12,110 Rare case reports describe eosinophilic pneumonia associated with rheumatoid arthritis, sometimes as a presenting manifestation (Figure 17) (see chapter 15).111,112 Biopsies typically show chronic eosinophilic pneumonia, although one case with acute eosinophilic pneumonia was described.113,114 Apical fibrobullous disease has been rarely described, similar in appearance to findings in ankylosing spondylitis.115,116 Patients with rheumatoid arthritis also

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Figure 16. Rheumatoid arthritis. Secondary amyloidosis. Amorphous eosinophilic deposits of amyloid are diffusely present within the wall of this small pulmonary artery. The interpretation of amyloid is confirmed by applegreen birefringence under polarized light after staining for Congo red (inset).

Figure 18. Rheumatoid arthritis. Methotrexate-associated diffuse large B-cell lymphoma, EBV-positive. This endobronchial biopsy shows diffuse permeation of the endobronchial wall by large B-lymphocytes. The inset shows positive staining of the cells for CD20. The cells were also positive for Epstein-Barr virus by in situ hybridization.

appear to be at increased risk for the development of lung cancer31 and lymphoproliferative disorders. Some lymphoproliferative disorders represent EBV-associated large B-cell lymphomas related to longstanding methotrexate therapy. They may regress following discontinuation of this immunosuppression (Figure 18).117,118

Systemic lupus erythematosus From 50–70% of patients with systemic lupus erythematosus (SLE) have either lung or pleural involvement.119,120 Pleuritis accounts for most cases (61%), whereas pulmonary parenchymal manifestations are less common (18%).120 Parenchymal involvement tends to be more serious and sometimes

Chapter 21: The lungs in connective tissue disease Table 3 Pleuropulmonary pathology of systemic lupus erythematosus

Pleuritis Diffuse alveolar hemorrhage Without capillaritis With capillaritis Diffuse alveolar damage With capillaritis Without capillaritis Pulmonary hypertension Plexogenic arteriopathy Thrombotic arteriopathy Antiphospholipid antibody syndrome Pulmonary venoocclusive disease/pulmonary capillary hemangiomatosis Interstitial lung disease Organizing pneumonia pattern UIP pattern NSIP pattern Fibrosing NSIP pattern Cellular NSIP pattern LIP pattern Secondary complications Infection Pulmonary edema from congestive heart failure Shrinking lung syndrome Drug reactions Increased risk of malignancy (lung cancer) Metabolic disturbances (amyloidosis)

life-threatening. Parenchymal manifestations of SLE are diverse and may affect pulmonary vessels, airways and interstitium, either alone or in combination. In addition, patients with SLE are also at risk from secondary complications including infection, therapeutic or drug reactions, metabolic disturbances and an increased risk of malignancy. Table 3 lists the primary and secondary pleuropulmonary manifestations of SLE.

Pleuritis Pleuritis occurs in a majority of patients with SLE. Symptoms include pleurisy, dyspnea, cough and fever. Occasional patients are asymptomatic. Imaging studies often show a unilateral or bilateral pleural effusion. Pleural biopsies or autopsy studies typically show a thickened visceral pleura with fibrosis and chronic inflammation, consisting of lymphocytes, histiocytes and plasma cells. Pleural adhesions are also often present (see Chapter 36).49,120–123 Lupus pleuritis typically responds promptly to treatment with systemic steroids. Chest drainage or pleurodesis are only rarely needed.49

Figure 19. Systemic lupus erythematosus. Diffuse alveolar damage. Hyaline membranes partially line edematous alveolar septa. This histological pattern is often found in patients with acute lupus pneumonitis.

Acute lupus pneumonitis Approximately 1–4% of patients with SLE develop the clinical syndrome of acute lupus pneumonitis. In about 50% of these patients, it is the presenting manifestation. Clinical symptoms are nonspecific and similar to an acute infectious pneumonia. Patients develop the acute onset of cough, dyspnea, fever and occasionally hemoptysis. In some patients, these symptoms progress to acute respiratory failure. Hypoxemia and hypocapnia are present on arterial blood gases. Chest X-ray and CT scan show diffuse uni- or bilateral alveolar infiltrates with a predilection for the lower lobes. The mortality rate is approximately 50% and the disease may recur.49,124 The underlying histological correlates for the syndrome are varied, probably reflecting the nonspecific nature of the clinical symptoms. The most common histology is DAD (Figure 19).147 However, cellular interstitial pneumonia, organizing pneumonia and alveolar hemorrhage have also been reported.49,124 Immune complexes may be identified within alveolar walls.

Involvement of pulmonary vasculature Involvement of pulmonary vessels in SLE may be manifested by diffuse alveolar hemorrhage, either with or without associated capillaritis or DAD. Patients may also develop pulmonary capillaritis and thromboemboli, as manifestations of the antiphospholipid syndrome, or pulmonary hypertension as a chronic sequel.124

Diffuse alveolar hemorrhage Diffuse alveolar hemorrhage is a rare but sometimes fatal complication occurring in less than 2% of all SLE patients.125 Mortality rates range from 70% to 90% although the prognosis is improving. Clinical features include dyspnea, hemoptysis

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Figure 20. Systemic lupus erythematosus. Diffuse alveolar hemorrhage. The hemorrhage is relatively recent as there is no accumulation of hemosiderin-laden macrophages. There is no accompanying capillaritis. Diffuse alveolar hemorrhage in SLE may occur either with or without an associated capillaritis.

and radiological diffuse bilateral alveolar infiltrates developing over hours to several days.124,126 Most patients also have renal involvement.124,127,128 Although it usually occurs in patients previously diagnosed with SLE, diffuse alveolar hemorrhage may rarely be the presenting manifestation.124,129,130 A decreasing hemoglobin level is frequently present.124,126 Bronchoalveolar lavage is hemorrhagic and often contains numerous hemosiderin-laden macrophages.124,128 Histologically, diffuse alveolar hemorrhage complicating lupus usually occurs in one of four contexts. It may occur either alone (bland alveolar hemorrhage), or in association with pulmonary capillaritis, DAD or the antiphospholipid syndrome. Bland alveolar hemorrhage refers to a variable combination of erythrocytes and hemosiderin-laden macrophages extensively filling alveoli, without capillaritis or DAD (Figure 20). Historically, alveolar hemorrhage has been described as bland in approximately 70% of lung biopsies from SLE patients. More recent studies show an increasing frequency of associated capillaritis in such cases, approaching 80%. The difference in frequency may stem from sampling issues, greater awareness of capillaritis or revised histological criteria for the recognition of more subtle features of capillaritis.124,130 In some patients with SLE, alveolar hemorrhage is accompanied by a capillaritis. Capillaritis literally means “inflammation of capillaries” (see Chapter 19). Histologically capillaritis consists of neutrophilic inflammation and nuclear dust in alveolar septa, accompanied by variable deposits of fibrin and fibrinoid necrosis of septal walls (Figure 21). Sometimes the degree of inflammation is so intense that it spills over into the adjacent alveoli.130–132 The neutrophilic inflammation and nuclear dust is always concentrated in capillaries of alveolar septa rather than in alveolar spaces. In contrast, while a hemorrhagic pneumonia may also sometimes show neutrophilic inflammation within alveolar septa, the

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Figure 21. Systemic lupus erythematosus. There is diffuse alveolar hemorrhage along with intra-alveolar aggregates of hemosiderin-laden macrophages. There is an accompanying subtle neutrophilic capillaritis.

Figure 22. Systemic lupus erythematosus. Vasculitis. A mixed inflammatory infiltrate permeates the wall of this small pulmonary artery. Vasculitis, when present, often occurs in association with capillaritis.

inflammation is more prominent within alveolar spaces than the alveolar septa.130–132 In patients with diffuse alveolar hemorrhage, capillaritis may be focal, patchy and subtle.105,131 In addition to capillaritis, in some cases there may be an associated arteritis, involving small muscular arteries and arterioles (Figure 22).130 Biopsies of patients with SLE and alveolar hemorrhage are occasionally accompanied by features of DAD. A superimposed infectious pneumonia may also be present.124 Diffuse alveolar hemorrhage may occasionally be the presenting manifestation of the antiphospholipid syndrome. The antiphospholipid syndrome sometimes occurs in association with SLE. This possibility can usually be assessed with appropriate serological tests (see below).126,133,134

Chapter 21: The lungs in connective tissue disease

The differential diagnosis of alveolar hemorrhage in patients with SLE depends on the combination of histological features. Bland pulmonary hemorrhage occurs not only as a primary manifestation of SLE but also as a secondary complication, reflecting involvement of other organ systems. For example, coagulation disorders associated with uremia and left ventricular failure due to myocarditis or marantic endocarditis may complicate SLE and cause alveolar hemorrhage and hemoptysis.124–126,135 Similarly, alveolar hemorrhage may be a prominent component of some infectious pneumonias. Cytomegalovirus, legionella, herpes simplex virus 1 and 2 and Staphylococcus aureus have been reported in association with pulmonary hemorrhage in patients with SLE.124,125,136 Pulmonary hemorrhage and capillaritis are also reported as the presenting manifestation of a drug reaction to drugs such as phenytoin,137 retinoic acid,138 propylthiouracil,139,140 hydralazine and penicillamine.141 In patients with no prior history of SLE, other causes of pulmonary hemorrhage to consider include pulmonary vasculidites – most importantly Wegener granulomatosis, microscopic polyangiitis and Goodpasture syndrome (see Chapter 19). Other CTD may also rarely cause pulmonary hemorrhage including polymyositis,142 mixed connective tissue disease,104,143 scleroderma144 and rheumatoid arthritis.104 Deposition of immune complexes is thought to play a significant role in the pathogenesis of diffuse alveolar hemorrhage in SLE. Immune complexes can be demonstrated by immunofluorescence and ultrastructural studies in many (50 to 75%), but not all, cases.127,130,145,146 Immune complex deposits occur regardless of whether there is associated capillaritis or DAD. Granular deposits of IgG may be identified within alveolar septa on immunofluorescence often accompanied by C1q, C3 and less often by other immunoreactants, including IgM and IgA. Ultrastructurally, electron-dense deposits are present in alveolar walls – most often within the basement membrane but sometimes in a subendothelial or subepithelial location.127,130,145 Apoptotic cells and immune complexes in areas of alveolar hemorrhage have been documented in two patients by immunofluorescence and ultrastructure. In contrast, immune complexes were not found in lung biopsies from four SLE patients without alveolar hemorrhage. In this study, investigators postulate that bland alveolar hemorrhage in SLE is a form of alveolar capillary wall injury secondary to apoptosis precipitated by immune complex deposition. This alveolar wall injury and the resulting bland alveolar hemorrhage might be considered analogous to the microangiopathy seen in lupus nephritis.127

Pulmonary hypertension Five to fourteen percent of patients with SLE develop pulmonary hypertension at some time in the course of their disease in the absence of interstitial lung disease or pulmonary emboli. The frequency and severity of pulmonary hypertension tend to increase with time.49,148–150 The most common clinical symptom is dyspnea on exertion. Right ventricular hypertrophy

associated with right axis deviation is noted on electrocardiogram. Echocardiography may be helpful in detecting pulmonary hypertension, although cardiac catheterization is necessary for definitive diagnosis. Raynaud’s phenomenon is present in 75% of patients. Histologically, most cases of pulmonary hypertension show plexogenic arteriopathy with involvement of small pulmonary arteries by concentric laminar intimal fibrosis, medial hypertrophy and plexiform lesions.49,149,151–154 Less frequently a chronic thrombotic arteriopathy pattern has been reported, sometimes, in association with the antiphospholipid antibody syndrome (Figure 23).155–164 There are also isolated reports of pulmonary capillary hemangiomatosis and pulmonary venoocclusive disease.165,166 A recent study found significant occlusion of veins/preseptal venules in most patients with pulmonary hypertension associated with CTD, including one of two patients with SLE. The frequency of this finding was much greater than in controls with pulmonary arterial hypertension without CTD. These authors proposed that significant venoocclusive disease frequently complicates pulmonary hypertension in patients with underlying CTD.167 The pathogenesis of pulmonary hypertension in SLE and CTD in general is complex (see Chapter 18). Antiphospholipid antibodies, anti-endothelial cell antibodies, vasculitis and vasospasm are implicated.49,158,168 Pulmonary hypertension tends to worsen with time. Overall 2-year survival was less than 50% in one study.169 Treatment consists of oral anticoagulants and vasodilators. Isolated reports have shown a response to corticosteroids and cyclophosphamide.170

Interstitial lung disease The prevalence of interstitial lung disease in SLE increases with duration of disease. Interstitial lung disease occurs in about 1% of patients at diagnosis, 4% within 1 year of diagnosis and 8% after 12 years. Overall prevalence is estimated around 3%.171,172 Symptoms are similar to interstitial lung disease, occurring outside SLE, and consist of progressive exertional dyspnea and cough. The gradual appearance of these symptoms is in contrast to acute lupus pneumonitis, which has a more abrupt onset. Interstitial lung disease appears to arise in two distinct clinical settings: (1) as a long-term sequel of acute lupus pneumonitis or (2) insidiously over time, without an obvious precipitating cause.147,173–176 Morphological descriptions of interstitial lung disease in SLE are limited. Most studies of histopathological findings were published before the 2002 ATS/ERS classification of idiopathic interstitial pneumonias and describe fibrosis, interstitial inflammation and honeycombing. More recent publications describe pathological patterns of UIP, fibrosing and cellular NSIP, LIP and follicular bronchiolitis (Figures 24 and 25).2,177–179 An organizing pneumonia pattern (BOOP)

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(a)

(b)

(c)

Figure 23. Systemic lupus erythematosus. Pulmonary thromboembolic disease in a patient with pulmonary hypertension and lupus anticoagulant. (a and b) Recanalized thrombi fill several small pulmonary arteries. (c) An EVG stain highlights recanalized vascular channels within the arterial lumen.

Figure 24. Systemic lupus erythematosus. Fibrosing NSIP pattern. There is mild, but relatively uniform, fibrosis of alveolar septa.

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Figure 25. Systemic lupus erythematosus. Lymphocytic interstitial pneumonitis pattern. A dense polymorphous mononuclear cell infiltrate expands alveolar septa.

Chapter 21: The lungs in connective tissue disease

(a)

(b)

Figure 26. Systemic lupus erythematosus. Organizing pneumonia pattern. (a) Organizing buds of loose connective tissue consolidate lung. (b) Connective tissue is seen within alveolar spaces and respiratory ducts.

has also been described, sometimes as a presenting manifestation (Figure 26).180–182 As in patients with more acute expressions of SLE, immune complexes can be demonstrated in many cases by immunofluorescence for IgG, IgM and C3. The prognosis of patients with SLE and interstitial lung disease depends on the particular histological pattern of lung disease. Data are limited and based mostly on isolated case reports and small retrospective series. The prognosis of patients with a UIP or NSIP pattern is variable but tends to be better than for patients with idiopathic interstitial lung disease.177 In some cases, the interstitial lung disease may stabilize or slightly improve.173 Treatment generally consists of corticosteroids, sometimes combined with cyclophosphamide.173,176 Prognosis of most, but not all, patients with an organizing pneumonia pattern has been good, with response to corticosteroids and resolution of the infiltrates.180,181

Shrinking lung syndrome Shrinking lung syndrome is an uncommon manifestation of SLE characterized by shortness of breath, small lung volumes radiographically, a restrictive pattern on pulmonary function tests and elevated hemidiaphragms. Affected patients often have associated pleuritis and fever.183 First described in 1954,184–186 the cause is still controversial. Restriction of chest wall expansion probably plays a role.187 Restriction of chest wall expansion may result from either bilateral phrenic nerve paralysis or diaphragmatic dysfunction due to a primary diaphragmatic myopathy or fibrosis.188–192 The prognosis is generally good, with stabilization in most patients and gradual improvement in a few. Treatment with corticosteroids may be beneficial.183,193,194 A recent article reports remission with rituximab.195

Drug reactions Lupus-like drug reactions occur with various drugs, including hydralazine, procainamide and carbamazepine. These drugs may even cause lung disease (see Chapter 16).196–198

Antiphospholipid antibody syndrome The antiphospholipid syndrome refers to a pattern of venous and arterial thrombosis which occurs in association with antibodies to phospholipids. Recurrent miscarriages, thrombocytopenia, livedo reticularis and neurological symptoms are other features of this disorder. While initially described exclusively in association with SLE, it is now recognized that the antiphospholipid syndrome may occur in patients without lupus.199,200 Antiphospholipid antibody syndrome occurs in two settings – a primary form unrelated to other diseases, and a secondary type occurring in the spectrum of patients with SLE and other CTD (including Sjögren syndrome, systemic sclerosis, rheumatoid arthritis, and others). Up to 32% of patients with SLE have antiphospholipid antibodies.201,202 The antibodies in antiphospholipid antibody syndrome are heterogeneous and directed against a variety of antigens. Despite their name, these antibodies do not recognize phospholipid directly, but are directed against plasma proteins, which bind tightly to the anionic surfaces of phospholipids. Targeted plasma proteins include apolipoprotein H (formerly termed b2-glycoprotein I), which binds cardiolipin, and prothrombin, which binds phosphatidyl serine.203 Antibodies to other targets, some unidentified, are also present. Antiphospholipid antibodies are detected by both ELISA assays for anticardiolipin and antiphosphatidylserine, as well as the detection of lupus anticoagulants. The term “lupus anticoagulant” is not

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Chapter 21: The lungs in connective tissue disease

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Figure 27. Catastrophic antiphospholipid antibody syndrome (Asherson syndrome). There is marked pulmonary hemorrhage and hemosiderosis. Inset highlights hemosiderin on special stain for iron (Prussian blue). At autopsy the patient also had multiple brain infarcts and a renal thrombotic microangiopathy.

Figure 28. Catastrophic antiphospholipid antibody syndrome (Asherson syndrome). A peripheral pulmonary infarct is present.

limited to patients with SLE. It reflects the ability of antiphospholipid antibodies to inhibit clotting in phospholipidsdependent in vitro assays of clotting, such as the partial thromboplastin time (PTT). In the presence of such inhibition, in contrast to deficiencies of specific factors, clotting time fails to correct upon mixing with normal pooled plasma. Pulmonary manifestations of the antiphospholipid syndrome are varied. Pulmonary thromboembolism is most common (38.9%) and may be the presenting manifestation. It is usually a sequel of deep vein thrombosis.159,160,162,204 Pulmonary hypertension is another serious complication, which is estimated to occur in 3.5% of patients with primary antiphospholipid syndrome and 1.8% of patients with secondary forms of this condition.157–159 It may be a manifestation of chronic thromboembolic disease or in a primary form unassociated with chronic thromboembolic disease (Figure 23).158–162 Acute respiratory distress syndrome is also fairly common, most often in the setting of the catastrophic antiphospholipid antibody syndrome (see below).159,205 Histological features include hyaline membranes (DAD) accompanied by extensive small vessel thrombosis.159,162 Rare pulmonary manifestations include alveolar hemorrhage with or without capillaritis and fibrosis.134,160,162,206 Catastrophic antiphospholipid antibody syndrome (Asherson syndrome) is a term coined in 1992 to describe a subset of individuals with an accelerated form of the disease. Patients have an acute onset of multiple vascular occlusive events, which rapidly lead to multiorgan failure.207 Pathological features of the lungs of patients with catastrophic antiphospholipid antibody syndrome include a non-inflammatory thrombotic microangiopathy, sometimes accompanied by hyaline membranes (DAD) and/or intra-alveolar hemorrhage (Figures 27 and 28).156,205 Approximately half of afflicted individuals have SLE.205,208

Scleroderma (progressive systemic sclerosis) is a multisystem CTD characterized by fibrosis of the skin and internal organs. The disease predominantly affects middle-aged females.209 Two major forms of scleroderma have been described – a diffuse variant and a more indolent, limited variant (CREST variant). Multiorgan involvement is most prominent in the diffuse variant, with vascular and connective tissue fibrosis affecting the skin, gastrointestinal tract, lungs and kidneys. The limited\(CREST) variant is a more indolent form first described in the mid-1960s.210,211 In this form, cutaneous changes are restricted to the distal extremities without involvement of the trunk. This type of disease is also characterized by calcinosis cutis, Raynaud’s phenomenon, esophageal dysfunction, sclerodactyly and telangiectasia – hence the acronym CREST. Regardless of the form of disease, pulmonary involvement is a leading cause of morbidity and mortality.212,213 The predominant primary pulmonary manifestations of scleroderma include pulmonary hypertension and interstitial lung disease (Table 4).214,215 Pulmonary hypertension may occur as primary or secondary forms associated with interstitial lung disease. The primary form of pulmonary hypertension (12–16% of patients) is associated more frequently with the limited, rather than diffuse, variant of scleroderma. Interstitial lung disease affects patients with both the limited and diffuse variants of scleroderma and is detected in most patients by high-resolution CT scan or autopsy.214–216 Clinically significant interstitial lung disease occurs in approximately 30% to 40% of patients. Rarely other patterns of injury, such as organizing pneumonia, small airways disease and DAD, have been reported.35,215,217,218 Scleroderma is associated with a variety of autoantibodies, which tend to reflect different subsets of the disease. Autoantibodies most frequently found include anti-topoisomerase (also known as Scl-70), anti-centromere antibodies and

Scleroderma

Chapter 21: The lungs in connective tissue disease

anti-RNA polymerase I/III. Other less common, although still fairly specific, autoantibodies include anti-Th/To, antiU3-RNP and anti-PM/Scl autoantibodies. Autoantibodies which may be present but are not specific to scleroderma include anti-Ro (SS-A) and anti-La (SS-B), often found in Sjögren syndrome and SLE, anti-RNA polymerase II, often found in SLE and overlap syndromes, and anti-U1-RNP, found in mixed connective tissue syndrome.215

Interstitial lung disease in scleroderma The development of interstitial lung disease is strongly linked to the presence of anti-topoisomerase antibodies (Scl-70). In Table 4 Pleuropulmonary pathology of progressive systemic sclerosis (scleroderma)

Pleuritis Interstitial lung disease (listed in order of frequency) NSIP pattern Fibrosing NSIP pattern Cellular NSIP pattern UIP pattern Organizing pneumonia pattern Pulmonary hypertension Fibrointimal mucoid hyperplasia of muscular pulmonary arteries Early plexiform lesions Pulmonary veno-occlusive disease Secondary complications Aspiration pneumonia Pulmonary edema from congestive heart failure Drug reactions Increased risk of lung cancer

(a)

contrast, anti-centromere antibodies are associated with an increased risk of pulmonary hypertension, but an absence of fibrosis.215,219

NSIP and UIP patterns Initial studies of interstitial lung disease in scleroderma were based on autopsy series or open lung biopsies with fairly advanced fibrosis.216,217,220 These cases were described before the reports of NSIP and, accordingly, the histological features were thought to be identical to UIP. More recent studies have classified the pattern of interstitial lung disease according to the 2002 ATS/ERS consensus conference. While these studies confirmed the presence of a UIP pattern in a significant minority of patients, a surprising finding was that most cases had an NSIP pattern (Figure 29).221,222 In one recent series of surgical lung biopsies from 80 patients with scleroderma and interstitial lung disease, 47 had a fibrotic NSIP pattern (59%), 15 (19%) had a cellular NSIP pattern, and six (7.5%) each had a UIP pattern, endstage lung disease or other patterns.222 Studies of scleroderma patients with HRCT scans support these findings. In 225 patients with scleroderma, two-thirds of patients had HRCT features similar to a control cohort of NSIP patients. In comparison to a control group of patients with proven IPF, the HRCTs showed a greater degree of ground-glass opacities with less reticulation. However, a third of the scleroderma patients in this study showed increased, coarse, fibrosis with reticulation, which probably corresponds to a UIP pattern.223 The prognosis of patients with interstitial lung disease in scleroderma is significantly better than in patients with IPF. That patients with NSIP have a better prognosis than patients with UIP and that NSIP is the commonest interstitial lung disease in scleroderma partially explains this observation.2,221,224 However, in one series, scleroderma patients with a UIP pattern of fibrosis histologically had a similar prognosis (b)

Figure 29. Scleroderma. Fibrosing NSIP pattern. (a) Temporally uniform interstitial fibrosis is seen. (b) Fibrosis distorts alveolar spaces.

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(a)

(b)

Figure 30. Scleroderma. Pulmonary hypertension. (a) Marked intimal fibroplasia of a small pulmonary arteriole compresses the lumen of this arteriole. (b) EVG elastic tissue stain of a small pulmonary artery highlights the marked fibrointimal thickening and resulting luminal compromise.

to NSIP patients.222 These results support the findings of other studies that a UIP pattern, in the setting of CTD, has a better prognosis than in patients with IPF.74,225 Treatment of interstitial lung disease in scleroderma consists of immunosuppressant therapy and cytotoxic agents, particularly cyclophosphamide. In 158 patients with scleroderma-related interstitial lung disease, treatment with cyclophosphamide resulted in improved lung function, dyspnea and health status/disability. The improvements appeared to wane after discontinuation of therapy.226

Other patterns of parenchymal involvement Other patterns of interstitial lung disease may occur. An organizing pneumonia pattern218 and DAD have been rarely reported.34,35 Follicular bronchiolitis may occur in association with other patterns or rarely by itself.53,89,92,217,220 Pleural effusions and pleuritis likewise may be seen either alone or in association with parenchymal disease.217 Pulmonary involvement by scleroderma may also reflect disease involvement of other organ systems. For example, aspiration pneumonia may be a result of esophageal dysmotility.227 Infectious complications resulting from the use of immunosuppressant and cytotoxic agents, as well as aspiration, are also possible.

Pulmonary hypertension in scleroderma Pulmonary hypertension is the second major cause of morbidity and mortality in scleroderma. As noted above, pulmonary hypertension may occur as a secondary finding in the setting of interstitial lung disease or as a primary process in the absence of interstitial lung disease. It may be found in up to 60% of patients, when assessed by right heart cardiac catheterization.219

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Isolated pulmonary hypertension is present most frequently in patients with scleroderma compared to other CTD, and more often in the limited (CREST) form.217,219 Affected patients often have associated Raynaud’s phenomenon and anti-centromere antibodies serologically.219 Histologically, affected pulmonary muscular arteries show thickened media with intimal fibroplasia. The intimal fibroplasia consists of concentric deposits of myxoid acid mucopolysaccharide-rich collagen, admixed with spindle-shaped fibroblasts and myofibroblasts. This proliferation results in variable but sometimes marked (> 75%) or complete luminal occlusion (Figure 30).217 Fibrinoid necrosis is not identified. Early plexiform lesions in association with the marked intimal changes may rarely be seen. These changes are not as well developed or prominent as in idiopathic primary pulmonary hypertension. On the basis of staining for Factor VIII-related antigen within areas of concentric intimal fibrosis, investigators suggest endothelial cell proliferation plays a central pathogenetic role.228 Aside from the classic changes in pulmonary arteries, histological alterations in other vascular compartments have rarely been reported in association with pulmonary hypertension. A case of pulmonary venoocclusive disease in a woman with the limited form of scleroderma (CREST) has been reported. While pulmonary arteries in this case showed some mural thickening, no concentric intimal fibroplasia was identified.229 The prognosis of scleroderma patients with isolated forms of pulmonary hypertension is poor, with a 2-year survival of 40%. Patients tend to respond poorly to vasodilator drugs.

Lung cancer in scleroderma Patients with CTD have an increased incidence of malignancy. This association is particularly prominent in patients with

Chapter 21: The lungs in connective tissue disease

scleroderma. Scleroderma patients have up to a 4.9-fold increased incidence of lung cancer.32 In a series of 262 patients with scleroderma, investigators found a 5% incidence of lung cancer.33 Another noted a particularly high incidence of carcinoma (21%) in 17 patients with CREST variant scleroderma.217 All histological types are represented, including adenocarcinoma, squamous cell carcinoma and small cell carcinoma. The increased risk of carcinoma appears to be related to longstanding pulmonary fibrosis and immunological abnormalities. Interestingly, the development of lung cancer has not been associated with cigarette smoking in this setting.32

Sjögren syndrome Sjögren syndrome is a systemic autoimmune disorder, characterized by an abnormal infiltration and destruction of exocrine glands by a chronic inflammatory infiltrate. Destruction of exocrine glands leads to diminished or absent glandular secretions. This results in the principal symptoms of the disease, including mucosal and conjunctival dryness (sicca syndrome).230,231 Extraglandular manifestations of Sjögren syndrome may include articular involvement, Raynaud’s phenomenon and pulmonary involvement. Sjögren disease may occur by itself (primary Sjögren syndrome) or secondarily in association with another connective tissue disease (e.g. SLE or rheumatoid arthritis). Diagnosis of Sjögren syndrome is facilitated by the detection of characteristic autoantibodies. Affected patients characteristically have antibodies to the extractable cellular antigens Ro/SS-A and La/SS-B. Elevation of other autoantibodies, including antinuclear antibodies and rheumatoid factor, also occurs. Detection of an inflammatory infiltrate in a minor salivary gland biopsy may be helpful.230,232 The reported frequency of pulmonary involvement varies widely from 9 to 75% depending on detection methods.9,233–237 Pathological and clinical manifestations of pulmonary involvement by Sjögren syndrome are broad and include different patterns of interstitial lung disease, bronchiolar disease, pulmonary arterial hypertension and the secondary development of lymphoproliferative disorders (Table 5).

Interstitial lung disease The spectrum of interstitial lung disease in Sjögren syndrome includes patterns of NSIP, LIP, UIP and OP.

NSIP and LIP patterns NSIP and LIP are most common. Although early reports emphasized LIP as the principal manifestation, more recent studies performed after the description of NSIP238 have recognized it more frequently.9 Both cellular and fibrosing patterns of NSIP are reported, although it is unclear which pattern predominates.2,9,239,240 LIP is considered a pattern of hyperplasia of the BALT, which develops as a reaction to varied stimuli. In addition to

Table 5 Pleuropulmonary pathology of Sjögren syndrome

Interstitial lung disease (listed in order of frequency) NSIP pattern Cellular NSIP pattern Fibrosing NSIP pattern LIP pattern UIP pattern Organizing pneumonia pattern Airway Tracheobronchial sicca (xerotrachea) Chronic bronchiolitis Vascular Pulmonary arterial hypertension Intimal and medial hyperplasia of pulmonary arteries with plexiform lesions Secondary complications Pulmonary lymphoproliferative disorders Low-grade marginal zone B-cell lymphoma Nodular lymphoid hyperplasia (pseudolymphoma) Metabolic disturbances (amyloidosis)

Sjögren syndrome, the observation of LIP in association with HIV infection, EBV infection, common variable immune deficiency and others241–243 presumably reflects the effects of longstanding chronic antigenic stimulation.88 It is tempting to speculate that similar mechanisms in this context may underlie both LIP and the development of MALT lymphoma (see below). Radiographic features of LIP on HRCT include bilateral areas of ground-glass opacity and consolidation accompanied by centrilobular and subpleural nodules, patchy bronchovascular and interlobular septal thickening. Cysts or even bullae are often present and may rarely dominate the radiographic findings.9,244–247 LIP is characterized histologically by a dense interstitial mononuclear inflammatory infiltrate, consisting of lymphocytes and variable numbers of plasma cells and histiocytes. This infiltrate diffusely expands alveolar septa. In some cases, there are associated reactive lymphoid follicles present along bronchovascular bundles and/or pleura, representing a component of follicular bronchiolitis and/or lymphoid hyperplasia.248–250 In approximately 20% of cases, these two patterns overlap to such an extent that the distinction becomes somewhat arbitrary (Figure 31).90,250 Lung biopsies from patients with Sjögren’s may only demonstrate follicular bronchiolitis/ lymphoid hyperplasia. Minor changes include occasional isolated giant cells or loose non-necrotizing granulomas, intraepithelial lymphocytes, type II pneumocyte hyperplasia, focal lymphocytic infiltration of vessel walls, foci of organizing pneumonia and

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Figure 31. Sjögren syndrome. Lymphoid hyperplasia. (a) Numerous lymphoid follicles with reactive germinal centers are present around a bronchiole (follicular bronchiolitis) and along the pleura. There is also expansion of alveolar septa by a polymorphous mononuclear infiltrate. (b) Reactive germinal centers with adjacent bronchiole.

Figure 32. Sjögren syndrome. Organizing pneumonia pattern. Organizing buds of loose connective tissue are present within alveolar spaces.

Figure 33. Sjögren syndrome. UIP pattern. There is a temporally heterogeneous pattern of fibrosis.

varying degrees of interstitial fibrosis.90,248–250 Amyloid may also be noted.249 The distinction between cellular NSIP and LIP patterns may be problematic and is based primarily on the degree of cellular infiltration. In LIP, the infiltrate is so dense that it markedly widens and distorts the underlying alveolar framework. Reactive lymphoid follicles are also often present in LIP. In contrast, alveolar septa in cellular NSIP are only modestly widened and reactive lymphoid follicles are either absent or rare.251

radiological and histological features of these patterns are similar to those in idiopathic forms of the disease.2,9,16,240

Other patterns of parenchymal involvement Less common histologies of interstitial lung disease in Sjögren syndrome include OP (Figure 32),9,252 UIP (Figure 33),9,240 and interstitial and nodular amyloidosis.9,13–15,18 The

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Airway disease

Tracheobronchial sicca (xerotrachea) Bronchi and bronchioles are commonly involved in Sjögren syndrome but have different clinical manifestations. Desiccation of the tracheobronchial tree (tracheobronchial sicca or xerotrachea) presumably underlies the common symptom of cough in patients without other parenchymal disease. Limited pathological studies of endobronchial and transbronchial biopsies report chronic mucosal and submucosal mononuclear infiltrates.253,254 There are increased numbers of CD-4 positive T-lymphocytes within the bronchial mucosal infiltrates compared to non-smoking controls.255 Thus, involvement of

Chapter 21: The lungs in connective tissue disease

extraglandular tissue by CD-4-positive lymphocytes may be important to the pathogenesis of tracheobronchial sicca. Whether destruction of tracheal and bronchial submucosal glands per se is also important is unclear and not well studied. In one report of six autopsied patients with Sjögren syndrome, hyperplasia rather than destruction of bronchial secretory cells and submucosal glands, compared to normal controls, was observed.256

Chronic bronchiolitis Chronic bronchiolitis is also a common histopathological finding and may underlie some of the obstructive symptoms attributed to small airways disease.2,235,253,257,258 In one study of open lung biopsies from 37 patients with CTD, chronic bronchiolitis was the commonest histopathological finding, affecting four of five patients with Sjögren syndrome.2 Follicular bronchiolitis is also commonly seen and may contribute to obstructive clinical manifestations.89,253 Middle lobe syndrome, presumably through impairment of mucociliary clearance, has also been reported (see Chapter 17).89,253,259

Pulmonary arterial hypertension Pulmonary hypertension is rare in patients with Sjögren syndrome, with fewer than 40 patients reported. In the largest series of nine patients, pulmonary arterial hypertension was more often associated with Raynaud’s phenomenon, cutaneous vasculitis and interstitial lung disease. Sjögren syndrome patients with pulmonary arterial hypertension also had more frequent antinuclear, anti-Ro/SSA and anti-RNP autoantibodies.260 Histological findings consist of intimal and medial hypertrophy of small arteries accompanied by plexiform lesions (see Chapter 18).260–262 Deposition of immunoglobulins and complement in the pulmonary arterial walls has been noted.260–262

Marginal zone B-cell lymphoma and nodular lymphoid hyperplasia Patients with Sjögren syndrome are estimated to have a relative risk 44 times the normal population of developing non-Hodgkin lymphoma. In the lung, patients are at increased risk of the development of lymphoma of BALT. As with lymphomas developing at other sites in patients with Sjögren syndrome, this type of lymphoma is considered a variant of extranodal marginal zone B-cell lymphoma.23,26 In seven patients with Sjögren syndrome who developed BALT lymphoma (extranodal marginal zone B-cell lymphoma), histological and radiological features were similar to those in patients without Sjögren syndrome.23 In addition to typical histological features, occasional cases are accompanied by amyloid deposits.263 Development of a higher-grade component (diffuse large B-cell lymphoma) may occur.25,26 In the absence of a higher-grade component, most patients follow an indolent course with partial or complete remission.23

In addition to low-grade lymphoma of BALT, pulmonary nodular lymphoid hyperplasia (“pseudolymphoma”) has been reported in Sjögren syndrome patients.264 Nodular lymphoid hyperplasia, or localized BALT hyperplasia, consists of a masslike proliferation of reactive lymphocytes, often containing numerous reactive follicles with intense interfollicular plasmacytosis and a variable degree of interfollicular fibrosis. In contrast to low-grade BALT lymphoma, the proliferation in nodular lymphoid hyperplasia is always polyclonal on both molecular and immunohistochemical studies (see Chapter 34).265

Polymyositis/dermatomyositis Polymyositis (PM) and dermatomyositis (DM) are systemic inflammatory diseases involving skeletal muscles and other organs, including the lungs. Characteristically patients present with proximal muscle weakness, increased serum skeletal muscle enzymes (creatinine phosophokinase (CPK)), characteristic findings on electromyography and infiltrates of inflammatory cells within skeletal muscle fibers. In addition to these features, patients with DM have a characteristic skin rash, consisting of a purple discoloration of the eyelids (heliotrope rash) or an erythematosus dermatitis over the extensor surface of the metacarpophalangeal and proximal interphalangeal joints of the fingers (Gottron’s papules).266–268 Occasional cases of DM may occur without muscular findings, so-called amyopathic DM.269,270 Traditionally, PM and DM are considered part of the same spectrum of disease. Several recent reports have challenged that assumption.271–274 Pulmonary manifestations of PM and DM are fairly common (Table 6). In one recent series of 81 consecutive patients, pulmonary manifestations occurred in 61% of patients.268 Pulmonary manifestations broadly include Table 6 Pleuropulmonary pathology of polymyositis/dermatomyositis

Interstitial lung disease NSIP pattern Cellular NSIP pattern Fibrosing NSIP pattern UIP pattern Organizing pneumonia pattern DAD pattern (predominantly in patients with dermatomyositis) Other rare manifestations Acute fibrinous and organizing pneumonia pattern Pulmonary capillaritis and diffuse alveolar hemorrhage Vasculitis (active or healed) Plexogenic arteriopathy Secondary complications Aspiration pneumonia Respiratory compromise due to hypoventilation Increased risk of lung cancer

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Chapter 21: The lungs in connective tissue disease

(a)

(b)

Figure 34. Polymyositis. Fibrosing NSIP pattern. (a) Fibrosis is advanced but relatively uniform and lacks the heterogeneity of a UIP pattern. (b) Early honeycomb lung is noted.

interstitial lung disease and the secondary manifestations of aspiration, hypoventilation and associated malignancies.

Interstitial lung disease Interstitial lung disease is a common complication of PM/DM. In one study it was seen in 39% of consecutive patients.268 Histological patterns of involvement include NSIP (cellular, cellular and fibrotic and fibrotic) (Figure 34), UIP, OP and DAD.271,275–279 LIP has also rarely been reported.275 While most prior studies emphasized UIP as a common pattern of pulmonary involvement,278 more recent ones have shown that NSIP occurs with equal, if not greater, frequency. Several recent series have reported NSIP in 36% to 81% of lung biopsies, while UIP was noted in 5% to 45% of patients.271,275,277,279 Both cellular and fibrosing patterns of NSIP have been reported (Figure 34). The prognostic significance of a UIP pattern is unclear. Some reports have suggested a worse prognosis, others propose a prognosis similar to patients with NSIP.271,277 Organizing pneumonia, previously reported as bronchiolitis obliterans and organizing pneumonia (“BOOP”), may also occur and has rarely been reported as the presenting feature of pulmonary involvement by PM (Figure 35).275–280 Aside from those patients with DAD, the prognosis of patients with NSIP versus those without an NSIP pattern is unclear. No survival difference at 3 years (89% in patients with NSIP vs. 80% in patients with non-NSIP) was noted in one limited series.275 Some reports have suggested a somewhat better prognosis in patients with organizing pneumonia.278 DAD is a rare complication in patients with PM/DM. Some suggest this complication occurs predominantly in patients with DM rather than PM.271,281 This finding lends further support to the idea that these diseases represent

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Figure 35. Dermatomyositis. Organizing pneumonia pattern. As in other connective tissue diseases, an organizing pneumonia pattern may occur within polymyositis/dermatomyositis. Buds or loose organizing connective tissue are present within alveolar airspaces.

distinct entities.268,272 DAD has been associated with pneumomediastinum/subcutaneous emphysema in some patients.268,282,283 Prognosis is uniformly poor despite therapy.268,272,278,281 Polymyositis/dermatomyositis patients with antihistidyl transfer ribonucleic acid (tRNA) synthetase antibodies (antiJo-1) comprise a subgroup of patients with increased risk of interstitial lung disease.266,284 However, this is not a specific finding as interstitial lung disease also occurs in patients who lack these antibodies.266,268,277 UIP is the most common pattern in this subgroup, followed by NSIP and OP. DAD also appears increased in frequency sometimes as an acute exacerbation of another pattern.285

Chapter 21: The lungs in connective tissue disease

Other rare pulmonary manifestations Other primary pulmonary manifestations are rare in the setting of PM/DM. Acute fibrinous and organizing pneumonia (AFOP), considered a histological variant of DAD,286 was reported in a child with juvenile dermatomyositis.287 Pulmonary capillaritis and diffuse alveolar hemorrhage has also been noted in isolated case reports.142,288 Active and healed vasculitis is infrequently seen at autopsy.289 Although development of secondary pulmonary hypertension due to interstitial lung disease is not uncommon, reports of primary pulmonary hypertension including plexogenic arteriopathy are very rare.290,291 Aspiration pneumonia is a frequent secondary complication, occurring in up to 17% of patients.292 The propensity to aspiration pneumonia is thought to be due to abnormalities in swallowing and regurgitation, caused by dysfunction of pharyngeal and esophageal skeletal muscles.266 Respiratory compromise due to hypoventilation is uncommon in PM/DM, occurring in less than 5% of patients.266,293,294 It is thought to be due to weakness of the inspiratory and expiratory respiratory muscles, with consequent restrictive lung function abnormalities. Chest radiographs show an elevated hemidiaphragm with reduction in lung volume and basal atelectasis. These changes predispose to infection.266 Several studies have shown an association between DM/PM and cancer.20–22,295 The risk of cancer is greatest at or in the first 2 years following diagnosis but is not increased in subsequent years. This supports the view that DM or PM may occur as a paraneoplastic syndrome. In lung cancer, DM/PM has been associated with all the major histological types of cancer, including small cell, adenocarcinoma, squamous carcinoma and others.19 In one report, it was associated with a pulmonary inflammatory pseudotumor (myofibroblastic tumor).296 Interestingly, DM/PM may regress after successful treatment of the tumor.296,297

Mixed connective tissue disease Mixed connective tissue disease refers to a disorder with overlapping features of SLE, scleroderma and PM/DM.298 Immunologically, antibodies against the U1-ribonucleoprotein complex (U1-RNP) are a characteristic feature.299 The existence of mixed connective tissue disease has been somewhat controversial although most authors now accept it as a distinct entity.300–302 Clinical features of mixed connective tissue disease include sclerodermatous skin changes, Raynaud’s phenomenon, arthritis and myositis.301 Pulmonary manifestations include interstitial lung disease, pleural effusions and pulmonary hypertension.37,300,303,304 While showing characteristic antibodies directed against the U1-ribonucleoprotein complex (U1-RNP), patients generally do not have anti-Sm or anti-native DNA antibodies, as are more typical with SLE.219,305

Table 7 Pleuropulmonary pathology of mixed connective tissue disease

Pleuritis Interstitial lung disease NSIP pattern UIP pattern Organizing pneumonia pattern Vascular Pulmonary hypertension Intimal and medial hypertrophy of pulmonary arteries with plexiform lesions Thrombotic arteriopathy Necrotizing vasculitis Pulmonary veno-occlusive disease Alveolar hemorrhage with or without capillaritis Secondary complications Aspiration pneumonia due to dysfunction of esophageal skeletal muscle Hypoventilation due to dysfunction of diaphragmatic skeletal muscle

Pulmonary manifestations are common, occurring in between 20 and 85% of patients (Table 7).306 Pathological descriptions, however, are sparse and features have not been fully characterized.

Interstitial lung disease Early reports of biopsies or autopsies of patients with interstitial lung disease reported fibrosis or UIP.67,304,307 Based on isolated reports and radiological findings, it is likely that NSIP, as in other CTD, is a more common pattern (Figure 36).37,39,177,303,308,309 An organizing pneumonia pattern has rarely been reported.310 DAD may rarely occur, sometimes as a presenting manifestation (Figure 37).34,311,312

Pulmonary hypertension Pulmonary arterial hypertension is also a common pulmonary manifestation, occurring in 10–45% of patients.304 It is an important cause of morbidity and mortality and may not respond to therapy.219 Histological features consist of intimal proliferation and medial hypertrophy of pulmonary arteries. Plexiform lesions are present in some patients.307,313,314 Necrotizing vasculitis has rarely been reported.307,315–317 A single case of pulmonary veno-occlusive disease has been reported in this entity.318

Other pulmonary manifestations Other common features of pulmonary involvement include pleural effusion (50%) and pleurisy (20%).304 As in PM,

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(a)

(b)

Figure 36. Mixed connective tissue disease. Fibrosing NSIP pattern. (a) There is a pattern of relatively uniform fibrosis, which expands alveolar septa. (b) Scattered inflammatory cells are seen.

of the 11 patients who died. Interestingly, the presence of anticardiolipin antibodies was strongly associated with pulmonary hypertension-related deaths. No thromboembolic events were noted in the patients with anticardiolipin antibodies, either clinically or at autopsy.314 Another study also noted anticardiolipin antibodies in 7/48 patients with mixed connective tissue disease but not thromboembolic events or other features of the antiphospholipid syndrome.322 As in SLE, these observations suggest a possible role for antiphospholipid antibodies in the development of pulmonary hypertension, independent of their association with thromboembolic disease.219,314

Undifferentiated connective tissue disease Figure 37. Mixed connective tissue disease. Diffuse alveolar damage. Hyaline membranes are present and focally incorporated into overlying edematous alveolar septa. There is mild thickening of alveolar septa by a loose fibromyxoid connective tissue.

aspiration pneumonia and hypoventilation may occur from dysfunction of the esophageal and diaphragmatic skeletal muscles, respectively, and lead to infection. Alveolar hemorrhage is a rare pulmonary manifestation of mixed connective tissue disease.143,319,320 In some cases there is an associated necrotizing capillaritis.104 One case of alveolar hemorrhage was reported in association with the development of serum MPO-ANCA.321 Prognosis of patients with mixed connective tissue disease is mixed. In a series of 47 patients with disease ranging from 3 to 29 years, 62% had a favorable outcome, whereas 38% of patients either had persistent active disease or had died. Pulmonary hypertension was a major contributing factor in nine

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Undifferentiated connective tissue disease (UCTD) describes a group of patients with some clinical and serological features of a CTD, which are nonetheless insufficient for a diagnosis of a specific type.37,323 In one cohort at a tertiary referral center, these patients represented about 18% of newly referred patients with CTD. A proportion of these patients (average of 30%) evolve into specific defined connective tissue disease (e.g. SLE, rheumatoid arthritis, others) usually during the first year after presentation. A significant proportion of patients, however, will remain undifferentiated when followed over a long period of time. The most common clinical features of UCTD are mild and include polyarthralgia, arthritis, Raynaud’s phenomenon, mucocutaneous manifestations (malar rash, oral apthous ulcers), sicca symptoms, autoimmune thyroid disease and leukopenia. In contrast to other defined CTD, renal or central nervous system manifestations are usually absent.323 Antinuclear antibodies are the most common serological abnormality, occurring in between 58% and 100% of patients. The most frequent specificities include anti-Ro/SSA and anti-RNP followed by anti ds-DNA.323

Chapter 21: The lungs in connective tissue disease Table 8 Pulmonary pathology of undifferentiated connective tissue disease

Table 9 Pulmonary pathology of ankylosing spondylitis

NSIP pattern

Apical fibrobullous disease

UIP pattern (rare)

Superimposed infection (aspergillomas, nontuberculous mycobacteria)

Organizing pneumonia pattern (rare)

Chronic pleuritis

Follicular bronchiolitis pattern (rare)

(a)

(b)

Figure 38. Undifferentiated connective tissue disease. Idiopathic fibrosing NSIP pattern. (a) This is an example of a case previously interpreted as idiopathic fibrosing NSIP. Recent studies have suggested that cases such as these may represent pulmonary manifestations of undifferentiated connective tissue disease. (b) Although the alveolar walls are of variable width, normal lung is not appreciated.

Proposed diagnostic criteria for UCTD are signs and symptoms of a CTD which nonetheless do not fulfill criteria for a defined CTD. Patients should have positive antinuclear antibodies and a disease duration of at least 3 years. This final criterion intentionally excludes those patients who evolve into defined CTD. Of note, these criteria consider patients with less than 3 years follow-up as early UCTD.324

Interstitial lung disease A recent study of idiopathic interstitial pneumonias suggests that a significant proportion of patients with idiopathic NSIP fulfill clinical criteria for UCTD (Table 8, Figure 38). In a study of idiopathic interstitial lung disease with underlying UCTD, one group found that NSIP was the predominant pattern, occurring in 83% of the patients biopsied (15/18). In contrast, UIP was present in 19 (86%) of the 22 biopsies available from a control group of 47 patients with idiopathic interstitial lung disease without UCTD. NSIP was present in only two (9%) of the control group biopsies. The study did not distinguish between cellular and fibrotic NSIP patterns.325 In addition to pathological features of NSIP, the clinical and radiographic features of ILD in patients with UCTD were also similar to those described in idiopathic NSIP. Most patients with UCTD were female (63% vs. 23% in the control group), with a younger age of onset (50 vs. 65 years) and lower

incidence of smoking history (43% vs. 75%). HRCT studies frequently showed ground-glass opacities extending beyond areas of reticulation in the UCTD group. Honeycombing and traction bronchiectasis were more frequent in the control group.325 While NSIP appears characteristic of patients with interstitial lung disease with UCTD, other patterns have also been noted with a much lower frequency. UIP, OP and non-classifiable fibrosis were noted in biopsy samples.325 Follicular bronchiolitis has also been described in a UCTD patient (Table 8).92

Ankylosing spondylitis Ankylosing spondylitis is a chronic inflammatory CTD that affects predominantly the spine and sacroiliac joints. On occasion, it may affect other peripheral joints, as well as extraarticular organs, such as the eyes, lungs and cardiovascular system. It is more common in men by a ratio of 3:1 and is strongly associated with the HLA-B27 allele. Pulmonary manifestations are usually asymptomatic. Complications (e.g. aspergillomas) may lead to symptoms in occasional patients. Initial pathological studies suggested that the most common pulmonary manifestation was apical fibrobullous disease, occurring in about 1% of patients (Table 9). This disease evolves over many years and is often

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Chapter 21: The lungs in connective tissue disease

asymptomatic.326,327 Occasionally patients develop superimposed aspergillomas or non-tuberculous mycobacterial infections, such as Mycobacterium avium intracellulare, Mycobacterium kansasii and Mycobacterium scrofulaceum, which may ultimately result in upper lobe cavitation.327,328 Chronic necrotizing aspergillosis was reported in one patient.329 Patchy chronic pleuritis was another less specific finding.327 More recent imaging studies with HRCT confirm an overall low incidence of apical fibrobullous disease but show a much higher frequency (53–88%) of other asymptomatic, although nonspecific, parenchymal abnormalities.330–333 These abnormalities include parenchymal bands, linear opacities, emphysema, bronchiectasis and thickening of the bronchial wall and interlobular septa.330,332 Parenchymal bands and linear opacities are considered nonspecific findings within the radiographic spectrum of interstitial lung disease.332 While these changes are frequent on HRCT, conventional chest X-rays are most often normal.332,334 HRCT changes are statistically associated with dorsal spine involvement and restrictive defects at pulmonary function testing. Due to extreme spinal rigidity, prone-position HRCT scans are unattainable in many ankylosing spondylitis patients. Thus, the parenchymal abnormalities could be associated with decubitus atelectasia (gravitationally dependent opacities).332 Pulmonary manifestations in most patients with ankylosing spondylitis are asymptomatic. Nevertheless, approximately half the patients will have a subclinical restrictive pattern on pulmonary function tests. The restrictive pattern is not associated with the parenchymal changes on HRCT,335,336 but rather correlates with impairment of chest wall expansion, caused by increased stiffness of the spine, and ankylosis of costovertebral joints.332,337

Relapsing polychondritis Relapsing polychondritis is a rare multisystem disease characterized by inflammation and progressive destruction of cartilaginous tissue in 1923 by Jaksch-Wartenhorst.338 It typically affects cartilage-rich tissues, including the ears, nose, joints and respiratory tract.339–341 The most common presentation is auricular chondritis, which causes red ears simulating an auricular cellulitis.341 Males and females are affected equally. It may occur at any age, although peak prevalence is between 40 and 60 years old.342 A significant proportion of cases (25 to 30%) are associated with other inflammatory conditions, including rheumatoid arthritis, Sjögren syndrome, Behçet disease, SLE, ankylosing spondylitis, polyarteritis nodosa, Reiter syndrome and Wegener granulomatosis.339 Pulmonary involvement in relapsing polychondritis commonly affects the trachea and bronchi. Approximately 25% of patients present with respiratory involvement, while 50% develop it at some point in the disease.339,343 Involvement may be limited to focal areas or diffusely involve the tracheobronchial tree. Symptoms of affected patients include dyspnea, wheezing, cough, choking, stridor and aphonia.339,344,345

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Figure 39. Relapsing polychondritis. Narrowing and irregularity of the trachea (black arrows) and left mainstem bronchus (white arrows) are readily demonstrated on this 3D external rendering viewed from an anterior perspective. (Reproduced with permission, from Lee KS, et al. Relapsing polychondritis. Prevalence of expiratory CT airway abnormalities. Radiology 2006;240:565–73.)

Figure 40. Relapsing polychondritis. Dynamic collapse of the trachea of a 64year-old male is demonstrated on CT scan at the end of inspiration and end of expiration. There is calcification and thickening of the cartilaginous portions of the trachea (arrow) with sparing of the posterior wall (arrowhead). (Reproduced with permission, from Prince JS, et al. Nonneoplastic lesions of the tracheobronchial wall: radiologic findings with bronchoscopic correlation. Radiographics 2002;22:S215–S230.)

Tracheobronchial stenosis is a severe complication and cause of respiratory distress. Tracheal narrowing and irregularity may be identified on conventional chest X-ray or CT scan (Figure 39).346 Other more nonspecific findings include pneumonia, atelectasis, widening of the aortic arch and prominence of the aorta.339 On CT scan, destruction of cartilage rings with thickening of the tracheal wall is characteristic. In contrast to other diseases, there is sparing of the posterior membrane of the trachea. Airway collapse may sometimes be demonstrated on dynamic imaging during expiration (Figure 40).342 Endotracheal or endobronchial biopsies may help confirm the disease. Histological features include a vigorous mixed inflammatory infiltrate within affected cartilage, consisting of lymphocytes, macrophages, plasma cells and eosinophils (Table 10). Lacunar chondrocytes are damaged with shrunken,

Chapter 21: The lungs in connective tissue disease

pynknotic nuclei. There is loss of basophilic staining of the cartilage matrix with indistinct borders with adjacent connective tissue. Over time there is replacement of cartilage by variable irregular fibrosis and calcification.339,345,347–349 Prognosis of patients with relapsing polychondritis is variable and treatment is individualized according to the extent and severity of the disease.339 The mortality rate was 20% in one series of 62 patients with serious airway complications.345 Medical treatment consists of a variable combination of nonsteroidal anti-inflammatory drugs, dapsone and corticosteroids. Surgical treatment is necessary in some patients to maintain airway patency. Surgical approaches have included tracheostomy, airway stents, tracheoplasty and laryngotracheal reconstruction with variable results.339

Behçet syndrome Behçet syndrome is a multisystem disease of unknown etiology first described in 1937 in patients with recurrent genital and oral ulcers and uveitis (see Chapter 19).350 It has since become apparent that patients may have systemic involvement of other organs including, skin, joints, large vessels, lung, brain and gastrointestinal tract.351–356 Criteria for diagnosis established by the 1990 International Study Group for Behçet disease require the presence of recurrent oral ulceration accompanied by two other findings: recurrent genital ulcerations, eye lesions, skin lesions or a positive pathergy test.357 The disease is most prevalent in Turkey and adjacent areas, although it may occur elsewhere. Males and females appear to be affected

Table 11 Pulmonary pathology of Behçet syndrome

Transmural vasculitis (lymphocytic, neutrophilic) involving veins and arteries Thrombosis in arterial aneurysms and vessels Infarcts Pulmonary artery to bronchial fistulas

Table 10 Pulmonary pathology of relapsing polychondritis

Pulmonary artery aneurysms

Mixed inflammatory infiltrate within tracheal and bronchial cartilage

(a)

equally although the disease is often more severe in males. The disease usually affects young to middle-aged adults in their third and fourth decades of life. There is a strong association with carriers of the HLA-B51 allele.356 Pulmonary manifestations of Behçet syndrome are uncommon but are estimated at anywhere between 1% and 18% of affected patients.357,358 The commonest pulmonary manifestations are pulmonary artery aneurysms. Aneurysms are often multiple. The diameter of aneurysms ranges from 1 to 7 cm. The most common location is in the right lower lobar pulmonary artery followed by the left and right main pulmonary arteries. They are best detected radiographically by spiral CT (Figure 41).359 They may be associated clinically with hemoptysis. In situ thrombosis in both aneurysms and in normal vessels may occur and in some cases lead to infarcts.356,358,359 Pulmonary artery-bronchial fistulas may occur.360,361 Pathological descriptions of Behçet syndrome in the lung are rare (Table 11). As in other organ systems, vasculitis is a cardinal histological feature.356,361 In one case report, a transmural vasculitis involving large, medium-sized and small arteries and veins was seen (Figure 42). The vasculitis was predominantly lymphocytic although occasionally mixed with

Septal capillaritis (rare)

(b)

Figure 41. Behçet syndrome. (a) Contrast-enhanced chest CT scan (mediastinal window) shows vasculitis and thrombosis in multiple peripheral pulmonary arteries. Note the large aneurysm on the left side and the smaller ones on the right side (arrows). (Reprinted with permission from Hiller N, et al. Thoracic manifestations of Behcet’s disease at CT. Radiographics 2004;24:801–8). (b) Contrast-enhanced chest CT scan (lung window) again shows the aneurysms (thin arrows). Note also the small, peripheral round lesion in the lingula (thick arrow), a finding that is probably due to vasculitis or infarction. (Reprinted with permission from Hiller N, et al. Thoracic manifestations of Behcet’s disease at CT. Radiographics 2004;24:801–8).

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Chapter 21: The lungs in connective tissue disease Table 12 Pulmonary pathology of pyoderma gangrenosum

Acute inflammation with sterile abscesses Absence of vasculitis or granulomatous inflammation

Figure 42. Behçet syndrome. A lymphocytic vasculitis is present within this large pulmonary artery.

other cells, including plasma cells, eosinophils, macrophages and neutrophils. Vascular inflammation was generally transmural, although with a subintimal tendency in larger vessels. Areas of necrotizing arteritis with neutrophilic inflammation were also present, but less common. Older lesions of healed vasculitis consisting of segmental replacement of the media and elastica by granulation tissue could be demonstrated on elastic tissue stain. In addition to the vasculitis, there were recent and organizing thrombi within normal and aneurysmally dilated pulmonary arteries and within areas of phlebitis. Focal pulmonary infarcts were present.360 In addition to the vasculitis, a septal capillaritis has also been reported.362,363 Immune complexes have been identified in one report both within small vessels and within the sera.362 Recurrent exacerbations and remissions are characteristic of Behçet syndrome. The development of pulmonary artery aneurysm is associated with a poor prognosis and is a principal cause of death in this disease.356 Recent studies, however, are encouraging as pulmonary artery aneurysms have regressed or resolved completely following long-term immunosuppressive treatment.364 Hughes-Stovin syndrome is an extremely rare disorder thought by some authors to be an incomplete form of Behçet syndrome.365–368 First described in 1959, it consists of multiple pulmonary artery aneurysms associated with systemic thrombosis.369 The pulmonary manifestations may be identical to those of Behçet syndrome although patients do not have recurrent oral or genital ulcers.365,367

Pyoderma gangrenosum Pyoderma gangrenosum is an uncommon condition of unknown etiology, which typically presents as rapidly developing cutaneous ulcers often associated with fever. It is idiopathic in 50% of cases but may also be associated with a variety of systemic conditions. It occurs in 30–60% of patients with

832

Figure 43. Pyoderma gangrenosum. Middle-aged adult with pyoderma gangrenosum. Bilateral nodular densities are present on this CT scan.

inflammatory bowel disease but also occurs in association with CTD, Behçet syndrome, hematological conditions, underlying visceral carcinomas and HIV infection.370 Lesions typically develop on the lower extremities but may also involve the thorax and upper extremities as well. Skin biopsies typically show marked neutrophilic infiltration, which undermines the adjacent skin. The findings are similar to those of an abscess, although infectious agents are not identified either on culture or on special stains. Pulmonary involvement by pyoderma gangrenosum is rare. Isolated case reports usually note the development of multiple pulmonary nodules on chest CT (Figure 43), some of which show central cavitation and necrosis.371–373 Other findings include bronchiolar micronodules, cystic lesions or patchy infiltrates with pleural effusion.372,374 Occasional reports describe a cavitating pneumonia or cavitating lung lesions, which can mimic carcinoma.375,376 Lung biopsies have shown necrosis with acute inflammation and parenchymal abscess (Table 12).371–373, 377 Tracheobronchial disease may also occur, with reports of scattered white and yellow nodules along the trachea and bronchi on fiberoptic bronchoscopy. Biopsies of these nodules showed acute inflammation with neutrophils and histiocytes (Figure 44).374,378 Special stains and cultures for microorganisms are negative. The principal differential diagnosis in these cases is infection, which can look histologically identical and must be excluded in all cases. Patients usually also have characteristic skin lesions which are an important clue to the diagnosis.

Chapter 21: The lungs in connective tissue disease Table 13 Pulmonary pathology of Sweet syndrome

Patchy interstitial and intra-alveolar neutrophilic exudates

Figure 44. Pyoderma gangrenosum. Same patient as in Figure 43. Transbronchial biopsy shows neutrophilic inflammation adjacent to fragments of alveolated pulmonary parenchyma. These features are not diagnostically specific as they could be seen in an infectious abscess. The nodular densities present on CT scan in this patient resolved with steroid therapy.

Wegener granulomatosis is also a consideration.374,376,379,380 The typical suppurative, granulomatous appearance of Wegener’s is absent in reported cases of pulmonary involvement by pyoderma gangrenosum. Biopsies from either skin or lung lack vasculitis. Serological assessment for ANCA as well as assessment for other features and the location of Wegener granulomatosis (e.g. head and neck lesions, renal involvement) is also helpful in excluding this possibility.374 Both cutaneous and pulmonary manifestations of pyoderma gangrenosum usually respond dramatically to steroids.371–374,377

Sweet syndrome Sweet syndrome (acute febrile neutrophilic dermatosis) is a rare cutaneous disorder characterized by the recurrent eruption of erythematosus plaques and nodules classically on the face and extremities accompanied by fever and leukocytosis.381,382 While the disease is usually limited to the skin, it may rarely involve non-cutaneous sites such as the oral mucosa, joints, lungs, liver and kidneys.383 It may be idiopathic or associated with a variety of other conditions, including gastrointestinal or upper respiratory tract infection, malignancy and various types of CTD (e.g. rheumatoid arthritis, Sjögren syndrome, SLE, and pustular psoriasis among others). Approximately 20% of cases are associated with an underlying malignancy, often hematological (e.g. myeloproliferative or myelodysplastic disorder). Skin biopsies show dense dermal neutrophilic inflammation, accompanied by variable amounts of leukocytoclasis.381,382,384 The etiology of Sweet syndrome remains undiscovered although it is responsive to treatment with corticosteroids.383 Pulmonary involvement is rare, with only isolated cases reported.385–391 Patients usually develop bilateral infiltrates

on chest X-ray388 associated with a concurrent or preceding cutaneous eruption.391 Reported symptoms include cough,387 dyspnea,386,388 and chest pain.392 Some cases occurred in association with an underlying myelodysplastic or myeloproliferative syndrome or acute myelogenous leukemia.383,385,388,393 Features on open and transbronchial lung biopsies (Table 13) consist of patchy interstitial and alveolar, predominantly neutrophilic exudates. Small numbers of admixed eosinophils and lymphocytes may also be present, as well as foci of hemorrhage and hemosiderin-laden macrophages.386,388,394 Patchy mucosal and submucosal infiltrates within bronchi and bronchioles have also been observed. These infiltrates consist of mostly neutrophils admixed with smaller numbers of lymphocytes, plasma cells and eosinophils.386,392 In one patient, these corresponded to pustules identified on fiberoptic bronchoscopy in the left and right mainstem bronchi.392 Rare associated patterns of pulmonary involvement include OP395,396 and pleural effusions.387,392,394 As pulmonary involvement by Sweet syndrome is very rare, it is important to exclude infection before accepting the diagnosis. Special stains and correlation with culture results and clinical features should be performed on all cases.391 The biopsies should be carefully assessed for any signs of viral cytopathic change. Similar to the cutaneous lesions, patients generally respond to corticosteroid treatment, but may relapse.386–389 Occasional patients have developed acute respiratory distress syndrome with DAD and died.388,392

Pulmonary involvement by inflammatory bowel disease The pulmonary manifestations of ulcerative colitis (UC) and Crohn disease (CD) are varied, with some overlap, but nonetheless appear distinctive.397,398 Respiratory changes are rare although the incidence appears somewhat greater in UC than in CD. While usually occurring at the time of or after the initial diagnosis of inflammatory bowel disease, the changes may occasionally precede the diagnosis. Clinicopathological patterns of respiratory involvement can broadly be grouped into upper airway disease, small airways disease and parenchymal disease (Table 14).397,398 Upper airway manifestations which affect both UC and CD reflect sequellae of ongoing mucosal inflammation and include glottic/subglottic stenosis, tracheal inflammation and stenosis, and bronchiectasis. Scarred bronchial granulomas may be present in CD, but have not been identified in UC.397,398 Small airways may be affected in both UC and CD. A fairly common pattern present in patients with CD but not observed in UC consists of a chronic bronchitis/bronchiolitis with nonnecrotizing granulomatous inflammation. Biopsies show mild

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to moderately dense chronic inflammation of bronchioles and the peribronchiolar interstitium associated with intramural and peribronchiolar non-necrotizing granulomas.397,399 An acute necrotizing bronchiolitis may be seen in both UC and Table 14 Clinicopathological patterns of pulmonary involvement in inflammatory bowel disease

Ulcerative colitis Crohn’s Upper airway Glottic or subglottic stenosis

∎

∎

Tracheal inflammation/stenosis

∎

∎

Bronchiectasis

∎

∎

Scarred bronchial granulomas

∎

⌧

Granulomatous bronchiolitis

⌧

∎

Necrotizing bronchiolitis

⎕

⎕

Organizing pneumonia

∎

∎

Obliterative bronchiolitis

⎕

⎕

Diffuse panbronchiolitis

⎕

⌧

Cellular NSIP

⎕

⎕

Eosinophilic pneumonia

⎕

⎕

Sterile necrobiotic nodules

⎕

⎕

Small airway

Parenchymal

∎, fairly common; ⎕, occurs but is less common or rare; ⌧, not reported. This table has been modified and reproduced with the permission of the European Respiratory Society. The table has not been reviewed by European Respiratory Society prior to release, therefore the European Respiratory Society may not be responsible for any errors, omissions or inaccuracies, or for any consequences arising there from, in the content. Modified from reference 397, table 1, with permission.

(a)

CD and may be associated with an aseptic bronchopneumonia.397–399 An organizing pneumonia pattern has also been observed in both diseases but in CD may be associated with rare granulomas or isolated giant cells.397–399 Rare cases of a diffuse panbronchiolitis pattern have been reported in UC but not in CD.397 Obliterative bronchiolitis or permanent scarring of the airways has been reported in both disorders.397,400 Several patterns of parenchymal involvement occur in both UC and CD. These include a pattern of cellular nonspecific interstitial pneumonia, eosinophilic pneumonia (pulmonary infiltrates with eosinophilia) and sterile necrobiotic nodules. In CD, the pattern of cellular NSIP has been reported in association with rare giant cells.399 Both UC and CD may show necrobiotic nodules.397,401 Early nodules consist of acute fibrinopurulent inflammation while later nodules are larger with central necrosis and cavitation resembling necrotic granulomas (Figure 45a). In some cases, these nodules are present in association with cutaneous lesions of pyoderma gangrenosum and may pre-date the diagnosis of inflammatory bowel disease.401,402 One author observed a patient with CD who had pulmonary necrotizing granulomas with an associated granulomatous vasculitis and focal adjacent nonnecrotizing granulomas (Figure 45). In such cases infection should be rigorously excluded by culture and molecular biological techniques, prior to attributing the cause to inflammatory bowel disease. The differential diagnosis of pulmonary involvement by UC and CD includes infection, sarcoidosis, hypersensitivity pneumonitis and Wegener granulomatosis. These can usually be separated by careful consideration of the clinical context, histological features, culture and special stains.397,399 Wegener granulomatosis may be particularly difficult to distinguish when patients have cavitary lesions or necrobiotic nodules. This task is made more complicated as many patients with (b)

Figure 45. Pulmonary involvement with Crohn disease. (a) Wedge biopsy with necrotizing granuloma. The outline of a necrotic vessel is present within the central area of necrosis. Infection needs to be rigorously excluded in cases such as this before considering this possibility. (b) Granulomatous vasculitis is also noted. Clinical history is required to avoid a mistaken diagnosis of Wegener granulomatosis.

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inflammatory bowel disease have ANCA.403 However, ANCAs in patients with Wegener granulomatosis are usually c-ANCA directed against proteinase 3, while ANCAs associated with inflammatory bowel disease are usually p-ANCA and directed against a variety of antigens.397

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other rheumatic diseases. N Engl J Med 1976;295(21):1149–54. 300. Maddison PJ. Mixed connective tissue disease: overlap syndromes. Baillieres Best Pract Res Clin Rheumatol 2000;14(1):111–24. 301. Venables PJ. Mixed connective tissue disease. Lupus 2006;15(3):132–7. 302. Aringer M, Smolen JS. Mixed connective tissue disease: what is behind the curtain? Best Pract Res Clin Rheumatol 2007;21(6):1037–49. 303. Bodolay E, Szekanecz Z, Devenyi K, et al. Evaluation of interstitial lung disease in mixed connective tissue disease (MCTD). Rheumatology (Oxford) 2005;44(5):656–61. 304. Prakash UB. Respiratory complications in mixed connective tissue disease. Clin Chest Med 1998;19(4):733–46, ix. 305. Evans J. Antinuclear antibody testing in systemic autoimmune disease. Clin Chest Med 1998;19(4):613–25, vii. 306. Sullivan WD, Hurst DJ, Harmon CE, et al. A prospective evaluation emphasizing pulmonary involvement in patients with mixed connective tissue disease. Medicine (Baltimore) 1984;63(2):92–107. 307. Wiener-Kronish JP, Solinger AM, Warnock ML, et al. Severe pulmonary involvement in mixed connective tissue disease. Am Rev Respir Dis 1981;124(4):499–503. 308. Saito Y, Terada M, Takada T, et al. Pulmonary involvement in mixed connective tissue disease: comparison with other collagen vascular diseases using high resolution CT. J Comput Assist Tomogr 2002;26(3):349–57. 309. Kozuka T, Johkoh T, Honda O, et al. Pulmonary involvement in mixed connective tissue disease: highresolution CT findings in 41 patients. J Thorac Imaging 2001;16(2):94–8. 310. Katzenstein AL, Myers JL, Prophet WD, Corley LS, Shin MS. Bronchiolitis obliterans and usual interstitial pneumonia. A comparative clinicopathologic study. Am J Surg Pathol 1986;10:373–81. 311. Parambil JG, Myers JL, Aubry MC, Ryu JH. Causes and prognosis of diffuse alveolar damage diagnosed on surgical lung biopsy. Chest 2007;132(1):50–7. 312. Scully RE, Mark EJ, McNeely WF, Ebeling SH, Phillips LD. Case records of the Massachusetts General Hospital.

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Weekly clinicopathological exercises. Case 20–1997. A 74-year-old man with progressive cough, dyspnea, and pleural thickening. N Engl J Med 1997;336(26):1895–903. 313. Hosoda Y, Suzuki Y, Takano M, Tojo T, Homma M. Mixed connective tissue disease with pulmonary hypertension: a clinical and pathological study. J Rheumatol 1987;14(4):826–30. 314. Burdt MA, Hoffman RW, Deutscher SL, et al. Long-term outcome in mixed connective tissue disease: longitudinal clinical and serologic findings. Arthritis Rheum 1999;42(5):899–909. 315. Ueda N, Mimura K, Maeda H, et al. Mixed connective tissue disease with fatal pulmonary hypertension and a review of literature. Virchows Arch A Pathol Anat Histopathol 1984;404(4):335–40. 316. Eulderink F, Cats A. Fatal primary pulmonary hypertension in mixed connective tissue disease. Z Rheumatol 1981 Jan-Feb;40(1):25–9. 317. Kobayashi H, Sano T, Ii K, et al. Mixed connective tissue disease with fatal pulmonary hypertension. Acta Pathol Jpn 1982;32(6):1121–9. 318. Zhang L, Visscher D, Rihal C, Aubry MC. Pulmonary veno-occlusive disease as a primary cause of pulmonary hypertension in a patient with mixed connective tissue disease. Rheumatol Int 2007;27(12):1163–5. 319. Sanchez-Guerrero J, Cesarman G, Alarcon-Segovia D. Massive pulmonary hemorrhage in mixed connective tissue diseases. J Rheumatol 1989;16(8):1132–4. 320. Germain MJ, Davidman M. Pulmonary hemorrhage and acute renal failure in a patient with mixed connective tissue disease. Am J Kidney Dis 1984;3(6):420–4. 321. Kitaura K, Miyagawa T, Asano K, et al. Mixed connective tissue disease associated with MPO-ANCA-positive polyangiitis. Intern Med 2006;45(20):1177–82. 322. Komatireddy GR, Wang GS, Sharp GC, Hoffman RW. Antiphospholipid antibodies among anti-U1–70 kDa autoantibody positive patients with mixed connective tissue disease. J Rheumatol 1997;24(2):319–22. 323. Mosca M, Tani C, Bombardieri S. Undifferentiated connective tissue

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diseases (UCTD): a new frontier for rheumatology. Best Pract Res Clin Rheumatol 2007;21(6):1011–23. 324. Mosca M, Neri R, Bombardieri S. Undifferentiated connective tissue diseases (UCTD): a review of the literature and a proposal for preliminary classification criteria. Clin Exp Rheumatol 1999 Sep-Oct;17(5):615–20. 325. Kinder BW, Collard HR, Koth L, et al. Idiopathic nonspecific interstitial pneumonia: lung manifestation of undifferentiated connective tissue disease? Am J Respir Crit Care Med 2007;176(7):691–7. 326. Campbell AH, Macdonald CB. Upper lobe fibrosis associated with ankylosing spondylitis. Br J Dis Chest 1965;59:90–101. 327. Rosenow E, Strimlan CV, Muhm JR, Ferguson RH. Pleuropulmonary manifestations of ankylosing spondylitis. Mayo Clin Proc 1977;52(10):641–9. 328. Levy H, Hurwitz MD, Strimling M, Zwi S. Ankylosing spondylitis lung disease and Mycobacterium scrofulaceum. Br J Dis Chest 1988;82(1):84–7. 329. Pamuk ON, Harmandar O, Tosun B, Yoruk Y, Cakir N. A patient with ankylosing spondylitis who presented with chronic necrotising aspergillosis: report on one case and review of the literature. Clin Rheumatol 2005;24(4):415–9. 330. El Maghraoui A, Chaouir S, Abid A, et al. Lung findings on thoracic highresolution computed tomography in patients with ankylosing spondylitis. Correlations with disease duration, clinical findings and pulmonary function testing. Clin Rheumatol 2004;23(2):123–8. 331. Souza AS Jr, Muller NL, Marchiori E, Soares-Souza LV, de Souza Rocha M. Pulmonary abnormalities in ankylosing spondylitis: inspiratory and expiratory high-resolution CT findings in 17 patients. J Thorac Imaging 2004;19(4):259–63. 332. Sampaio-Barros PD, Cerqueira EM, Rezende SM, et al. Pulmonary involvement in ankylosing spondylitis. Clin Rheumatol 2007;26(2):225–30. 333. Senocak O, Manisali M, Ozaksoy D, Sevinc C, Akalin E. Lung parenchyma changes in ankylosing spondylitis: demonstration with high resolution CT

and correlation with disease duration. Eur J Radiol 2003;45(2):117–22. 334. Kiris A, Ozgocmen S, Kocakoc E, Ardicoglu O, Ogur E. Lung findings on high resolution CT in early ankylosing spondylitis. Eur J Radiol 2003;47(1):71–6. 335. Fenlon HM, Casserly I, Sant SM, Breatnach E. Plain radiographs and thoracic high-resolution CT in patients with ankylosing spondylitis. AJR Am J Roentgenol 1997;168(4):1067–72. 336. Turetschek K, Ebner W, Fleischmann D, et al. Early pulmonary involvement in ankylosing spondylitis: assessment with thin-section CT. Clin Radiol 2000;55(8):632–6. 337. van Noord JA, Cauberghs M, Van de Woestijne KP, Demedts M. Total respiratory resistance and reactance in ankylosing spondylitis and kyphoscoliosis. Eur Respir J 1991;4(8):945–51. 338. Jaksch-Wartehnorst R. Polychondropathia. Wein Archives of Internal Medicine 1923;6:93–100. 339. Lee-Chiong TL Jr. Pulmonary manifestations of ankylosing spondylitis and relapsing polychondritis. Clin Chest Med 1998;19(4):747–57, ix. 340. Gergely P Jr, Poor G. Relapsing polychondritis. Best Pract Res Clin Rheumatol 2004;18(5):723–38. 341. Rapini RP, Warner NB. Relapsing polychondritis. Clin Dermatol 2006 Nov-Dec;24(6):482–5. 342. Prince JS, Duhamel DR, Levin DL, Harrell JH, Friedman PJ. Nonneoplastic lesions of the tracheobronchial wall: radiologic findings with bronchoscopic correlation. Radiographics 2002;22 Spec No:S215–30. 343. Staats BA, Utz JP, Michet CJ Jr. Relapsing polychondritis. Semin Respir Crit Care Med 2002;23(2):145–54. 344. Michet CJ Jr, McKenna CH, Luthra HS, O’Fallon WM. Relapsing polychondritis. Survival and predictive role of early disease manifestations. Ann Intern Med 1986;104(1):74–8. 345. Eng J, Sabanathan S. Airway complications in relapsing polychondritis. Ann Thorac Surg 1991;51(4):686–92. 346. Lee KS, Ernst A, Trentham DE, et al. Relapsing polychondritis: prevalence of

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expiratory CT airway abnormalities. Radiology 2006;240(2):565–73.

manifestations of Behçet disease at CT. Radiographics 2004;24(3):801–8.

347. Boulware DW, Weissman DN, Doll NJ. Pulmonary manifestations of the rheumatic diseases. Clin Rev Allergy 1985;3(2):249–67.

360. Slavin RE, de Groot WJ. Pathology of the lung in Behçet’s disease. Case report and review of the literature. Am J Surg Pathol 1981;5(8):779–88.

348. Burlew BP, Lippton H, Klinestiver D, Haponik EJ. Relapsing polychondritis: new pulmonary manifestations. J La State Med Soc 1992;144(2):58–62.

361. Uzun O, Erkan L, Akpolat I, et al. Pulmonary involvement in Behçet’s disease. Respiration 2008;75(3):310–21.

349. Tanoue LT. Pulmonary involvement in collagen vascular disease: a review of the pulmonary manifestations of the Marfan syndrome, ankylosing spondylitis, Sjögren’s syndrome, and relapsing polychondritis. J Thorac Imaging 1992;7(2):62–77. 350. Behçet H. Über rezidivierende Aphtöse, durch ein Virus verursachte Geschwüre am Mund, am Auge und an den Genitalien. Dermatologische Woochenschr 1937;105:1152–7.

362. Gamble CN, Wiesner KB, Shapiro RF, Boyer WJ. The immune complex pathogenesis of glomerulonephritis and pulmonary vasculitis in Behçet’s disease. Am J Med 1979;66(6):1031–9. 363. Green RJ, Ruoss SJ, Kraft SA, et al. Pulmonary capillaritis and alveolar hemorrhage. Update on diagnosis and management. Chest 1996;110(5): 1305–16. 364. Tunaci M, Ozkorkmaz B, Tunaci A, et al. CT findings of pulmonary artery aneurysms during treatment for Behçet’s disease. AJR Am J Roentgenol 1999;172(3):729–33.

351. Shimizu T, Ehrlich GE, Inaba G, Hayashi K. Behçet disease (Behçet syndrome). Semin Arthritis Rheum 1979;8(4):223–60.

365. Erkan D, Yazici Y, Sanders A, Trost D, Yazici H. Is Hughes-Stovin syndrome Behçet’s disease? Clin Exp Rheumatol 2004 Jul-Aug;22(4 Suppl 34):S64–8.

352. O’Duffy JD, Carney JA, Deodhar S. Behçet’s disease. Report of 10 cases, 3 with new manifestations. Ann Intern Med 1971;75(4):561–70.

366. Emad Y, Ragab Y, Shawki Ael H, et al. Hughes-Stovin syndrome: is it incomplete Behçet’s? Report of two cases and review of the literature. Clin Rheumatol 2007;26(11):1993–6.

353. Chajek T, Fainaru M. Behçet’s disease. Report of 41 cases and a review of the literature. Medicine (Baltimore) 1975;54(3):179–96. 354. Lakhanpal S, Tani K, Lie JT, et al. Pathologic features of Behçet’s syndrome: a review of Japanese autopsy registry data. Hum Pathol 1985;16(8):790–5.

367. Durieux P, Bletry O, Huchon G, et al. Multiple pulmonary arterial aneurysms in Behçet’s disease and Hughes-Stovin syndrome. Am J Med 1981;71(4):736–41. 368. Francois MF. Is Hughes-Stovin syndrome a particular expression of Behçet’s disease? Chest 1983;83(2):288.

355. Matsumoto T, Uekusa T, Fukuda Y. Vasculo-Behçet’s disease: a pathologic study of eight cases. Hum Pathol 1991;22(1):45–51.

369. Hughes JP, Stovin PG. Segmental pulmonary artery aneurysms with peripheral venous thrombosis. Br J Dis Chest 1959;53(1):19–27.

356. Erkan F, Gul A, Tasali E. Pulmonary manifestations of Behçet’s disease. Thorax 2001;56(7):572–8.

370. Powell FC, Su WP, Perry HO. Pyoderma gangrenosum: classification and management. J Am Acad Dermatol 1996;34(3):395–409; quiz 10–2.

357. Criteria for diagnosis of Behçet’s disease. International Study Group for Behçet’s Disease. Lancet 1990;335(8697):1078–80. 358. Uzun O, Akpolat T, Erkan L. Pulmonary vasculitis in Behçet disease: a cumulative analysis. Chest 2005;127(6):2243–53. 359. Hiller N, Lieberman S, Chajek-Shaul T, Bar-Ziv J, Shaham D. Thoracic

gangrenosum. Presse Med 2007;36(10 Pt 1):1395–8. 373. Kasuga I, Yanagisawa N, Takeo C, et al. Multiple pulmonary nodules in association with pyoderma gangrenosum. Respir Med 1997;91(8):493–5. 374. Wang JL, Wang JB, Zhu YJ. Pyoderma gangrenosum with lung injury. Thorax 1999;54(10):953–5. 375. Brown TS, Marshall GS, Callen JP. Cavitating pulmonary infiltrate in an adolescent with pyoderma gangrenosum: a rarely recognized extracutaneous manifestation of a neutrophilic dermatosis. J Am Acad Dermatol 2000;43(1 Pt 1):108–12. 376. Field S, Powell FC, Young V, Barnes L. Pyoderma gangrenosum manifesting as a cavitating lung lesion. Clin Exp Dermatol 2008;33(4):418–21. 377. Vignon-Pennamen MD, ZelinskyGurung A, Janssen F, Frija J, Wallach D. Pyoderma gangrenosum with pulmonary involvement. Arch Dermatol 1989;125(9):1239–42. 378. Kanoh S, Kobayashi H, Sato K, Motoyoshi K, Aida S. Tracheobronchial pulmonary disease associated with pyoderma gangrenosum. Mayo Clin Proc 2009;84(6):555–7. 379. Micali G, Cook B, Ronan S, Yadgir J, Solomon LM. Cephalic pyoderma gangrenosum (PG)-like lesions as a presenting sign of Wegener’s granulomatosis. Int J Dermatol 1994;33(7):477–80. 380. Wong YW, Lyon CC, Benbow EW, Bradley BL, Beck MH. Pyoderma gangrenosum in a thoracotomy wound associated with a pulmonary cavitating lesion. Clin Exp Dermatol 2003;28(3):274–6. 381. Sweet RD. An acute febrile neutrophilic dermatosis. Br J Dermatol 1964 AugSep;76:349–56. 382. von den Driesch P. Sweet’s syndrome (acute febrile neutrophilic dermatosis). J Am Acad Dermatol 1994;31(4):535–56; quiz 57–60.

371. Kruger S, Piroth W, Amo Takyi B, Breuer C, Schwarz ER. Multiple aseptic pulmonary nodules with central necrosis in association with pyoderma gangrenosum. Chest 2001;119(3):977–8.

383. Fett DL, Gibson LE, Su WP. Sweet’s syndrome: systemic signs and symptoms and associated disorders. Mayo Clin Proc 1995;70(3):234–40.

372. Chahine B, Chenivesse C, Tillie-Leblond I, et al. Pulmonary manifestations of Pyoderma

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397. Camus P, Colby TV. The lung in inflammatory bowel disease. Eur Respir J 2000;15(1):5–10. 398. Camus P, Piard F, Ashcroft T, Gal AA, Colby TV. The lung in inflammatory bowel disease. Medicine (Baltimore) 1993;72(3):151–83. 399. Casey MB, Tazelaar HD, Myers JL, et al. Noninfectious lung pathology in patients with Crohn’s disease. Am J Surg Pathol 2003;27(2):213–9.

393. Soderstrom RM. Sweet’s syndrome and acute myelogenous leukemia: a case report and review of the literature. Cutis 1981;28(3):255–7, 60.

400. Bentur L, Lachter J, Koren I, et al. Severe pulmonary disease in association with Crohn’s disease in a 13-year-old girl. Pediatr Pulmonol 2000;29(2):151–4.

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Chapter

22

Benign epithelial neoplasms and tumor-like proliferations of the lung Douglas B. Flieder

Introduction Despite the tremendous number of malignant epithelial lung neoplasms worldwide, benign epithelial tumors and tumor-like lesions are rare. Endobronchial and peripheral parenchymal lesions are both seen. While voluminous epidemiological and molecular information is lacking, lesional morphologies are well described. Recognizing these lesions as benign is of paramount importance.

Bronchial inflammatory polyps Introduction Although the upper respiratory tract is a common location for inflammatory polyps, for unknown reasons these nonneoplastic lesions are exceedingly rare in major bronchi or smaller-caliber airways. The World Health Organization (WHO) prefers the term bronchial inflammatory polyp to emphasize the non-neoplastic nature of the lesion.1 Fibroepithelial polyp, though commonly used, is a misnomer since the lesion has no true epithelial component. Nevertheless, the lesion is best discussed in this chapter along with true benign epithelial tumors.

Classification, cell of origin, pathogenesis and etiology Inflammatory polyps are regenerative lesions representing exuberant, non-resolving, localized tissue repair following injury. As in all organs, if regeneration does not follow injury, a fibroproliferative tissue response patches the wound. This repair relies on tissue fibroblasts and vascular endothelial cells, which form granulation tissue. The leaky blood vessels allow protein and red cell extravasation into the extravascular space and form a scaffold for fibroblasts and subsequent fibrillar collagen deposition.2 In most instances a scar forms but in some situations the granulation tissue persists. Inflammatory polyps are an example of this persistent exuberant tissue repair process, following mucosal erosion or ulceration.3 The bronchial mucosa is susceptible to many insults and

Table 1 Etiologies of inflammatory polyps

Burns Thermal Chemical Infections Fungi Histoplasma capsulatum Aspergillus Actinomyces Cocciodioides Mycobacteria Mycobacterium avium-intracellulare Mycobacterium tuberculosis Bacteria Pseudomonas aeruginosa (Cystic fibrosis-associated bronchiectasis) Sinusitis Asthma Hypersensitivity pneumonitis-associated Reflux/aspiration Foreign bodies Broncholiths Prolonged intubation and mechanical ventilation with suction

immunological, infectious and environmental causes abound. Neonates, infants and children are particularly susceptible to prolonged intubations, mechanical ventilation and suction while infections, reflux and aspiration as well as thermal and chemical inhalation injuries are common factors in adults (Table 1).4–24 Of note, polyps may develop months after a respiratory problem, such as smoke inhalation, chemical inhalation or aspiration.9,12

Genetics No information is available.

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Figure 2. Inflammatory polyp. This 10 cm. yellow, white and gelatinous endobronchial lesion was resected after 40 years of recurrent pulmonary infections and at least one episode of hemoptysis. (Courtesy of I. Dincer, MD, Istanbul, Turkey.) Figure 1. Inflammatory polyp. Sagittal reformatted multislice computed tomogram demonstrates a right main bronchus mass with right lower lobe bronchiectasis. (Courtesy of I. Dincer, MD, Istanbul, Turkey.)

Special clinical features Inflammatory polyps are exceedingly rare to the point that many high-volume bronchoscopists may never encounter one during their entire career.25 Lesions may be seen in both adults and children and no gender difference has been reported. While associated with a myriad etiologies (Table 1), tobacco is not implicated. Solitary polyps are more common than multiple tumors and a right-sided predominance is probably due to anatomic considerations, rendering this half of the bronchial tree more susceptible to disease.8,18,19,22,25–28 Presenting symptoms in adults run the gamut from wheezing, heartburn, shortness of breath, dyspnea on exertion, orthopnea and hemoptysis to fever, cough and pleuritic chest pain.25,26,28,29 Both adults and children may also present with intermittent respiratory distress and/or lung collapse.19,30–32 Decreased respiratory sounds, inspiratory crackles, elevated erythrocyte sedimentation rates, obstructive findings on pulmonary function tests and decreased peripheral blood oxygen saturation may be encountered.

Radiographic findings Chest X-rays and computed tomography (CT) usually demonstrate segmental parenchymal infiltrates or atelectasis. An endobronchial mass may be seen on CT scan, depending on the size of the lesion (Figure 1). Protean manifestations include deviation of the trachea, lobar collapse, air-fluid levels, bronchiectasis or contralateral lung/lobar hyperinflation.8,19,25–27,31

Macroscopic pathology Endobronchial inflammatory polyps range from 0.2 cm to 10 cm in length with diameters up to 1.5 cm (Figure 2).26,31 Lesions,

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Figure 3. Inflammatory polyp. Polypoid endobronchial lesions are composed of granulation tissue lined by respiratory epithelium.

either solitary or multiple, may be sessile or pedunculated and are often gelatinous. The sessile lesions are usually pearly gray, white or tan while larger and pedunculated tumors are usually tan, lobulated and hyperemic. The polyps may resemble mucus plugs.19 The surrounding mucosa may be normal or boggy and hyperemic. Distal lung parenchyma may feature pus-filled airways, parenchymal consolidation or bronchiectasis.

Histopathology Whether biopsied or resected, microscopy usually demonstrates polypoid granulation tissue lined by benign respiratory epithelium (Figure 3). The substance of the lesion usually

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Figure 4. Inflammatory polyp. Edematous granulation tissue features many thin-walled vessels and edema.

Figure 5. Inflammatory polyp. Smooth muscle, seromucinous glands, often dilated, and lymphoplasmacytic infiltrates are common findings. Overlying respiratory epithelium may be distorted.

metaplasia is usual. Pseudoepitheliomatous hyperplasia may also be seen, often adjacent to mucosal erosions with adherent fibrinous exudates.28

Cytology Bronchoalveolar lavage and brushing specimens from substantial sized polyps, include metaplastic squamous epithelium.26,28 Relevant is the absence of cytomorphological features suggestive of malignancy.

Immunohistochemistry No information is available. Figure 6. Inflammatory polyp. Longstanding lesions often feature stromal fibrosis and calcification. (Courtesy of I. Dincer, MD, Istanbul, Turkey.)

features edematous pale to myxoid, light-blue, hypocellular mesenchyme with widely spaced, thin-walled and focally patulous arborizing blood vessels along with sparse intervening bland spindle cells (Figure 4). Endothelial cells may be prominent and native smooth muscle, as well as seromucinous glands, may be incorporated into the polyp base or stalk (Figure 5). Stromal fibrosis and calcification are frequently seen in long-standing lesions (Figure 6). Scattered neutrophilic, eosinophilic and/or dense lymphoplasmacytic infiltrates are common. Xanthogranulomatous inflammation may also be present. Overlying mucosa and thickened basement membrane are usually distorted and invaginated, while squamous

Electron microscopy No information is available.

Molecular findings No information is available.

Clinicopathological correlation Since neither imaging studies nor bronchoscopic visualization allow for diagnosis, all endobronchial polypoid lesions, even if incidental and apparently innocuous, should be biopsied. Incidental small inflammatory polyps usually do not recur. Although large polyps must be entirely removed to prevent further obstructive sequela, bronchoscopic sampling should be

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performed first, since alternative therapies might obviate the need for lobectomy.

Differential diagnosis Although the bronchoscopic differential diagnosis for inflammatory polyps includes a host of benign and malignant neoplasms and rare non-neoplastic processes, such as amyloid, morphological considerations are limited to papillomas and perhaps rare mesenchymal tumors. Squamous, glandular and mixed papillomas are neoplastic epithelial proliferations with true fibrovascular stalks, unlike the epithelial-covered, usually bulbous “granulation tissue” polyps (see below). Rare endobronchial hamartomas, leiomyomas and lipomas all feature true mesenchymal elements rather than edematous fibrovascular cores (see Chapter 33).

Prognosis and natural history Inflammatory polyps can be lethal in infants and adults alike, when they cause significant airway obstruction or secondary infectious pneumonias. However, the lesions are slow-growing and without malignant potential. Individuals with documented lesions have been followed for up to 30 years before requiring their removal.26,31 Treatment modalities depend on the underlying etiology. Those associated with infection may be treated with antimicrobials, while aerosolized or systemic corticosteroids may be efficacious in patients with inhalation-injury-induced lesions or asthma.7,17,18,33 Flexible or rigid bronchoscopic extirpation with laser or electrocautery is preferable to surgical resection when possible.5,25,27,34

Bronchial papillomas Introduction Bronchial papillomas are very rare lung tumors yet still account for up to 8% of all benign lung neoplasms. Unlike inflammatory polyps these lesions are true neoplasms. Confusion regarding classification appears to be a thing of the past, as one can categorize endobronchial and even endobronchiolar papillomas according to number, location and histology. The WHO recognizes squamous cell, glandular and mixed histologies.35 The latter two types are very uncommon and thus the scope of the following discussion centers on squamous cell papillomas.

Squamous cell papillomas Classification, cell of origin, pathogenesis and etiology Squamous cell papillomas can be solitary or multiple, exophytic or inverted. Animal studies conducted in 1933 suggested an infectious, probably viral, etiology.36 Human papilloma virus (HPV) probably has a pathogenetic role in the development of laryngotracheal papillomatosis (respiratory papillomatosis) and in a large percentage of solitary squamous papillomas.37–43 Infection probably occurs during a vaginal birth or later in life,

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through infected secretions. In utero transmission has also been reported.44–48 First-born children are at higher risk than subsequent births, perhaps since primigravid mothers are more likely to have a long second stage of labor, resulting in prolonged fetal exposure to virus.49,50 HPV probably infects bronchial reserve cells.51,52 The virus then either remains latent or viral DNA is expressed. HPV proteins E6 and E7 interfere with cell cycle proteins p53 and retinoblastoma, causing increased cell proliferation and perhaps malignant transformation.47,53–59 Since so many babies are exposed to HPV during childbirth but very few develop papillomas, unrecognized genetic factors must play a role in tumorigenesis, progression and malignant transformation.60 Active areas of investigation include the role of cytokines and expression of major histocompatibility complex antigens in cell-mediated immunity dysfunction.61,62 Of note, an etiological role for tobacco smoke has not been established though more than half of individuals with solitary squamous cell papillomas are tobacco smokers.37 Tobacco and radiation exposure are implicated in carcinomatous transformation of laryngotracheal papillomatosis.63

Genetics No information is available.

Special clinical features Solitary squamous papillomas commonly afflict men in their sixth or seventh decades of life. Patients usually present with obstructive symptoms, including dyspnea, hoarseness, wheezing and chest pain, as well as fever and productive cough, indicating pneumonia. At least 25% of lesions are incidental radiographic findings.64 Respiratory papillomatosis has a bimodal age distribution with the first peak at 5 years of age (so-called juvenile onset) and the second peak between ages 20 and 30 (adult onset). It affects both boys and girls but adult disease usually affects men.46,65 The juvenile form has an incidence of 4/100 000 in the USA and is the commonest laryngeal neoplasm in children, while the adult form affects only 1/100 000 Americans. The younger the child is at his/her first presentation, the more severe the disease. The disease usually recurs many times over many years, becomes widespread and extends into the bronchial tree in up to 5% of patients.66,67 Papillomas extend into alveolar parenchyma in less than 1% of juvenile cases.66–68 Any process that leads to squamous metaplasia of the bronchial tree, such as gastrointestinal reflux, predisposes the patient to florid spread of papillomas.69 Parenchymal involvement is often associated with the development of squamous cell carcinoma.70

Radiographic findings Solitary endobronchial papillomas are rarely seen on chest radiographs; large lesions may manifest with pneumonia, emphysema, bronchiectasis, atelectasis or as a hilar mass.37,71,72

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Figure 8. Pulmonary papillomatosis. Normal lung is reduced to a bronchiectatic cavity filled with white-tan friable papillary fronds.

Figure 7. Lung involvement with tracheobronchial papillomatosis. This computed tomogram shows not only a narrowed trachea, but significant pulmonary centrilobular nodules. Central cavitation is noted bilaterally. (Courtesy of C. Derkay, MD, Norfolk, Virginia, USA.)

High-resolution CT (HRCT) findings demonstrate endobronchial protuberances, nodular airway thickening or masses.73,74 Additionally, air-trapping, atelectasis, consolidation and bronchiectasis may be noted.75 When lung is involved with tracheobronchial papillomatosis, chest radiographs and CT scans feature diffuse, ill-defined, non-calcified, parenchymal centrilobular opacities and multiple, well-defined, 0.5 to 5.0 cm cavitated thick-walled nodules (Figure 7).76 A lobar predilection is not noted. Air-fluid levels are not uncommon in the cysts and represent either post-obstructive pneumatoceles or cavitating papillomas.63,77 Computed tomograms with reconstructed images (so-called virtual bronchoscopy) identify small mural nodularities. This modality reduces the risk of downward spread of virus, noted in bronchoscopy patients.77

Macroscopic pathology Solitary squamous cell papillomas arise from mainstem bronchi or major subdivisions. Tumors range from 0.7 to greater than 9.0 cm with a reported median size of 1.5 cm.37 Sessile or exophytic tumors have polypoid, tan-white, glistening and friable excrescences protruding into bronchi. Bronchiectasis develops proximal and distal to the papilloma on account of the tumor and consequent post-obstructive atelectasis/pneumonia. Large lesions may fill bronchiectatic airways and surrounding lung may form a rim of firm white fibrous tissue. Distal parenchyma may be atelectatic, consolidated or feature honeycomb change. Multiple airway papillomas are similar to solitary lesions but when confluent impart a velvety appearance to the bronchial

Figure 9. Solitary squamous cell papilloma. This endobronchial papilloma demonstrates classic architectural features – arborizing fibrovascular cores covered by a proliferative squamous epithelium. Adjacent mucosa features squamous metaplasia.

mucosa.63 Parenchymal involvement may form single or multiple, well-circumscribed, expansile, tan-white, papillary aggregates, surrounded by consolidated lung (Figure 8). Cavitation and surrounding lung fibrosis are also not uncommon; the latter may even suggest tumor invasion. The mass lesions can grow to large sizes and essentially replace entire pulmonary segments.

Histopathology Squamous cell papillomas feature arborizing loose fibrovascular cores covered by stratified squamous epithelium (Figures 9–11).

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Figure 10. Solitary squamous cell papilloma. Endobronchial biopsy fragments show fronds cut en face. Each fragment is remarkably similar in size and with regard to amount of epithelial proliferation.

(a)

Figure 11. Solitary squamous cell papilloma. Squamous epithelium matures toward the surface of each frond. Parakeratosis is common.

(b)

Figure 12. Solitary squamous cell papilloma. (a) Binucleate cells and wrinkled nuclei suggest human papilloma virus infection. (b) In situ hybridization staining for HPV 6/11 highlights many infected cells.

Exophytic lesions have orderly epithelial maturation extending from the basal layer to the superficial flattened and usually keratinized surface cells. Parakeratosis is also common. The underlying basement membrane is intact. Acanthosis and nonkeratinized surfaces are often seen, along with intraepithelial

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neutrophils. Less than 25% of solitary papillomas demonstrate morphological features consistent with HPV infection, including binucleate forms, wrinkled nuclei and perinuclear halos (Figure 12).37 Occasional dyskeratotic cells, large atypical cells and mitotic figures, above the basal layer, can be seen. The

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Figure 13. Inverted squamous cell papilloma. Not unlike urinary bladder inverted papillomas, endobronchial lesions are exophytic with invaginated nests of bland squamous mucosa. The neoplasm may colonize seromucinous glands.

(a)

Figure 14. Inverted squamous cell papilloma. This rare neoplasm is non-keratinizing and always surrounded by basal lamina. Inflamed stroma accompanies the cellular proliferation.

(b)

Figure 15. Lung involvement with squamous cell papilloma/papillomatosis. (a) Papillary fronds fill a bronchiectatic cavity. (b) Underlying alveolar spaces are filled with similar epithelium.

degree of dysplasia should be graded according to the current WHO classification (see Chapter 23). In rare instances atypia reaching the level of carcinoma in situ may be present and in such instances a diagnosis of carcinoma arising in a squamous papilloma should be rendered. The amounts of stroma and stromal lymphoplasmacytic infiltrates vary from very scant to prominent, while the adjacent respiratory mucosa may be normal, inflamed, hyperplastic or metaplastic. The involved airway(s) may be bronchiectatic. Inverted squamous cell papillomas demonstrate exophytic and focal random invaginations of squamous epithelium (Figure 13). Neoplastic cells often extend into seromucinous

glands. These rare lesions are non-keratinizing and show orderly maturation from the basal layer upward. Central whorls may develop but keratin balls are not seen. Basal lamina always invests the endophytic nests. The stroma is often expanded with lymphocytes and plasma cells (Figure 14). Alveolar parenchymal involvement has two overlapping forms. Well-circumscribed, solid, intra-alveolar nests of cytologically bland, non-keratinizing squamous cells may fill alveolar spaces. These are lined by hyperplastic pneumocytes, or solid nests of squamous cells can obliterate alveolar parenchyma, resulting in large cystic collections of neoplastic cells (Figures 15 and 16). In these cases the cyst lining cells are

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Figure 17. Fine-needle aspirate of squamous cell papilloma. This Papanicolau (PAP)-stained sample demonstrates a cluster of squamous cells with well-defined cytoplasmic borders, dense cytoplasm, and a suggestion of perinucelar halos. (Glass slide courtesy of S. Ali, MD, Baltimore, Maryland, USA.)

Figure 16. Lung involvement with squamous cell papilloma/papillomatosis. Cytologically bland tumor fills alveolar spaces. Bronchiolar epithelium is hyperplastic. Distinguishing this process from invasive squamous cell carcinoma can be very difficult.

Figure 19. Fine-needle aspirate of squamous cell papilloma. Papillae are lined by crowded nonkeratinizing oval and spindly cells with scant dense cytoplasm in this PAP-stained case. (Glass slide courtesy of S. Ali, MD, Baltimore, Maryland, USA.)

Cytology Figure 18. Fine-needle aspirate of squamous cell papilloma. This PAP-stained sample of a pulmonary parenchymal lesion demonstrates three-dimensional branching papillae. (Glass slide courtesy of S. Ali, MD, Baltimore, Maryland, USA.)

neoplastic but lack cytological features of malignancy. The surrounding lung may be inflamed and/or fibrotic but infiltrative tumor tongues indicative of invasive carcinoma are not appreciated. Bronchial and alveolar parenchymal involvement with laryngotracheal papillomatosis is morphologically similar to the isolated lesions; however, virtually all lesions feature viral cytopathic effect.

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Sputum, bronchial washings and touch imprint reports indicate that sheets and single squamous cells have abundant thick, dense, and glassy cytoplasm with sharp margins (Figure 17).72 Perinuclear halos and degenerative vacuoles are occasionally seen but keratohyaline granules are absent. Multinucleation may be seen and nuclei vary from small and pyknotic to large with evenly distributed coarse chromatin. Smaller basal cells with scant basophilic cytoplasm and round regular nuclei are also noted along with background neutrophils.78,79 Fine-needle aspiration samples demonstrate well-developed, three-dimensional, branching papillae, lined by crowded non-keratinized bland, round to oval and short spindly cells with scant dense cytoplasm (Figures 18 and 19). Scattered single cells are also

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

described. All cells have enlarged nuclei, anisonucleosis and prominent nucleoli.80 Irregular nuclear contours may be observed. Keratinization, mitoses, karyorrhexis and necrosis are not seen.81

Immunohistochemistry Immunohistochemical and in situ DNA hybridization studies documenting either the presence or absence of HPV and particular subtypes are not diagnostically necessary. However, positivity appears to correlate with the morphological presence of koilocytosis.37 It has been suggested that viral subtyping may hold prognostic value (see below). A similar claim has been made regarding p53 and topoisomerase alpha II expression for laryngotracheal papillomatosis.82

Electron microscopy Ultrastructural examination of a solitary squamous cell papilloma harboring an unspecified HPV virus detected by immunohistochemical stain, revealed 45 to 55 nm in diameter, electron-dense, intranuclear particles with a suggestion of lattice arrangement.83

Molecular findings No information is available.

Clinicopathological correlation Viral subtyping indicates that HPV subtypes 6b and 11 are identified in up to 70% of the solitary papillomas and almost all laryngotracheal papillomas.37,40,53,84 Cases demonstrating HPV-11 behave more aggressively than those infected with HPV-6 and these children are at higher risk of obstructive airway disease.53,63,85 While malignant transformation is more common in patients with a history of smoking or radiation therapy, infection with HPV-11 may be an early event associated with progression of recurrent respiratory papillomatosis to carcinoma.68,86 The more oncogenic subtypes, 16 and 18, have also been reported in a smaller percentage of laryngotracheal papillomatosis.65

Inverted papillomas with focal mild cytological atypia are virtually indistinguishable from invasive squamous cell carcinoma; parenchymal destruction, overt pleomorphism and significant hyperkeratosis all favor a diagnosis of carcinoma. Recognizing even focal carcinoma within an otherwise benign squamous papilloma warrants a diagnosis of carcinoma.56 Distinguishing a benign papilloma from carcinoma may not be possible on small biopsy or cytology samples.

Prognosis and natural history Surgically resected solitary squamous cell papillomas do not recur, but up to 20% of patients treated with endoscopic removal experience local recurrences.37,40 Although some suggest that these papillomas may transform into or overlie carcinomas in up to 40% of cases, such findings suggest erroneous diagnoses of papillomas, due either to incomplete excisions or misinterpretation of pathology material.38,39,55,56,71,72 Incompletely excised lesions harboring HPV should be either surgically resected or monitored closely. Despite generalizations regarding HPV subtypes and clinical behavior, the predictive value of HPV subtyping in predicting future development of carcinoma remains unproven.68,86–90 The natural history of laryngotracheal papillomatosis is variable and uncertain. The disease can undergo spontaneous remission, persist as stable disease, or progress. The more aggressive form usually affects children. Extralaryngeal spread is noted in approximately 30% of children and less than 20% of adults. The most frequent sites of spread in decreasing order of frequency are the oral cavity, trachea, bronchi, and esophagus.45,50,91 The development of pulmonary involvement heralds the insidious loss of pulmonary function, culminating in respiratory failure. Enlarging lesions lead to bronchiectasis, abscesses and recurrent pneumonia.92 Malignant transformation, though much discussed in case reports and small series, does not appear to exceed 2%.41,91,93 The disease is incurable despite surgical and adjuvant treatment modalities, including antiviral therapies and Cox-2 inhibitors.94,95 HPV vaccines are theoretically very promising.63

Differential diagnosis

Glandular and mixed squamous cell and glandular papillomas

The major differential considerations for squamous cell papillomas are inflammatory polyp and squamous cell carcinoma. As discussed above, inflammatory polyps lack a true papillary architecture, stromal cores and proliferative epithelium. Squamous cell carcinomas can be endobronchial and papillary but cytological features of overt malignancy are usually appreciated, even when stromal invasion is not obvious. Unlike papillomas, well-differentiated squamous cell carcinomas lack orderly epithelial maturation and often feature keratinization. Difficulties arise when one misinterprets entrapped seromucinous glands for invasion into the polyp stalk.

As no more than a dozen glandular papillomas are described in the thoracic literature, comments are very limited.37,96–99 Patients, both men and women, are often in their seventh decade of life and less than one-half are tobacco users. No specific etiologies are associated with this neoplasm. While lesions may be incidental findings on screening CT scans, patients may present with obstructive symptoms or bloodtinged sputum.37,97 A reported case of extreme FDG-PET avidity in a mixed histology papilloma raised the possibility of malignancy.100 Tumors may be endobronchial or, unlike squamous cell papillomas, endobronchiolar and range in size from 0.8 to 4.0 cm with a mean of 1.2 cm. Endobronchial and

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Figure 20. Glandular papilloma. These rare tumors often resemble upper respiratory tract Schneiderian papillomas. Complex fibrovascular cores are lined by proliferative columnar epithelium. (Courtesy A. Khoor, MD, Jacksonville, Florida, USA.)

peripheral well-demarcated, white, semifirm lesions feature papillomatous epithelial fronds with vascular or hyalinized stromal cores. The complex architecture may suggest a jigsaw arrangement but bronchial wall or parenchymal invasion is absent (Figure 20). Tumors may closely resemble Schneiderian papillomas of the upper respiratory tract. Stratified or pseudostratified columnar epithelium may form micropapillae but desquamated cells are not seen (Figure 21). The glandular epithelium may be ciliated, cuboidal or columnar, and interspersed mucin-rich cells are often identified. A basal cell layer is often apparent and can be highlighted with either p63, K903 or cytokeratin17 immunohistochemical stains.97 Focal clear cell change can be observed. Neoplastic epithelium may rarely extend along adjacent airway mucosa. Cytological atypia, necrosis and mitoses are not present and HPV studies are to date negative.37,97 Electron microscopy studies reveal normal ultrastructural glandular features, including unremarkable cilia processes.97 The differential diagnosis includes primary and metastatic papillary adenocarcinoma and various adenomas. Since cytology and biopsy samples cannot discern these rare lesions from very common adenocarcinomas, surgical resections are required. Mucus gland adenomas feature mucusfilled cysts and tubules, while papillary adenomas are true pulmonary parenchymal, rather than endobronchial lesions. All but one reported case was treated surgically and the only recurrence and death was in the patient unable to tolerate anything more than bronchoscopic treatments.37 Mixed squamous cell and glandular papillomas, formerly known as transitional papillomas, are the rarest of all solitary endobronchial papillomas, yet mixed histology is not uncommon in laryngotracheal papillomas involving bronchi.37,99,101 In all types the squamous epithelium is probably a metaplastic phenomenon. While the demographic information and the median tumor size mirrors values observed in squamous cell

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Figure 21. Glandular papilloma. Uniform columnar cells with eosinophilic cytoplasm, round regular nuclei and interspersed mucin-rich cells are surrounded with inflammatory cell-rich mucin while stromal cores contain many plasma cells.

Figure 22. Mixed squamous cell and glandular papilloma. Inflamed arborizing papillary cores are lined with discrete foci of glandular and squamous epithelium.

papillomas, most of the epithelium is glandular. The glandular epithelium may be cuboidal or pseudostratified columnar, with or without cilia, while the squamous epithelium is acanthotic and focally keratinizing (Figures 22 and 23).

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Figure 24. Bronchial brushing of a mixed squamous cell and glandular papilloma. Squamous cells have dense cytoplasm and central round nuclei, while rare glandular cells are columnar with occasional cilia in this PAP-stained sample. (Courtesy K. Kadota, MD, Kagawa, Japan.)

Figure 23. Mixed squamous cell and glandular papilloma. Glandular mucosa is identical to that seen in glandular papillomas. Accompanying inflammation suggests the squamous component is metaplastic.

Glandular atypia is considered reactive, while squamous atypia is believed to be neoplastic. Neither mitoses nor necrosis are appreciated. The fibrovascular cores are inflamed with lymphocytes and plasma cells.101 Transbronchial brush and intraoperative imprint cytology clearly demonstrate both squamous and glandular cells. Squamous cells are both keratinized and non-keratinized, while columnar cells are both ciliated and non-ciliated (Figure 24). Interestingly, viral cytopathic effect is not seen and HPV DNA has not been reported in the squamous component.37,101 Complete surgical resection appears curative.37

Multifocal micronodular pneumocyte hyperplasia Introduction Multifocal micronodular pneumocyte hyperplasia (MMPH) is a benign epithelial proliferation most frequently associated with tuberous sclerosis complex (TSC)-related pulmonary disease. Also described as “acinar atypical adenomatoid proliferation of epithelium”, “multiple adenomatoid lesions”, “micronodular pneumocyte hyperplasia”, “multifocal alveolar hyperplasia” and “micronodular hyperplasia of type II pneumocytes”, these peculiar lesions may affect up to one-half of TSC patients.102–104 The WHO defines MMPH as “a multifocal micronodular proliferation of type II cells with mild thickening of the interstitium”.1 The morphological differential diagnosis is lengthy and often challenging.

Classification and cell of origin Though described in the literature as a pulmonary hamartomatous manifestation of TSC, MMPH is best considered an epithelial tumor-like proliferation with a pronounced stromal response.104–107 The epithelial component derives from type II pneumocytes and/or Clara cells.108–110 Alveolar septal thickening may be a reactive response to the epithelial proliferation or altered hemodynamic or ventilatory forces. Little is known about the stromal component, aside from the fact it lacks the morphological and immunohistochemical characteristics of perivascular epithelioid cell tumor (PEComa) cells seen in other TSC-related neoplasms, including angiomyolipoma (AML) and lymphangioleiomyomatosis (PLAM) (see Chapter 33).

Genetics Tuberous sclerosis complex results from mutations in the tumor suppressor genes TSC1, encoding hamartin, on chromosome 9q34, and TSC2, encoding tuberin, on chromosome 16p13.3.111,112 TSC1 and TSC2 mutations are reported in some patients with MMPH.102,104 Strong in situ hybridization expression for TSC2 mRNA and immunohistochemical expression for tuberin in lesional epithelial cells are also noted.102,104,113

Special clinical features Multifocal micronodular pneumocyte hyperplasia is predominantly diagnosed in individuals with TSC. Most, but not all, patients are premenopausal women, with a mean age of 37 years.102,104,114,115 Approximately half of MMPH patients, without TSC, have PLAM. The remainder, men and women, have neither TSC nor PLAM.104,109,116,117 Lesions are often

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Figure 25. Multifocal micronodular pneumocyte hyperplasia. Minute welldemarcated lesions owe their nodularity to thickened alveolar septa and intraalveolar macrophages.

Figure 26. Multifocal micronodular pneumocyte hyperplasia. Lesional epithelial cells grow along alveolar walls and feature eosinophlic cytoplasm and round nuclei with vesicular chromatin. Nucleoli may be apparent.

incidental, radiographic ground-glass opacities or microscopic findings noted at the time of a pneumothorax or hemothorax, secondary to PLAM or sometimes as part of systemic screening in TSC patients.104,106,108,110,118,119 Clinical manifestations from isolated MMPH include dyspnea, exertional dyspnea and cough.104,115,117

center of the lesions may form papillae. Lepidic extension beyond the nodules is not seen. The enlarged cuboidal epithelial cells have abundant eosinophilic, faintly granular cytoplasm and vesicular nuclei with smooth nuclear contours, occasionally prominent nucleoli, and rare eosinophilic inclusions (Figure 26). Rare multinucleated cells are noted but marked atypia, mitoses or necrosis are absent. Thickened and mildly fibrotic alveolar septa contain scattered lymphocytes and increased reticulin fibers but lack smooth muscle proliferation (Figure 27). Alveolar macrophages also contribute to the nodular character of the lesion by filling airspaces lined by the epithelial cells. In many instances close study of adjacent alveolar parenchyma reveals subtle interstitial thickening, lined by hyperplastic type II pneumocytes.

Radiographic findings Chest radiographs demonstrate bilateral diffuse fine nodular opacities, while HRCT reveals scattered 0.3 to 1.0 cm shadows, ground-glass opacities or nodules.107,120–122 Less commonly one sees many 0.1 to 0.3 cm miliary nodules.110,122,123 Nodules are more common in individuals with pulmonary cysts, i.e. PLAM, than in otherwise normal lung.102 A lobar predilection is not seen and lesions are both peripheral and central.

Macroscopic pathology Most reported lesions lack macroscopic descriptions but several case reports describe 0.2 to 0.5 cm randomly distributed small, white, solid and firm nodules.106,117,124 A solitary 0.8 cm lesion was recently reported.125

Histopathology Well-demarcated nodules range from 0.1 to 1.0 cm but most are smaller than 0.5 cm.104,110,117,123 The pneumocyte proliferation has a nodular low-magnification appearance owing to the epithelial cells, intra-alveolar macrophages and mild interstitial thickening (Figure 25). Crowded epithelial cells in the

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Cytology No information is available.

Immunohistochemistry Lesional cells stain with antibodies directed against pancytokeratins, EMA, TTF-1, surfactant apoproteins and occasionally peanut and Ulex europaeus I agglutinin. The cells are non-reactive with vimentin, desmin, smooth-muscle actin, muscle-specific actin, S100 protein, HMB-45, melan-A, Clara cell protein, estrogen and progesterone receptors.104,108,110,113,124,126 Ki-67 shows less than 5% nuclear staining and epithelial cells are not immunoreactive with p53 antibodies.104,108–110,117 The thickened alveolar stroma also fails to react with desmin, smooth-muscle actin, muscle-specific actin, S100 protein and HMB-45 antibodies.

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

proliferations, very difficult. Healing infectious and postinflammatory processes are usually less circumscribed, feature airspace fibrin and inflammatory cells, if not fibromyxoid connective tissue, and greater cytological atypia than appreciated in MMPH. Atypical adenomatous hyperplasia (AAH) has the same size range as MMPH but is less well circumscribed, completely lepidic in its growth pattern without solid or papillary areas and features a greater degree of cytological atypia. Multifocal micronodular pneumocyte hyperplasia also features more pronounced alveolar septal thickening and intra-alveolar macrophages. Non-mucinous adenocarcinoma in situ (AIS) features pure lepidic growth and malignant cytological characteristics.

Prognosis and natural history Multifocal micronodular pneumocyte hyperplasia is almost always an incidental radiographic or microscopic finding with no clinical significance. Lesions followed with serial radiographs appear stable.104 One report of florid lesions leading to respiratory failure and death must temper our assessment regarding its biological behavior.117 Figure 27. Multifocal micronodular pneumocyte hyperplasia. Alveolar septa are widened by fibrosis and reticulin. Such areas often contain intra-alveolar macrophages.

Electron microscopy Ultrastructural studies confirm the epithelial nature of the proliferation and describe cuboidal cells resting on a basal lamina with desmosomes and hemidesmosomes.108,109,117 Osmiophilic lamellar inclusions and surface microvilli indicate type II pneumocyte differentiation. One study also identified electron-dense, membrane-limited, secretory granules with a granular matrix and abundant rough endoplasmic reticulum, suggestive of Clara cell differentiation.109

Molecular findings Although allelic loss in the region of the TSC2 gene was not identified in a single LOH study,113 the same laboratory group recently reported LOH in eight separate lesions from eight patients.127 In seven cases LOH was detected in the TSC2 gene and in one case in the TSC1 gene. In addition, activation of mTOR-related proteins was also noted.

Clinicopathological correlation While the finding of multiple scattered subcentimeter nodules on radiographic studies is usually a very worrisome sign of either infection or malignancy including metastatic disease, in patients with clinical manifestations of TSC or PLAM such a finding suggests a diagnosis of MMPH. Nevertheless, these individuals, as well as those without TSC or PLAM require excision of at least one lesion to confirm the diagnosis.

Differential diagnosis Lack of awareness regarding MMPH makes its distinction from inflammatory reactions, as well as other pneumocyte

Papillary adenoma Introduction

The WHO defines papillary adenoma as “a circumscribed papillary neoplasm consisting of cytologically bland cuboidal to columnar cells lining the surface of a fibrovascular stroma”.35 Synonyms include bronchiolar apocrine tumor, bronchiolar adenoma, papillary adenoma of type II pneumocytes, type II pneumocytic adenoma, adenoma of type II pneumocytes with oncocytic features, and peripheral papillary tumor of type II pneumocytes.128–133 Papillary adenomas are so rare that most lung pathologists never see a case in their daily practice.

Classification, cell of origin and etiology This benign peripheral lung tumor is believed to arise from a multipotential stem cell or immature bronchioloalveolar cell.118,133–135 As such, tumor cells may manifest type II pneumocyte or Clara cell features. The etiology is unknown but similar morphological lesions are genetically and/or chemically induced in mice.136 Tobacco use does not appear to be an etiological factor.

Genetics No information is available.

Special clinical features These adenomas have been reported in both sexes in patients ranging from 7 to 60 years of age (mean 32 years). A slight male predominance is noted. Multiple tumors were resected from a teenage boy with von Recklinghausen disease, but a clear association is not recognized.137 Patients are asymptomatic.

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Figure 29. Papillary adenoma. A single layer of columnar cells lines fibrovascular cores. Tumor cells are uniform and cytologically bland. Only the peripheral location allows distinction from a glandular papilloma.

Figure 28. Papillary adenoma. This well-circumscribed parenchymal lesion has an obvious papillary configuration.

Radiographic findings Chest radiographs disclose well-defined pulmonary nodules. Computed tomography reveals a smooth circumscribed parenchymal nodule, without pleural indentations or spiculations, which may bulge into a small bronchus.135,138

Macroscopic pathology Solitary tumors measure from 1.0 to 4.0 cm, while the lesions in the von Recklinghausen disease patient ranged from 0.05 to 0.2 cm.137 No lobar predilection is reported. Well-circumscribed and occasionally encapsulated tumors have tan, gray, soft to firm, granular and spongy cut surfaces. Focal hemorrhage has been noted but necrosis is absent. Lesions are not endobronchial but may protrude into adjacent small airways.

Histopathology Papillary adenomas are always well circumscribed, and encapsulation or infiltration into surrounding alveolar parenchyma may rarely be seen (Figure 28).130 Tumors have obvious papillary architecture and focal, solid areas can be observed. A single layer of tumor cells rest on prominent fibrovascular cores, which may be edematous and inflamed or focally hyalinized (Figure 29). Elastic fibers are absent from the stromal cores and scattered mast cells may be seen.133 Cuboidal to columnar cells can appear pseudostratified and micropapillary in tangential sections. The cytoplasm ranges from eosinophilic to foamy or clear (Figure 30). Basally located round to oval nuclei with uniformly distributed chromatin may contain

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Figure 30. Papillary adenoma. In this example tumor cells feature foamy cytoplasm. Stromal inflammation and intra-alveolar macrophages are also seen.

eosinophilic intranuclear inclusions but significant cytological atypia or mitoses are not present (Figure 31). Rare interspersed ciliated cells or even surfactant whorls may be seen (Figure 32). Psammoma bodies and intracellular mucin are absent.

Cytology Transbronchial needle aspiration and imprint material from one case reveals sheets and clusters of cuboidal and columnar cells without cilia or appreciable nuclear atypia against a

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Figure 31. Papillary adenoma. Basally located tumor cell nuclei are round and regular. There is no suggestion of malignancy in this field.

Figure 32. Papillary adenoma. In addition to foamy cytoplasm, luminal surfactant whorls can be seen. This nonspecific finding suggests a type II pneumocyte origin for the tumor.

background of degenerating epithelial cells. Round to oval nuclei demonstrate fine chromatin with occasional nucleoli.138

evaluation is also likely to diagnose adenocarcinoma rather than suggest a benign entity.

Immunohistochemistry

Differential diagnosis

Tumor cells stain positive with antibodies directed against broadspectrum cytokeratins, CEA, TTF-1, surfactant apoproteins A and B and sometimes Clara cell protein (CC-10), indicating both type II pneumocyte and Clara cell differentiation.129,135,139,140 Neuroendocrine markers are consistently negative.141 Tumors have a proliferative index of less than 2%.135,141

Papillary adenoma must be discerned from benign and malignant tumors. Pulmonary and metastatic papillary adenocarcinomas may not always be infiltrative but are always proliferative lesions with cellular crowding and cytological atypia. Mitoses and necrosis are also commonplace. Of note, metastatic papillary thyroid carcinoma features nuclear grooves and thyroglobulin positivity. Papillary carcinoid tumor may be architecturally identical to papillary adenoma, but granular cytoplasm and round regular nuclei with granular chromatin allow distinction. Neuroendocrine immunohistochemical stains (chromogranin and synaptophysin) may be required in rare instances (see Chapter 31). Sclerosing hemangioma is less of a practical consideration given its typical heterogeneous architectural patterns and dual cell population (see below). Papillary cystadenoma, a recently described entity arising in the lung of patients with von Hippel-Lindau disease, enters the differential diagnosis. In this lesion, papillae are lined with bland multilayered epithelial cells with focally clear cytoplasm and nuclear enlargement. Unlike papillary adenoma, this cystadenoma is associated with many microcysts accompanied by fibrous stroma and thin-walled vascular channels.143

Electron microscopy Ultrastructural studies performed on many cases reveal several findings. Tumor cells contain a moderate number of mitochondria, rough endoplasmic reticulum, blunt surface microvilli, as well as various numbers of different-sized osmiophilic lamellar bodies and membrane-bound, electron-dense granules.129–131,133,134,140–142 Intranuclear inclusions are composed of 50–60 nm tubular structures.

Molecular findings Flow cytometric nuclear DNA analysis of tumor cells reveals a diploid pattern and a low S-phase fraction.130

Clinicopathological correlation This rare peripheral lung tumor is asymptomatic but causes both the patient and the clinician anxiety, since a pre-resection diagnosis is virtually impossible. Intraoperative frozen section

Prognosis and natural history Although several case reports suggest that tumor cells infiltrate surrounding alveolar parenchyma and examples of

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Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

lymphatic and venular spread are documented, there are no reports of either metastatic spread to loco-regional lymph nodes or distant sites.129,141 Thus, surgical resection is curative.

Alveolar adenoma Introduction First mistakenly considered a pulmonary lymphangioma in 1974, alveolar adenoma is an exceedingly rare, benign, peripheral lung tumor.144 Although the clinico-pathological qualities are now well-defined, tumor histogenesis is uncertain.

Classification and cell of origin Many opinions regarding the nature of this benign tumor have been offered, including suggestions that the lesion is an epithelial tumor, a mesenchymal tumor, a biphasic tumor or even a sclerosing hemangioma variant.145–152 Although definitive evidence is entirely lacking, the WHO considers alveolar adenoma a benign peripheral lung tumor composed of proliferating alveolar pneumocytes and septal mesenchyme.35 No etiological agents are suggested.

Genetics Cytogenetic studies undertaken on a 6.0 cm tumor allowed for 54 metaphase investigations. Forty-four had normal karyotypes and ten (19%) showed a pseudodiploid karyotype: 46, XX, add(16)(q24). Fluorescence in situ hybridization characterized the add(16)(q24) as der(16)t(10;16)(q23;q24). This non-balanced translocation is of uncertain significance.152

Special clinical features Alveolar adenomas are reported in 39–74-year-olds. A slight female predominance is noted and virtually all individuals are asymptomatic.146,153 Of the less than 30 reported cases, only one suggested multifocality.154 However, in this individual only one of three lesions was surgically resected and histologically studied.

Radiographic findings Chest radiographs almost always demonstrate round, welldefined, isolated, uniform, peripheral and often subpleural shadows or nodules without notching or calcification.144,149,151 One case manifested as a cystic lesion mistaken for a pneumatocele.147 Computed tomograms show non-enhancing, partially cystic lesions while magnetic resonance imaging shows central low signal intensity and a peripheral high signal thin rim enhancement on T1-weighted images, consistent with central fluid accumulation154–157 PET report from a 1.1 cm tumor demonstrated no isotope uptake.158

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Figure 33. Alveolar adenoma. This well-circumscribed tumor features larger cystic spaces in the center of the lesion rather than around the periphery.

Macroscopic pathology Tumors measure from 0.7 to 6.0 cm, and may involve any lung lobe but a left lower lobe predilection is noted.153 Resected lesions are well-demarcated with smooth, lobulated, spongelike, soft to firm pale yellow, gray to tan cut surfaces. Tumors may be predominantly cystic with mural nodules.154

Histopathology Alveolar adenomas are well-circumscribed, unencapsulated, multicystic masses that compress adjacent lung tissue (Figure 33). Connections with the bronchial tree are not observed. Cystic spaces are often larger in the center of the lesion than at the periphery, where they may be more uniform, rigid and cribriform. The ectatic spaces contain periodic acid-Schiffpositive, granular eosinophilic material, foamy histiocytes, hemosiderin and erythrocytes (Figure 34). A single layer of cytologically bland flattened, cuboidal or hobnail cells lines the cysts (Figures 35 and 36). The nuclei are flat to round with even chromatin and inconspicuous nucleoli. Intranuclear pseudoinclusions may be seen. Squamous metaplasia is rare and mucin is absent from these cells. At the edge of the tumor, lesional cells become contiguous with attenuated normal lung pneumocytes. Cellular stratification, budding, micropapillae and cytological atypia are not present and only a rare mitotic figure may be seen. Cystic spaces are formed by alveolar septal-like connective tissue of varying thickness and cellularity (Figure 36). When prominent, the interstitium may compress the epithelial-lined cysts to mere slits or lead to papillary formation. The mesenchymal cells may

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Figure 34. Alveolar adenoma. Ectatic spaces are filled with periodic acid-Schiff-positive material, hemosiderin and foamy macrophages. In areas stroma is inconspicuous.

Figure 35. Alveolar adenoma. Spaces are lined by a single layer of bland cuboidal to columnar cells. Macrophages are apparent.

or myxoid stroma. A case with interstitial mature adipocytes has been reported.147 Collagen and reticulin fibers are present but elastic fibrils are sparse in comparison to normal alveolar interstitium.146 Central scars composed of granulation tissue and blood can be seen.

Cytology A single report describes the intraoperative cytological features of an alveolar adenoma.159 The paucicellular sample featured small clusters of monolayered epithelial cells against a proteinaceous fluid background. Polygonal epithelial cells featured a moderate amount of well-defined cytoplasm and round nuclei with central inconspicuous nucleoli. Macrophages, lymphocytes and mast cells were also noted. While such findings may aid in excluding a diagnosis of malignancy, a diagnosis of alveolar adenoma on cytological preparations is not possible.

Immunohistochemistry Figure 36. Alveolar adenoma. Stroma can be prominent. While bland, the mesenchymal spindle cells often compress cysts and suggest the presence of a papillary proliferation.

be spindled, oval or blunt-ended with open chromatin and inconspicuous nucleoli. These mesenchymal cells are admixed with tortuous capillaries, lymphocytes, plasma cells, occasional mast cells and histiocytes in an often edematous

Lesional epithelial cells stain for broad-spectrum cytokeratin, CAM5.2, cytokeratin 7, CEA, EMA, surfactant proteins and TTF-1 but are negative for Clara cell antigen (CC10), neuroendocrine markers NSE, chromogranin and synaptophysin, desmin, ER and PR. A single case also stained for cytokeratin 20.158 Interstitial cells are vimentin-positive with very focal smoothmuscle actin and muscle-specific actin reactivity.146,160,161 The case with mature adipocytes also reported interstitial S100 protein positivity.147 Several reported tumors reveal proliferation indices of less than 1% in the epithelial and mesenchymal cells.146,149,153,155

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Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Electron microscopy Ultrastructural examinations confirm the pneumocytic nature of the epithelial cells.146,149,151 Compact hobnail cells feature blunt surface microvilli, intracytoplasmic lamellar bodies and thin basement membrane. Interspersed flat cells with attenuated cytoplasm characteristic of type I pneumocytes are also seen. Cell attachments of the zonula adherens type are noted. The interstitium contains fibroblasts and inflammatory cells embedded in a collagenous stroma.146,162

Molecular findings Flow cytometric nuclear DNA analysis of tumor cells reveals a diploid pattern and a low S-phase fraction.149,153 Laser capture microdissection of both epithelial and mesenchymal elements from four alveolar adenomas revealed different findings. One alveolar adenoma demonstrated microsatellite and loss of heterozygosity alterations only in the epithelial component. Loss of heterozygosity was noted at four of 13 sites (chromosome 4 (MH34 and SHGC4), 5q22–23 (MCC), and 17 (p53)) while microsatellite alterations were seen at three additional sites (chromosomes 9p22 (p16), 11q24–25, and 17p13 (p53)). The interstitial mesenchymal cells featured the same molecular pattern as control lymphocytes. These findings from this case suggest the epithelial and mesenchymal components do not arise from a common progenitor cell.147 However, allelic losses were not identified in three additional cases investigating markers adjacent to MYCL-1, MCC, p16, PTEN and p53.163

Clinicopathological correlation Alveolar adenoma is a rare entity unlikely to be diagnosed on anything less than a surgical resection specimen.146 Preoperative percutaneous needle biopsies demonstrate uniform spindle cells, suggesting a diagnosis of hamartoma or a nonspecific mixture of histiocytes and bland spindle cells.150,162 As discussed above, intraoperative cytological findings are nonspecific. With increased use of improved imaging modalities, one wonders whether greater numbers of this lesion will be seen.

Differential diagnosis The differential diagnosis includes developmental and neoplastic entities. Congenital cystic adenomatoid malformation is rarely encountered in adults and features immature and malformed lung with interstitial fibrosis and honeycomb change (see Chapter 3). Atypical adenomatous hyperplasia and non-mucinous AIS are never discrete nodules, feature lepidic growth of atypical pneumocytes and lack a spindle cell component. Rare pulmonary lymphangiomas can only be discerned on the basis of immunohistochemical studies; lymphangioma lining cells are D240 and factor VIII positive, while cytokeratins and TTF-1 are negative. Hamartomas may be considered, especially in cases with a prominent mesenchymal component, yet alveolar adenoma is a multicystic lesion,

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lacking cartilage and ingrowth of bronchiolar epithelium along its periphery. Sclerosing hemanigoma features varied architectural patterns and two different epithelial cell populations (see below). Metastatic spindle cell sarcomas, including endometrial stromal sarcoma and benign metastasizing leiomyoma, often feature cystic change, but these neoplasms lack the multicystic architecture of alveolar adenoma and epithelial lining cells (see Chapter 33).

Prognosis and natural history Alveolar adenomas are benign slow-growing tumors without apparent metastatic potential. Several unresected lesions did not grow over a 15 month observation period.154 Surgical excision is curative.

Sclerosing hemangioma Introduction While papillomas, papillary adenoma and alveolar adenoma are rare epithelial neoplasms, no uncommon lesion generates as much interest as sclerosing hemangioma. Although the clinical, morphological and immunohistochemical aspects of the tumor are well described, these features, as well as tumor histogenesis, remain the subjects of an overwhelming number of primary journal publications around the world. Before their introduction as “sclerosing hemangioma” in 1956, these tumors were reported as “pulmonary histiocytomas”, “plasma cell granulomas”, “xanthomatous pseudotumors” and “alveolar angioblastomas”.164,165

Classification, cell of origin, pathogenesis and etiology The original investigators noted morphological similarities between the lung tumor and sclerosing hemangioma of the skin, and thus proposed an endothelial cell origin for the lesion.164 Evidence suggesting mesenchymal, mesothelial and neuroendocrine cell origins followed in subsequent years.165–169 Current scholarship proposes sclerosing hemangioma is a clinically benign neoplasm, rather than a hamartoma, and originates from primitive respiratory epithelium.169–174 The evidence supporting this claim derives from immunohistochemical, ultrastructural and molecular findings.163,170,173,175–179 Estrogen and progesterone receptors on at least a percentage of sclerosing hemangioma tumor cells suggest a possible etiological role, i.e. a stimulatory role, for these hormones in tumor development.180–182 Aberrant mammalian target of rapamycin (mTOR) signaling may also feature in the development of this tumor. As proposed, elevated vascular endothelial growth factor (VEGF) levels secondary to mTOR signaling dysregulation might partially explain tumor vascularity.183 The WHO classification retains the term “sclerosing hemangioma” for historical reasons, although pneumocytoma or

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

variations thereof would more accurately reflect the cellular origin of the neoplasm.35,184

Genetics Two case reports noting an association between sclerosing hemangioma and familial adenomatous polyposis (FAP) might not seem worthy of inclusion in a textbook; however, several aspects of these cases and other studies suggest a possible link between the two conditions.185,186 FAP patients have germline mutations in the APC gene at 5q21, upregulated Wnt signaling, and nuclear and cytoplasmic β-catenin is an oncogenic transcriptional activator for Wnt signaling. The lung sample from one of the FAP patients contained morular lesions, histomorphologically similar to lesions noted in FAPassociated thyroid carcinomas.187 Also, as is the case with FAP-associated intestinal adenomas, adenocarcinomas and thyroid carcinomas, aberrant expression of nuclear and cytoplasmic β-catenin was demonstrated in sclerosing hemangioma cells and morular lesions, as well as other sclerosing hemangiomas.185,186,188,189 Finally, 67% of sclerosing hemangiomas in one study demonstrated allelic loss for microsatellite marker D5S.615 on chromosome 5q, adjacent to the tumor suppressor gene APC, suggesting the APC gene may play a role in tumor pathogenesis.163 Perhaps a subset of patients with sclerosing hemangioma have an attenuated FAP phenotype.

Special clinical features Over 250 reported cases of sclerosing hemangioma delineate the clinical features of this tumor. While exceedingly rare in the Western hemisphere, it is the second commonest benign lung tumor in East Asia, with approximately as many cases a year as typical carcinoid tumor.190 The tumor is much commoner in women than men (almost a 5:1 ratio) and affects all age groups, ranging from 4 years of age through to the ninth decade of life.164,170,191–193 The mean age of afflicted individuals is 46 years. Most patients are non-smokers. Patients are usually asymptomatic but up to 20% complain of chest pain or cough or experience hemoptysis.170,192 Location and size of the tumor determine symptoms.

Radiographic findings Radiographs demonstrate that greater than 95% of patients have solitary peripheral lung nodules, while the remainder have multiple lesions.194–196 Lesions are almost always either slow-growing or stable on follow-up radiographs for many years.195,197,198 Solitary, well-circumscribed lesions on chest radiographs have been reported to occupy an entire hemithorax and develop within an extralobar sequestration.199,200 Rare tumors are noted in interlobar fissures, visceral pleura and mediastinum.201–203 Computed tomograms demonstrate round to oval nodules with smooth edges; lesions enhance with intravenous contrast and calcifications are often noted. Peritumoral inhomogeneous enhancement

Figure 37. Sclerosing hemangioma. This cream-tan firm 2.9 cm solitary tumor compresses surrounding lung parenchyma. Calcium specks are apparent on the cut surface. A carcinoid tumorlet is adjacent to the tumor (arrowheads).

may be seen.204–207 Magnetic resonance imaging studies reveal mixed areas of high and low signal intensity on both T1- and T2-weighted images.208,209 Several PET studies demonstrate intermediate-level, standardized uptake values of 18 F-fluorodeoxyglucose.186,210,211

Macroscopic pathology Sclerosing hemangiomas are usually intrapulmonary masses, ranging from 0.3 to 20 cm in greatest dimension.170,192,193 Most tumors are less than 3.0 cm in greatest dimension, although tumors as large as 20 cm are reported in individuals who were followed for up to 47 years prior to surgical resection.193,200,212 Solitary well-defined homogeneous masses show no lobar predilection. Endobronchial and endobronchiolar extension are more commonly seen than pure endobronchial growth.170,193,213,214 Visceral pleural and interlobar fissure involvement is infrequent.170,192 Most mediastinal lesions represent extension of lung tumors yet bona fide primary mediastinal tumors occur. Pericardial adherence has also been reported.164,202,215 While the great majority of lesions are well-circumscribed solid tumors with a thin fibrous pseudocapsule, tumors may be entirely cystic, either uni- or multiloculated, and mimic hydatid cysts radiographically.216,217 Cut surfaces reveal bulging tumors with variegated appearances. Solid to partially cystic masses range from gray to tan-yellow to focally mottled red-brown with a fleshy, rubbery or firm consistency (Figure 37). Cut surfaces may have a gritty or granular consistency, secondary to punctate calcifications.

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Figure 38. Sclerosing hemangioma. The unencapsulated tumor demonstrates a variegated low-magnification architecture. Papillary, solid and sclerotic areas are apparent.

Multiple sclerosing hemangiomas manifest with several different gross patterns but the individual lesions are indiscernible from solitary tumors. Numerous smaller satellite lesions may surround a dominant tumor or multiple small lesions may aggregate to form one or more coin lesions. These patterns produce radiographic consolidations, while rare instances of ipsilateral and/or bilateral lung involvement raise the specter of metastatic disease.170,194,218–220

Histopathology Unencapsulated well-demarcated tumors are composed of two neoplastic cell populations, arranged in up to four distinct architectural patterns (Figure 38). While each tumor is particular, characteristic features are noted in all. More than 50% of tumors feature papillary, sclerotic, solid and hemorrhagic areas, while most of the remainder have three of these patterns (Figure 39). No more than 5% are composed of only two patterns and no tumor has only one pattern. The two types of neoplastic cells are cuboidal or “surface” and round or “stromal”. The cuboidal tumor cells line papillae and cysts and may form tubules, whereas round cells fill papillary cores and form sheets in solid areas (Figure 40). The papillary pattern is seen in most cases and is also most plentiful in these tumors. In a sense, the other architectural patterns are variants of this form. This morphology features closely packed projections of cellular or hyalinized stalks lined by the cuboidal cells. These cells approximate type II pneumocyte morphology, i.e. cuboidal with voluminous eosinophilic cytoplasm, dark round nuclei and occasional intranuclear

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inclusions. Vacuolated cytoplasm, intracytoplasmic hemosiderin, multinucleation, and mild to moderate cytological atypia may be seen (Figure 40). The papillary stalks are often filled with the second tumor cell type, the round cell. These round cells are slightly smaller than the cuboidal cells and have indistinct cell borders, abundant pale eosinophilic or clear cytoplasm, and centrally located bland nuclei with fine dispersed chromatin. Nucleoli are not obvious. On occasion, cytoplasmic vacuoles create a signet-ring type morphology (Figure 40). Some cells contain glycogen but not mucin. Papillae need not contain round cells but may be sclerotic with foci of calcification. Psammoma-like bodies may be seen. Dense eosinophilic sclerotic papillae often aggregate into large sheets and entrapped airways and alveolar walls may bear superficial resemblance to invasive carcinoma. Sclerotic regions always abut papillary, solid or hemorrhage areas. The so-called solid pattern features sheets or nests of round cells. These sheets of cells may feature only clear cytoplasm. Occasional cells may have pyknotic nuclei or hyperchromasia but mitotic figures are absent. Round cells spilling out from papillary cores may account for this solid morphology. Hemorrhagic areas feature large dilated blood-filled spaces or irregular clefts with erythrocytes mimicking true vascular channels. Such areas superficially resemble cavernous hemangiomas. Single layers of cuboidal cells usually line these spaces, while intervening stroma contains closely packed round cells. This pattern probably develops from large distorted papillae. Many secondary findings may be seen in addition to the heterogeneous tumor growth patterns. Scattered lymphocytes, plasma cells, mast cells and esoinophils may be found amidst the cellular and sclerotic tumors. Clusters of xanthomatous histiocytes along with cholesterol clefts fill distorted airspaces but tumor cell necrosis is quite uncommon (Figure 41). On occasion one sees lamellar whorls of extracellular surfactant (Figure 41). Intratumoral mature adipose tissue is an infrequent finding (Figure 42).192 A peripheral granulomatous reaction and even intratumoral collections of multinucleated giant cells have been noted.221 This finding is probably a sarcoidosis-like reaction to tumor (see Chapter 13). Given the striking range of tumor variability, it should not be surprising that rare sclerosing hemangiomas demonstrate additional unusual morphological patterns and associations. A socalled cystic pattern features practically empty cystic spaces along with thin septa and solid areas. The septa and solid areas contain the two tumor cell types.170,196,216,222 This is probably a degenerative phenomenon. Very rare cases of sclerosing hemangiomas with focal areas resembling alveolar adenoma and papillary adenoma have also been reported and illustrated.150,193,194,196,223,224 Exceedingly rare reports of atypical adenomatous hyperplasia and mucinous metaplasia or hyperplasia adjacent to or associated with sclerosing hemangiomas are also noted but no associations with carcinomas are known.193,194,223,224 Infrequently, sclerosing hemangiomas are associated with neuroendocrine proliferations ranging from neuroendocrine cell hyperplasia to carcinoid tumorlets and typical carcinoid

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

(a)

(b)

(c)

(d)

Figure 39. Sclerosing hemangioma. The majority of tumors feature four architectural patterns. (a) Papillary fronds are the most plentiful. (b) Sclerosis with calcification is likely a degenerative phenomenon. (c) The solid pattern may cause diagnostic uncertainty. (d) Hemorrhagic areas resemble true vascular channels.

tumor (Figure 43).167,169,170,192,196,224,225 These lesions may be mixed or separate micronodules in patients with many radiographic abnormalities. The neuroendocrine proliferations may be hyperplastic, secondary to tumor encroachment on airways, or represent differentiation of primitive respiratory cells toward a neuroendocrine phenotype (see below).

When sclerosing hemangiomas are multiple, tumors may have varying histologies.218,220 With regard to small satellite lesions, one sees mostly papillary areas with little solid or hemorrhagic and no sclerotic architectural patterns.170,223,224 The cubodial (surface) cells may predominate in these incipient lesions.170,192,223

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Figure 40. Sclerosing hemangioma. Tumors are composed of two distinct cell types. The “surface” or cuboidal cells line papillae and pseudovascular channels and form tubules. These resemble type II pneumocytes with eosinophilic cytoplasm, round nuclei and scattered intranuclear pseudoinclusions. Clear cell change is not uncommon. “Stromal” or round cells fill papillae and proliferate in sheets. These have indistinct cell borders, lighter cytoplasm and irregular nuclei with vesicular chromatin. Occasional cytoplasmic vacuoles suggest signet-ring morphology.

Figure 42. Sclerosing hemangioma. Intratumoral adipose tissue probably represents a stromal metaplasia. Note the scattered “stromal” or round cells.

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Figure 41. Sclerosing hemangioma. Cholesterol clefts, multinucleated giant cells and surfactant bodies are not uncommon secondary findings. Sheets of “stromal” or round cells are apparent.

Figure 43. Sclerosing hemangioma. This sclerosing hemangioma demonstrates an intimate association with a typical carcinoid tumor. Papillary fronds lined by “surface” or cuboidal cells are filled with “stromal” or round cells in the upper left, while the spindle cell carcinoid tumor is apparent in the right lower area. The tumors may be more closely related than we currently believe.

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

(a)

(b)

Figure 44. Fine-needle aspirate of sclerosing hemangioma. This H&E-stained sample features (a) a papillary arrangement of several “surface” or cuboidal cells and (b) a sheet of “stromal” or round cells with indistinct cytoplasm. Two “surface” or cuboidal cells are seen on the far left. Note the larger size of these in comparison to the “stromal” or round cells.

Sclerosing hemangiomas may rarely breach the visceral pleura or metastasize to locoregional lymph nodes.203,226 Lymph node metastases are predominantly microscopic findings although focal subcapsular white areas may be noted macroscopically.226 Rarely, extracapsular extension may be observed. Metastases can feature mixtures of all four tumor patterns and both types of tumor cells. Metastases may demonstrate different proportions of growth patterns, as compared with the primary lesions, and solid growth is common in metastases.196,226 Not unlike the lung lesions, metastases have bland cytology. The obvious presence of cuboidal/surface cells in intrapulmonary and lymph node metastases supports the contention that this cell type is neoplastic.170,223 Intraoperative frozen section diagnosis of sclerosing hemangioma is possible if one appreciates the macroscopic and microscopic circumscribed nature of the tumor and identifies at least three architectural patterns.227 Imprint cytology may be helpful but cytological atypia may force one to defer a diagnosis (see below).227–230 Requesting additional tumor fragments when one is considering this diagnosis may allow for a definitive diagnosis. Intraoperative differential diagnoses include but are not limited to adenocarcinoma and typical carcinoid tumor.227,231

Cytology Most cytological descriptions of sclerosing hemangioma are based on case reports. Unfortunately, almost all reports are fine-needle aspirate (FNA) or intraoperative touch imprint

cases and many of the cases were not initially recognized as sclerosing hemangioma. Bronchial washings of an endobronchial tumor demonstrated moderately cellular, non-necrotic smears with separate clusters and sheets of the two distinct cell populations.213 Clusters of small to medium cells with oval to round nuclei, evenly dispersed chromatin, inconspicuous nucleoli and scanty cyanophilic cytoplasm (round cells) separate from sheets of round to polygonal cells with centrally located nuclei, coarse chromatin, and eosinophilic cytoplasm and occasional well-defined borders (cuboidal cells). Fine-needle aspirate samples also feature the two cell populations (Figure 44). However, the so-called cuboidal cells may appear spindled and may be arranged in a crisscross pattern (Figure 45).232 Tumor cells may be admixed with hyalinized stromal tissue fragments or even form acini. Intranuclear inclusions can be seen in the cuboidal cells.172 These aspirates also contain blood, foamy macrophages, multinucleated giant cells, and clumps of hemosiderin, and sometimes orange lamellar concretions are identified.233,234 Cell blocks often demonstrate more than one of the four architectural patterns, suggesting the diagnosis.233,235 Intraoperative cytology smears range from hypocellular to hypercellular with varying amounts of background blood, hemosiderin and foamy macrophages.227–229 Papillary and solid architectural patterns are common, while sclerotic and hemorrhagic patterns are infrequent. Papillary structures lined by cubodial cells and solid sheets of round to polygonal cells may demonstrate mild cytological atypia. Significant cytological atypia is rare.236

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Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung Table 2 Immunohistochemical profile of sclerosing hemangioma

EMA

Cytokeratin

CAM5.2

CK7

CK20

CEA

TTF-1

ProSpA

ProSpB

Clara cell protein

Surface/cuboidal

þ

þ

þ

þ



/þ

þ

þ

þ

þ/

Round/stromal

þ



/þ

/þ





þ







Notes: þ denotes positive,  denotes negative, þ/ denotes more likely positive than negative, /þ denotes more likely negative than positive.

Figure 45. Fine-needle aspirate of sclerosing hemangioma. The “surface” or cuboidal cells in this H&E-stained sample form an irregular three-dimensional cluster. Such a pattern probably represents a compressed papillary frond devoid of “stromal” or round cells.

Astute pathologists may on occasion render a diagnosis of sclerosing hemangioma on fine-needle aspiration (FNA) or transbronchial needle aspiration samples. While the presence of blood and round cells, and absence of necrosis are helpful features, attention to the polymorphous nature of the samples and bland tumor cytology is necessary.236 The cytological differential diagnosis is essentially limited to adenocarcinoma and typical carcinoid tumor. Such a diagnosis greatly depends on the quantity of the sample and quality of the staining.172,234

Immunohistochemistry The immunohistochemical profile of sclerosing hemangioma is not only helpful in diagnosis, but also clarifies tumor histogenesis (Table 2). Both cell types are EMA and TTF-1 positive (Figure 46). The cuboidal cells also stain for pancytokeratin, surfactant proteins A and B and Clara cell protein, while the round cells are usually negative for these markers (Figure 46).170,175,177,179,181,196,212,237,238 Only a small percentage of the round cells stain for cytokeratin 7 and Cam5.2. TTF-1, a critical determinant of lung development present in fetal terminal airways as early as 11 weeks gestation, is expressed in both

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neoplastic cells. This finding suggests a lung origin for both cell types.239 The fact that surfactant proteins, first expressed by fetal lung between gestational weeks 15 and 18, are only expressed in the cuboidal/surface cells but not in the round/stromal cells suggests that the round/stromal component is a more primitive respiratory epithelial cell than the cuboidal/surface cell.238–243 It is likely these tumor cells differentiate divergently from a common progenitor cell.170,175 Neuroendocrine findings in this tumor have been a contentious issue in recent years. Although one research group reported significant neuroendocrine marker positivity in many cases and suggested that the tumor should be renamed benign neuroendocrine tumor of the lung, others only note scattered individual cells positive for synaptophysin or chromogranin A.167,170,244 These cells may be entrapped non-neoplastic bronchial epithelial cells or entwined carcinoid tumorlets.170,181,196,223,224 However, neuroendocrine staining of true tumor cells might represent individual cell differentiation toward a neuroendocrine phenotype. Sex steroid hormone receptor expression in sclerosing hemangioma is an intriguing finding. Although several investigators failed to demonstrate either estrogen or progesterone receptors via immunohistochemistry or enzyme immunoassays, more recent studies demonstrate strong progesterone receptor protein labeling in most if not all round/stromal cells.170,179–181,245,246 Estrogen receptor-β was also expressed in over 90% of cases in one study and in a minority of cases in others.170,180,182,245 These positive findings suggest a role for these hormones in the pathogenesis of the tumor and might explain the striking gender difference.

Electron microscopy In keeping with the plethora of publications regarding the histogenesis of sclerosing hemangioma, many ultrastructural studies report a variety of observations. The suggestion that neoplastic cells contain Weibel-Palade bodies has not stood the test of time and late twentieth-century as well as early twenty-first-century studies indicate that both tumor types are epithelial.170,171,238,247–252 The surface or cuboidal cells feature abundant apical and lateral microvilli, poorly formed desmosomal junctions, abundant rough endoplasmic reticulum, mitochondria, glycogen, and lamellar bodies.168,170,177,237 These findings indicate type II pneumocyte derivation. The stromal or round cells are smaller with fewer intracytoplasmic organelles. In addition to few mitochondria and sparse rough endoplasmic reticulum, lipid vacuoles, microtubules and scattered electron-dense bodies (dense core granules) of varying sizes are noted.167 Lamellar bodies are absent in this cell type. These cells

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Chromogranin

Synaptophysin

ER

PR

CD34

Factor 8

S100 protein

VIM















/þ



/þ

/þ

þ/







þ/

Data from references 167,170,175,177–182,196,212,238,244–246.

(a)

(b)

Figure 46. Immunohistochemical staining of sclerosing hemangioma. (a) Both tumor cells stain with anti-TTF-1 antibodies while (b) only the “surface” or cuboidal cells stain with anti-cytokeratin antibodies.

are thus likely to be primitive respiratory epithelial cells.237,248,250 These dense core granules represent neurosecretory granules or immature lamellar bodies. Even if they indicate neuroendocrine differentiation, the quantities are not abundant enough to suggest that this cell is a neuroendocrine cell, rather than a primitive multipotential epithelial cell.170,237

Molecular findings Clonal analysis studies, based on the human androgen receptor gene, the phosphoglycerate kinase gene, as well as p16 and Rb loci, demonstrate that both tumor cell types show the same loss of alleles or an unbalanced methylation pattern, i.e. monoclonality. These findings confirm the neoplastic nature of the tumor and indicate that both cells derive from a common precursor.173,176,253 The p16 and Rb allelic losses also suggest a possible link between this clinically benign tumor and early lung adenocarcinoma, i.e. AIS. In fact, sclerosing hemangioma and AIS share several patterns of allelic loss for microsatellite markers located on chromosomal regions with tumor suppressor genes MYCL-1, MCC/APC, PTEN as well as p16 and Rb.163,176,183 These findings, while nonspecific, are fascinating given that both sclerosing

hemangioma and AIS usually affect middle-aged, non-smoking Asian women. However, p53 protein expression and gene mutations are less common in sclerosing hemangioma, compared to AIS. Data demonstrating normal EGFR and HER2 gene copy number with FISH, and the lack of EGFR, HER2 and K-RAS mutations by direct sequencing suggest major differences between sclerosing hemangioma and lung adenocarcinoma, including AIS.163,176,253 Proteomic analysis of 12 tumors identified five downregulated proteins including antioxidant proteins, peroxiredoxin II and glutathione S-transferase. The same authors also reported increased expression of matrix metalloproteinase 9 and tubulin-α in a subsequent study.190,254 Such findings foreshadow future developments in tumor research.

Clinicopathological correlation Interestingly, approximately 2 to 4% of patients with sclerosing hemangioma are found to have hilar or mediastinal lymph node metastases at the time of surgical exploration and resection.170,192,193,226,255,256 A single report of pleural dissemination is also noted.257 This cohort ranges in age from 10 to 67 years and tumors range from 1.5 to 9 cm in greatest

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dimension. Most nodal metastases involve peribronchial or hilar stations and mediastinal lymph node involvement is rarer.169,170,193,196,212,226,255,256,258 While this metastatic potential suggests that sclerosing hemangiomas have malignant potential, these tumors are clinically benign since neither parenchymal metastases nor direct tumor mortality have been reported.

Differential diagnosis Numerous malignant and benign primary pulmonary and metastatic carcinomas enter the morphological differential diagnosis. This differential is not trivial as almost half of cases submitted for expert consultation in one large series carried incorrect diagnoses.192 Since lung adenocarcinomas may be well circumscribed with pure papillary or mixed papillary and solid architecture, bland nuclei and intranuclear inclusions, one should predicate a diagnosis of sclerosing hemangioma on the presence of at least two if not three architectural patterns, in addition to identifying the two cell populations. Both metastatic papillary thyroid carcinoma and the papillary variant of mucoepidermoid carcinoma lack additional architectural patterns while the latter also features an intimate admixture of squamous and mucinous cells, not seen in sclerosing hemangioma. Although TTF-1 will not discriminate metastatic papillary thyroid carcinoma from sclerosing hemangioma, thyroglobulin may. This stain may be useful since both papillary thyroid carcinoma and sclerosing hemangioma may demonstrate nuclear pseudoinclusions and psammoma bodies. Typical carcinoid tumor, including the papillary subtype, lacks architectural variety and features only one cell type with round regular nuclei and finely granular chromatin. Strong chromogranin and synaptophysin staining are also seen in typical carcinoid tumors but not in sclerosing hemangiomas. As discussed in the histopathology section, this distinction may be impossible on intraoperative frozen section. In this setting, identifying three architectural patterns in the well-circumscribed sclerosing hemangioma suggests the correct diagnosis. When uncertain, however, a definitive diagnosis should be deferred since an erroneous interpretation of typical carcinoid tumor will lead to an unnecessary lobectomy and lymphadenectomy.231,259 Clear cell tumors such as metastatic renal cell carcinoma and clear cell “sugar” tumor of lung (CCTL) may mimic sclerosing hemangioma round/stromal cells, but these tumors feature sinusoidal vascular patterns and cytological atypia, rather than blood lakes, and the bland two cell population. Immunohistochemical stains, including RCC and CD10 for renal cell carcinoma and HMB-45 for CCTL, are also useful. Exceedingly rare papillary adenoma and alveolar adenoma can also lead to diagnostic difficulties. While classic cases of these tumors lack the variegated patterns and two types of epithelial cells characteristic of sclerosing hemangioma, the focal presence of papillary adenoma-like and alveolar adenoma-like regions within sclerosing adenomas cannot be discounted. These similarities have led some to suggest that

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the lesions are simply morphological expressions of a single primitive tumor. Lastly, one should also exclude the possibility that small sclerosing hemangioma lymph node metastases are not benign mesothelial inclusions. While both tumor cells and mesothelial cells are cytologically bland, only the mesothelial cells stain with calretinin.

Prognosis and natural history Sclerosing hemangioma is a clinically benign neoplasm. Neither parenchymal metastases nor deaths have been reported, even as lesions have been clinically followed for up to 47 years before surgical removal.195,197,200,233 Nevertheless, the tumor may invade visceral pleura, disseminate in the pleural cavity, can be multifocal in up to 4% of cases, and metastasizes to locoregional lymph nodes in up to 4% of patients.170,193,195,203,218–220,226,257,258 Surgical removal is considered curative, even though a single case report noted a staple line recurrence 10 years following the original wedge resection.260

Pulmonary hyalinizing granuloma Introduction Pulmonary hyalinizing granuloma (PHG) is a peculiar fibrosing process that garners the attention of internists, radiologists and pathologists owing to its many clinical and pathological associations. Although well documented in the world literature, virtually all PHG publications are case reports. The term granuloma is a misnomer, since epithelioid cells are not part of the lesion and giant cells are almost never seen. This entity mimics metastatic carcinoma clinically and radiographically, and unfortunately requires excision for diagnosis.

Clinical details, including epidemiology and etiology Pulmonary hyalinizing granulomas usually afflict adults with a mean age in the fifth decade of life. Teenagers and septuagenarians are not spared and a male predominance is noted.261–263 Patients complain of cough, shortness of breath, chest pain and infrequently hemoptysis, dysphagia, fatigue, weight loss, sinusitis or sore throat. Twenty-five percent of patients are asymptomatic.263–265 The etiology is not known. An association with tobacco use is not seen but associations with other fibrosclerosing entities and immune phenomena are well documented, suggesting an underlying immunological process. Almost one-fourth of reported series observe concomitant sclerosing mediastinitis, retroperitoneal fibrosis, or systemic idiopathic fibrosis.261,263,266–268 Identical lesions involving the tonsils, larynx, kidneys or subcutis have also been reported.261,269,270 These diseases may precede, occur at the same time, or follow the onset of pulmonary lesions.271 Cases are reported in patients with multiple sclerosis, human immunodeficiency virus/acquired immunodeficiency syndrome following highly active antiretroviral therapy (immune reconstitution inflammatory syndrome), Castleman disease,

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

idiopathic thrombocytopenic purpura, posterior uveitis, Morvan syndrome (Morvan fibrillary chorea – neuromyotonic discharges, cramping, weakness, pruritus, hyperhidrosis, insomnia and delirium), multiple myeloma and small lymphocytic lymphoma of the lung.272–277 In addition, a myriad serological associations have also been reported in up to 60% of patients. Elevated titers to antinuclear, anti-smooth muscle, antimicrosomal and antithyroglobulin antibodies and increased levels of rheumatoid factor lead the list.263,268,278 Also, patients with Coombs’positive hemolytic anemia, lupus anticoagulant, antineutrophil cytoplasmic antibodies and polyclonal hypergammaglobulinemia, as well as elevated interleukin-2, interleukin-6 and C-reactive protein levels, are noted.266,272,277,279–282 Circulating immune complexes have also been identified in some instances.263,278 Whether autoimmunity is a pre-existing and predisposing factor or occurs after development of the disease is unknown. Only rare patients have positive tuberculin, histoplasmin or coccidioidin skin tests.263,281

Figure 47. Pulmonary hyalinizing granuloma. Tumors are often multiple. Individual lesions are well-circumscribed, firm and white-tan.

Genetics No information is available.

Radiological details Chest radiographs usually demonstrate randomly distributed, bilateral, multilobar nodular lesions with well-defined borders measuring less than 4.0 cm.261,283–285 Pulmonary infiltrates are rarely noted.263,286 Up to one-third of cases are unilateral and most of these are solitary.263 Coarse calcifications are occasionally seen but cavitation is rare.281,287 Serial radiographs may reveal slow growth.263 Computed tomography shows homogeneously enhancing, well-demarcated, slightly irregular nodules.282,284,288 Ground-glass opacities and peribronchovascular interstitial thickening surround many lesions and locoregional lymph nodes are often enlarged.282 Gallium scans show uptake in lesions while 18F-fluorodeoxyglucose positron emission tomography may show increased uptake.262,267,288,289

Macroscopic pathology They are sharply circumscribed firm white-tan rubbery masses, which range from 0.5 to 15 cm in greatest dimension with a mean size of 3.0 cm (Figure 47).261–263 Lesions usually “shell out” from surrounding lung.

Histopathology At low magnification, lesions are characterized by fairly wellcircumscribed nodules of irregular haphazard or whorl-like dense eosinophilic lamellar collagen. Normal lung parenchyma is essentially obliterated by these so-called storiform or parallel collagen arrangements (Figure 48). Lesions may have a zonal quality. Their centers are paucicellular, while the edges contain lymphocytes, plasma cells with germinal centers and scattered spindle cells.

Figure 48. Pulmonary hyalinizing granuloma. Haphazardly arranged lamellar collagen obliterates large areas of lung parenchyma. Entrapped airways with bronchus-associated lymphoid tissue are all that remain of the native lung.

Lymphoplasmacytic infiltrates in the center of lesions are mostly confined to perivascular and peribronchial/bronchiolar sheaths but retraction artifact between collagen bundles may contain inconspicuous spindle cells, sparse T-lymphocytes, polytypic plasma cells, karyorrhectic nuclei or microscopic calcification (Figure 49). Broad areas of necrosis or calcification are rare. Collagen surrounds entrapped small blood vessels and compresses airways. Larger blood vessels within the lesions may be overrun by collagen and only apparent on elastic tissue stains. Remaining arteries feature obvious intimal fibroelastosis and medial hyalinization without fibrinoid necrosis. The tumoral interface with lung parenchyma may be rounded or irregular. In addition to germinal centers and

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Clinicopathological correlation Pulmonary symptoms are due to location of the mass(es). Central masses may compress large airways, leading to obstructive symptoms. Peripheral lesions are likely to be silent unless very large. Fatigue and weight loss suggest the presence of a systemic illness perhaps related to altered immune status. Identification of multiple bilateral lung nodules raises the possibility of multifocal lung cancer or metastatic disease. Unfortunately, neither bronchoscopic nor CT-guided needle aspirates nor core biopsies can provide a definitive diagnosis. In fact, lesional tissue is more likely to be considered nonspecific fibrosis rather than suggestive of this rare entity. One should not be comfortable with a diagnosis of PHG on anything less than a completely removed lesion.

Differential diagnoses

Figure 49. Pulmonary hyalinizing granuloma. Collagen bundles may be separated by edema, lymphocytes and plasma cells.

scattered inflammatory cells, bronchovascular structures abutting the lesion demonstrate peribronchiolar inflammation. In most cases arterioles at the edge have transmural lymphoplasmacytic infiltrates without fibrinoid necrosis. Adjacent pulmonary parenchyma often features organizing pneumonia and diffuse hyperplasia of the bronchus-associated lymphoid tissue. Overlying visceral pleura may be thickened and adherent.

Cytopathology No information is available.

Immunohistochemistry No information is available.

Electron microscopy The hyaline lamellae are composed of electron-dense, compact, amorphous material and swollen collagen fibrils. Fibrillar amyloid is not seen. Spindle cells contain fibroblastic and myofibroblastic ultrastructural organelles.269,290,291

Pathogenesis Clinical and morphological associations with fibrosing mediastinitis and idiopathic retroperitoneal fibrosis suggest that these entities share a common pathogenesis. Many documented immunological abnormalities in PHG patients further suggest an autoimmune process. An exaggerated immune response appears likely in susceptible individuals, but inciting antigens are not known. Elevated patient serum or tumor IgG4 levels have not been reported.292,293

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Clinicopathological correlation is required for many entities in the differential diagnosis. Fungal infections, especially histoplasmosis and coccidiomycosis, may show hyaline fibrosis. In fact, old granulomas may become incorporated into the fibrosis. This fibrosis has parallel bundles rather than the haphazard arrangements seen in PHG. Clinical history, hilar lymph node calcification on chest radiographs and necrotizing granulomatous inflammation on tissue samples all favor an infectious etiology. Inflammatory myofibroblastic tumors are usually seen in young individuals with solitary nodules. This lesion is far more cellular than PHG, with spindle cells, lymphoplasmacytic infiltrates and foamy histiocytes (see Chapter 33). Intrapulmonary solitary fibrous tumors are also single lesions and more cellular than PHG. This collagen-rich mesenchymal tumor, unlike PHG, also features storiform and hemangiopericytomatous architectural patterns (see Chapter 33). Nodular amyloid may bear a morphological resemblance to PHG; however, the waxy amorphous eosinophilic material also features giant cells, metaplastic bone and cartilage, as well as Congo red positivity (see Chapter 34).

Prognostic factors and natural history The prognosis of PHG is uncertain but appears to depend at least in some part on the extent of disease. Individuals with solitary lesions appear cured by surgical resection.263 Almost one-half of patients with bilateral multifocal disease experience progressive radiographic enlargement and/or coalescence of lesions with increasing shortness of breath. Recurrences following complete surgical extirpation are rare.263 Individuals with progressive or recurrent disease often have a long and protracted clinical course. Those with associated sclerosing mediastinitis, retroperitoneal fibrosis or other fibrosclerosing entities often succumb to those more infiltrative processes. Case reports of lesional regression and clinical improvement following glucocorticoid therapy are noted.265,267,276,280

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Pulmonary endometriosis Introduction Although endometriosis affects up to 15% of women of childbearing age, extrapelvic disease is decidedly rare. Recognized in the early twentieth century, pleuropulmonary endometriosis continues to hold the attention of pulmonologists 100 years later.294,295 Pulmonary disease is usually discussed in combination with pleural endometriosis under the term “thoracic endometrial syndrome”.296,297 This constellation includes four recognized clinical and radiographic entities, namely, catamenial pneumothorax, catamenial hemothorax, catamenial hemoptysis and lung nodules. However, different clinical and pathogenetic aspects allow separation between the pleural and pulmonary diseases. Pleural endometriosis is discussed in Chapter 36.

Clinical details, including epidemiology and etiology While pulmonary endometriosis is most often diagnosed in women in their third to fourth decades of life, ages range from the teenage years through to the eighth decade.297–299 Cases have been reported in postmenopausal women on hormone replacement therapy, as well as in women who did not receive hormone replacement therapy after hysterectomies and oophorectomies.300,301 Pulmonary endometriosis is diagnosed in women with symptomatic pelvic endometriosis, but this is not a prerequisite.297,302 Additional extrapelvic endometriosis may also be appreciated and the presentation may mimic metastatic cancer.303 Prior pregnancies, vaginal births or other uterine surgeries or pathologies, including pelvic inflammatory disease, are not strongly associated with endobronchial disease but such histories are commonly elicited from those with parenchymal disease.304,305 Lung disease usually presents with catamenial hemoptysis.297,299,306,307 Massive hemoptysis is an exceedingly rare event and death has been reported. Symptoms may not occur with every menstrual cycle.297,308 As such, the temporal association with menses may not be appreciated immediately and diagnostic delays of up to 16 years have been noted.309,310 Parenchymal nodules may also come to clinical attention as incidental radiographic findings. Pleural endometriosis often presents with pneumothorax (see Chapter 36).311

Genetics No information is available.

Radiographic details Chest radiographs during menses may demonstrate opacities or infiltrates.306,307,309,312–314 Many chest films are normal. Computed tomography usually demonstrates well-demarcated consolidations or ground-glass opacities.309,313,315 Nodules, thin-walled cavities and bullae are less common findings.316,317

Figure 50. Pulmonary endometriosis. This computed tomogram taken during the patient’s menses demonstrates a 5.5 cm left lower lobe cavitary lesion with smooth borders extending to the lateral pleural and diaphragmatic surfaces. (Courtesy of L. Haramati, MD, New York, New York, USA.)

Nodules may be solitary or multiple (Figure 50).310 All these lesions often change in size and form during the menstrual cycle, owing to the change in size of the lesion and secondary hemorrhage. Magnetic resonance imaging with contrast demonstrates little lesional contrast in pre- or post-menstrual phases but an increased size and significant contrast during menstruation.309 Tracheobronchial disease may demonstrate bronchial wall thickening prior to menses with the development of a surrounding infiltrate during menses.299,302,314,315,317 Intense 18F-FDG uptake on PET-CT has been reported in a single case of pulmonary nodular endometriosis.318

Macroscopic pathology Endobronchial endometriosis during menses may appear as diffuse hyperemia with mucosal oozing or multiple bilateral purple-red submucosal lesions (Figure 51).298,302,314,319–321 Endobronchial polypoid masses or diverticulae are exquisitely rare.299,322 From 0.5 to 5.5 cm often multifocal parenchymal nodules are semifirm tan to brown pseudoencapsulated or welldemarcated lesions often surrounded by blood (Figure 52).301 Cavitation is rare.318 Right sided and lower lobe predominance is noted.297,301

Histopathology Not unlike benign endometrium and pelvic endometriosis, pulmonary endometriosis usually features proliferative endometrial glands and stroma in varying proportions. The secretory pattern is rarely observed. Pseudostratified and columnar glandular epithelium lines alveolar spaces. Round to oval stromal cells with little cytoplasm and indistinct cell borders along with extravasated erythrocytes, hemosiderinladen macrophages and plasma cells populate the interstitium

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Figure 51. Pulmonary endometriosis. This bronchoscopic view demonstrates purple-red submucosal patches in the left upper lobe bronchus. The area bled easily during the procedure. (Courtesy of O. Elbek, MD, Gaziantep, Turkey.)

Figure 53. Pulmonary endometriosis. Proliferative-phase endometrial glands feature larger cells than adjacent bronchiolar epithelium (arrowheads). Stroma can have pronounced smooth muscle.

(Figure 53). The glandular epithelium may demonstrate typical endometrial metaplasia, including focal tubal, mucinous or even clear cell metaplasia. While mitoses are apparent in virtually each gland, crowding, tufting, squamous metaplasia and

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Figure 52. Pulmonary endometriosis. This formalin-fixed well-circumscribed friable mass vaguely resembles endometrium from hysterectomy specimens. (Courtesy of L. Haramati, MD, New York, New York, USA.)

cytological atypia are absent. Stroma usually features prominent spiral-arteriole-type blood vessels and may feature smooth muscle differentiation or pseudodecidual change. Periglandular cuffing and hypercellularity are absent. Microscopic nodules are usually lymphangitic and often centered on bronchovascular bundles but larger “coin lesions” replace normal alveolar parenchyma. Nodules of 0.1–0.4 cm diameter are usually surrounded by benign lymphoplasmacytic infiltrates, erythrocytes, hemosiderin-laden macrophages and hemosiderin (Figure 54). Parenchymal scarring, arterial wall intimal proliferation and recanalization as well as fibrotic hemosiderin-rich micronodules may be appreciated (Figure 55). Rare cases of endometriosis emboli in small pulmonary arteries feature almost complete obliteration of vascular lumens with epithelial cells and prominent fibroelastosis.309 The epithelial cells may line the vascular spaces.299 Mass lesions are unencapsulated but well circumscribed and feature small to large glands randomly distributed throughout cellular stroma and blood lakes (Figure 56). Presurgical fine-needle aspirates may distort the regular edge of the lesion and impart a “pseudoinvasive” pattern. Ectopic pulmonary deciduosis is also a recognized entity with a striking lymphangitic distribution (Figure 57).301 These nodules feature cells with distinct cell borders, abundant eosinophilic and focally basophilic granular cytoplasm and small regular nuclei (Figure 58). A glandular component is absent. Of note, stroma-rich, epithelium-poor pulmonary endometriosis has also been reported (see differential diagnosis section below).323

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

Figure 55. Pulmonary endometriosis. Burnt-out foci of endometriosis present as hemosiderin-rich stromal nodules. Figure 54. Pulmonary endometriosis. The infiltrative process often settles in bronchovascular bundles. Surrounding airspaces are filled with blood and hemosiderin.

Figure 57. Ectopic pulmonary deciduosis. This rare process also has a lymphangitic distribution.

Cytology Figure 56. Pulmonary endometriosis. Tumoral endometriosis features glands of varying sizes and shapes along with typical stroma. These unencapsulated well-circumscribed lesions may be difficult to distinguish from welldifferentiated fetal adenocarcinoma.

Bronchial brushings, washings and fine-needle aspiration procedures may yield diagnostic samples.298,324–326 Clusters of columnar and cuboidal epithelial cells forming microacini contain regular nuclei and scattered cytoplasmic vacuoles. Bundles of round stromal cells may also be appreciated against a bloody background, including hemosiderin-laden macrophages. However, poorly

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Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung Table 3 Differential diagnosis of pulmonary endometriosis

Epithelial neoplasms Lung adenocarcinoma Metastatic adenocarcinoma Mesenchymal neoplasms Metastatic endometrial stromal sarcoma Metastatic smooth muscle neoplasms including benign metastasizing leiomyoma Biphasic neoplasms Well-differentiated fetal adenocarcinoma Biphasic pulmonary blastoma Metastatic adenosarcoma Metastatic carcinosarcoma Pulmonary hemorrhage syndromes Congenital cystic adenomatoid malformation Bullae

Figure 58. Ectopic pulmonary deciduosis. Lesional cells have distinct cell borders, abundant eosinophilic and focally basophilic granular cytoplasm and small regular nuclei.

preserved epithelial cells resemble benign mesothelial cells, while endometrial stromal cells are similar to lymphoid cells, macrophages and mesothelial cells. Thus, these specimens are extremely difficult to evaluate. Cell block preparations from aspirates are more likely to be informative.

Immunohistochemistry Pulmonary endometriosis demonstrates the same immunohistochemical staining pattern as uterine endometrium. The epithelium stains with antibodies directed against cytokeratin, CK7, ER and PR, while the stroma stains with vimentin, CD10, ER and PR and focally with smooth-muscle actin, musclespecific actin, and desmin.298,301 Decidual tissue is vimentin, smooth-muscle actin, muscle-specific actin, desmin, ER and PR positive, while cytokeratin-negative.301

Electron microscopy No information is available.

Pathogenesis It remains unknown whether pleural endometriosis results from coelomic metaplasia or retrograde menstruation with transdiaphragmatic passage and implantation of endometrium inside the thoracic cavity. However, pulmonary endometriosis is considered to be due to lymphatic or vascular embolization of endometrial tissue.295,327 Trauma or manipulation of uterine tissue, including pregnancy and prior surgery, predispose to microembolization.294 Endometrium might also enter the

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lymphovascular circulation, via foci of pelvic endometriosis. Multifocality and the lower lobe predominance also support an embolic process given that perfusion is greatest in the lower lung fields.301 Rare cases with endovascular endometrial glands provide further evidence to support an embolic etiology.

Clinicopathological correlation Hemoptysis results from rupture of capillaries and other small blood vessels entrapped by or directly involved by endometrial tissue. Since the ectopic endometrium cycles according to systemic hormone levels, fluid shifts during the cycle and tissue breakdown during menstruation cause both hemorrhage and tissue protusion into airways.304 Menstrual-induced increases in prostaglandin F2 levels, a potent bronchoconstrictor, may also play a role.328

Differential diagnosis The differential diagnosis for parenchymal endometriosis includes primary and metastatic neoplasms, as well as pulmonary hemorrhage syndromes and other non-neoplastic processes (Table 3). Distinctions may be impossible on biopsies or fine-needle aspirates. While the biphasic nature of endometriosis allows distinction from adenocarcinomas, discerning endometriosis from well-differentiated fetal adenocarcinoma (WDFA) can be difficult (see Chapter 27). However, pulmonary masses are common in WDFA and rare in endometriosis. Endometrial glands are almost always in the proliferative phase and often feature tubal, mucinous or clear cell metaplasia. Cytoplasmic glycogen and squamous morules are absent. Metastastic uterine tumors, including biphasic adenosarcoma and carcinosarcomas, are overtly malignant but low-grade endometrial stromal sarcomas and metastatic smooth muscle tumors,

Chapter 22: Benign epithelial neoplasms and tumor-like proliferations of the lung

especially with cystic change, are very challenging considerations that require a full clinical history. Again these tumors are usually larger than endometriotic lesions but in small metastases the problem remains. One must also distinguish entrapped respiratory epithelium in these malignancies from the glandular component of endometriosis. Unfortunately immunohistochemical stains are not helpful owing to significant cross-reactivity between primary and metastatic carcinomas, as well as metastatic sarcomas from the female genital tract. Within the proper clinical context, pulmonary hemorrhage syndromes also enter the differential diagnosis. Lobules filled with erythrocytes and hemosiderin-laden macrophages raise the possibility of pulmonary infarction and systemic diseases, such as Wegener granulomatosis, Goodpasture syndrome and idiopathic pulmonary hemosiderosis. Hemorrhage adjacent to a neoplasm must also be considered. Biopsies should be carefully scrutinized for minute foci of endometriosis. Lastly, pulmonary ectopic deciduosis may be mistaken for squamous cell carcinoma, adenocarcinoma and epithelioid hemangioendothelioma. Squamous cell carcinoma features cytological atypia, while signet ring adenocarcinoma shows

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Prognostic factors and natural history Although not usually considered life-threatening, the morbidity of pulmonary endometriosis necessitates medical intervention. Bronchiectasis is a possible long-term complication.320 Medical therapy aims to suppress the endometrial implants with hormonal alterations. Options include oral contraceptives, progestational drugs, anovulatory agents and gonadotropin-releasing hormone (GnRH) agonists.329 Unfortunately, recurrence rates exceed 50%.297 Localized endobronchial endometriosis may be managed with Nd-YAG laser.314 Bronchial artery embolization has also recently been reported in this setting.330 Surgical resections should be as limited as possible and video-assisted thorascopic surgery is often the method of choice.299,331,332 Hysterectomy with bilateral salpingo-oophorectomy is the definitive treatment of endometriosis.

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and interlobar fissures: CT findings. J Comput Assist Tomogr 1994;18:34–8. 202. Sakamoto K, Okita M, Kumagiri H, et al. Sclerosing hemangioma isolated to the mediastinum. Ann Thorac Surg 2003;75:1021–3. 203. Shimosato Y. Lung tumors of uncertain histogenesis. Semin Diagn Pathol 1995;12:185–92. 204. Cheung YC, Ng SH, Chang JW, et al. Histopathological and CT features of pulmonary sclerosing haemangiomas. Clin Radiol 2003;58:630–5. 205. Hung JJ, Liu JS, Hsu WH. Sclerosing hemangioma with an air halo. J Thorac Cardiovasc Surg 2008;136:1365–7. 206. Nam JE, Ryu YH, Cho SH, et al. Air-trapping zone surrounding sclerosing hemangioma of the lung. J Comput Assist Tomogr 2002;26:358–61. 207. Takatani H, Ashizawa K, Kawai K, Kohno S. Pulmonary sclerosing hemangioma manifesting as a nodule with irregular air clefts on high-resolution CT. AJR Am J Roentgenol 2007;189:W26–8. 208. Fujiyoshi F, Ichinari N, Fukukura Y, et al. Sclerosing hemangioma of the lung: MR findings and correlation with pathological features. J Comput Assist Tomogr 1998;22:1006–8. 209. Fujiyoshi F, Nakajo M, Ikeda K, et al. A case of sclerosing hemangioma of the lung: correlation of MR images with pathological findings. Radiat Med 1995;13:85–8. 210. Hara M, Iida A, Tohyama J, et al. FDG-PET findings in sclerosing hemangioma of the lung: a case report. Radiat Med 2001;19:215–8. 211. Lin KH, Chang CP, Liu RS, Wang SJ. F-18 FDG PET/CT in evaluation of pulmonary sclerosing hemangioma. Clin Nucl Med 2011;36:341–3. 212. Chan AC, Chan JK. Pulmonary sclerosing hemangioma consistently expresses thyroid transcription factor-1 (TTF-1): a new clue to its histogenesis. Am J Surg Pathol 2000;24:1531–6. 213. Devouassoux-Shisheboran M, de la Fouchardiere A, Thivolet-Bejui F, et al. Endobronchial variant of sclerosing hemangioma of the lung: histological and cytological features on endobronchial material. Mod Pathol 2004;17:252–7. 214. Wani Y, Notohara K, Tsukayama C, Okumura N. Sclerosing hemangioma

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260. Wei S, Tian J, Song X, Chen Y. Recurrence of pulmonary sclerosing hemangioma. Thorac Cardiovasc Surg 2008;56:120–2.

272. Atagi S, Sakatani M, Akira M, Yamamoto S, Ueda E. Pulmonary hyalinizing granuloma with Castleman’s disease. Intern Med 1994;33:689–91.

261. Engleman P, Liebow AA, Gmelich J, Friedman PJ. Pulmonary hyalinizing

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unusual association with multiple sclerosis. South Med J 1995;88:1076–7.

posterior uveitis. Tohoku J Exp Med 2004;204:93–7.

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286. Russell AF, Suggit RI, Kazzi JC. Pulmonary hyalinising granuloma: a case report and literature review. Pathology 2000;32:290–3.

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287. Ramirez J, Mehta JB, Taylor RA, Byrd RP Jr, Roy TM. Symptomatic pulmonary hyalinizing granuloma. South Med J 1998;91:867–9.

276. Satti MB, Batouk AA, Abdelaziz MM, Ahmad MF, Abdelaal MA. Pulmonary hyalinizing granuloma. Bilateral pulmonary nodules associated with chronic idiopathic thrombocytopenic purpura. Saudi Med J 2005;26:1459–63. 277. Winger DI, Spiegler P, Trow TK, et al. Radiology-Pathology Conference: pulmonary hyalinizing granuloma associated with lupus-like anticoagulant and Morvan’s Syndrome. Clin Imaging 2007;31:264–8. 278. Schlosnagle DC, Check IJ, Sewell CW, et al. Immunologic abnormalities in two patients with pulmonary hyalinizing granuloma. Am J Clin Pathol 1982;78:231–5. 279. Gorini M, Forloni F, Pezzoli A, Pezzica E. Pulmonary hyalinizing granuloma. A limited form of Wegener’s granulomatosis? Ann Ital Med Int 1998;13:176–9. 280. Hashimoto S, Fujii W, Takahashi T, et al. Pulmonary hyalinizing granuloma with hydronephrosis. Intern Med 2002;41:463–6. 281. Patel Y, Ishikawa S, MacDonnell KF. Pulmonary hyalinizing granuloma presenting as multiple cavitary calcified nodules. Chest 1991;100:1720–1. 282. Shibata Y, Kobayashi T, Hattori Y, et al. High-resolution CT findings in pulmonary hyalinizing granuloma. J Thorac Imaging 2007;22:374–7. 283. Basoglu A, Findik S, Celik B, Yildiz L. Pulmonary hyalinizing granuloma mimicking lung carcinoma. Thorac Cardiovasc Surg 2006;54:282–3. 284. Colen RR, Nagle JA, Wittram C. Radiologic-pathologic conference of the Massachusetts General Hospital. Pulmonary hyalinizing granuloma. AJR Am J Roentgenol 2007;188:W15–6. 285. Esme H, Ermis SS, Fidan F, Unlu M, Dilek FH. A case of pulmonary hyalinizing granuloma associated with

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Chapter

23

Pulmonary pre-invasive disease Keith M. Kerr

Introduction Primary carcinoma of the lung remains globally the most frequent cause of death from malignancy. Although the incidence of this most fatal of diseases is falling in the USA and some western European countries, it is rising in other parts of the world. The enormous increase in tobacco consumption in Asia portends a rise in this disease in those affected countries. Primary lung cancer will be a global problem for many years to come. As well as a geographic shift in incidence, the most striking demographic change, at least in “Western” populations, has been the decline in lung cancer in males, yet a rise in women. This is almost certainly due to gender differences in smoking habits in previous decades.1 In addition there has been a shift in the reported incidence of different cell types. In Western populations squamous cell carcinoma and possibly small cell carcinoma (SCLC) have declined, while adenocarcinoma has risen in frequency. While some classification bias may account for a proportion of this change,2 these shifts have been attributed to changes in smoking habits, changes in cigarettes themselves and, given their apparent greater propensity to develop adenocarcinoma, the increase in the number of women who smoke. This apparent shift from central “bronchogenic” carcinomas to those usually more peripheral adenocarcinomas signals biological differences between these tumor types. Central squamous cell carcinomas have long been strongly associated with cigarette smoking and with pre-invasive changes in bronchial epithelium, namely squamous dysplasia (SD) and carcinoma in situ (CIS). There is no recognized progenitor lesion for SCLC, although squamous SD/CIS has been suggested as a possible precursor. Adenocarcinomas, though still associated with tobacco consumption, show weaker links. Their putative pre-invasive precursors, atypical adenomatous hyperplasia (AAH) and localized non-mucinous bronchioloalveolar carcinoma (LNMBAC – adenocarcinoma in situ (AIS)), are, in comparison to squamous SD/CIS, relatively recently described. SD/CIS and AAH/LNMBAC-AIS are the morphological representations of two biologically separate carcinogenic path-

ways in the human lung. The emergence of the AAH/ LNMBAC-AIS pathway, in particular, has been an important advance in our understanding of how some adenocarcinomas develop. An appreciation of this duality in pulmonary carcinogenesis is vital, given the increasing interest in developing strategies for the early detection of lung cancer through screening. The earliest screening trials failed because of the low sensitivity for detection of early disease but also, at least in part, because the technology employed (sputum cytology and chest radiography) was biased in favor of detecting central bronchogenic tumors.3 More recent trials based on high-resolution computed tomography (HRCT) scanning have seen more success but mostly in populations where peripheral adenocarcinomas are prevalent (see Chapter 24).4–6 In an ideal world a protocol for early lung cancer detection would cover both early bronchial lesions as well as small peripheral parenchymal abnormalities. As well as SD/CIS and AAH, the WHO classification of lung tumors also recognizes diffuse idiopathic pulmonary neuroendocrine cell hyperplasia (DIPNECH) as a third preinvasive lesion with the potential to give rise to invasive malignancy.7 As a rare precursor of some carcinoid tumors, this lesion is discussed in Chapter 31. Other pathological processes besides SD/CIS and AAH/ LNMBAC-AIS, many of which are recognized clinicopathological entities in the human, carry an increased risk of the subsequent development of lung carcinoma. These are listed in Table 1. In all these lesions, an abnormal epithelial proliferation serves as a precursor lesion for lung cancer development. The presence of, in some instances, molecular changes associated with the more usual forms of lung cancer supports this hypothesis. These lesions are discussed in relevant chapters elsewhere in this text.

Bronchial carcinogenesis Malignant change in the bronchial epithelium leading to bronchogenic carcinoma is the archetypal manifestation of lung carcinogenesis. These lesions have been recognized for a long time and animal models have been extensively studied. It

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 23: Pulmonary pre-invasive disease Table 1 Pre-existing lung lesions associated with lung cancer development

a

Disease or lesion

Lung cancer type evolving

References

Type 1 congenital cystic adenomatoid malformations (CCAM)

Lepidic pattern mucinous (bronchioloalveolar) adenocarcinoma

367–377

Juvenile tracheobronchial squamous papillomatosis

Squamous cell carcinoma

378–384

Bronchopulmonary sequestration

Squamous cell carcinoma

385

Bronchiectasis

Squamous cell carcinoma

386,387

Bronchogenic or other lung cysts

Adenocarcinoma ? Lepidic pattern mucinous (bronchioloalveolar) adenocarcinoma

388,389

Idiopathic lung fibrosis

All types (?Adenocarcinoma more common)

232, 390–416

Connective tissue diseasesa

All types (Adenocarcinoma probably more common)

417–428

Asbestosis

All types (?Adenocarcinoma more common)

429–442

Silicosis

All types (?Adenocarcinoma more common)

443–449

There is debate as to whether pulmonary fibrosis is required in various connective tissue diseases to confer an increased risk of lung cancer. This is also the case in relation to asbestos exposure and lung cancer. Scleroderma seems to carry a particularly high risk of developing lung cancer.420

is only relatively recently that some understanding of the molecular biology of this process has emerged. A number of morphological changes may occur in the bronchial epithelium as a result of chronic irritation or injury. These changes are not, in and of themselves, pre-malignant. They may co-exist, through a common causation, with the mutagenic changes required for malignant transformation. However, they may also represent a cell population more likely to undergo malignant change. Reactive upregulation of cell proliferation, an apparent prerequisite for carcinogenesis in any epithelium, may play a role. There is thus an ill-understood relationship between what is apparently reactive hyperplasia and metaplasia, both essentially physiological adaptive processes, and dysplasia, which is the morphological manifestation of dysregulated cell morphogenesis and proliferation. The boundaries between these are not clear. The morphological changes in the bronchial epithelium are the result of genetic changes, due to chronic exposure to irritants and/or carcinogens. Animal models have been used to study the development of carcinoma in tracheobronchial epithelium.8,9 Aside from ethical issues, these studies have the advantage of observing changes longitudinally in the same subject. This was more or less impossible in humans, until lung cancer screening and autofluorescence bronchoscopy became available in the 1990s (see below). The applicability of animal studies to humans has been questioned.9 Apart from the obvious biological differences, most animal studies involve much shorter time courses and higher carcinogen exposures than seen in human subjects. Regular exposure of carcinogens in the hamster induces mucous cell hyperplasia, basal cell hyperplasia, squamous metaplasia and, eventually, carcinoma in situ.10 Other studies

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in rodents have described three phases in airway epithelium experimentally exposed to carcinogens; an acute toxic phase, a pre-neoplastic (sub-acute toxicity) phase and frank neoplasia.9 Similar findings are described in dogs.8 In the first stage, acute cellular damage leads to mucous and basal cell hyperplasia, and subsequent squamous metaplasia. These changes appear to be reversible, when the injurious stimulus is removed. With more severe exposure, squamous metaplasia is more widespread and persistent, with discrete, well-formed orthokeratotic patches and less inflammation (the sub-acute phase). Cytological atypia may develop in these lesions. Ultimately, some lesions transform into either squamous carcinoma in situ (CIS) or invasive carcinoma. Interestingly, even CIS lesions are reversible if carcinogens are withdrawn.11 In a dog model, where animals were exposed to tobacco smoke, possibly recapitulating the exposure levels of the bronchial epithelium in human smokers, the early reactive changes seen in the epithelium were the same as in the high-dose experiments described above. However the atypia was much slower to develop, less widespread, and progression to invasive carcinoma uncommon.12

Etiology of SD/CIS As alluded to above, carcinogens in tobacco smoke are thought to account for 85–90% of human lung cancers. A doseresponse relationship between tobacco consumption and lung cancer risk is well recognized. Other factors associated with lung cancer include exposure to naturally occurring radon gas and occupational exposure to a variety of chemical compounds, as well as asbestos. The role of atmospheric air pollution is not clear (see Chapter 24).

Chapter 23: Pulmonary pre-invasive disease

The association between tobacco smoking and central bronchial carcinogenesis is far stronger than the association with adenocarcinomas.13 Tobacco smoke contains over 4000 different chemicals, at least 50 of which are recognized carcinogens.14,15 Of these, the polycyclic aromatic hydrocarbons (PAH) and the nitrosamines are probably the most important groups. PAHs appear particularly important in bronchial carcinogenesis. A person’s inherited genotype may well confer variance of risk from the effects of smoking tobacco and certain carcinogens therein. Polymorphisms of the nicotinic acetylcholine receptor gene cluster at 15q24–25 seem to increase the risk in some patients. This is probably by driving nicotine addiction and increasing tobacco consumption (see Chapter 24).16 Inherited polymorphisms of xenobiotic metabolizing enzymes (XMEs) are also associated with increased lung cancer risk. Phase 1 enzymes, such as the cytochrome P450s (CYPs), paradoxically activate pro-carcinogens to active carcinogens. Phase 2 XMEs, including glutathione S-transferases (GSTs), detoxify and promote the elimination of active compounds. Inheritance of more active phase 1 and less active phase 2 enzymes increases tissue levels of active carcinogens, promoting the various carcinogenic effects of these compounds on bronchial epithelium.15 Certain polymorphisms of CPY1A1 and the GST M1-null phenotype are associated with an increased risk of bronchial squamous cell carcinoma.17 Bronchial squamous carcinogenesis is probably the result of sequential accumulation of between three and 12 critical genetic abnormalities.18 It seems likely a fully fledged invasive malignant phenotype requires a number of specific genetic changes in the target cell population. These changes probably need to occur over time in a particular order, should be nonfatal to the affected cells, and heritable in daughter cell populations. It is presumed the gradual accumulation of genetic alterations is reflected in recognizable morphological changes in the “host” epithelium. These morphological changes are the pre-invasive lesions described in the next section. The statistical odds of the correct number and order of mutations and other alterations in gene expression occurring in a cell population which remains viable are hard to quantify but are undoubtedly enormous. Thus, as Sir Richard Doll stated, getting cancer is “largely a matter of luck: bad luck if the several necessary changes all occur in the same stem cell, when there are several thousand cells at risk.”19,20 The complete set of changes would lead to the full expression of malignancy in perhaps only one or occasionally more than one “focus of cells.” Increasingly larger numbers of stem cells, and correspondingly larger numbers of hyperplastic or dysplastic lesions arising from those stem cells, are found with progressively fewer genetic abnormalities. This is the basis of the concept of a field change of carcinogenesis, within which an invasive tumor may be found, often associated with several foci of pre-invasive change.21 These pre-invasive lesions are probably dependent on one or more altered stem cells leading to the development of a genetically divergent clonal “patch” of

daughter cells, from which the morphologically recognizable pre-invasive lesion develops. Clonal “patches” measuring up to 7 cm have been detected in human head and neck mucosa.21 It has been suggested that in the human bronchus, these clonal patches may comprise anywhere from 40 000 to 360 000 cells;22,23 this would equate to an area of between 4 and 80 mm.2 The population of stem cells supporting the human bronchial epithelium is different from that serving the bronchiolo-alveolar epithelial compartment. The former are probably located amongst CK5 and CK14-expressing basal cells at the submucosal gland duct junctions and intracartilaginous boundaries.23 These are the likely progenitors of basal cell hyperplasia, SD and CIS. The presence of two discrete stem cell populations in the lung is an apposite observation in the discussion of central and peripheral pulmonary carcinogenesis. This concept suggests the morphologically malignant tumor is merely the “tip of the iceberg” having accumulated many genetic abnormalities, while many underlying morphologically normal and probably functioning cells harbor only a few alterations. There is abundant evidence that, in tobacco smokers, morphologically normal bronchial epithelium shows limited genetic changes associated with malignant transformation (see below).24–27 Pre-invasive genetic changes may be present in morphologically normal bronchial epithelium in “at risk” individuals. Detailed study of several hundred autopsy cases provided early insights into the extent of SD/CIS in tobacco smokers’ lungs. Lesions ranging from basal cell hyperplasia and squamous metaplasia to dysplasia and CIS were widely distributed and more extensive, frequent and atypical in heavier smokers.28–31 The tracheal epithelium was relatively spared but the most severe abnormalities were found in airways, which had already developed invasive carcinoma. Most changes were potentially reversible on smoking cessation, whilst progression of disease, if it occurred, took years.30,31 In uranium miners with lung cancer, the rates of CIS in the bronchi were extremely high; 96% in the miners and 92% in a non-mining control group.32 Such findings probably reflect extensive and detailed examination of the airways only possible at autopsy. They also suggest a less demanding set of criteria for reaching a diagnosis of CIS. SD/CIS was reported in 40%, and basal cell hyperplasia and squamous metaplasia even more frequently in smokers without carcinoma.30 Cytological atypia in squamous cells may be detected in sputum samples, especially in high-risk groups, such as heavy tobacco smokers or uranium miners exposed to high levels of environmental radon gas.33–35 The efficiency of this modality in detecting SD/CIS is probably low. Findings in screening studies have varied greatly, probably being a function of how “high-risk” groups were defined and differing diagnostic criteria. Mild and moderate/severe dysplasias were reported in between 48% (mild) and 3.5% to 26% (moderate/severe) of subjects, respectively.34,35 In an autofluorescence bronchoscopy (AFB) biopsy study of 207 high-risk patients, sputum cytology detected none of the dysplasias and only 32% of the

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Figure 1. Goblet cell hyperplasia. In this region of bronchial respiratory epithelium in a smoker’s airway the number of goblet cells is increased above normal.

metaplasias identified by combined AFB and biopsy (see Chapter 24).36 In a study based on standard white light bronchoscopy (WLB) in over 100 heavy smokers, squamous metaplasia was more frequent and extensive in those with the heaviest exposure.37 AFB is superior in detecting bronchial, pre-invasive lesions, although there are issues with specificity (see below).38 AFB is a key recommended diagnostic modality for SD/CIS detection in recently published guidelines.39 In one high-risk cohort screened by AFB, moderate dysplasia was found on biopsy in 14% of participants, severe dysplasia in 6.5% and CIS in 1.8%, while another found low-grade atypia in 14% and high-grade changes in 4.9%.40 Women had a lower prevalence of high-grade changes (14% versus 31%) and fewer lesions than males.41 The greater prevalence of SD/CIS in males was also noted in one autopsy study.42 Paris et al. found SD/CIS in 241 patients screened by AFB. They noted active smokers rather than former smokers, occupational exposure to chemical carcinogens, and a long history of asbestos exposure were all associated with bronchial SD/ CIS.43 In a very large study of possibly less “at risk” patients, Haussinger et al. found high-grade SD/CIS in 5.1% of participants having AFB, twice the rate of disease detected in a group having only WLB.44 Two-thirds of cohorts with more risk factors had squamous metaplasia or worse.36,40 SD/CIS appears to be associated most strongly with chronic exposure to tobacco smoke and some forms of environmental irradiation (see Chapter 24). Certain occupational chemical exposures may also be implicated but the role of environmental/urban air pollution remains unclear (see Chapter 24).34,45 SD/CIS has a tendency to be more frequent and of higher grade in males, although this may be a historical point reflecting former smoking habits. These habits are not reflected in contemporary populations, where there are fewer gender differences in tobacco consumption.

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Figure 2. Basal cell hyperplasia may be diagnosed when the layers of basal cells number three or more.

Morphologically recognizable pre-invasive bronchial lesions It is believed that one of the earliest changes occurring in clonal patches is the upregulation of cell proliferation. Since hyper-proliferating epithelia are more likely to undergo malignant transformation, cell division seems to be a prerequisite for some of the genetic lesions required for the malignant genotype. Epithelial hyperplasia probably represents the earliest recognizable pre-invasive change in bronchial epithelium. Squamous metaplasia has long been recognized as a step in this progression towards squamous dysplasia and carcinoma in situ (SD/CIS) of the bronchial epithelium.

Mucous cell (goblet cell) hyperplasia

Any of the so-called “mucous membranes”, including the respiratory epithelium, respond to chronic irritation by increasing mucus production. Apart from changes in bronchial glands, this hypersecretory state, seen in chronic bronchial asthma and chronic bronchitis, is also based on an increase in the number of bronchial mucus-secreting goblet cells. In the tobacco smoker’s bronchial epithelium there may be a general increase in these cells. However, small patches of densely crowded goblet cells, devoid of atypia, may also be seen as small epithelial tufts slightly proud of the bronchial epithelial surface (Figure 1). This is a well-recognized, smokingrelated change.46 Whether this is a pre-invasive lesion which may progress to malignancy or, more likely, a reactive change without pre-invasive potential, is debated.47

Basal cell hyperplasia Basal cell or reserve cell hyperplasia (BCH) is indicated by the presence of three or more layers of basal cells (Figure 2).48 Occasionally basal cells may almost completely replace

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Figure 3. A comparative illustration of normal respiratory epithelium with a single layer of basal cells (a) showing that basal cells do not stain with cytokeratin CK7 (b) whilst differentiated respiratory columnar cells do stain. In contrast the basal cell layer is well stained by CK5/6 (c) and p63 (d). Basal cell hyperplasia (e) is nicely demonstrated on the CK7 (f), CK5/6 (g) and p63 (h) stains.

differentiated columnar cells and care has to be taken not to overdiagnose severe dysplasia or “basaloid” carcinoma in situ. The basal cells show no atypia and mitoses are scarce. If the respiratory epithelium is cross-cut, an erroneous impression of basal cell hyperplasia may be formed. Basal cells can be demonstrated in bronchial epithelium using antibodies to p63 protein or cytokeratin 5/6 (Figure 3). By definition the expanded layers of basal cells lack any evidence of squamous differentiation. Cases are encountered where intercellular bridges may be seen between the basal cells, while a layer of differentiated, though possibly rather attenuated, columnar cells remains on the epithelial surface. Although this finding does not fulfill the definition of squamous metaplasia (see below), a term such as immature squamous metaplasia is applicable (Figure 4).

Squamous metaplasia Squamous metaplasia is defined as the replacement of the respiratory epithelium by a full-thickness squamous epithelium with a basal cell zone, an intermediate zone of larger cells

Figure 4. Immature squamous metaplasia. Intercellular bridges between basal and intermediate cells, yet differentiated columnar cells are present on the surface.

with intercellular bridges and a maturing superficial cellular layer (Figure 5). This superficial layer may feature surface keratinization. There is no atypia. Squamous metaplasia is

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Chapter 23: Pulmonary pre-invasive disease Table 2 Factors associated with bronchial squamous metaplasia

Airborne irritants

Tobacco smoke28,29,37 Irradiation (radon gas)51 Marijuana smoke50 Air pollution49

Chronic lung inflammation

Tuberculosis Bronchiectasis Pneumoconiosis52 Chronic lung cavities Airways draining septic foci

Endobronchial lesions

Benign tumors Carcinoid and other tumors

Chronic trauma

Tracheostomy Prolonged endobronchial intubation Retained inhaled foreign body

Others

Vitamin A deficiency53,54

Figure 5. Complete full-thickness squamous metaplasia. The cell flattening and cytoplasmic eosinophilia are characteristic.

Figure 6. Squamous metaplasia overlying bronchial carcinoid tumour is typically thin.

the result of prolonged chronic irritation of the bronchial mucosa and tobacco smoking is an important cause.28,29,37 Other factors implicated include atmospheric pollution,49 smoking marijuana,50 irradiation51 and chronic lung diseases with bronchial involvement, such as bronchiectasis, tuberculosis and pneumoconiosis (Table 2) (see Chapter 24).52 Vitamin A deficiency may cause both squamous metaplasia and BCH in the airway epithelium.53,54 Squamous metaplasia is well recognized in the mucosa overlying slow growing lesions, such as carcinoid tumors (Figure 6) or endobronchial hamartomas, in mucosa lining chronic lung cavities (Figure 7), and in airways draining areas of chronic lung suppuration and where there is chronic trauma (tracheostomy, prolonged endobronchial intubation, retained inhaled foreign body). Squamous metaplasia may also be encountered in a more subacute setting, associated with pneumonia, where BCH and loss of differentiated cells may be seen.42

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Figure 7. Mature, non-dysplastic squamous metaplasia lining a chronic lung cavity. Note the pus in the cavity lumen.

Squamous metaplasia is an adaptive phenomenon representing an epithelium better able to cope with the airway microenvironment. While it is assumed squamous metaplasia is the precursor lesion for SD/CIS, this may be only partially true.55,56 While squamous metaplasia and SD/CIS may have common etiological factors in tobacco smoke or environmental irradiation, squamous metaplasia is not an obligatory intermediate stage in the development of SD/CIS, nor does squamous metaplasia always progress to dysplasia and invasive disease.

Squamous dysplasia and carcinoma in situ Squamous dysplasia and carcinoma in situ in the bronchial mucosa are recognized as the precursor lesions for

Chapter 23: Pulmonary pre-invasive disease Table 3 Histological features of bronchial squamous dysplasia and carcinoma in situ

Epithelial cytology

Epithelial architecture

Mild dysplasia

Moderate dysplasia

Severe dysplasia

Carcinoma in situ

Mild increase in cell size, minimal pleomorphism Mild variation of N/C ratio

Mild increase in cell size, pleomorphism moderate Moderate variation of N/C ratio

Finely granular chromatin, minimal nuclear angulation, nucleoli inconspicuous or absent Vertically oriented nuclei in lower third Mitoses absent or very rare

Finely granular chromatin; nuclear angulations, grooves and lobulations present, nucleoli inconspicuous or absent Vertically oriented nuclei in lower two-thirds Mitotic figures in lower third

Marked increase in cell size and pleomorphism N/C ratio often high and variable Chromatin uneven and coarse, prominent nuclear angulations and folds, nucleoli frequent and conspicuous Vertically oriented nuclei in lower two-thirds Mitotic figures in lower two-thirds

Marked increase in cell size and pleomorphism N/C ratio often high and variable Chromatin uneven and coarse, prominent nuclear angulations and folds, nucleoli variable. Nuclei haphazardly orientated relative to epithelial surface Mitotic figures throughout epithelium

Mild increase in epithelial thickness Complete epithelial maturation Superficial flattening of epithelial cells Prickle cell zone often present Basilar zone expanded into lower third

Moderate increase in epithelial thickness Partial epithelial maturation Superficial flattening of epithelial cells Prickle cells confined to upper third of epithelium Basilar zone expanded into lower two-thirds

Marked increase in epithelial thickness Little epithelial maturation Superficial flattening of epithelial cells Prickle cell zone rare Basilar zone expanded well into upper third

Epithelium ranges from greatly thickened to thinner than normal No maturation. Epithelium would be same if inverted Superficial flattening minimal or absent Prickle cell zone absent Cellular crowding throughout epithelium

Reproduced with permission from Kerr KM. Precursors of malignancy. In: Pulmonary Pathology (Foundations in Diagnostic Pathology), Eds. Zander D, Farver C. Churchill Livingstone Elsevier, Philadelphia, 2008.

“bronchogenic” carcinoma, principally squamous cell carcinoma, but possibly also SCLC. Much credit for this hypothesis goes to Oscar Auerbach and colleagues, who, beginning in the 1950s, carried out very extensive studies of the airways of lungs obtained at autopsy from tobacco smokers.28–32,42 Recently, increasing interest has been sparked by AFB, used to detect bronchial epithelial lesions and as a tool in lung cancer screening.57 The 1999 and 2004 World Health Organization (WHO) classifications of lung cancer provide criteria for diagnosing three grades of dysplasia and carcinoma in situ in the bronchus (Table 3).55 Prior schemata were complex and difficult to implement29,30 and many pathologists simply applied criteria from other organ systems. The system for bronchial dysplasia, endorsed by the WHO, borrows many principles from the descriptions of intraepithelial neoplasia in the cervix and larynx and features three grades of dysplasia (mild, moderate and severe) and a separate category of carcinoma in situ. At other sites, where both squamous and glandular epithelial dysplasia are considered (cervix, esophagus, colon, etc.), there has been a trend towards using a two-grade system of highand low-grade dysplasia. This has been suggested by some for bronchial disease35 and there is molecular biological evidence in support of this (see below).

Macroscopic features of SD/CIS Most SD/CIS lesions are invisible on gross examination. Those which can be seen present subtle changes only apparent to experienced observers. Even where early bronchial neoplasia was diagnosed by bronchial cytology or biopsy, and the lung subsequently resected, an active search for the responsible lesion revealed 39% of CIS lesions and 17% of early invasive carcinomas had no grossly visible mucosal lesions.58 In surgical or autopsy specimens, areas of carcinoma in situ are most frequently found at airway bifurcations,59,60 perhaps because airborne carcinogens are preferentially deposited here as a result of turbulent airflow. These lesions may cause mucosal pallor and a loss of the usual mucosal transparency. The mucosa can have a nodular or granular character, obliterating the fine pattern of ridges and rugae present in normal respiratory mucosa and mucosal pits at bronchial gland duct openings.56 Even in the absence of such a mass lesion causing bronchial obstruction, extensive and circumferential SD/CIS can create sufficient disruption of the mucociliary escalator function that bronchial clearance is compromised, leading to a risk of retention pneumonia. Most SD/CIS lesions are relatively small. Woolner et al.58 showed bronchial CIS lesions had a mean diameter of 9 mm

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Figure 8. Paired images from normal “white light” (pink/red images) and autofluorescence bronchoscopy (AFB) images. Panels a and b show the loss of the “normal” green coloration over this endobronchial squamous cell carcinoma, which retains a pinkish hue. Panels c and d illustrate a similar property with a small patch of moderate dysplasia on a carina. Unfortunately, this optical change is not specific and may be seen with histologically normal mucosa (e and f). (Images courtesy of Dr Robert Rintoul, Cambridge, UK.)

(range 2–17 mm) and a thickness of 2–4 mm with focal areas extending up to 7 mm. Nagamoto et al. described 19 cases of isolated bronchial CIS detected by sputum cytology in a cancer screening program.60 Their maximum lesion diameter was 12 mm, while 21% of the lesions were under 4 mm in diameter. Autofluorescence bronchoscopy has allowed endoscopic identification of bronchial dysplasia, otherwise invisible to the most experienced bronchoscopist using standard WLB approaches (see below). Lam et al. found just over half SD/CIS lesions detected in this way measured 1.5 mm or less in diameter, while almost 45% measured between 1.6 and 4 mm in diameter.61 Autofluorescence bronchoscopy involves the use of a blue or violet light source and special imaging sensors to highlight dysplastic areas, otherwise invisible by white light. The lesions do not reflect green light waves to the same degree as red waves and are thus visible (Figure 8).62 WLB has been reported with 65% sensitivity for detecting SD/CIS,

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while autofluorescence techniques showed 90% sensitivity.63 In a series of bronchial CIS detected using sputum cytology, only 29% of patients had lesions visible by standard WLB. The use of AFB in these patients showed a 6.3-fold improvement in detection of moderate or severe dysplasia and an even better performance, by a factor of 2.7, in the detection of CIS lesions.64 In a large prospective randomized trial comparing WLB against WLB and AFB to detect SD/CIS, AFB was again demonstrated to be superior.44 The detection rate for CIS was more than doubled using AFB. While AFB has a much greater sensitivity than WLB for detecting SD/CIS in the bronchial mucosa, it suffers from poor specificity. Hirsch et al.35 reported 68.8% sensitivity for AFB in detecting “high-grade” dysplasias, compared to 21.9% for standard WLB, but also found 31% of cases of severe dysplasia in their study were missed by AFB. Lam et al.64 found almost half of the AFB-abnormal areas detected were histologically

Chapter 23: Pulmonary pre-invasive disease

Figure 9. Mild dysplasia. Compare this image with Figure 5.

normal when biopsied (Figure 8), and another third of cases showed only inflammation, epithelial hyperplasia or metaplasia. Another study demonstrated that, even in a high-risk cohort of patients, 67% of the biopsies taken from AFB-abnormal areas failed to demonstrate SD/CIS or invasive disease.65 With its limited ability to detect very small invasive carcinomas, 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) failed to identify SD/CIS lesions identified at AFB.66 Given the concept of field carcinogenesis, SD/CIS lesions should be frequently multicentric. In a small AFB study of 28 patients with high-grade SD or CIS, 23% of patients had multifocal bronchial disease, as opposed to only 7% of patients when assessed by WLB.67

Microscopic features of SD/CIS There are four grades of disease identified in the current WHO classification of bronchial squamous dysplasia, including squamous carcinoma in situ.55 The features described are based on evaluation of full-thickness squamous change in the bronchial mucosa (Table 3). Not all the features for a particular grade will be present in every lesion and this is a biologically continuous evolution of disease. The choice of boundaries for division into the four “stages” is somewhat arbitrary and driven by practical expediency, as well as experience of squamous dysplasia at other anatomical locations. Thus, there may be considerable overlap in the occurrence of various features and variation between different microscopic zones within any given lesion. The author’s practice is to grade lesions on their most advanced feature(s). For the purposes of grading, and in keeping with practice in other organ sites, the dysplastic squamous epithelium is assessed by the presence of features in the upper, middle and lower thirds of the epithelium. The assessment of cell size and maturation, nuclear features, cell orientation and epithelial thickness are all subjective. Not all cases of a particular grade of disease look the same; in some cases maturation and the squamous phenotype are obvious with surface keratin, large cells and eosinophilic cytoplasm, in others the cells are smaller with a more basaloid appearance. In addition

Figure 10. Moderate dysplasia. Nuclear irregularity is the key feature in this case.

dysplastic epithelium is shed easily, meaning there are often only several layers left on the histology section. In mild dysplasia the changes are confined to the lower third of the bronchial squamous epithelium. There is expansion of the basal cell layer into the lower third of the epithelium, where the nuclei may be oriented vertically (Figure 9). Above the lower third there is retention of the prickle cell layer and complete maturation of the cells superficially. The epithelium may be a little thickened. The cytological changes are very subtle. Cell size increase, variation in nuclear/cytoplasmic ratio and nuclear angulation are all slight. The chromatin is finely granular, while nucleoli and mitoses are inconspicuous or absent. Moderate dysplasia often has the expanded basal layer of cells with vertically oriented nuclei, occupying the lower twothirds of the epithelium. There may be epithelial thickening but the maturation is partial, with some superficial flattening of cells and prickle cells in the superficial third of the epithelium. The cells are larger than in mild dysplasia and there is comparatively more pleomorphism. Nuclear alterations, such as angulations, grooving and lobulation, are easier to see (Figure 10). Chromatin remains, in general, finely granular but mitoses are usually present in the lower third of the epithelium. Any extension of the basilar zone maloriented cells into the upper third of the epithelium deserves classification as severe dysplasia. This is usually accompanied by a marked thickening of the epithelium and little maturation with only rare prickle cells. Cytologically there is a notable increase in cell size, nuclear pleomorphism and nuclear/cytoplasmic ratio. Nuclear contour irregularity is the norm and chromatin is often coarse and irregular. Nucleoli are frequent, and mitoses may be found in the lower two-thirds of the epithelium (Figure 11). The most notable feature of carcinoma in situ is the lack of maturation and chaotic orientation of cells such that, were the epithelium inverted, it would look similar. Many cases show marked epithelial thickening but this is not universal and some examples of thin CIS are encountered. Crowding and overlapping are prominent. Cytological abnormalities are generally

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Figure 11. Severe dysplasia. Note the mitosis high up in the epithelium.

extreme, mirroring those in poorly differentiated invasive squamous cell carcinoma. Nuclear orientation is haphazard and mitoses may be present at any level (Figure 12). Kayser et al. observed that SD/CIS lesions close to a concurrent tumor tend to be of higher grade than lesions located further from the neoplasm. In addition, chromosomal aberrations in morphologically normal bronchial epithelium (see below) are more numerous nearer to associated carcinomas.68,69 They postulated that pre-neoplastic lesions may be “partly induced by the tumor itself”. This is a somewhat controversial suggestion and cannot account for pre-neoplastic lesions occurring in lungs without invasive carcinoma. Auerbach et al. noted that as the degree of atypia increases so does basement membrane thickness.29 Others described a tendency for greater variability in the thickness of the basement membrane with increasing grades of dysplasia.70 In the highest grades of atypia, the variation in basement membrane thickness may include areas where it appears to be absent.71 In practice the author does not find these changes in basement membrane helpful in diagnosis, including in the recognition of invasion (see below). Fissler-Eckhoff et al. showed that neovascularization of the subepithelial stroma increased as the grade of dysplasia increased.71 However, no such association was found in another study of microvascular density in pre-invasive bronchial lesions.72 In some cases this process of new vessel growth may manifest as capillary vessels protruding into the overlying epithelium. Such vascular buds retain a covering of epithelium, although they may be thin. Such lesions have been referred to as micropapillomatosis (Figure 13)8,72,73 but the term angiogenic squamous dysplasia (ASD) has also been suggested.74 In ASD lesions the degree of atypia is variable but may be difficult to determine, given the thinning of the epithelium over the vascular buds. The vessels in the buds are often invested in a thick layer of eosinophilic basement membranelike material (Figure 13). Despite the term angiogenic squamous dysplasia, overlying epithelium may be metaplastic without architectural or cytological atypia. In the author’s

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Figure 12. Carcinoma in situ. There is no maturation of this epithelium, which would look identical if inverted.

experience, similar vascular changes with intraepithelial projections may be seen in respiratory epithelium lacking dysplasia or squamous change, so that this pattern of microarchitecture does not guarantee the presence of dysplasia. In an AFB study of heavy cigarette smokers without lung cancer, 34% of patients had ASD lesions, while they were absent in the non-smoking control group.74 The same authors detected ASD in 60% of their patients with bronchial squamous cell carcinoma and have suggested this change, i.e., neovascularization, is a precursor to stromal invasion. While ASD is detectable by AFB, until its biological significance is more clearly determined, its recognition is of uncertain clinical utility.75 The possible molecular mechanisms behind ASD are discussed in the molecular biology section below.

Exfoliative cytology and the diagnosis of SD/CIS Atypical squamous cells may be found in sputum, bronchial washings or bronchial brush samples. The cytological features of these cells have been matched to those found in histopathological samples and classification systems for bronchial cytological atypia devised.8,33,76,77 Metaplastic cells are recognized individually, in small clusters or forming flat sheets. They are smaller than oral squames, which may be present in sputum samples, but larger than basal cells, and have round to oval

Chapter 23: Pulmonary pre-invasive disease

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Figure 13. Angiogenic squamous dysplasia (ASD), understandably also called micropapillomatosis in some cases (a), shows invagination of capillary vessels which are invested in a thickened, hyaline basement membrane (b).

Figure 14. A group of metaplastic squamous cells from a bronchial brushing sample. A small cohesive sheet of cells with abundant cytoplasm and round/oval regular nuclei with indistinct nucleoli lack nuclear irregularity of shape, outline or chromatin structure which might imply dysplasia. (Case courtesy of Dr Mary McKean, Aberdeen Royal Infirmary, UK.)

nuclei (Figure 14).55 Basophilia or orangeophilia (Orange G staining) may be seen. Increasing nuclear abnormalities and variations in both cell size and nuclear/cytoplasmic ratio, as in the histological classification of SD/CIS, facilitate the recognition of dysplasia. Mild dysplasia has been characterized by the presence of a “thickened” cytoplasm and slight hyperchromasia. More frequent orangeophilia and hyperchromasia is found in moderate dysplasia. In severe dysplasia and CIS, the changes are more exaggerated, with uneven, coarse chromatin and

thickened nuclear membranes.78 Nucleoli become more obvious in severe atypia, and in CIS multinucleate cells and emperipolesis may be identified. Sputum examination or bronchial cytology has at least the potential to identify and even allow grading of SD/CIS. Studies of the sensitivity and specificity of these modalities in diagnosing SD/CIS are few. There are considerable issues around sampling error and attributing the dysplastic cells in a cytological preparation to any particular lesion identified at a subsequent histological examination. Older lung cancer screening studies suggest that the efficiency of sputum cytology in detecting pre-invasive lesions is quite low.57 Woolner et al.79 demonstrated that sputum cytology had a combined sensitivity of 78% for detecting CIS and invasive carcinoma. Another study suggested that even established invasive squamous cell carcinomas causing chest radiographic abnormalities were undetected by sputum cytology in 40% of cases.80 Combined WLB/AFB in a cohort of patients with “moderate” sputum atypia revealed severe dysplasia in 9%, CIS in 3% and early squamous carcinoma in 4%, implying a substantial false-positive rate.81 In a further AFB study, conventional sputum cytology failed to detect any of the dysplasias and two-thirds of the squamous metaplasias identified by this bronchoscopic method.36 As with many other areas of diagnostic cytopathology, the success of the test is probably very dependent on the skill and experience of the pathologist/cytologist. The absence of architecture, which is an integral part of the defining criteria for dysplasia, makes cytological diagnosis challenging and risky. There are numerous artifacts and non-neoplastic processes, which may generate cytological atypia, leading to false-positive diagnosis of dysplasia or malignancy.82–84 Although there are published criteria for the distinction of CIS from invasive squamous carcinoma on cytology,76 this may be a difficult exercise.84 It is also a challenging task in biopsy material, where the pathologist has the additional advantage of architecture.

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Problems in the histological assessment of SD/CIS The diagnosis and classification of SD/CIS in the bronchus can be very demanding. Pathologists who work in centers where AFB is performed have the greatest experience. In centers where bronchial biopsy samples are derived solely from WLB, pathologists will see dysplasia and CIS in cases where an experienced bronchoscopist identifies and samples a lesion; otherwise the presence of SD/CIS in samples may be serendipitous. In addition, many bronchoscopists do not perform biopsies in the absence of visible lesions. If tissue blocks of airways uninvolved by tumor are taken during the grossing of lung resection specimens bearing squamous cell carcinomas, SD/CIS lesions will be encountered. Despite the plethora of literature describing a multitude of molecular biological changes in SD/CIS, there are remarkably few published studies of the applicability of the WHO criteria for SD/CIS and interobserver variability in diagnosis. One study suggested that reasonable intra-observer consistency could be achieved (mean weighted Kappa (K(w)) values 0.71) while inter-observer agreement was less (K(w) 0.55), with no difference between application of a full five-point grading system (squamous metaplasia, three dysplasia grades and CIS) versus a three-grade system.85 This study was based on examination of fixed photographic images. Unfortunately, no data were provided on a two-point system of high grade (moderate dysplasia or worse) versus low grade (mild dysplasia or hyperplasia). This two-tier grading system has been implemented by some authors. Venmans et al. noted sufficient interobserver variability between pathologists in reporting grades of SD and CIS in AFB-derived bronchial biopsy samples that this could impact the interpretation of AFB studies.86 Application of the WHO criteria for the diagnosis of SD/ CIS assumes the lesion shows full-thickness change, including an increase in the number of cell layers. It is relatively easy to visually divide a thickened stratified epithelium into upper, middle and lower zones for assessment of the distribution of the changes indicated in Table 3. Not infrequently the atypical epithelium is not particularly thickened, or may be thinner than even normal respiratory epithelium. Division of such epithelium into three tiers is extremely difficult. Obliquely cut or poorly oriented epithelium may be impossible to assess. Atypia may often be seen in an epithelium that retains a layer of differentiated respiratory epithelial cells (columnar, goblet or ciliated cells) on the surface. These cells are often attenuated. A mucin stain or a cytokeratin 7 may assist in demonstrating these residual cells (Figure 3). Squamous change with prickle cells may be seen in zones of basal cell hyperplasia, with retention of the overlying columnar respiratory epithelial cells; referred to earlier as immature or incomplete squamous metaplasia. Atypia may be seen in basal cell hyperplasia or in immature squamous metaplasia and fullthickness squamous metaplasia is not a prerequisite for the development of bronchial dysplasia. While dysplasia may be encountered in the classic full-thickness squamous epithelium,

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it is the author’s impression that dysplasia in a partially altered epithelium is at least as frequent (Figure 15). The irritant and carcinogenic effects of tobacco smoke may act independently of each other. This means that foci of squamous metaplasia and basal cell hyperplasia may occur in the same patient and each can undergo dysplastic change.8 Such suggests carcinoma in situ could arise de novo from respiratory epithelium without passing through a dysplasia phase. Others suggest that based upon observations in animal models, hyperplastic goblet cells may transform into atypical squamous cells.47 The possibility of dysplasia arising in a range of different lesions in the bronchial mucosa may account for the range of bronchial dysplastic lesions described by Wang et al.87 These authors noted basal cell and bronchial epithelial dysplasia with transitional features, as well as classic squamous dysplasia and columnar cell dysplasia. The significance of these different lesions and the utility of differential keratin expression in discriminating between them require further study. This range of appearances probably indicates that dysplasia may supervene, presumably as a result of carcinogen action on different reactive or hyperplastic bronchial lesions. The resulting dysplasia may be difficult to grade if the architecture does not match the classic full-thickness squamous lesion described by the WHO. In bronchial biopsies it is common for the surface bronchial epithelium to be ragged, with some areas completely missing and others having lost some of the superficial cell layers. This may be more likely to happen if the bronchoscopist washes or brushes the mucosa before taking biopsies, or it could be due to inadvertent trauma from the bronchoscope. If the partial loss of cells occurs in a dysplastic epithelium, application of diagnostic criteria may again be difficult. If the superficial respiratory epithelial glandular cells remain, these are more likely to separate at their irregular interface with the atypical cells beneath (Figure 15). The diagnosis of SD/CIS may be challenging, especially when the pathologist has limited experience. If one cannot apply the diagnostic criteria in full, a more pragmatic approach is required. Most of the above-mentioned pitfalls involve a loss of epithelial architecture, in which case the pathologist has to rely more heavily on the cytological features of the (residual) dysplastic epithelium. In such cases the author often adopts a two-tier categorization, classifying cases as simply low grade (mild dysplasia or less) or high grade (moderate dysplasia or more severe grades).

Differential diagnosis of SD/CIS As with the assessment of other pathological conditions which involve a spectrum and likely transition of a disease process, there are important differential diagnoses. These are especially between mild dysplasia and a variety of non-dysplastic, reactive/hyperplastic changes in the bronchial epithelium, and between CIS and invasive disease.88

Chapter 23: Pulmonary pre-invasive disease

(a)

(c)

(b)

(d)

Figure 15. These images show clear evidence of atypical squamous epithelium surmounted by differentiated respiratory epithelium. Apart from the presence of ciliated cells and goblet cells, the eosinophilia of the residual respiratory epithelial cells is characteristic and should not be mistaken for keratinization when these residual cells become atrophic. In (a) and (b) the degree of atypia is rather less than is evident in (c) and (d). In these cases it is difficult, if not impossible, to apply the WHO criteria for grading “squamous” dysplasia.

The distinction between mild dysplasia and squamous metaplasia or even marked examples of basal cell hyperplasia can be impossible. If the expanded zone of vertically oriented basal cells is distinctive, then a diagnosis of dysplasia is relatively straightforward. Unfortunately, this scenario is rare and the cytological changes attributed to mild dysplasia are very subtle. Chronic irritation and inflammation of the bronchial mucosa may induce basal cell hyperplasia and some reactive atypia (Figure 16). A diagnosis of mild dysplasia is probably unwise when there is appreciable airway inflammation. Knowledge of the clinical details, the exact source of a biopsy and information on any accompanying lesion accounting for the reactive change or squamous metaplasia help in reaching the correct diagnosis. Even epithelium which is obviously pseudostratified respiratory in type may show foci of atypia in a range of inflammatory situations. More specific examples

include atypia from viral cytopathic effect, radiotherapy or cytotoxic chemotherapy. Given the probable low rate of progression and propensity for regression of mild dysplasia, the distinction between mild dysplasia and reactive change is probably of negligible clinical significance (see below). Endobronchial atypical squamous papillary lesions lacking invasion pose an uncommon clinical problem (see Chapter 22).89 Such is their rarity that it may be difficult to come to any definite diagnosis. While a diagnosis of CIS would not be unreasonable, the WHO classification considers such squamous papillary lesions “invasive”. Since the cytological features of CIS are identical to those of invasive carcinoma, this distinction may be impossible. If the biopsy sample comprises relatively small fragments of “malignant-looking” squamous epithelium, devoid of any non-epithelial stromal tissue, the histopathologist has no way

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(a)

(b)

Figure 16. These are examples of reactive squamous epithelia in the lung, occurring in situations where there is cavitation and/or chronic inflammation. An old tuberculous cavity containing an aspergilloma (a) and bronchiectasis (b), both showing squamous metaplasia. Note intraepithelial inflammatory cells in both images.

Figure 17. Severe dysplasia and carcinoma in situ extending from the bronchial surface down the bronchial gland duct to involve bronchial gland acini.

of discerning in situ from invasive disease. A number of features may favor one or the other but a “suggestive” rather than definitive report may be most appropriate. It is essential to know whether the bronchoscopist saw an endobronchial mass. Large irregular chunks of aberrant epithelium are probably derived from invasive disease. CIS generally has a smooth straight or undulating interface with the subepithelial stroma. If stripped off the mucosal surface, the shape of the strips of CIS is retained. If there is a distinct zonation within the aberrant epithelium, different areas showing markedly different patterns of squamous carcinoma, the disease is more likely to be invasive. Vascularization of the epithelium with thin slivers of barely perceptible stroma also suggests invasion. Care is needed to distinguish this from the tufts of plump capillaries invested in a hyaline basement

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membrane-like layer seen in ASD. Necrosis is not seen in CIS. Knowledge of the presence of a radiological mass in the region of the biopsy may be of diagnostic help. Invasive disease and CIS often coexist in a biopsy sample. In these instances, CIS is only of academic interest since clinical management is determined by the invasive carcinoma. CIS adjacent to invasive carcinoma confirms the bronchial origin of the carcinoma.90 Severe dysplasia or CIS may extend from the bronchial surface to involve bronchial gland ducts or the gland lobules (Figure 17). In the author’s experience, this change is rarely seen without a nearby invasive carcinoma, but it is important to appreciate as a possible pitfall for overdiagnosis of invasion. Attention to the location of the change (adjacent to cartilage), the shape of the atypical cell groups (tubular when replacing ducts or round lobules replacing the seromucous glands) and any accompanying residual seromucous gland cells identify this as in situ disease (Figure 18). Clearly the diagnosis of invasive, as opposed to in situ, carcinoma usually has clinical implications. In difficult cases, clinicopathological correlation is important. Pure in situ change without invasive disease is a rare occurrence. In equivocal cases it may be best to indicate that the features are “at least” those of CIS but indicate any suspicion of invasion as appropriate. Mutual understanding between the requesting physician and the pathologist of all the issues surrounding the diagnosis of SD/CIS is clearly important.

Molecular biology of bronchial pre-invasive lesions A large amount of literature has been published on many aspects of the molecular biology of pre-invasive lesions in the bronchus. Apart from studies of specific oncoprotein expression using immunohistochemistry, other molecular biological techniques have been employed to investigate epigenetic

Chapter 23: Pulmonary pre-invasive disease

In tumor biology, whenever there is increased cell production by mitosis, there is also increased cell loss by apoptosis. However, an imbalance in favor of cell production causes cell populations to expand and lesions to grow. Tormanen et al. demonstrated that apoptosis increased three-fold in squamous metaplasia and four-fold in dysplasia. Additional studies failed to demonstrate an association between apoptotic levels and expression of the apoptosis-related proteins, bcl2, bax or p53.103

p53 and related proteins

Figure 18. Carcinoma in situ involving the bronchial surface, gland duct and acini. The lobulated architecture of the deep clusters of atypical cells and the admixture of glandular cells prevents misinterpretation as invasive carcinoma.

changes in gene expression, gains and losses of genetic material through “loss of heterozygosity” (LOH) studies and comparative genomic hybridization, expression array analyses, and gene mutation studies.91–94

Hyperproliferation Increased cell proliferation is a consistent event in early neoplastic change. Some “genetic accidents”, such as translocations and mutations, occur during gene replication and cell division and lead to genomic instability. Basal cell hyperplasia has been discussed as a likely important precursor for the subsequent development of dysplasia. Mitotic activity is an important diagnostic feature of SD/CIS. Several studies demonstrate that the proliferative compartment of bronchial epithelium is expanded in SD/CIS. This finding mirrors the basal zone expansion observed on standard histological sections. The proportion of epithelium containing “cycling” cells, as measured by the expression of proliferating cell nuclear antigen (PCNA) or the anti-MIB1 (Ki67) antibody, rises from that seen in normal pseudostratified epithelium to higher levels in both low- and high-grade dysplasia.95–99 Tan et al. found that the minichromosome maintenance 2 protein (MCM2) gave consistently higher indices in lesions when compared to Ki67 and concluded it was a more complete cell cycle marker.100 Boers et al. showed no difference in the MIB1 index in squamous metaplasia or dysplasia in those with, versus those without, invasive carcinoma.97 Interestingly, Khuri et al. showed the PCNA index fell on smoking cessation, combined with 13-cis-retinoic acid therapy. These changes accompanied the reversal of squamous metaplasia.101 A relationship between tobacco consumption and proliferative index in bronchial epithelium in smokers has also been recorded. Ki67 indices remain elevated in former smokers though below levels seen in active smokers.102

Located at chromosome 17p13, P53 is a tumor suppressor gene (TSG). p53 protein acts as a transcription factor controlling a number of genes. It negatively regulates the anti-apoptotic Bcl2 and positively regulates pro-apoptotic bax. p53 also positively regulates P21 (waf1), which inhibits the CDK4/CyclinD1 complex phosphorylation of Rb (retinoblastoma) and thus induces G1/S arrest. Bcl2 and bax form complexes and the bcl2:bax ratio may regulate apoptosis. The TSG functions of P53 are thus mediated by preventing cell proliferation and promoting apoptosis. The P53 gene responds to DNA damage by inducing either DNA repair or cell death, hence the name “guardian of the genome”. Hypoxia or some oncogenes (MYC, RAS, E2F1) can mediate a similar effect in stimulating p53 activity. P53 is itself regulated by mdm2, p14 and homolog protein p63.94,104 The function of p53 is directly altered by gene mutation. Changes in upstream regulators or downstream effecter proteins can emulate the same effect. p53 protein is normally either undetectable or found at very low levels in “normal” cells, using immunohistochemistry. Altered p53 protein has a much longer intracellular half-life than labile wild-type p53 protein, resulting in detectable quantities in cell nuclei. Most anti-p53 antibodies bind the wild type as well as mutant proteins. P53 mutation is one of the commonest alterations in human cancer and gene function may also be lost by deletion (LOH). After initial studies demonstrated elevated p53 protein levels in both SD105 and CIS,106 subsequent work showed increasing expression of p53 protein in nuclei progressing from normal respiratory epithelium, through basal cell hyperplasia and squamous metaplasia, to the dysplasias and CIS.70,95,107–113 While most normal, hyperplastic and metaplastic epithelia do not stain for p53, between 11 and 30% of mild dysplasias, 25–50% of moderate dysplasias and 50–78% of severe dysplasias show stainable p53. CIS shows high levels, similar to those seen in severe SD. Oddly in three studies where SD and CIS were scored separately, the proportion of CIS positive for p53 was less than in severe SD.70,107,113 Very low numbers (1–5%) of p53-positive cells have been found in normal bronchial epithelium, in either patients with concurrent invasive carcinoma or those with a very high risk of developing lung cancer.108,113 The significance of expression at this level is uncertain. It probably does not reflect pathological alteration of P53 but may be a physiological change in p53

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metabolism or a reflection of sensitive immunohistochemical detection. Concordance of p53 status between carcinoma and concurrent SD/CIS is high.70,108,111,114 Discordance most likely manifests as p53-negative SD/CIS but p53-positive carcinoma. Brambilla et al. also found that SD/CIS close to p53-positive cancer was more likely to be positive than SD/CIS more remote from the tumor.111 This is consistent with the concept of field carcinogenesis. Most authors believe SD/CIS associated with invasive carcinoma is more likely to be p53-positive than SD/CIS occurring in isolation, without concurrent carcinoma. This raises issues around the molecular basis of progression of SD/CIS (see below). Only relatively few lesions, generally high-grade squamous dysplasias, have P53 mutations.105,106,115 Kohno et al. showed P53 mutation and 17p LOH (see below) in 9% of dysplasias (two of 22 cases), 17p LOH but no dysplasia in another two cases, but squamous metaplasias showed neither abnormality.116 P53 mutations have been found, in some studies, to be widespread in the airways, suggesting their association with clonal expansion of cells in the “at risk” epithelium.117 An area of dysplasia with one p53 mutant allele transformed after 9 months into a tumor homozygous for the same mutation.118 On the other hand, other studies have shown heterogeneity in genetic alterations between synchronous lesions in the same lung, including differences in P53 status.22,119,120 Less literature exists on other p53-associated proteins. P63 protein is highly expressed and the P63 gene at 3q27 is frequently amplified in most squamous cell carcinomas. Gene copy number is often found increased in severe dysplasia and CIS but not in lesser stages of disease. This finding corresponds to an excess of p63 protein expression, above that found in normal basal cells.121 While there is no demonstrable relationship between p53, bcl2 and bax expression in SD/CIS,108,111 some studies have shown an increase in bcl2 expression in SD/CIS over normal cells. 122 Others have either failed to demonstrate bcl2 in squamous metaplasia,97 or shown a degree of positivity in 30–50% of the lesions of basal cell hyperplasia, squamous metaplasia or dysplasias, but with no difference between the grade of the lesions.110,111 There appears to be an increasing diminution in bax expression as lesions become more aberrant. Thus the bcl2:bax ratio increases with disease progression, mediating a pro-survival, anti-apoptotic state in higher grade disease.111 Although p21 (waf1/cip1) is upregulated in pre-invasive lesions compared to normal respiratory epithelium, there is no consistent association between expression and grade of lesion.111 P53 regulators, mdm2, p14arf and nucleophosmin (NPM), have been studied in bronchial pre-invasive lesions.123 While both NPM and p14arf are diffusely expressed in normal epithelium, mdm2 staining was increased in mild SD. Nucleolar redistribution of NPM was seen in moderate SD. In severe SD, in association with high mdm2 expression, p14arf expression

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was either lost or relocalized to nucleoli. Nucleolar relocalization of p14arf was associated with the same finding for NPM. The precise role of these changes in altered p53 regulation remains unclear.

P16 (ink4a)–CyclinD1–CDK4–RB pathway The retinoblastoma (RB) pathway plays a major role in controlling cell progression from G1 into S-phase, and thus cell division. The CDK4/CyclinD1 complex phosphorylates Rb; phosphoRb promotes G1-S transition by releasing bound E2F, which is an important transcription factor essential for G1-S transition104 and cell proliferation. A pro-oncogenic state is thus promoted by loss of p53/p21 (waf1/cip1) mediated inhibition (see above), loss of p16 (ink4a) mediated inhibition of CDK4/CyclinD1 activity, or by an overexpression of CyclinD1 itself. Loss of Rb protein also promotes cell proliferation by freeing E2F. Loss of p16 function may be realized through deletion or mutation of the gene. Promoter hypermethylation of P16 also inhibits gene expression. Loss of Rb protein is rare in NSCLC, is never seen in bronchial pre-neoplastic lesions, but is frequently detected in SCLC (see below).94,112 Loss of p16 has been shown in 12% of moderate SD and 30% of severe SD.124 Breuer et al. studied 23 pre-invasive lesions across the spectrum of change and found loss of p16 in only two SD/CIS cases.125 Cyclin D1 overexpression may occur earlier than p16 loss. Brambilla et al.124 showed overexpression of cyclin D1 in 6% of hyper/metaplastic lesions, 17% of mild SD, 46% of moderate SD and 38% of CIS lesions. Lonardo et al. found almost identical rates of staining and also showed that in high-grade SD, cyclin E was overexpressed in 33% of cases, something not seen in lower-grade disease. Cyclin E is influential in promoting G1-S transition.112 Jeanmart et al. found cyclin E overexpressed in lower grade lesions.126 The polycombgroup gene, BMI-1, is a possible negative regulator of p16 and 11/23 preinvasive lesions were positive for this protein. There was no relationship between BMI-1 positivity and p16 loss.125 The CDNK4A/P16INK4a gene is located at 9p21 and hypermethylation of its promoter is a common mechanism of (epi)genetic silencing in NSCLC, apparently related to tobacco smoking.127 Belinsky et al. found that CIS lesions associated with squamous cell carcinomas had P16 methylation in 75% of cases, as did the carcinomas. They showed P16 hypermethylation in 50% of all CIS, 24% of squamous metaplasias and 17% of basal cell hyperplasias.128 A similar progressive increase in P16 hypermethylation was found by Lamy et al.; in 5% of normal epithelial samples, 21% of lesions showing metaplasia or mild/moderate SD, and in 50% of severe SD/CIS lesions, all obtained via AFB.129 The p16 protein expression in these lesions mirrored the methylation changes; the latter persisting long after smoking cessation. Breuer et al., however, only found P16 hypermethylation in one CIS lesion but not in the 22 other lower-grade lesions examined.125

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P16 gene function may also be lost through deletion of the gene (LOH). It is found in around 80% of CIS lesions, 30–75% of dysplasias and in some studies in 20% of hyper/metaplastic lesions, and in 15% of morphologically normal respiratory epithelium from smokers.120,130–132 Loss at 9p21 was found in the squamous lining of a lung cyst, associated with squamous cell carcinoma.133 Loss of function of P16 seems to be important in the evolution of bronchial SD/CIS. It may be an early lesion occurring in morphologically normal epithelium but is very frequent in high-grade lesions. The mechanism of the loss may vary but is probably a combination of gene deletion and promoter hypermethylation.

Tyrosine kinase (TK) signaling pathways Among the EGFR family of transmembrane TK receptors, EGFR (EGFR1) and HER2 (EGFR2 or ERBB2/NEU) are the most frequently studied in lung cancer. Following ligand binding, heterodimers form, including with the remaining two family members HER3 and 4. This activates their internal domain tyrosine kinases through phosphorylation. Downstream effects include promotion of cell cycle progression, cell division and angiogenesis, and inhibition of apoptosis (the effect is to some extent ligand-dependent) through activation of several signaling pathways, including Ras/Raf, PI3-kinase/ AKT/PTEN, SRC, STAT and MAP/kinase. EGFR protein has been demonstrated immunohistochemically in early lesions, such as hyperplasia and metaplasia, in morphologically normal mucosa of smokers and in increasing amounts in SD/CIS.101,134–136 The amount of EGFR expression correlates with cell cycle activity101 and expression is diminished on smoking cessation. Piyathilake et al. found a little more HER2 protein staining in the normal or hyperplastic epithelium of smokers but it appeared not to be a significant factor in SD/CIS.135 This finding was supported by Meert et al., who found no EGFR expression in most low-grade lesions, while high-grade SD/CIS was mostly positive for EGFR.137 Merrick et al., in a large study, also demonstrated progressively more EGFR expression as lesion atypia increased, as well as a correlation with Ki67 expression.138 They failed to demonstrate any link between HER2 expression and grade of disease. These authors speculated about the potential for EFGR TK inhibitors (EGFR TKI) as chemopreventative agents. Meert et al. also showed increased EGFR expression but only at the severe SD/CIS stage. These authors associated EGFR expression with angiogenesis (see below).72 A limited increase in EGFR gene copy number has been described in 70% of SD/CIS lesions, though it was unrelated to grade of disease.139 There are no reports of EGFR mutations in SD/CIS. EGFR TKIs (tyrosine kinase inhibitors) are an important therapeutic option for some patients with lung cancer. Response to these drugs is strongly related to the presence of certain EGFR mutations, and to a lesser extent to EGFR gene amplification. EGFR protein levels do not predict response.

The data on EGFR status in SD/CIS and the lack of EGFR mutations in squamous cell carcinomas suggest this molecular mechanism may be largely irrelevant in squamous carcinogenesis in the lung.140 This issue is discussed further in relation to adenocarcinogenesis (see below). K-RAS mutation does not seem to be a factor in the evolution of SD/CIS. Unlike the case of peripheral lung adenocarcinogenesis (see below), there have been only a few studies of K-RAS mutations in bronchial pre-invasive lesions. These have failed to demonstrate K-RAS mutation in the bronchial lesions.141 A serine/threonine protein kinase B (AKT) is a downstream effector of PI3-kinase and is active in its phosphorylated form pAKT. Phosphatase and tensin homolog (PTEN) is an inhibitor of PI3-kinase. Overactivity of PI3-kinase may result from gene amplification. This pathway can regulate several cellular processes, such as cell proliferation, apoptosis, as well as cytoskeletal organization.104 Tobacco smoke appears to upregulate AKT in respiratory epithelial cells. Tsao et al. detected pAKT in 27% of normal bronchial mucosal samples, 44% of hyperplasias and 88% of dysplasias.142 Massion et al. found both overexpression of pAKT and amplification to be concordant and a feature of severe SD.143

Angiogenesis and related factors The induction of new vessel growth is important in the development of invasion. The appearance of new vessel growth in the tissues deep to SD/CIS is of interest as a possible harbinger of invasion. In fact, an “angiogenic switch” might be crucial at this stage of disease evolution.144 A progressive increase in vascularization has been documented deep to squamous metaplasia, dysplasia and CIS.71,145 Both these studies describe the evolution of capillary clusters just deep to the more atypical epithelia, with the growth of intraepithelial capillary sprouts, reminiscent of ASD. This process may be mirrored by an increase in vascular endothelial growth factor (VEGF) expression.145 Lantuejoul et al. also found a progressive increase in VEGF expression with increasing grades of dysplasia. Interestingly, they did not find especially high amounts in ASD.146 Merrick et al. also showed progressive VEGF protein expression, as well as similar changes in VEGF mRNA also with increasing grades of dysplasia.147 They also showed expression of VEGF165 mRNA changed to the 121 isoform during disease progression. A progressive increase in VEGF receptors neuropilin 1 (NP1), kinase insert domain receptor (KDR) and fms-related tyrosine kinase 1 (flt1) was also demonstrated. These authors found the highest levels of angiogenic stimuli in ASD cases. The tumor-promoting effects of VEGF binding to receptors NP1 and NP2 may be abrogated by competitive binding of other ligands, such as class 3 semaphorins (SEMA). SEMA3F may thus act as a TSG (tumor suppressor gene). Semaphorin gene loci are found at 3p21.3.148 Loss of SEMA3F expression has been demonstrated in pre-invasive bronchial lesions,146 perhaps as a result of the 3p LOH, a frequent finding in this

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disease (see below). NP1 and 2 expression increases with disease grade.146 Meert et al. could not demonstrate a significant increase in subepithelial microvascular density until invasion was seen, except when ASD was present.72 COX-2-derived prostaglandin E2 may be important in tumor angiogenesis and COX-2 is believed to be implicated in the genesis of many human cancers. In a study of 106 AFBderived samples of pre-invasive bronchial lesions, low-grade changes and moderate SD did not express COX-2. However, 57% of severe SD and 63% of CIS overexpressed this enzyme.149

Cell immortalization Cell senescence may be prevented by the activation of the telomerase ribonucleoprotein complexes. These are TTAGGG repeats, which are important in chromosome end stabilization. Activated telomerase can prevent the progressive, “physiological” telomere shortening, which occurs after each cell division. Short telomeres are identified as “damaged DNA” and can trigger p53-mediated apoptosis.94 Activation of telomerase is a frequent finding in most human tumors. The earliest study of telomerase activity in SD/CIS found human telomerase reverse transcriptase (hTERT) was increased four-fold over normal in all pre-invasive lesions but 40-fold in invasive carcinoma.150 Capkova et al. reported similar rises in hTERT expression.151 Yashima et al. showed that, while normal respiratory epithelium showed weak expression of human telomerase RNA component (hTERC) in 20% of cases, expression was strong in 70–80% of hyperplasias and dysplasias, and in 95% of CIS. There is a progressive increase in hTERT mRNA with increasing grade of lesion.152,153 The latter authors also showed an association with telomerase activity and cell cycle activity, bcl2:bax ratio rise and p53 expression. These findings suggest resistance to apoptosis, increased cell proliferation and cell immortalization all occur in a coordinated fashion.

Transcription factors and other intracellular effectors Nuclear factor-kappaB (NF-kappaB) is a key transcription factor active in oncogenesis, regulating many important signaling pathways. A progressive rise in NF-kappaB p65 protein was demonstrated with progression from normal and reactive epithelium to SD/CIS. Upstream stimulatory factors USF-1 and USF-2 also regulate transcription. While USF-2 protein expression appeared limited to normal bronchial ciliated cells, it was also strongly expressed in 72% of bronchial dysplasias.154 Bolon et al. failed to demonstrate any dysregulation of transcription factor c-Ets-1 in SD/CIS.155 Capello et al. found heat shock proteins (Hsp) 10 and 60 were each expressed in half the normal bronchial and hyperplastic samples examined, but in only 3% of metaplasias and 1% of dysplasias.156 Heterologous nuclear ribonucleoprotein (hnRNP) B1 is present in most squamous cell carcinomas. It was detected in normal respiratory epithelium in cancer-bearing lungs and in 63% of SD/CIS lesions,157 making it a putative

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marker in sputum screening for lung cancer.158 Maspin, one of several serpin genes, located at 18q21.3, is overexpressed in pre-invasive bronchial lesions. Associated invasive squamous cell carcinomas show a wider expression of several serpins.159

Other tumor suppressor genes Besides P53, there are a number of TSGs, which have been studied in pre-invasive bronchial squamous lesions. Many are implicated in LOH studies and are discussed below. Some have been studied using immunohistochemistry to detect protein gene product. Retinoic acid receptor (RAR) function is often lost in carcinogenesis and loss of RAR-b expression has been shown in SD/CIS, usually in high-grade lesions.160 Another 3p TSG, Fragile Histidine Triad (FHIT), spans the FRA3B fragile site at 3p14.2. FHIT is thought to have a role in regulating cell proliferation and apoptosis, and is frequently lost in human cancers.161 The importance of this loss is not clear since gene mutations are very rare. Nonetheless, FHIT protein was lost from 60% of moderate SD and all severe SD and CIS lesions.162 A number of studies demonstrate LOH at 3p14.2. In some this loss was an early phenomenon, even in normal or hyperplastic epithelium, while in other studies it only appeared in severe dysplasia.132,161–166

Human papilloma virus, cell adhesion molecules and other assorted factors Human papilloma virus (HPV) probably has no role in the development of SD/CIS since HPV DNA is reported as absent or very uncommon in pre-invasive lesions.167,168 There are reports of HPV DNA in a small proportion of invasive squamous cell carcinomas but other studies have failed to find HPV DNA in invasive tumours (see Chapters 24 and 28). If HPV has a genuine role in the evolution of occasional squamous cell carcinomas of the lung, it may be that these arise independently of SD/CIS. Bronchial epithelial basal cells consistently express CD44v6. This adhesion molecule splice variant may also be found in squamous metaplasia169 and is detected extensively throughout the cell layers in SD/CIS.170 Kato et al. found there was a significant reduction in the expression of E-cadherin and/or its intracytoplasmic binding molecules, alpha and beta catenin and plakoglobin, as pre-invasive lesions increased in grade from metaplasia, and dysplasia to CIS.171 Cytokeratin (CK) expression has been studied in pre-invasive lesions. Basal cells express CK 5 and 6; 34betaE12, which decorates CKs 1, 5, 8 and 14, does the same but also stains some more differentiated cells. CK7 tends to stain only differentiated columnar cells. CKs 4, 6 and 17 may be lost as metaplasia and dysplasia supervene, but CK10 and 14 can appear as disease progresses.172,173 Galateau-Salle et al. found there was reciprocity of matrix metalloproteinase (MMP) 9 and MMP1 expression during bronchial pre-invasive disease progression with MMP9 increasing and MMP1 falling as grade increased.174 MMP2 and its inhibitor, TIMP1, showed no

Chapter 23: Pulmonary pre-invasive disease

variation in expression. Bolon et al. found minimal MMP1 or MMP7 expression but noted MMP3 in about a third of lesions of all grades and MMP11 in dysplasia and CIS, but not in hyperplasia or metaplasia.155 Sanchez-Mora et al. showed loss of histo-blood group antigens, A and B, particularly in high-grade SD and when cell cycle activity was increased.175

Studies of genomic instability and other global expression data Nuclear aberration is a histological hallmark of cancer. Changes in the size, shape and staining characteristics of nuclei are integral features of the grading system for SD/CIS. These nuclear features are, to some extent, a function of the DNA content of the nuclei. While the invasive phenotype is associated with the most extensive genomic alterations, numerous changes to chromosomes or even individual genes may be found in all stages of pre-invasive bronchial disease. Morphologically normal respiratory epithelium in smokers shows a significant number of changes compared to non-smokers’ mucosa. DNA aneuploidy increases with grade of pre-invasive bronchial lesion. Hirano et al. found aneuploidy absent in normal bronchial epithelium but present in 8% of low-grade SD, 33% of high-grade SD and all invasive carcinomas.95 These changes were related to proliferative activity in the lesion. As well as confirming the grade-associated increase in aneuploidy from normal, through hyperplasia and metaplasia to SD/CIS, Smith et al. also demonstrated concordance between the frequency of aneuploidy in pre-invasive lesions and concurrent cancer in the same lung.176 Chromosomal aneusomy was demonstrated using fluorescence in situ hybridization (FISH) probes to centromeres 6, 5p15.2, 7p12 (EGFR) and 8q24 (MYC) in atypical and normal bronchial epithelial cells in sputum from heavy smokers, some of whom had carcinoma.177 The same group used the same probes in SD/CIS biopsy samples to show aneusomy in 64% of SD/CIS lesions from lungs with, and 31% of SD/CIS lesions in lungs without, concurrent cancer.178 Zojer et al. also demonstrated chromosome 7 aneusomy in squamous metaplasia associated with carcinoma.179 Pelosi et al. found 3q24 amplification and chromosome 3 polysomy in 27% of high-grade SD/ CIS lesions.180 Comparative genomic hybridization (CGH) has been used to demonstrate chromosome 3 changes in SD/CIS lesions derived from AFB examinations,181 and amplification at 8p21 and 8q22 in CIS.182 CGH was also used to demonstrate genomic gains at 1q25–32, 12q23–24.3 and 17q12–22 in CIS lesions but not in dysplasia or metaplasia.26 LOH studies provide insight into the genomic changes noted during bronchial carcinogenesis. Many of these changes relate to smoking, are present at lower levels even in morphologically normal respiratory mucosa and increase in frequency and extent as morphological changes occur and the grade of lesion increases.132,183–185 These studies demonstrate multiple SD/CIS lesions in bronchi appear to be clonally unrelated and may persist long after smoking cessation.132,184 The risk of

lung cancer also persists after smoking, diminishes slowly but never reaches never-smoker levels. Concordance between losses in invasive squamous cell carcinoma and associated SD/CIS appears to be high. Changes in chromosome 3p can be found in morphologically normal bronchial mucosa in smokers. Such losses are also found in pre-invasive lesions; in such cases the losses are greater and more widespread.25,132,184–186 Among the earliest losses are in 3p14.2 (FHIT), 3p21 (RASSF1A, FUS-1, SEMA3B), 3p22–24 (BAP-1) and 3p25. In a comparative study of 3p losses, while 47% of smokers showed loss in 3p, none of the never-smokers did.184 Normal and hyperplastic epithelium showed losses in 31% and 42% of cases respectively.132 Hung et al. found more 3p losses in early lesions; in 76% of hyperplastic foci, 86% of SD lesions and all CIS.186 Wistuba et al. also reported more 3p loss in up to 78% of SD/CIS and noted that loss at 3p12 (DUTT1) appeared in SD.132 Loss at 9p21 (P16ink4a) also appears to be an early change and while 17p13 (P53) loss has been described in basal cell hyperplasia, it is more frequently found in high-grade dysplasia and CIS.132 Deletions at 8q21–23 have been described at an early pre-dysplastic stage,24 though usually in conjunction with 3p and 9p losses. Losses at 13q14 (RB) and 5q (APC) are much less frequent during bronchial carcinogenesis and generally appear late.132 Bronchial epithelial cells from heavy smokers show promoter hypermethylation of the P16 gene (see above), as well as other potential TSGs, such as RARbeta, FHIT, RASSF1A and H-cadherin.25 Methylation of ECAD, MGMT and DAPK has also been found in smokers’ bronchial epithelium,187 and in morphologically normal bronchial resection margins in cancer resection specimens.188 These are just some examples of the vast amount of data which has shown silencing of several genes through promoter hypermethylation, at the very earliest stages of bronchial carcinogenesis. Epithelial microdissection and expression array analysis of morphologically normal bronchial epithelium from smokers and non-smokers reveal differential expression of 23 genes; 10 of these genes are XMEs (xenobiotic metabolizing enzymes), two are oncogenes (FGFR3 and LMO3) while HLF is a TSG.128 Zhang et al. found 591 differentially expressed genes between smokers and non-smokers, but only 145 genes differed between current and former smokers, implying that many smoking-induced genetic alterations persist on smoking cessation.189 Other global expression studies came to similar conclusions.190 These techniques were used to compare gene expression in morphologically normal bronchial epithelium and concurrent squamous cell carcinomas from current and ex-smokers.191 The authors found that 246 genes, mainly those related to oxidative stress responses, were differentially expressed in normal mucosa between active and ex-smokers. Many of the changes noted in current smokers’ normal epithelium were also found in their tumors. In ex-smokers, the tumors had gene profiles very similar to those found in current smokers’

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tumors. In addition, there were more differences between tumor and normal mucosa in ex-smokers. These findings suggest that while smoking-related changes persist in exsmokers’ mucosa, some smoking-induced changes revert to “normal”. Proteomic analysis of a range of pre-invasive bronchial lesions demonstrates increases in the number of expressed proteins with increasing lesion grade. Between 30 and 50 differentially expressed proteins were identified in metaplasias, dysplasias and invasive carcinomas.192 Rahman et al. identified specific proteomic profiles, which allowed the segregation of histologically classified pre-invasive bronchial lesions.193 MicroRNAs (miR) are short non-coding RNAs involved in post-transcriptional regulation of gene expression. Mascaux et al. found differential expression of 69 different microRNAs during progression of bronchial carcinogenesis.194 MiR-32 and miR-34c expression declined in linear fashion from normal epithelium to invasive disease, miR-142–3p and miR-9 were variably expressed and some, such as miR-199a and miR-139, were stage-specific. The significance of these new approaches is yet to be realized. There is an enormous amount of data available on the molecular changes found during the proposed progression of bronchial epithelial transformation. This reflects the evolution of carcinogenesis in this pulmonary epithelial compartment. Less is known about which are the key changes, although studies produce consistent findings for certain genes or loci and suggest there is a particular order in which changes occur. Among the most interesting aspects of this are how these changes may be exploited to either treat SD/CIS or prevent its occurrence, how markers may be used in early detection/ screening for lung cancer and, finally, how they may be used to predict disease progression.

Predicting the progression of bronchial pre-invasive disease It is impossible to directly observe the progression of preinvasive bronchial lesions into invasive squamous cell carcinoma. The belief this occurs in the bronchus is partly predicated upon comparison with similar progression of disease at other sites. The association of SD/CIS with concurrent squamous cell carcinoma and the identification of appropriate molecular biological changes in putative precursor lesions are commonly seen. Longitudinal studies of human subjects who are at known risk of developing bronchogenic carcinoma (smoking history, other known exposures, history of previous lung or head and neck cancer, etc.) or who have morphological evidence of preinvasive disease are difficult to conduct and interpret. The introduction of AFB has improved this situation. Until AFB was available, sputum cytology was the only feasible methodology for regular detection and follow-up of SD/CIS but this approach is far from ideal. It has poor sensitivity, there are issues with diagnostic accuracy and results are prone to sampling error. Users have no way of knowing the exact source of

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Table 4 Summary of findings on studies of SD/CIS progression

Pre-invasive lesions may progress to invasion The risk of progression is probably higher for CIS than severe SD and still less for lower grades of change A significant proportion of lesions of any grade, including CIS, can regress to a lesser grade or disappear completely Lesion stability in CIS is just as frequent as progression or regression (data are confounded by some short follow-up periods) Low grade lesions are more likely to regress or remain stable rather than progress Accurate and consistent pathological assessment of these lesions is vital for meaningful study of this disease

atypical cells in a sputum sample. Nonetheless, some useful data may be derived from this body of literature. Patients with sputum atypia suggesting pre-invasive disease are at increased risk of developing invasive carcinoma. One study using sputum cytology found 17% of patients with squamous metaplasia and 33% of those called dysplastic subsequently developed carcinoma.195 Another showed invasive carcinoma developing over a period of 4–6 years follow-up in 46% of patients with severe sputum dysplasia.196 In a 17-yearlong sputum cytology study screening uranium miners, many of whom also smoked, Saccomanno et al. found that the mean age at which participants showed signs of various stages of disease showed the expected rise with grade. CIS appeared anywhere from after months of screening up to after 10 years of screening. The average time to develop invasive carcinoma after detection of moderate or severe atypia or CIS was 4.8, 2.9 and 2.5 years respectively.33 Another screening study in a smoking population found the risk of developing invasive carcinoma over a 9-year follow-up period was 4% for lowgrade atypia or normal sputum, 11% for moderate atypia and 43% in severe atypia.34 Biopsy-proven dysplasia in four chromate workers was followed 7 to 13 months later by invasive carcinoma in three patients with severe dysplasia, while the patient with mild dysplasia took 82 months to develop invasion.114 The ability of AFB studies to detect and locate SD/CIS has offered at least a theoretical chance of longitudinal lesion observation. A substantial literature has built up in this area and been reviewed elsewhere.57,197 The “take-home” messages from these studies are summarized in Table 4. The largest AFB study followed 104 patients over 2 years and found no clear trend. Almost as many lesions of either low or high grade either regressed or remained stable as those that progressed.198 All of Venmans et al.’s patients with severe SD or CIS ultimately developed invasive disease after 6 years of follow-up.199 Most patients in another study developed invasive disease with increasing probability, as lesion grade increased.200 A further study showed most CIS lesions remained stable over 2 years.62 Moro-Sibilot et al. found in patients with severe SD or CIS that

Chapter 23: Pulmonary pre-invasive disease

the risk of invasive carcinoma was 17% at 1 year and 63% after 3 years.201 In this study continued smoking had no effect on outcome. George et al. found that high-grade lesions, including CIS, left untreated had a 33% risk at 1 year and 54% risk at 2 years of developing invasion, while none of the low-grade lesions progressed.202 Pasic et al. found patients with a larger number of suspicious lesions on baseline AFB were more likely to subsequently develop invasive carcinoma.203 A regression rate of 54% for all lesions and non-sequential changes in lesions (in as much as could be assessed in an AFB study) were reported by Breuer et al. These authors also found a significantly higher rate (13.4%) of lesion progression to CIS or invasion for severe SD compared with lesser grades.204 However, this study found that on a per patient basis, the progression rates of 26–39% for patients with low- versus high-grade changes were not significantly different. The authors concluded that lesion grade was not a useful clinical parameter for predicting outcome. It should be noted that these studies are hampered by the low specificity of AFB but more critical is the unknown effect of biopsy on any SD/CIS lesion. Apart from the chance the lesion may be either missed by the follow-up biopsy procedure or completely removed in the original sample, it is conceivable the tissue reaction to biopsy could alter the natural history of the lesion. Some studies attempt to measure actual lesion outcome, in others the endpoint is the outcome for the patient. In some of the studies the higher-grade lesions were treated.86 As alluded to earlier and emphasized by Banerjee,197 there is a lack of discussion of the pathological diagnosis of SD/CIS in these trials and few published data on the consistency of histological diagnosis of SD/CIS. Several molecular biological studies of SD/CIS have related molecular/genetic factors to the risk of lesion progression and are summarized in Table 5. The literature is not conclusive on any of these and is, in some cases, contradictory. There is the possibility that pharmacological intervention could abrogate the effect of a significant molecular change. For example, retinoid treatment may be able to reverse a loss of RARbeta in smokers and, at least theoretically, help reduce disease progression.205 The potential for molecular markers to prognosticate on lesions and select those which should be treated is interesting but more data are required before any test is put into clinical use.

Bronchial pre-invasive lesions: comment and conclusion This section reviewed the background and biochemical basis of bronchial carcinogenesis, with a focus on the development of squamous cell carcinoma. Other types of lung carcinoma appear to arise from the bronchial epithelium. Basaloid carcinoma and the basaloid variant of squamous cell carcinoma are closely related lesions which have been associated with a basaloid pattern of CIS.206 Basaloid carcinoma and possibly a proportion of bronchial large cell carcinomas may well be examples of de-differentiated squamous cell carcinomas. They have lost the light microscopic features of squamous

Table 5 Potential molecular factors predicting risk of SD/CIS progression

Factor

Reference

Reduced surfactant protein D in BAL fluid

450

Overexpression of p53 protein or P53 mutation

97,111,126,200,451

Overexpression of cyclin D1 and/or cyclin E

124,126

High bcl2:bax ratio

126

LOH at the FHIT locus

451

P16 loss

124

Increase in hTERT mRNA

452,453

differentiation but retain both ultrastructural207 and immunohistochemical208 evidence of this lineage. It is also possible that some bronchial carcinomas originate de novo from bronchial epithelium without a recognizable precursor (the so-called parallel theory of carcinogenesis).24 Small cell lung carcinomas (SCLC) account for around 25– 30% of bronchogenic carcinomas yet there is no known preinvasive lesion for this tumor type. It has been suggested that SCLC may arise through some “sudden chromosomal change” within an SD/CIS lesion.33 SD/CIS may be associated with SCLC but this could be due to a common etiology (tobacco smoke). The morphologically normal bronchial epithelium in lungs of SCLC patients is “genetically scrambled” with much greater allelic losses, compared to morphologically normal bronchial epithelium seen in those with NSCLC.25,209,210 “Parallel” carcinogenesis may well be relevant in the evolution of SCLC. The P16-CyclinD1-CDK4-RB pathway is inactivated early in squamous carcinogenesis by loss of P16 function and overexpression of cyclinD1, but not through loss of RB, which is common in SCLC. The particular mode of inactivation of this pathway may determine which tumor type arises from the same epithelial compartment, whether or not through a phase of SD/CIS. Adenocarcinomas arising from the bronchial mucosa are rare.211,212 It is not known whether these arise from the surface epithelium or from bronchial glands and there is no known pre-invasive lesion. Evolving data on the molecular profiles of adenocarcinomas have the potential to support an origin of the lesion from different parts of the bronchial tree, but established facts are lacking.213

Carcinogenesis in peripheral lung epithelium The bronchioloalveolar epithelial compartment is distinct from that lining the more central, cartilaginous airways. Bronchiolar epithelium, at least in non-smokers, does not contain mucin-secreting goblet cells but harbors secretory Clara cells. Cytokeratin 5 and 14-positive cells are also less frequent, in comparison to central airway epithelium.

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Chapter 23: Pulmonary pre-invasive disease

Alveolar epithelium is morphologically and functionally very different from both bronchial and bronchiolar epithelium. While stem cells amongst CK5/14-positive basal cells in bronchial epithelium are the likely progenitors of SD/CIS,23,214 in the peripheral bronchioloalveolar epithelial compartment cell populations are rather more complex and some differentiated cells appear to have stem-like properties. A population of variant Clara cells at the bronchioloalveolar duct junction (BADJ) co-expressing the Clara cell marker CC10 (Clara cell secretory protein – CCSP) and surfactant proteins might function as bronchioloalveolar stem cells.23,214 Stem cells are thought to be present in bronchiolar epithelium in so-called neuroepithelial bodies (NEB), which contain cells expressing calcitonin-gene related peptide (CGRP), as well as other hormones and cytokines. These cellular microfoci are innervated and also contain cells which are CCSP-positive. Some cells co-express both CGRP and CCSP.215 There is evidence the stem cells of the BADJ and NEB lack the cytochrome P450 2F2,215 possibly of relevance in relation to metabolism of tobacco carcinogens. All bronchioloalveolar epithelial cells, including those with neuroendocrine features express thyroid transcription factor 1 (TTF1). This transcription factor appears to be extremely important in the normal development and differentiation of peripheral lung epithelium and is a lineage marker for bronchioloalveolar epithelium.216,217 In recent years this bronchioloalveolar epithelial compartment has been referred to in the context of pulmonary adenocarcinogenesis as the terminal respiratory unit (TRU).218 BADJ stem cells are very obvious candidates for the origin of foci of atypical bronchioloalveolar epithelial cells occurring in the centriacinar region, and referred to as atypical adenomatous hyperplasia (AAH). AAH is included in the WHO classification of lung tumors as a putative precursor of lung adenocarcinoma.219

Morphologically recognizable pre-invasive bronchioloalveolar lesions In contrast to the evidence for a pathway of central bronchial carcinogenesis leading to bronchial squamous cell carcinoma, the origins of peripheral pulmonary adenocarcinoma are less well understood. Adenocarcinomas are associated with various causes of diffuse pulmonary fibrosis, but the so-called “scar cancer” hypothesis has largely been refuted. Until the 1980s, the so-called “scar cancer” theory held sway as a putative pathway for peripheral lung adenocarcinoma development.220–225 The consistent presence of a dense fibrous core lacking tumor cells in the center of peripheral adenocarcinomas was thought to represent a pre-existing scar, which acted as a nidus of tumor development (Figure 19).223 Hyperplastic type II pneumocytes at the scar margins reinforced this view.224 Some authors questioned the scar cancer theory.226,227 Shimosato et al.228,229 established the basis of the argument against the

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Table 6 Factors important in refuting the “scar cancer” hypothesis

Feature

References

Adenocarcinomas are common in scar-free lungs

228,229

The relative sizes of the tumors and the scars are usually in proportion

228,229

Some scars contain psammoma bodies, implying previous papillary tumor in that area

228,229

Both pulmonary and extra-pulmonary metastases of lung adenocarcinoma often show central scarring

228,229

Sequential radiology shows scar post-dates emergence of tumor

454

Scars rarely in evidence on review of radiology prior to tumor diagnosis

454

Myofibroblast and collagen content of tumor scar implies new fibroplasia

455–457

Figure 19. Peripheral parenchymal-type adenocarcinoma with central collapse and scarring. Note the increase in anthracotic pigmentation centrally, as well as the approximation of airways and vessels.

“scar cancer” hypothesis, the essence of which, together with other factors, is given in Table 6. In 1959 Yanagisawa published, in Japanese, an autopsy study in which histogenesis of lung described pulmonary lesions in association with and possibly related to the carcinoma. Weng et al. regarded these lesions as most probably what we now refer to as AAH.230,231 The older literature refers to alveolar epithelial regeneration and hyperplasia as possible precursors of lung adenocarcinoma,224,232 but these descriptions seem to concern the epithelial changes associated with pulmonary inflammation and fibrosis. In 1982 Shimosato et al. described five patients from a collection of 1015 who had surgically resected lung cancer, where focal lesions of atypical alveolar cell hyperplasia were identified incidentally.229 The term “atypical alveolar cuboidal

Chapter 23: Pulmonary pre-invasive disease

Figure 20. A centriacinar focus of AAH photographed whilst the lung cut surface was flooded with water. Note the location of the lesion, in close association with the bronchiole.

cell hyperplasia” was used to describe these lesions, which measured from 1.5 to 13 mm in diameter. Shimosato et al. speculated that “one can assume that some peripheral adenocarcinomas arise without any association with pre-existing scar tissue as an in-situ carcinoma from almost normal-appearing bronchioloalveoli. It is not certain whether or not cancer develops through a stage of atypical hyperplasia”. Six years later Miller et al. described five cases from a series of 57 resected adenocarcinomas, where the lung parenchyma surrounding the tumor showed several foci of “bronchioloalveolar cell atypia or dysplasia”.233 From this observation and a follow-up study of additional cases 2 years later,234 Miller made the connection between these atypical foci and bronchioloalveolar carcinoma (BAC) as it was understood at the time. These authors proposed the existence, as in the colon, of an adenoma–carcinoma sequence in the lung periphery. Their preferred term for this atypical lesion was bronchioloalveolar cell adenoma. The currently favored term is atypical adenomatous hyperplasia, although throughout the early 1990s a number of publications referred to the same lesions as “alveolar epithelial hyperplasia”,235 “alveolar atypical hyperplasia”236 or “atypical bronchioloalveolar cell hyperplasia”.231 Ullman et al. described a lesion they termed bronchiolar columnar cell dysplasia (BCCD) and postulated that this may be a possible precursor lesion for the development of lung adenocarcinoma.237 This lesion was defined as “cytological and structural atypia confined to bronchiolar epithelium.” A rather surprising 11 cases of BCCD were identified in 25 consecutive lung specimens resected for adenocarcinoma and the study reports 14 patients with BCCD. Suggested criteria for the diagnosis are disordered epithelial architecture with a range of cytological abnormalities. Chromosomal aberrations were reported in these lesions (see below). In the only other published study of BCCD, lesions were detected in 30.2% of adenocarcinoma-bearing lungs and 17.6% of those with

Figure 21. This AAH lesion shows distinct alveolar spaces within the lesion. Such a gross appearance is typical of AAH but is not specific. Many such lesions prove to be fibro-inflammatory at microscopy.

squamous cell carcinoma.238 BCCD was suggested as a possible precursor of both these tumor types. BCCD remains an obscure, poorly described and little recognized lesion which has, judging by some of the published photographs, a questionable relationship with peribronchiolar metaplasia. This author has no experience with this lesion.

Atypical adenomatous hyperplasia AAH is now accepted as a putative precursor of peripheral, so-called parenchymal or TRU-type adenocarcinoma. In this pathway of pulmonary adenocarcinogenesis, there may be progression of AAH into lesions which currently would be classified as localized non-mucinous bronchioloalveolar carcinoma (LNMBAC) under the existing 2004 WHO classification.239 While BAC is discussed in detail in Chapter 27, some reference will be made to it here, especially since there are arguments to reclassify LNMBAC as adenocarcinoma in situ (AIS).240

Macroscopic features of AAH Most AAH lesions are incidental histological findings. Yet careful examination of the lung cut surface can identify AAH lesions. There are two main caveats though. The lung must be optimally prepared and sectioned, and many sampled lesions, thought to represent AAH, are nonspecific inflammatory or fibrotic areas. After adequate specimen distension and fixation visible AAH lesions are of millimeter size and present as soft, illdefined grayish white to pale yellow patches. Their identification is greatly improved by examining the lung cut surface under a bright light and periodically flooding the specimen with clean water (Figure 20). In larger AAH lesions, it may be possible to see a fine, stippled pattern of pits or depressions on the cut surface; these represent individual alveolar spaces (Figure 21).

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Chapter 23: Pulmonary pre-invasive disease

Figure 22. At very low magnification, AAH lesions may stand out against the normal background alveoli due to the slight thickening of the alveolar walls.

Figure 24. AAH showing small alveolar spaces and mild collagenous thickening of the interstitium.

The number of lesions detected is a function of the diligence of the pathologist, the adequacy of specimen preparation, the presence of normal anatomical structures, such as vessels, large airways and septa and co-existent pathological changes. Lesions are commoner in the upper lobes241 and in subpleural lung.235 The author finds most lesions in the most lateral two or three parasagittal sections of a large resection specimen.

Microscopic features of AAH The WHO defines AAH as “a localized proliferation of mild to moderately atypical cells lining involved alveoli and, sometimes, respiratory bronchioles, resulting in focal lesions in peripheral alveolated lung, usually less than 5 mm in diameter and generally in the absence of underlying interstitial inflammation and fibrosis” (Figure 22).219 Interestingly, in the 1999 WHO classification, where AAH was first recognized as a

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Figure 23. A small focus of AAH adjacent to a terminal bronchiole (top left). See also Figure 20.

putative adenocarcinoma precursor, lesions were stated to be always less than 5 mm in diameter. This was based on Miller’s original proposal that lesions more than 5 mm across should be considered bronchioloalveolar carcinoma (LNMBAC).234 It is now clear that AAH lesions may measure much more than 5 mm. This explains the modification in the 2004 WHO definition. Up to three-quarters of AAH lesions recognized on histological sections measure 3 mm or less in diameter. However, between 10% and 20% measure over 5 mm across, and the author and others have described lesions over 10 mm in diameter. In a study of 38 AAH lesions, Weng et al.231 found that 55% of lesions measured between 1 and 3 mm across with 25% under 1 mm, 10% between 3 and 5 mm in diameter and the remaining 10% measured between 5 and 10 mm across. In the author’s experience, two-thirds of lesions measured 3 mm or less, 17% and 9% were 3 to 5 mm and 5 to 10 mm across respectively, while 10% measured over 10 mm in diameter (largest 19 mm).91 The largest AAH lesion in Nakanishi’s study was 24 mm in diameter.235 Kitigawa et al. found AAH lesions ranged from 0.8 to 8 mm in diameter.242 Most lesions are located in the centriacinar region, close to terminal and respiratory bronchioles, although this may not always be appreciated in sections due to the plane of the section (Figure 23). At low magnification, AAH lesions are fairly discrete and stand out, due to slight thickening of the alveolar walls. The abnormal cell population may extend into the respiratory bronchiole. In most lesions the alveoli are of normal size but occasionally, especially when their walls are thickened, the spaces are small (Figure 24). In some lesions alveoli are large, giving the lesion a microcystic appearance. This may represent AAH arising in conjunction with centriacinar emphysema. Low-power microscopic examination of AAH shows the normally smooth outlines of the alveolar wall surface studded by a population of round to oval alveolar lining cells (Figure 25). Characteristically there are gaps between these cells and

Chapter 23: Pulmonary pre-invasive disease

Figure 25. In this example of AAH, as well as in Figure 24, the gaps between the alveolar lining cells can be appreciated.

Figure 26. In this example of AAH there is greater variability in the epithelial cell population but less alveolar interstitial thickening.

Figure 27. AHH with oval to round nuclei. Hobnail or peg cells are present. Figure 28. In AAH some cells may be quite large, yet apparently well separated from their neighbors. Alveolar macrophages often accumulate in the airspaces of AAH lesions.

Figure 29. Nuclear inclusions may be prominent in AAH. This is not a sign of malignancy.

there is marked heterogeneity in terms of cell size and shape (Figure 26). Low columnar cells and even hobnail or “peg” cells may be present. The intermittent cell layer of AAH shows little or no evidence of stratification. The nuclei are also usually oval to round (Figure 27). Their chromatin is generally quite dense, hyperchromatic and homogeneous, apart from intranuclear inclusions, which are frequent in larger cells (Figures 28 and 29). Weng et al. found inclusions in 25% of AAH cells.231 Some peg-shaped cells may have nuclei located in the apex of the cell (Figure 30). Larger cells with two or three nuclei may be encountered. Mitotic figures are virtually never encountered and if seen, careful consideration of an alternative diagnosis is warranted. Mucin-secreting goblet cells or ciliated cells are not seen in AAH.

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Chapter 23: Pulmonary pre-invasive disease

Figure 30. In AAH, nuclei may be located near the apex of peg-shaped cells. Double nuclei may also be seen (see also Figures 28 and 29).

Figure 31. In this example of AAH, there is quite marked collagenous thickening of the alveolar interstitium.

Figure 32. A more cellular AAH lesion. Cells are more crowded but this is still within limits acceptable as AAH.

Figure 33. A high-magnification view of the lesion in Figure 32. Although inter-cellular gaps are less obvious, the cells are still mostly cuboidal. Tall columnar cells are not a feature and nuclear features lack significant pleomorphism.

Immunohistochemical and ultrastructural studies suggest many of the cells in AAH lesions are either Clara cells or type II pneumocytes with electron-dense granules or nuclear branching microtubules and cytoplasmic lamellar bodies respectively.234,243–246 This fits with the hypothesis that AAH lesions may derive from variant Clara-like stem cells of the BADJ. Marked fibrosis is uncommon, although rare examples where there is focal or sub-total sclerosis with marked thickening of the alveolar walls may be encountered. Fibrosis and inflammation in AAH are limited to the involved area, as defined by the atypical epithelial cell component (Figure 31). Some lesions may show interstitial elastosis. Infiltration of the alveolar interstitium of the lesion by chronic inflammatory cells may be seen but this is rarely heavy; very occasionally a germinal center may be

914

encountered.234 Macrophage accumulation in lesional alveolar airspaces is not uncommon (Figure 28). The surrounding lung, by definition, is free of inflammatory or fibrotic changes. The histological appearances of AAH lesions can vary between lesions in the same patient or even within areas of the same lesion. This is in keeping with proposals concerning disease progression. Some AAH lesions show a more atypical cell population, with prominent columnar or hobnail type cells, increased cellular crowding and fewer gaps between cells (Figures 32–34). Occasional small foci of cellular tufting may be seen and all these changes may be focal in the lesion (Figure 35). These lesions tend to be larger than less cellular forms with a tendency towards more interstitial thickening. While some authors subclassify AAH into low- and highgrade lesions, the WHO recommends against any such attempt,

Chapter 23: Pulmonary pre-invasive disease

Figure 34. There is some nuclear overlapping in this AAH lesion, but gaps are noted between low columnar cells.

since there are no published criteria for making the distinction and it has no known clinical significance.231,247,248 Several studies noted a trend of increasing atypia and cellularity in AAH, as lesion diameter increased.91,234,248–250 Miller found marked atypia in two-thirds of lesions over 4 mm in diameter, while three-quarters of those under 3 mm across showed mild atypia.234 In this author’s own series, 16% of 111 AAH lesions were considered “high” grade and two-thirds of these measured over 5 mm in diameter.91 Koga et al. could not show a difference in size between what they considered low- and high-grade lesions; only 9% of their 119 lesions were regarded as high-grade.250

Issues in the diagnosis of AAH Since most AAHs are incidental microscopic findings, their identification depends on the absence of any intercurrent pathology obscuring the lesion. In lungs resected for carcinoma, any emphysema or obstructive pneumonia may make it impossible to identify lesions. Significant pathology is frequent in autopsy lungs, making identification a considerable challenge. Most AAH lesions are found in lungs resected for primary carcinoma, especially adenocarcinoma. The author has also found AAH lesions in wedge resections performed for the removal of both primary and metastatic carcinomas, volumereduction surgery specimens and lung biopsies taken for the diagnosis of benign lung disease, such as respiratory bronchiolitis-interstitial lung disease. AAH lesions have been found at autopsy.251–253 There is no stated minimum size for an AAH lesion but as a rule of thumb at least three or four alveolar spaces should be involved. Occasionally AAH-like areas may be seen involving alveolar walls in a more diffuse distribution. AAH-like areas may also be seen in continuity with lepidic components of an otherwise invasive adenocarcinoma. In these circumstances the author will not diagnose AAH but notes the fact in his

Figure 35. This AAH lesion shows cellular tufting, but features fall short of what would be required for a diagnosis of pure low-grade non-mucinous bronchioloalveolar carcinoma (LNMBAC)/adenocarcinoma in situ (AIS).

description. Such a finding is unusual, but not unexpected, given the putative role of AAH as an adenocarcinoma precursor. Practically speaking, AAH cannot be diagnosed on transbronchial biopsies, exfoliative (bronchial brushing or washing) or aspiration cytology. Theoretically a good transbronchial biopsy sample could include portions of an AAH lesion. Unless the limits of the lesion can be appreciated, considerable caution should be exercised before making any diagnosis of AAH. If only a few involved alveoli are present, distinction from a reactive hyperplastic lesion or even a lepidic pattern of adenocarcinoma may be impossible, depending on the degree of atypia present (see below). Diagnosis of AAH is not possible by cytology. Cytological samples may show atypical bronchioloalveolar cells but their origin cannot be ascertained. Even in a radiologically guided fine-needle aspiration cytology sample from an accessible “pure ground-glass” lesion (see below), such cells could never be diagnostic. The best the cytopathologist can offer in these circumstances is a diagnosis of “atypical bronchioloalveolar cell proliferation”.254

Differential diagnosis of AAH The differential diagnosis of AAH depends on the circumstances in each case. Various forms of reactive hyperplasia, benign tumours and the bronchioloalveolar pattern of adenocarcinoma should be discerned (Table 7).88 The distinction between AAH, reactive changes and LNMBAC-AIS is usually challenging. There are several different situations where reactive proliferation of bronchioloalveolar epithelium has to be distinguished from AAH. In many, it is obvious the epithelial proliferation is a reactive change associated with other pathologies. These include usual or nonspecific interstitial pneumonia, the proliferative phase of diffuse alveolar damage or

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Chapter 23: Pulmonary pre-invasive disease Table 7 Differential diagnosis of AAH

Possible alternative diagnosis

Important distinguishing features

Reactive hyperplasia – fibroinflammatory

Context. Evidence of inflammation, interstitial lung disease, organizing pneumonia. Continuous runs of low, cuboidal cells. Homogeneous cell population

Reactive hyperplasia – subpleural/paraseptal

Location. Homogeneous cell population

Peribronchiolar metaplasia

Continuous lining of columnar cells including ciliated cells. Mucigenic cells possible though rare. Scarring may be marked.

Papillary adenoma

Lesion size and architecture. True papillae. Columnar cells

Alveolar adenoma

Lesion size and architecture. Cystic spaces. Spindle cell-rich stroma

Micronodular pneumocyte hyperplasia

Associated with tuberous sclerosis. Compression of alveolar architecture, no atypia, alveolar macrophages

Minute meningothelial nodules

Location (paravenular). Plump interstitial spindle cells

Carcinoid tumorlets

Neuroendocrine cells. Can line alveolar walls in centriacinar location but elsewhere nodules of NE cells embedded in fibrous tissue is diagnostic (see Chapter 31)

Localized non-mucinous bronchioloalveolar carcinoma – adenocarcinoma in situ

Cytological atypia, cell size and density. Central fibroplasia. Lesion size.

Figure 36. Alveolar cell hyperplasia is noted in this case of nonspecific interstitial pneumonia.

other interstitial pneumonias (Figure 36). In these instances there is a clear underlying stimulus for the reactive proliferation. In AAH the abnormal population of enlarged alveolar lining cells “defines” the limits of the lesion; all associated interstitial scarring or inflammation is within the lesion. If interstitial fibrosis and/or inflammation extend beyond the alveoli lined by the abnormal epithelial cell population, a diagnosis of reactive hyperplasia is warranted. In such situations the reactive epithelial cell population is often rather bland and homogeneous, comprising cuboidal cells in continuous rows with small round nuclei (Figure 37). Intercellular gaps, hobnail cells and heterogeneity are absent. The presence of other pathology, such as inflammation, interstitial fibrosis, organizing pneumonia, etc., helps in reaching the correct

916

Figure 37. Reactive type II pneumocyte hyperplasia often shows regular cuboidal cells with foamy cytoplasm.

diagnosis (Figure 38). Similar reactive proliferations of alveolar lining cells may be seen partially lining alveoli which abut the pleura, pulmonary septa, bronchovascular bundles or irregular scars (Figure 39). These areas should not be confused with AAH. Small localized reactive lesions may be confused with AAH. Lung parenchyma surrounding or distal to a carcinoma or other obstructive bronchial lesion may show small inflammatory foci in which a few alveoli feature an alveolar cell proliferation. These lesions are often in the centriacinar region and demonstrate interstitial inflammation and airspaces filled with macrophages, some “foamy”, mononuclear cells and occasional neutrophils. Fibrin and fibromyxoid connective tissue plugs may also be present. This is a focal, nonspecific inflammatory lesion, probably related to infection or aspiration.

Chapter 23: Pulmonary pre-invasive disease

Figure 38. In this case the reactive type II pneumocyte hyperplasia is clearly associated with organizing pneumonia and interstitial inflammation. A diagnosis of AAH would not be tenable in this situation.

(a)

Figure 39. Reactive type II pneumocytes are commonly seen lining alveoli, which abut septa or large vessels, even in the absence of inflammation. The location, topography and cytology of this change should help avoid any confusion with AAH.

(b)

Figure 40. Peribronchiolar metaplasia (PBM) is another centriacinar epithelial proliferation which may be confused with AAH (a). The size and location of the lesions in PBM and AAH are similar but the former is easily distinguished since PBM shows proliferation of ciliated columnar bronchiolar cells lining the alveoli, which usually show fibrous thickening (b).

Chronic bronchiolar inflammation or injury may result in the replacement of peribronchiolar alveolar epithelium by bronchiolar lining cells; so-called peribronchiolar metaplasia (Figure 40). This change has also been referred to as “bronchiolization of alveoli” and Lambertosis (see Chapter 17). 255 The key to this diagnosis is the recognition of ciliated cuboidal cells (Figure 40). Rarely mucigenic cells are found (Figure 41). There may be some chronic inflammation and quite dense fibrous tissue is characteristic. Attention to the cell population lining the lesion airspaces ensures a correct diagnosis. Other lesions may be considered in the differential diagnosis of AAH. Papillary and alveolar adenomas are benign neoplasms, which occur very rarely in the lung parenchyma (see

Chapter 22). These lesions both measure greater than 1 cm and feature either true fibrovascular cores or cystic patterns, respectively. Micronodular pneumocyte hyperplasia (MNPH), a rare entity usually associated with tuberous sclerosis complex and pulmonary lymphangioleiomyomatosis, has a superficial resemblance to cellular forms of AAH (see Chapter 22).256–258 Kobashi et al. described multifocal MNPH in tuberous sclerosis, in which the differential diagnosis on high-resolution computed tomography scanning was multifocal AAH.259 Like AAH, these lesions are of millimeter size and well demarcated but show plump bland type II pneumocytes lining thickened distorted and compressed alveolar walls. The resulting small alveolar spaces tend to fill with macrophages.

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Chapter 23: Pulmonary pre-invasive disease

Figure 41. Very occasionally a PBM lesion will show mucigenic cells admixed with the ciliated cells. Care should be taken not to confuse this lesion with a mucinous adenocarcinoma.

Figure 42. LNMBAC/AIS. The cells in this lesion are more atypical, columnar and show some nuclear pleomorphism.

Table 8 Histological features of localized non-mucinous BAC (adenocarcinoma in situ – AIS)

1. Increased tumor cell height, greater than the height of columnar cells in associated or adjacent bronchioles 2. Cells growing in a so-called “picket fence” type pattern or the formation of true papillae 3. Cell stratification is marked 4. Cell density is high with notable nuclear overlapping 5. Nuclei have coarse chromatin and prominent nucleoli

The most important differential diagnosis is between AAH and a lepidic pattern of adenocarcinoma. Both comprise an atypical population of bronchioloalveolar cells originating from the TRU and lining native alveolar walls. At least some LNMBAC-AIS derive from AAH but the histological distinction between the two lesions is arbitrary. Minami et al.260 have suggested some criteria to make the distinction. Of the five histological features listed in Table 8, most AAH lesions would show, at most, only one, while three or more are likely in LNMBAC-AIS. A significant proportion of LNMBAC-AIS measure more than 1 cm in diameter and examples over 3 cm are unusual. The cell population lining the alveolar walls tends to be more uniform compared to the marked heterogeneity characteristic of AAH (Figures 42 and 43). The latter’s appearance may be enhanced by the intercellular gaps seen in AAH but not LNMBAC-AIS. The taller columnar cell population tends to make the edge of the lesion more abrupt in transition to normal alveoli, in contrast to AAH, which gradually disappear at the edges. The alveolar walls in LNMBAC-AIS may be thicker than in AAH and a significant interstitial lymphoid infiltrate is not unusual. While minimal alveolar collapse and

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Figure 43. Another example of LNMBAC/AIS. Cells are irregular, crowded, overlap and show nuclear pleomorphism. Interstitial lymphocytic infiltrates are common in LNMBAC/AIS and may be heavy. Compare with Figure 33.

fibroelastosis or sclerosis may be seen in AAH, a distinctive central nidus of new fibroblastic growth is much more likely to be encountered in a lesion which otherwise fulfills the above criteria for LNMBAC-AIS. Occasionally a lesion shows a transition to more AAH-like areas. Such transitional lesions are diagnostically problematic but consistent with the proposed step-wise progression of disease (Figure 44). The author’s practice is to consider such transitional lesions as LNMBAC-AIS. It is believed that LNMBAC-AIS undergoes a process of central fibro-elastosis and alveolar collapse, as a forerunner of invasion.240,261 Central neofibrogenesis is an indication of likely stromal invasion, even when invasive adenocarcinoma is not apparent.262–264

Chapter 23: Pulmonary pre-invasive disease

(a)

(b)

(c)

Figure 44. This lesion shows features, in the central portion, of a collapsed pattern of LNMBAC/AIS with flattened alveolar spaces and increased interstitial stroma (a). Entrapped carcinoma is composed of crowded and overlapping columnar cells (b), yet at the margins of the lesion the lesion has the features of low-grade AAH (c).

Prevalence of AAH Since AAH cannot be reliably detected and diagnosed in vivo, even with thin-slice high-resolution computerized tomography (HRCT), the background prevalence of AAH in any particular population is unknown. Identification of AAH has been a task solely for pathologists, either through careful searching for the lesions or by chance histological identification. Nevertheless, some interesting observations may be made. Detection of AAH during autopsy may give some indication of background prevalence. The earliest report of probable AAH lesions detected at autopsy was made by Yanagisawa.230 This study described lesions in 3.6% of 140 autopsies and was referred to by Weng et al.,231 who regarded these lesions as AAH. There have been three contemporary autopsy studies designed to detect AAH. Sterner et al. sought AAH in 100 consecutive autopsy examinations, where patients with any lung malignancy were excluded.251 Two patients in this study, both ex-smokers, had AAH. Between three and five tissue blocks were taken per case, none of the AAH lesions were identified grossly and all were described as “minute.” Lesion numbers were not given but by inference they were low. Yokose and colleagues conducted two autopsy studies seeking AAH.252,253 In the first, 3.4% of patients over 60 years of age and without malignancy, but 10.2% of patients with concurrent malignancy were identified with AAH in a study group of 241 cases.252 In the second study of 179 cases, ranging in age from 1 to 90 years, five males, aged 52 to 63 years, had AAH; one of these had an extra-thoracic malignant tumor.253 In these Japanese studies, up to seven tissue blocks per case were sampled and in most only one or two AAH lesions per case were identified. These are small lesion numbers, considering both lungs were available for examination. This may be a true reflection of prevalence but the number of tissue blocks taken per case is low relative to the amount of lung accessible, and identification of AAH in autopsy material is particularly difficult, given the frequency of concurrent pulmonary pathology. Surgically resected lung tissue offers the best opportunity for pathologists to identify AAH. Mention has been made of

the importance of well-prepared lung tissue, unobscured by other diseases, such as pneumonia, fibrosis, etc. Given surgical lung resection for benign disease is relatively uncommon and does not normally involve removal of much, if any, “normal” lung, such specimens offer little to the problem of discerning AAH prevalence. Most studies involved the identification of AAH in lungs resected for primary malignancy. Some data are presented in Table 9, taken from both prospective and retrospective studies. The prevalence of AAH lesions in lung-bearing primary carcinoma is greater, possibly the result of a carcinogen-induced field effect. Care must be exercised in drawing conclusions from these data, due to widely differing methods used to identify AAH lesions. Some figures are not primary source data but reports of data from elsewhere265,266 and others are not prospectively gathered. The value of a prospective active search for AAH lesions is reflected in the generally higher prevalence reported in such studies. The overall figure for AAH identified in all lung samples resected for malignancy is 22% (range 9.3– 49%).231,234,241,250,267 Not all studies describe clearly how the lesions were sought. Weng et al averaged 51 blocks per resected lobe or lung in their search for AAH.231 The study reporting the highest rates of AAH involved very close gross examination and averaged 60 tissue blocks from 4 mm thick lung slices.250 Miller used Bouin’s fluid to fix lungs and render AAH lesions yellow and easier to see.234 Chapman and Kerr sampled all visible lesions, as well as taking three to six random parenchymal blocks per case.267 Reports of AAH prevalence in lung resected for metastatic disease are few. Weng et al. found AAH in 4.8% of cases prospectively examined,231 whereas Morinanga and Shimosato reported AAH in 9.6% of metastatectomy specimens in a retrospective analysis.265 A prospective collection by Chapman and Kerr found AAH in 2/15 (13.3%) resections for metastatic carcinoma.267 The latter study also reported AAH in 1/5 resections for benign tumors. Atypical adenomatous hyperplasia is common in lungs bearing primary adenocarcinoma. Koga et al. reported the highest prevalence of AAH in a small case series and noted a particular

919

Chapter 23: Pulmonary pre-invasive disease Table 9 Studies reporting prevalence of AAH lesions in lung specimens resected for carcinoma

Prevalence of AAH in cases examined (%) Country of origin of study

No. of lungs examined

All cases

Cases with adenocarcinoma

Cases with large cell carcinoma

Cases with squamous cell carcinoma

Cases with other tumor histology

247

9.3%

15.6%

–

3%

–

165

16.4%

17%

23%

11.6%

20%

Scotland, UK363

554

12.1%

23.2%

12.5%

3.3%

5.4%

Japan334

508

23.2%

29.3%

11.8%

9.8%

19.5%

344

61

49%

57%

–

30%

29%

1535

22%

28.4%

15.8%

11.5%

18.4%

Japan361

203

13.9%

18.8%

–

5.9%

–

Japan

362

131

12.2%

19.2%

–

11.1%

–

Japan

328

70

21.4%

34.5%

10%

6.9%

–

Japan

364

2098

5.1%

7.8%

6.6%

1%

6.5%

2502

13.2%

20%

8.3%

6.2%

6.5%

Prospective studies Canada327 Japan

Japan

325

Total/ Average % Retrospective studies

Total/ Average %

association with the lepidic subtype of adenocarcinoma.250 Most resected adenocarcinomas are peripheral subpleural lesions, frequently devoid of surrounding pathology. Squamous carcinomas, with which AAH lesions seem less frequently associated, are often associated with parenchymal collapse or obstructive pneumonia. This facilitates AAH lesions being detected with adenocarcinoma and compromises the same task in squamous carcinoma resections. The reported prevalence of AAH in lung resected for large cell undifferentiated carcinoma is relatively high. There is ultrastructural and immunohistochemical evidence that possibly a substantial proportion of large cell carcinomas are “dedifferentiated” adenocarcinomas. Some of the Japanese studies report large numbers of adenosquamous carcinomas in their “others” categories (Table 9). It is not clear what criteria were used for this diagnosis and whether, as with large cell carcinoma, an association with adenocarcinoma is being disguised by diagnostic classification. In the author’s own series, adenosquamous carcinomas were very rare.267 It may be more meaningful to make comparisons within studies than between them. Regardless of the absolute percentages quoted, there seems to be consistently more AAH in association with adenocarcinoma, compared to squamous carcinoma. With the autopsy data in mind, it is possible the figures for AAH associated with squamous carcinoma quoted in some of the prospective studies in Table 9 may approximate

920

Table 10 Reported cases with large numbers of AAH lesions

AAH number per case

Reference

14

231

12, 19, 34, 42

267

161

331

125, 130

Author, unpublished

to a background prevalence of AAH, at least in tobacco smokers. In the five reported prospective studies, 72–81% of those cases with AAH also had adenocarcinoma. Although most reported cases of AAH, especially in earlier studies, are single lesions, this entity is probably a multifocal disease. On average in five studies, 48% (range 40% to 66%) of cases examined revealed multiple AAH lesions, mostly between two and six lesions per case.231,234,235,241,267 Higher numbers are also reported and listed in Table 10. Miller described some cases “studded” with lesions.234,268 In a more recent study of the author’s own AAH cases 13% of patients had six or more AAH lesions. There is a close association between large numbers of AAH lesions and adenocarcinomas, often with a BAC component. The associated tumors may be multiple and some of the lesions may be LNMBAC-AIS. In 70 patients with AAH, 14% had

Chapter 23: Pulmonary pre-invasive disease Table 11 Published cases of multiple synchronous primary adenocarcinomas and multiple AAH lesions

Travis et al. described rare neoplastic lepidic nodules in adolescent cancer patients.273

Synchronous adenocarcinomas

Etiology and other associations of AAH

Associated AAH lesions

Reference

6 4 3 3

42 8 34 19

267

4

2

458

4 4 3

3 2 6

459

3 2 2

2 3 2

242

2

161

331

2

12

460

2

3

250

multiple synchronous primary adenocarcinomas.267 Nakahara et al. found 21% of patients with a solitary adenocarcinoma had AAH, two-thirds of which were solitary lesions. However, these authors showed 46% of patients with multiple synchronous primary adenocarcinomas had AAH lesions, twothirds of which were multiple lesions in the resected cancerbearing lung.241 Table 11 lists studies where multiple AAH lesions and synchronous carcinomas are enumerated. There are also anecdotal reports of multiple AAH and adenocarcinomas being detected at CT scanning269 and successfully resected.270 There is no consistent association between AAH prevalence and gender. Two Japanese studies show no difference in AAH prevalence between males and females, either with lung cancer, or more specifically with adenocarcinoma.231,241 In the only published European study of AAH there was an increased prevalence of AAH in female cancer-bearers compared to males (19% versus 9.2%). This female preponderance was maintained in those with adenocarcinoma (30.2% versus 18.8%).267 It is equally difficult to come to any conclusion with regard to AAH and age. Since AAH lesions are never symptomatic, their discovery is incidental after presentation with other pathology. The exception is AAH lesions discovered during screening for lung cancer (see below). Thus, the published ages of AAH patients reflect the age ranges of associated diseases, i.e., lung cancer. There are two reports of AAH occurring in young patients; one in a 17-year-old male, where the lesion was discovered on CT scanning after presentation with spontaneous pneumothorax,271 the other in a 17-year-old female who had resected pulmonary metastases from osteosarcoma.272

It is impossible to ascribe an etiology to AAH. While most patients in two studies of Caucasians with AAH were smokers,236,267 two Japanese studies failed to show an association between smoking and AAH prevalence.241,274 Kitigawa et al. reported a possible association between multiple AAH lesions and adenocarcinoma and smoking.242 A variety of animal models suggest possible etiological factors for human AAH. When exposed to asbestos, rats develop pulmonary “adenomatosis” and adenocarcinomas. Thomas et al. reported a case of pulmonary adenocarcinoma and AAH in an asbestos-exposed patient with pleural mesothelioma275 but there is no other reported association with asbestos. A case-controlled study of AAH prevalence and occupation failed to identify an excess of likely asbestos exposure in the AAH group.276 In several Japanese studies there appears to be an association between AAH and a history of extra-thoracic malignancy.241,242,252,274 There is no association with a family history of malignancy.241,274 The opposite was reported in a European cohort of AAH patients; an association between AAH, especially multiple lesions, and a family history of malignancy, but no association with previous extra-thoracic tumors.277 This raises the intriguing possibility of a genetic predisposition to AAH development. A patient with Li-Fraumeni syndrome with three synchronous primary adenocarcinomas and a single AAH has been reported.278 Goto et al. describe a patient with familial adenomatous polyposis (FAP) who, in the absence of active gastrointestinal disease, presented with multiple pulmonary adenocarcinomas and multiple AAH lesions.279 There is an impression that AAH lesions are commoner, at least in Japanese if not in Asian populations in general. What might be particular to the “Japanese” genome, in terms of conferring an increased risk of AAH and adenocarcinoma, is not known. East Asian populations have a higher incidence of pulmonary adenocarcinoma, in comparison to Western cohorts.

Molecular biology of peripheral lung adenocarcinogenesis As described above with SD/CIS, there has been a large amount of work published on a range of molecular aspects of AAH. Many of the general comments made at the start of the molecular biology discussion of SD/CIS are relevant to AAH. Improved recognition of AAH, consistent classification of the lesion, and developments in molecular biological techniques have all facilitated many studies. Limitations to these studies include the difficulty in identifying lesions of sufficient quality and quantity. Almost without exception, the work on AAH has been carried out on formalin-fixed paraffin-embedded tissue from cancer-bearing lungs. In some of these studies the

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Chapter 23: Pulmonary pre-invasive disease Table 12 Cell cycle activity (proliferation index) in AAH and localized non-mucinous BAC (LNMBAC) lesions

“Low-grade” AAH

“High-grade” AAH

LNMBAC

Adenocarcinoma

Reference

0.59%

2.05%

6.08%

15.6%

248

0.73%

1.53%

3.7%

12.1%

294

1.8% 1.4%

3.5% 3.5%

2.2% 0.87% a b

2.56%

250 5%

12%

296

11.5%

49.5–65.5% (see text)

285b

a

These were described as “sclerosing BAC” lesions; if not truly invasive, then these lesions would be regarded by most as more advanced than LNMBAC. This study used minichromosome maintenance protein 2 (MCM2) rather than Ki67, as a cell cycle marker. There is evidence that MCM2 may detect more cycling cells and thus MCM2 indices tend to be higher than those for Ki67.

number of AAH lesions is quite small. Given the uncertain relationship between AAH and smoking, the relevance of gene expression profiling of “normal” peripheral lung parenchyma in smokers and non-smokers is unclear.

Cell morphometry and cytofluorimetry Nuclear size, shape and DNA content studies were amongst the earliest to attempt to show differences between normal bronchioloalveolar epithelium and AAH, and AAH and carcinoma. Several demonstrated that lesions regarded as AAH have a smaller mean nuclear area (MNA) than that for LNMBAC-AIS, with cut-off values fairly consistently between 40 and 50 mm2.235,247,249,280 Mori et al. made a more complex morphometric study of 12 different cellular and nuclear characteristics.281 Separation of AAH and LNMBAC-AIS was achieved for most cases. In several of these studies, lesions were encountered which showed intermediate features between AAH and LNMBAC-AIS, or a mixed cell population. Histological examination of such lesions suggested the existence of “more atypical” AAH lesions.281 These findings are consistent with the concept of disease progression. A similar intermediate position for AAH, between normal epithelium or reactive pneumocyte hyperplasia and LNMBACAIS, has been demonstrated in DNA content studies and, again, “transitional lesions” between AAH and LNMBACAIS were encountered.280,282 Aneuploidy was detected in in 36–54% of AAH lesions and 77–85% of LNMBAC-AIS. Another study failed to find aneuploidy in “low-grade” AAH, but detected it in a quarter of AAH lesions regarded as “highgrade” and in 35% of LNMBAC-AIS.283 This study also found argyrophilic nucleolar-organizer regions (AgNORs) progressively increased from AAH to LNMBAC-AIS.

Hyperproliferation Cell cycle activity, measured by PCNA-Ki67, is greater in AAH than in surrounding normal alveolar epithelium, and lower than in associated adenocarcinoma.236,284 Table 12 shows some of the data from several studies examining cell cycle activity in pre-invasive lesions in pulmonary

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245

adenocarcinogenesis. In some of these, the exact nature of the “adenocarcinomas” is unclear, with respect to current definitions of LNMBAC-AIS and adenocarcinoma. When MCM2 (see Table 12) was used to identify cells with cycling activity, the lower figure for MCM2 index in the adenocarcinomas given is the value for BAC components of mixed lesions. The higher value is for invasive adenocarcinoma.285 The range of values observed in the BAC component is extremely wide (20–80%, mean 49.5%), raising the possibility that this BAC component is biologically heterogeneous. Some BAC components could represent “residual” adenocarcinoma in situ, while others probably represent lepidic outgrowth of the invasive tumor.

p53 and related proteins Abnormal p53 protein expression has been demonstrated in AAH lesions but P53 mutation seems to be very rare. The first report of p53 expression in AAH found strong nuclear staining for p53 protein in 28% of the lesions. From 5% to 70% of the cells expressed p53.286 Several other studies followed, showing a wide range of findings (Table 13). These variations probably reflect different definitions of “positive staining”, different anti-p53 antibodies used, and variations in the immunohistochemistry process leading to differences in the sensitivity of protein detection. Higher levels of p53 expression are reported in more cellular, “higher-grade” AAH lesions.248,249,286 Although high levels of detectable p53 protein can be a reflection of mutant poorly degradable protein, most studies seeking P53 mutations in AAH failed to detect them (Table 13). P53 function may also be lost due to deletions of the relevant loci on 17p. These genomic studies have been performed on very few lesions. P21 (waf1/cip1) is upregulated by p53 and inhibits cell cycle progression by promoting G1 arrest. In the only study of this protein in AAH, no relation was found between p53 and p21 expression. However, several AAH lesions expressed p21, in contrast to reactive alveolar epithelium, which was devoid of this protein.287

Chapter 23: Pulmonary pre-invasive disease Table 13 Expression of p53 protein and P53 mutation or LOH in AAH

Reference Percentage of cases “positive” for p53 protein 28%a

286

19%

244

9%

248

25% (35% )

110

3%

461

17%

291

35%

284,462

b

Percentage of cases with P53 mutations or LOH No mutation

296c,287,461

5% (exon 8 mutation)

116

14% (exon 4 LOH)

287

6% 17p LOH

284d, 296c

LOH, loss of heterozygosity; AAH, atypical adenomatous hyperplasia. a A positive case required strong staining in over 10% of the cells. There was weaker staining of < 5% of the cells in a further 30% of AAH lesions. Higher levels of staining were seen in more cellular lesions. b The higher figure was observed in patients with pneumoconiosis. c This study showed p53 mutation in 16–33%, and 17p LOH in 11–36% of LNMBAC lesions. d This study reported 17p LOH in 17% of adenocarcinomas.

P63 protein is a homolog of p53 which probably has regulatory function over P53. The deltaN isotype is found in basal cells, including those of the bronchiolar epithelium, and probably maintains stem-like properties and proliferative capacity.288 P63 was found in 2/5 AAH lesions289 and focally in all eight lesions studied by Wu et al.290 These latter authors also found p63 in only 2/9 LNMBAC-AIS and none of the adenocarcinomas examined. p53 protein positively regulates proteins such as BAX, Fas and DR5, which promote apoptosis and negatively regulate anti-apoptotic bcl2. Bcl2 protein was not found in low-grade AAHs, but was expressed in 28% of high-grade AAHs and 48% of LNMBAC-AIS.291 In contrast Kayser et al. found bcl2 overexpressed in 70% of AAHs292 while Mori et al. detected this protein less frequently in LNMBAC-AIS than in AAH.245 Survivin is a member of the group of “inhibitor of apoptosis” (IAP) factors. This protein was detected in 9% of low-grade AAH, 89% of high-grade AAH and all LNMBAC-AIS.291 Akyurek et al. also reported survivin was absent from lowgrade but present in high-grade AAH.293 X-linked IAP (XIAP) was found in all AAH and LNMBAC-AIS lesions, and in 92% of adenocarcinomas.290

P16 (ink4a)–CyclinD1–CDK4–RB pathway This is an important pathway, regulating cell cycle progression in the development of non-small-cell carcinomas (see above).

The only immunohistochemical study of these markers in AAH showed upregulation of Cyclin D1 in AAH but less expression in LNMBAC-AIS and adenocarcinoma.294 The same authors reported little evidence of Rb loss in AAH or LNMBAC-AIS and only occasional loss of Rb in adenocarcinoma. Emerging data on P16 gene promoter hypermethylation suggest this is an alternative mechanism for dysregulating this pathway, as in SD/CIS.295 Amongst a number of genes, P16 showed the most marked change in methylation status, when comparing normal epithelium or AAH with adenocarcinoma. LOH at relevant chromosome loci is another mechanism by which some gene function may be lost from this pathway. Reported 9p LOH in AAH varies from 5% to 33% of cases.116,284,296 9p LOH was found in more atypical AAH lesions, and was more frequent in associated adenocarcinomas.116,284 Yamasaki et al. reported similar rates of LOH at D9S144 and IFNA (9p) – 7% and 33% respectively – in both AAH and LNMBAC-AIS, and also noted LOH at D13S176 (13q RB) in three of nine AAH.296 These changes are in keeping with those described in SD/ CIS and almost certainly reflect the evolution of key genetic changes in pre-invasive lesions.

Tyrosine kinase (TK) signaling pathways There has been a great deal of interest in recent years in these pathways, and EGFR-driven signaling in particular, in the development of pulmonary adenocarcinoma. This attention has been fostered by the advent of small molecule tyrosine kinase inhibitors, erlotinib and gefitinib (TKIs), as therapy for non-small-cell lung cancers.297 Sensitivity to these drugs is particularly associated with lepidic-pattern and papillary histology, female gender, East Asian ethnicity and a neversmoking background. The best predictor of good response to treatment, however, is the presence of certain sensitizing mutations in exons 18–21 of EGFR.298 Few studies of EGFR protein expression or gene amplification have been carried out in pre-invasive lesions of pulmonary adenocarcinomas. The findings are variable and most of these mutations have been deletions in exon 19 or the L858R point mutation in exon 21 (Table 14). Occasionally, even in the absence of TKI therapy, a T790M mutation in exon 20 (associated with TKI resistance) has been found in AAH.299 EGFR mutations were found in 43% of morphologically normal bronchial and bronchiolar epithelia in patients with EGFR mutated adenocarcinomas.300 The mutations detected were the same as those in tumor cells (see below). Yatabe et al. demonstrated that TRU-type adenocarcinomas, as defined by their morphology and TTF1 expression, showed EGFR mutations in 50% of cases, while only 13% of non-TRU adenocarcinomas were mutated.218 Ninety-four percent of EGFR mutant adenocarcinomas were TRU-type. In this same study 2/5 AAH lesions showed an EGFR mutation. In most Japanese studies there seems to be a consistent rise in mutation prevalence as the lesion grade increases. The single

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Chapter 23: Pulmonary pre-invasive disease Table 14 EGFR mutations in pulmonary adenocarcinogenesis: prevalence of EGFR mutations at various stages of disease evolution

a

AAH

LNMBAC/AIS

Early invasive adenocarcinoma

Overtly invasive adenocarcinoma

Reference

3%

10.8%

–

41.9%

463

44%

–

–

23%

299

32%

88%

–

464

25%

51%

36%

86%

304

10%

57%

–

90%

465

17%

100%

–

8%

301

a

Only three cases of LNMBAC were studied.

Table 15 KRAS mutations in AAH, LNMBAC/AIS and adenocarcinomas

Percentage of lesions studied showing KRAS mutation AAH

LNMBAC/AIS

Reference

Adenocarcinoma

0%

302

17%

141

34%

303 466

15%

35%

39%

42%

467b

11%

17%

301

2%

a

464

27%

16.7%

10%

463

33%

12%

8%

304

c

All TRU-type adenocarcinomas. Cases considered “central bronchial-type adenocarcinoma all lacked KRAS mutation. b Mutations were most often a G–T transversion at position 1 or 2. c Mutated cases were all “early invasive” adenocarcinomas. More overt, advanced cases lacked KRAS mutation. a

non-Japanese study did not study many lesions and cases were selected on the basis of lesion multiplicity, so it may not be a true reflection of a European patient cohort.301 Nonetheless these data suggest that EGFR mutations may be less important in Caucasian TRU-type adenocarcinogenesis than in Japanese patients. Many of these studies demonstrated there was frequent discordance in EGFR mutation status between AAH lesions as well as between AAH and adenocarcinoma in the same patient. While KRAS mutation does not appear to be a significant factor in SD/CIS and bronchial squamous cell carcinoma development, it is important in lung adenocarcinogenesis. Up to 40% of lung adenocarcinomas show point mutations in codons 12, 13 or 61, with codon 12 being the most frequently altered. G–T transversions are the most frequent change in adenocarcinomas and these are probably related to tobacco exposure. The earliest studies of KRAS mutation in AAH found either no or very few mutations but only small numbers of lesions were examined (Table 15).141,302,303 Interestingly, in

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cases with multiple AAHs, mutations could vary between lesions and there could be discordance between the AAH mutation and that present in an associated adenocarcinoma. Sartori et al. reported a 50% concordance between KRAS mutations in adenocarcinoma and associated AAH in four patients.301 Sakamoto et al. detected mutations of KRAS in 33% of AAH lesions, 12% of LNMBAC-AIS, 8% of early invasive adenocarcinomas but in none of the overt adenocarcinomas studied.304 The latter authors postulated that KRAS mutation may “cause” some AAH lesions but these do not progress to invasive carcinoma, unlike those with EGFR mutations (see above). This is a somewhat restricted view, reflecting the frequency of EGFR mutation, relative to KRAS mutation, in Japanese adenocarcinomas. The situation in “Western” patients is probably different, and the ethnicity of the study group as well as the methodology being used to detect KRAS mutation will probably influence the findings. In a study examining a number of gene polymorphisms in patients with adenocarcinoma and AAH, carriers of polymorphisms KRAS1 and KRAS-6 were more likely to have multiple AAH lesions.305 The HER2/Neu oncogene is frequently overexpressed in lung adenocarcinomas. HER2 protein (C-erbB-2) was demonstrated immunohistochemically in 7% of AAH lesions, all relatively high-grade lesions.286 Mori et al. also found HER2 protein in some AAH and more in LNMBAC-AIS.245 Awaya et al. found no HER2 protein in AAH, occasional expression in LNMBAC-AIS and more frequent expression in adenocarcinomas, especially in invasive tumors, as opposed to BAC components of mixed lesions. This study failed to demonstrate HER2 gene amplification, using CISH, in any lesion other than invasive adenocarcinoma. Sartori et al. were unable to demonstrate HER2 mutation in any adenocarcinoma or AAH lesion from 13 patients.301 HER2 mutation is an uncommon lesion, reported in only around 4% of lung adenocarcinomas. These data provide a fascinating but unclear insight into the possible role of the EGFR and related TKI pathways in peripheral TRU-type adenocarcinogenesis. There is much to be learned. The mutual exclusivity of EGFR and KRAS mutations in individual adenocarcinomas, if not patients, seems to “hold up” in the pre-invasive stages of disease. The precise role

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of tobacco smoking, gender and ethnicity in determining whether KRAS or EGFR plays a dominant part in disease progression is yet to be determined. There is limited evidence that EGFR mutations can be acquired or lost, as tumors progress (they are not obligatory at the pre-invasive stage). These mutations may be differentially expressed in histologically different areas of the same tumor.306 In Japanese cases, there is a suggestion that while KRAS may “cause” some AAH to develop, it is those with EGFR mutations which progress to LNMBAC-AIS and invasive carcinoma. This is not the case in Caucasians but whether this is related to smoking, other environmental factors, constitutive genomics or other unknown factors remains to be determined. It seems likely HER2 is not a major player in this pathway of carcinogenesis but few have studied this gene. The BRAF gene is occasionally mutated in lung adenocarcinomas but there are no reports, to date, of this mutation in AAH.

Angiogenesis and related factors There are no published studies of angiogenic factor expression in AAH but Kayser et al. demonstrated increased vascular density in AAH compared to normal lung.292 COX2 expression has been variably reported in AAH; it was found in 22% of lesions in one study307 and in over 80% of lesions in another, which also demonstrated abundant expression in LNMBAC-AIS and adenocarcinoma.308

Cell immortalization Data on aspects of telomerase biology in AAH and LNMBACAIS are sparse. Levels of hTERC and hTERT were reported in 27% of “low-grade” AAHs, 75% of “high-grade” AAHs and in 98% of LNMBAC-AIS lesions.309 Telomeric repeat binding factors 1 and 2 (TERF1, TERF2) are telomere-specific binding proteins, which play a role in controlling telomere length. TERF1 mRNA appears to increase in expression from “lowgrade” AAH (36% of cases), through “high-grade” AAH (65%) to LNMBAC-AIS (88% of cases). TERF2 was consistently highly expressed (82–88%) at all stages.309 Mre11 is a protein with a role in telomere length maintenance. Both Mre11 protein and mRNA were consistently highly expressed in all lesions.310 Telomeric DNA expression appeared reduced in both AAH and LNMBAC-AIS in similar amounts, compared to normal alveolar epithelium.311

Transcription factors and other intracellular effectors Eukaryotic initiation factor 4E (eIF4E) regulates protein synthesis and is upregulated in malignancy. Expression of eIF4E increases from normal epithelium through AAH and LNMBAC-AIS to invasive carcinoma.312 While not a transcription factor, large amino acid transporter type 1 (LAT1) is also related to increased intracellular metabolism. Both LAT1 protein and mRNA are found in increasing amounts between low- and high-grade AAH and LNMBAC-AIS.313 Transcription factor NF-kappaB regulates many signaling pathways and is found in greater amounts in AAH than

normal epithelium. Higher levels are detected in adenocarcinomas, especially those with mutated EGFR or KRAS genes.314 Given the association of AAH and adenocarcinoma with female gender, levels of estrogen receptor (ER) in pre-invasive lesions are of interest. Omoto et al. showed ERbeta expression in normal lung, AAH and adenocarcinomas.315 ERalpha was not detected.

Other tumor suppressor genes Apart from the data on P53 in adenocarcinogenesis, little work has been done on other TSGs in AAH and LNMBAC-AIS. FHIT protein is abundantly expressed in AAH and LNMBAC/AIS but significant numbers of invasive adenocarcinomas lose expression, more so in the invasive than in the lepidic components of mixed lesions.316 LOH at 3p14.2 (FHIT) was not detected in AAH.296 P27 protein is an inhibitor of cyclin-dependent kinase 2. Loss of p27 function promotes cell cycle activity and carcinogenesis. P27 protein is variably expressed in AAH and concurrent adenocarcinomas, with a high concordance of expression in associated lesions. Jab1 and Skp2 are p27 degradation pathway proteins. Jab1 was not found in normal alveolar epithelium but was highly expressed in 36% of AAH lesions and in 54% of adenocarcinomas. Jab1 and Skp2 expression correlated with Ki67 index and p27 levels.317 LKB1 (serine/threonine kinase 11 – STK11) is a TSG mutated in approximately 10% of lung adenocarcinomas. One-third of cases show biallelic inactivation of the gene. While strong LKB1 protein staining was observed in peripheral lung epithelia, 10% of AAH lesions and 26% of adenocarcinomas showed marked reduction in staining.318 High-grade AAHs showed more frequent loss of LKB1 staining (21% of cases) than low-grade AAH (5% of cases).

Lineage markers, cell adhesion molecules, matrix metalloproteinases and other factors Given the likely histogenesis of AAH and LNMBAC-AIS, it is no surprise that AAH lesions express surfactant apoprotein A (SPA – a marker of type II pneumocytes).243,319 These studies also demonstrated less staining with SPA in LNMBAC-AIS and adenocarcinomas. Yet it is surprising to note that these same studies failed to find staining in AAH for UP1, a Clara cell marker, found in 20% of LNMBAC-AIS and 70% of invasive adenocarcinomas.243 Equally expected is the regular expression of TTF1 in AAH and LNMBAC-AIS lesions.212,216 The v6 isotype of adhesion molecule CD44 is particularly expressed in lung epithelium. CD44v6 was found in 64% of AAH lesions and 56% of LNMBAC-AIS; rather less was seen in invasive adenocarcinomas (24%).316 In the same study E-cadherin and Beta-catenin were found in 30% and 34% of AAH lesions respectively in comparison to 60–80% of LNMBACAIS and adenocarcinomas. Awaya et al., however, reported the opposite, with E-cadherin and beta-catenin expressed in over

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90% of AAHs and 70% of LNMBAC-AIS, with much lower expression in invasive lesions,320 but criteria for scoring a case “positive” differed between these studies. AAH lesions express more galectins 1, 1b, 3b and 7b than normal alveolar epithelium.292 A variety of matrix metalloproteinases and their inhibitors have been studied in lung adenocarcinogenesis. MT1-MMP is absent in AAH but its inhibitor, TIMP1, is always present in AAH.321 MMP2 and TIMP2 were found in 30–40% of AAH lesions in two studies316,321 but MMP2 was not found in AAH in a third investigation.322 MMP2 was also upregulated in LNMBAC-AIS, showing evidence of collapse, compared to non-collapsed lesions; MMP9 did not show the same change.323 Kumaki et al. showed MMP2 and TIMP2 expression were preserved or increased as lesions became invasive321 but loss of these markers with the onset of invasion was demonstrated by others.316,322 MMPs 3, 7 and 9 have also been variably demonstrated in AAH and LNMBAC-AIS.321,322 These authors also showed, using collagen type IV staining, that basement membranes are intact in AAH and LNMBACAIS but focally absent in invasive adenocarcinomas. Carcinoembryonic antigen (CEA) is reported in a minority of AAH lesions.236 Expression increases as the lesions morphologically become progressively more atypical. Up to 35% of AAHs, 67% of LNMBAC-AIS and 77% of adenocarcinomas demonstrate immunohistochemical staining.235,249,319,324 Blood group antigens A, B and H diminish in expression from low- to high-grade AAH.235 MUC1 expression was decreased but MUC2, MUC5AC, MUC6 and depolarized MUC1 were all increasingly expressed in the transition from AAH to LNMBAC-AIS through to invasive adenocarcinoma.325 Mention has been made of the upregulation of XMEs in the morphologically normal peripheral lung epithelium in smokers. CYPs 1A1–2, 2B1–2 and 2E1 are also overexpressed in AAH and adenocarcinomas but not in normal pulmonary epithelium.319

Studies of clonality, genomic instability and other global expression data Clonality in AAH was first demonstrated using a cytofluorimetric approach.282 Polymorphisms of the X-linked human androgen receptor gene (HUMARA) were used to show clonality in AAH.326 This work also showed different polymorphisms in multiple AAH lesions in the same patient. Aneuploidy in chromosome 7 has been demonstrated in one of two AAH lesions studied using FISH.179 Ullmann et al. used comparative genomic hybridization (CGH) to show chromosomal gains and losses were less frequent in “low-grade” AAH but commoner in “high-grade” AAH. There were similar patterns in LNMBAC-AIS and concurrent adenocarcinomas.327 As with SD/CIS, studies of 3p LOH show losses in AAH lesions. From 10 to 18% of AAH lesions demonstrate 3p LOH.116,284,296 Kitaguchi et al. found 3p and 17p loss in the

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same AAH lesion.284 Yamasaki et al. failed to demonstrate LOH at D3S1234 (3p14.2 FHIT) but found LOH at D3S1300 in 25% of AAH, and in LNMBAC-AIS with and without collapse lesions.296 Marchetti et al. also found 3p14.2 LOH in 43% of LNMBAC-AIS,328 but Takamochi et al. did not.329 Given micronodular pneumocyte hyperplasia is found in tuberous sclerosis (see above) the status of the tuberous sclerosis genes (TSC1 and 2) in AAH is of interest. TSC1 is located at 9q34 while TSC2 is at 16p13.3. 9q LOH was found in 39% of AAHs with 9q34 LOH present in 25% of lesions,329 while 16p LOH was found in 22% and, specifically, 16p13.3 LOH is seen in 6% of AAHs. These losses were more frequent and extensive in concurrent adenocarcinomas. Adenocarcinomas showing these losses may be more likely to be associated with multiple AAH lesions.330 Anami et al. also demonstrated LOH at D9S51 (9q) in 1/6 AAH lesions from the same patient.331 The data suggest that either TSC1 or another gene close to 9q34 is a TSG. The same group responsible for much of this work on 9q and 16p LOH in AAH also suggest that gene silencing in this region may be the result of hypermethylation.329 A gene close to TSC1, rather than TSC1 itself, is the most likely candidate as a TSG in this region. Anami et al. found LOH at 17q (D17S791) in 2/6 AAH lesions in the same patient and in two concurrent adenocarcinomas.331 Nomori et al. found in 3/9 AAH lesions LOH at 13q (RB) while LOH was absent at 3p (FHIT), 5q (APC), 9p (P16), 17p (P53) and 22q (BandM).330 When compared to AAH, more loss is found in more advanced lesions. Aoyagi et al. examined eight key chromosomal loci in LNMBAC lesions with and without evidence of collapse or early invasion.332 They found deletions at 5q (APC), 9p (P16), 11q (Int-2) and 13q (RB) were relatively early changes in lung adenocarcinogenesis. They also demonstrated an increase in the frequency of 17p LOH as architectural collapse and fibroelastosis develops in LNMBAC-AIS lesions. Losses at 3p, 17p, 18q (SMAD4) and 22q (BandM) occurred later as signs of early invasion began to appear. Gradowski et al. examined the LOH patterns in AAH lesions from lungs bearing carcinoma, and from lung tissue excised for benign disease, using 21 polymorphic microsatellite markers situated on 3p, 5q, 7p, 9p, 10q and 17p.333 Most losses were found in 3p and 10q but no difference in the overall pattern of loss could be demonstrated in the AAH lesions derived from these two patient groups. The clonal relationship between AAH and concurrent adenocarcinoma has been examined, using EGFR or KRAS mutations or HUMARA polymorphisms (see above). Morandi et al. took a different approach to this question by using patterns of mutations in mitochondrial DNA and LOH. In a majority of the informative cases, differences in mutation or LOH pattern were found, suggesting that concurrent AAH and adenocarcinoma arise independently.334 Yohena et al. provided evidence that LOH at some loci may be associated with smoking. LOH at D3S1234 (FHIT) is most commonly implicated.335 Apart from the hypermethylation of

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P16 in AAH discussed above, other genes such as TIMP3, DAPK, MGMT, RARbeta, RASSF1A, hTERT and genes in the Wnt signaling pathway also showed variable but generally increasing methylation from AAH through adenocarcinoma.295,336 Gene expression profiling studies of LNMBAC-AIS and adenocarcinomas have shown, amongst many other things, upregulation of Dicer, a key effector protein for microRNA function. Immunohistochemistry demonstrates that Dicer is also upregulated in AAH and LNMBAC-AIS, while expression of this protein is diminished in early invasive and advanced adenocarcinoma.337 In another expression array study, 183 differentially expressed genes were found between normal lung and LNMBAC-AIS.338 Only 15/183 genes were upregulated and of these, only multidrug resistance protein 3 (MRP3) was solely upregulated in tumor cells. MRP3 was shown using immunohistochemistry to be overexpressed in AAH, compared to normal type II pneumocytes. Even more MRP3 was found in LNMBAC-AIS. The development of an AAH cell line is an interesting development with the potential to provide great insight into the molecular biology of this lesion.339 This model has so far allowed identification of tumor-associated calcium signal transducer 2 (TACSTD2) and S100 calcium binding protein A2 as unique molecular markers in lung adenocarcinogenesis.

The clinical relevance and prognosis of AAH AAH lesions cause no loss of respiratory function and no symptoms. Their importance is purely as a putative precursor of LNMBAC-AIS and invasive adenocarcinoma. As such they represent at least a potential risk factor for the development of this increasingly common form of lung cancer. The risk of AAH progressing to LNMBAC-AIS and invasive disease is unknown. It is impossible to identify AAH lesions with certainty in vivo. Developments with HRCT scanning are of interest but there is, at the time of writing, no reliable way of identifying AAH “in situ”. Thus, there are problems in designing longitudinal studies of lesions, to see how, if at all, they progress (see below). The only published work which even approaches this question concerns follow-up studies on patients who had AAH diagnosed on lung tissue resected for other reasons. The central premise which underpins these studies is the presumption that if the patient had AAH in their resected lung, there will be AAH remaining in the lung or lobes left behind after surgery. Thus the patient is at risk of developing, and dying from a second lung adenocarcinoma. Follow-up studies published to date have mostly used postoperative survival as their endpoint. Apart from the study by Takigawa et al., which showed a non-significant trend towards better survival in patients with AAH,274 all the other studies have shown no difference in survival.250,267,340,341 The author has reviewed the follow-up of a cohort of 128 patients from northeast Scotland who had AAH in their original resection.268 In this age/sex/surgery/tumor

histology case-controlled follow-up study of AAH patients, no difference in postoperative survival between the AAH and the control group was found, yet a trend towards poorer survival was seen in those patients with larger numbers of AAH lesions. The author has had the opportunity to examine completion pneumonectomies, lobectomies for second metachronous tumors, and autopsy lung tissue from a postoperative death, from patients whose original lobectomy showed AAH. Lesions were found in the second samples but in two cases (autopsy and completion pneumonectomy) there was a contrast between the large amount of disease in the original and relative lack of disease in the subsequent sample. This is no more than anecdotal comment, but the prospect of a lobectomy not only removing the primary tumor but the larger “at risk” zone which has undergone greater field cancerization than the rest of the lung is intriguing. If this zone is resected with the tumor, then the postoperative risk of second tumors may be less than assumed. One of the most important factors pertaining to preinvasive lesions is the risk of subsequent invasion. This remains largely unknown for AAH and also LNMBAC-AIS. Understanding the pathology of these lesions, as well as the structural changes which immediately precede and accompany invasion, has informed the interpretation of HRCT scans of small solitary pulmonary nodules. It remains to be seen whether these studies allow longitudinal study of AAH and LNMBAC-AIS to learn about rates of, and thus risk factors for, progression. AAH is not visible on chest radiographs; detection is either incidental or as a finding during HRCT-based lung cancer screening programs.342–344 Since both AAH and LNMBAC-AIS lesions contain alveolar air, they may present as pure “ground-glass” opacities (GGO) on HRCT (Figures 45 and 46).345,346 Studies have attempted to distinguish between AAH and LNMBAC-AIS by CT, but reliable separation is lacking.347 Given the considerable “gray area” which exists pathologically around AAH versus LNMBAC-AIS, this is hardly surprising. More recent work, with advanced scanners, shows promise of better radiological distinction between lesions.348 With very small lesions there are particular problems with specificity. Of 139 lesions of 5 mm or less detected using an extremely sensitive 1.25 mm slice multidetector-row HRCT scanner, two-thirds corresponded to normal anatomy or motion artifacts. Twelve percent of lesions were inflammatory and 5% were intrapulmonary lymph nodes; only 5% were AAH lesions, while 3.5% were LNMBAC-AIS.349,346,350

Peripheral lung adenocarcinogenesis: comment and conclusions This section has reviewed the structural pathology, prevalence, associations and molecular biology of AAH. All these factors provide evidence that this lesion is a pre-invasive lung lesion; a progenitor of pulmonary adenocarcinogenesis. These lesions

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Figure 46. This larger pure GGO lesion was LNMBAC/AIS on resection. The GGO texture and shade are the result of the persistence of air in the lesion due to the presence of alveoli. (Image courtesy of Dr Keiko Kuriyama, Osaka, Japan.) Figure 45. High-resolution computed tomography (HRCT) scan showing a small peripherally located pure ground-glass opacity (GGO). On resection this lesion proved to be AAH. Pure GGO lesions may also be the result of inflammatory or other nonspecific changes in the lung (see text). (Image courtesy of Dr Keiko Kuriyama, Osaka, Japan.)

may be difficult to diagnose, especially if pathologists’ practice does not facilitate their identification. Issues in the differential diagnosis of AAH have been discussed; this is especially relevant if experience of a particular lesion is limited. In this proposed adenoma-carcinoma sequence of adenocarcinogenesis affecting the TRU epithelium there is a transition from AAH to LNMBAC-AIS. The histological distinction between these two lesions is difficult and arbitrary. They are both part of a spectrum of what could be considered pulmonary alveolar intraepithelial neoplasia (PAIN) and until differential risks of progression are recognized, distinction between these two lesions is arguably of lesser importance than the identification of invasive adenocarcinoma. It is probable that only a minority of AAH lesions progress; in those cases with multiple lesions, the number of AAH lesions usually far exceeds that of LNMBAC-AIS. The fate of AAH lesions which do not progress is unknown. They may regress and revert to “normal”, as with some SD/CIS lesions. The author has observed several cases where some AAH lesions show partial or near complete sclerosis, the alveolar walls becoming markedly thickened by hyaline connective tissue and elastosis. This process may be associated with chronic inflammation and “burnt out” alveolar scars may result. The AAH/LNMBAC-AIS/invasive adenocarcinoma sequence may not be the only avenue to peripheral pulmonary adenocarcinoma. We have no idea of how many

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adenocarcinomas evolve this way. There are similarities between this process and adenocarcinogenesis in association with pulmonary fibrosis, in terms of a proliferative alveolar epithelium undergoing malignant transformation. In the latter situation, other types of lung carcinoma also develop (see above). It is also possible that some adenocarcinomas may arise “de novo” from bronchioloalveolar epithelium. Such would represent another example of the “parallel theory” of tumor evolution.25

Pulmonary pre-invasive disease: final comments This chapter has reviewed a great deal of information related to pre-invasive diseases of the lung, which may be precursors for the development of lung cancer. There are at least three well-described pathways of tumor development, which have recognizable morphological changes. One of these, concerning DIPNECH, is discussed in Chapter 31. Other lesions, such as some proposed patterns of bronchial dysplasia and bronchiolar columnar cell dysplasia, have been suggested as alternative pre-invasive lesions. These deserve consideration but require a greater collective experience and understanding before their true role can be understood. Central bronchial carcinogenesis is typified by the proposed progression of epithelial hyperplasia/metaplasia leading to SD/ CIS and ultimately invasive squamous cell carcinoma. These pathological lesions are well described, the diagnostic challenges arising from their classification are numerous and much is known about the molecular alterations found in this disease complex. Less is understood of the important facets of SD/CIS molecular biology. Currently there is focus on the possibility of

Chapter 23: Pulmonary pre-invasive disease

using such molecular markers to facilitate early detection of disease; in practice this would probably be a sputum-based test.351,352 A number of factors have been considered as candidate test targets and gene methylation has emerged as one promising biomarker.353 Other approaches include image analysis-based cytometry, possibly augmented by some molecular markers to identify abnormal sputum cells.354,355 The analysis of exhaled breath for a chemical signature of lung cancer also has its advocates.356 Despite much activity on many fronts, a test ready for deployment seems distant.357 Another possible way of exploiting the molecular characteristics of bronchial, or indeed any pulmonary pre-invasive disease, is through the development of chemopreventative agents.358 These agents are similarly “under development” and the subject of various trials.359 The concept of peripheral lung adenocarcinogenesis has evolved more recently than its counterpart in the central airways. The AAH/LNMBAC-AIS/invasive adenocarcinoma pathway is now accepted as the origin of some, possibly many, lung adenocarcinomas. There has been a substantial rise in the amount of literature on this subject in the last 10 years, the most recent work concentrating on various aspects of the molecular biology of the process. As with SD/CIS, we await developments which might exploit this new knowledge, towards better methods of screening or chemoprevention. In many ways this is a more difficult pathological system to study than SD/CIS since it poses greater problems in terms of detection, clinical accessibility of disease and availability of pathological material for study. Among the many fascinating but still largely unanswered questions in this area concern issues around who actually develops pre-invasive lung diseases, what determines which lesions will progress, and which type of invasive tumor develops. Elements of environment and lifestyle expose the lung epithelium to a range of carcinogens. It appears that pre-invasive changes such as SD/CIS and AAH/LNMBACAIS are the earliest morphological expression of the molecular evolution of malignancy, at least in part resulting from carcinogen-induced damage. These two major pathways of lung carcinogenesis reflect a common response (malignant transformation) in two separate epithelial compartments. This response seems to largely determine the “histotype” of the resultant tumor. The bronchial epithelium gives rise to several tumor types; it may be that certain molecular events determine

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which tumor type arises from a common epithelial precursor. Equally, in some cases, the tumor type identified at clinical presentation may be the result of neoplastic progression and/ or dedifferentiation after the lesion becomes malignant. There is evidence that lung adenocarcinomas in particular may have considerable capacity to evolve as the lesion grows. Apart from issues around detection of disease, risk of progression is a key factor in the clinical management of pre-invasive lung disease. Yet very little is known of these risks. Longitudinal studies of SD/CIS and especially AAH/ LNMBAC-AIS are difficult to conduct. Many studies are confounded by various factors, including the understandable ethically driven desire and need to treat disease, rather than to observe its potential evolution into something with metastatic ability. Whether molecular factors can give any insight into the risk of progression remains to be seen.360 Researchers are, however, in something of a “Catch 22” situation; on the one hand it seems that even carcinoma in situ can spontaneously regress. On the other, potential overtreatment of dysplasia or in situ disease risks unnecessary morbidity while depriving clinicians of the opportunity of answering some fundamental questions. Constitutive genetics also have an influence on tumor development. There are gender associations with SD/CIS and AAH/LNMBAC-AIS. This may be the result of differences in carcinogen exposure but may also reflect differences in susceptibility. Inherited polymorphisms of various XMEs and other genes may determine susceptibility in different lung epithelial compartments as certain polymorphisms and certain carcinogens appear to be associated with the development of different lung cancer cell types.361–366 Smoking prevalence is declining in many Western countries but is booming in many of the most populous nations, such as China and India. In the West non-smoking-associated lung cancers will assume a relatively greater importance. In Asia in particular, the interplay between increasing exposure to tobacco carcinogens in populations with an apparently greater propensity to developing peripheral TRU-type adenocarcinomas will be observed with interest and concern. If screening for lung cancer is to be deployed effectively in these various populations, account must be taken of both central and peripheral lung carcinogenesis in each patient.

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Chapter

24

Epidemiological and clinical aspects of lung cancer Erik Thunnissen, Michael Unger and Douglas B. Flieder

Introduction Lung cancer is the leading cause of cancer death in the world; over 1.3 million worldwide deaths were recorded in 2008.1 These statistics are astounding given the rarity of lung cancer during the first half of the twentieth century, when lung cancer had a lower incidence than liver, prostate, colon, stomach, uterine, breast and even ovarian cancer. Only a sound understanding of the complex epidemiological, etiological, and molecular-pathological aspects of lung carcinoma will enable clinical and scientific progress against this deadly disease, regardless of technological advances. This chapter aims to elucidate the epidemiological, etiological and clinical aspects of lung cancer.

Epidemiology Incidence and mortality Lung cancer is the most common and deadliest cancer in the world. Estimated numbers of lung cancer cases worldwide increased 51% since 1985.1 The incidence information is collected routinely by cancer registries and expressed as an absolute number of cases per year or as a rate per 100 000 persons per year. The latter provides an approximation of the average risk of developing a cancer. Excluding non-melanoma skin cancer, approximately 7.5 million people died of cancer worldwide in 2008.2 Of these deaths 1.37 million were lung cancer deaths (20%). The worldwide incidence of lung carcinoma in 2008 reached 1 608 823 cases, representing 12.7% of newly diagnosed cancer cases.1,3 Lung cancer gender distribution is 68% males and 32% females. An age-standardized rate (ASR) is a summary measure of a rate that a population would have if it had a standard age structure. Standardization is necessary when comparing several populations that differ with respect to age. This is because age has such a powerful influence on the risk of cancer. The ASRs are 34 and 13.5 new cases per 100 000 males and females, respectively.3 For males, the lung cancer rate (16.5%) is followed by prostate (13.6%), colon-rectum (10.0%), stomach (9.7%) and liver (7.9%) (Figure 1). For

Lung Prostate Colorectum Stomach Liver Esophagus Bladder Non-Hodgkin lymphoma Leukemia Lip, oral cavity Kidney Pancreas Larynx Brain, nervous system Incidence

Other pharynx

Mortality

0

10

20

30

40

Age standardized rate per 100,000

Figure 1. Age-standardized world cancer incidence in men by site. Almost one million new lung cancer cases are diagnosed each year. Data from Globocan 2008.2

females the most frequent cancer is breast (22.9%) followed by colon-rectum (9.4%), cervix (8.8%), lung (8.5%) and stomach (5.8%)3 (Figure 2). The total burden of lung cancer is slightly greater in less well-developed countries, compared to more developed regions. However, the incidence ASR for lung cancer is higher in more developed countries, 47.4/100 000 men and 18.6/100 000 women, than in less developed countries, 27.6/100 000 men, and 11.1/100 000 women. Approximately 15% of the global lung cancer cases in men and 53% in women do not appear to be caused by smoking. The overall number of non-tobacco-related lung cancer cases in

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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2008 is estimated to be 430 000. In global terms, for men and women combined, lung cancer in never-smokers ranks in seventh place, after cancer of the lung due to smoking, and cancers of the stomach, colon–rectum, liver, breast and esophagus. Geographic trends in lung cancer incidence and mortality basically reflect regional differences in smoking behavior and Breast Cervix uteri Colorectum Lung Stomach Corpus uteri Ovary Liver Thyroid Leukemia Non-Hodgkin lymphoma Esophagus Pancreas Brain, nervous system Incidence Mortality

Kidney 0

30 10 20 Age standardized rate per 100,000

Figure 2. Age-standardized world cancer incidence in women by site. Almost 400,000 new lung cancer cases are diagnosed each year. Data from Globocan 2008.2

40

in wood fires in homes. Developed countries had 723 000 new cases and 599 000 deaths in 2008. In the same year developing nations, including China, reported 883 000 new cases and 778 000 deaths. These data are a reflection of future trends, since the rising incidence and mortality of lung cancer will produce huge epidemics in developing countries in the coming decades (Figure 3). In China about half of the male population smokes, and 70% of the households use solid fuels (wood, crop residues and coal) for heating and cooking.4 Tobacco smoking and pollution in the home from solid-fuel usage are the most important global risk factors for chronic obstructive pulmonary disease (COPD) and lung cancer. These two diseases account for a significant proportion of deaths from these diseases in developing countries.4,5 The impact of COPD and lung cancer burden in China is enormous.5 Lin and colleagues calculate that complete gradual cessation of smoking and solid-fuel use by 2033 could avoid 26 million deaths from COPD and 6.3 million deaths from lung cancer.4 Head and neck cancer is caused by tobacco smoking and alcohol consumption. In patients with previous primary head and neck cancer, subsequent lung cancer contributes to the highest proportion of the second primary cancers, with a 20-year cumulative risk of 13%.6 The risk of dying from lung cancer is highest in North America, Australia, New Zealand, Europe (particularly central and eastern Europe) and South America, while rates in China, Japan, and southeast Asia are rising steeply (Figure 4). The lowest rates are observed in southern Asia and sub-Saharan Africa, at least in part due to poor data collection. Declining lung cancer rates were first observed in the United Kingdom, and then in Finland, Australia, Netherlands, New Zealand, the USA, Singapore, Denmark, Germany, Italy and Sweden. This decline is related to the reduction in smoking, especially in the older generations.7 Figure 3. American and Chinese smoking rates.

% smoking population 80 70 60 50

Smoking men USA Smoking women USA

40

Smoking men China Smoking women China

30 20 10 0 1930

946

1940

1950

1960

1970

1980

1990

2000

Chapter 24: Epidemiological and clinical aspects of lung cancer Figure 4. American and Chinese lung cancer mortality rates.

Mortality rate/100,000 90 80 70 60 Mortality men USA

50

Mortality women USA Mortality men China

40

Mortality women China

30 20 10 0 1930

1940

1950

1960

1970

1980

1990

2000

Figure 5. Nations with greatest numbers of male smokers ( 1000). Data from Globocan 2008.2

Gender In 2002, 965 241 men and 386 891 woman were stricken with lung cancer and 848 132 and 330 786, respectively, died worldwide.1,3 In men the estimated number of lung cancer cases had increased by 44% since 1985, due to smoking in a growing and aging population. In reality, there has been a 3.3% decrease in the actual age-standardized incidence. In men the lung cancer death rate in the USA showed in 1975–1982 an increase of þ1.7%, but changed to 2.0% in the period 1994–2005.8 In the European Union, the lung cancer mortality rate per 100 000 men declined 1.6% from the period

1990 to 2000, despite the addition of central and eastern European nations, who have the highest lung cancer rates in Europe.9–12 In 1996, tobacco smoking among Chinese men reached levels seen in the USA in 1950. Currently, the countries with the highest number of smoking males are China, India and Indonesia (Figure 5). With 300 million current male smokers, lung cancer will soon be responsible for more than 2 million deaths per year in Chinese men.4 Whether the incidence of lung cancer in never-smoking women is higher than in never-smoking men is a matter of debate and currently not clear.13–15 Some, but not all,

947

Chapter 24: Epidemiological and clinical aspects of lung cancer

case-control and cohort studies suggest that smoking causes a larger relative increase in lung cancer risk in women than men.16–20 However, results from cohort studies generally find similar incidence rates in men and women with comparable smoking histories.14,15,21 In women, the estimated numbers of lung cancer cases worldwide has increased 76% since 1985.1 While reported cases have perhaps peaked in the UK, most Western countries show a rising incidence and mortality trend, which may begin levelling off.9–12 The highest incidences are noted in North America and northwestern Europe, and the greatest rises are seen in southern and eastern Europe.9–12 The 600% increase in the death rate from lung cancer in American women from 1930 through to 1997 corresponds to the availability and social acceptability of tobacco use among women. This is now responsible for the current lung cancer epidemic in women. The smoking prevalence in American men decreased by nearly 50% from its peak in the 1960s and the death rate in men from lung cancer has decreased slightly. In the same period, the smoking prevalence in American women has only decreased by 25%.22 Although the annual percentage change in the lung cancer incidence rate for US women fell from 5.5% for the period 1975–1982 to 1.0% for the period 1991–1998, a decline for the subsequent period 1998–2006 (0.6%) was reached.8,23 In the European Union, the lung cancer mortality rate per 100 000 women rose 1.2% from 1990 to 2000 and a striking 38% in women younger than 55 years. In the USA it is estimated that 48% (107 280) of newly diagnosed lung cancers in 2009 were in women. This incidence is significantly less than that attributed to breast cancer including in situ carcinoma (192 370). However, more women were predicted to die of lung cancer (70 490) in 2009 than the combined mortality of breast (40 170) and colorectal (24 680) carcinoma, the next two most common carcinomas in US women.8 In the Netherlands before 2008 more women died of breast than of lung cancer. Due to a decline in breast cancer incidence and an increase in lung cancer incidence in women, this order is reversed: more women died in 2008 of lung cancer than of breast cancer.24 In the USA more women over 40 years of age die of lung cancer than from breast cancer.8 Moderate but rising rates are reported in China, where an estimated 20 million women started smoking since the 1990s.25,26 In addition, smoking among women in Japan doubled to 18% during the 5-year period 1986–1991. In many developing countries where female smoking is not common, lung cancer rates are very low. An upward trend is predicted for the female lung cancer epidemic within the next two to three decades in Spain and France, in contrast to other European countries.27 A significant percentage of non-smoking women also develop and die of lung cancer. Lung carcinoma is the third commonest cancer amongst Chinese women in Singapore and constitutes almost 10% of all cancers in this group.28 However, only 3% of Chinese women, aged 18 to 64, in Singapore smoke. The population risk for lung cancer attributable to smoking

948

approaches 80% for Hawaiian women, 45% for Japanese women living in Hawaii, and 15% for Chinese women living in Hawaii, indicating that, especially for the latter two, other factors than cigarette smoking play a role in the development of lung cancer.29 The proportion of Japanese women with lung cancer in Japan who are never-smokers is 70%, whereas the proportion of women over 20 who smoke is around 10%.30 Lung cancer incidence rates are higher and more variable among women in East Asia than in other geographic areas with low female smoking.14,31 The environmental tobacco smoke and other environmental carcinogens, including cooking oil vapor, and coal burning-devices may play a role in development of lung cancer.4,32 Lung cancer in neversmokers is an important public health issue.31

Age Non-small cell lung cancer can occur in newborns and octogenarians, but most cases are diagnosed in the sixth and seventh decades of life.33–36 The diagnosis rises exponentially and plateaus at the age of 80 in men and 70 in women, before decreasing. The median age of lung cancer diagnosis in the general population of the USA is 71 years.8,37 No more than 10% of lung cancers are diagnosed in individuals under 50 years of age and in less than 2% of individuals under 45 years of age.38–40 There is a greater representation of African-Americans, Asians and Pacific Islanders in the 80% power to detect risk of 1.5 Caucasians  45 years of age Lack of homogeneity across all studies No differences by smoking status, sex, histology and ethnicity Publication bias for small studies with positive findings Larger studies only (>500 cases) Non-smokers Asian population indoor air pollution (coal) Publication bias for small studies with positive findings and ethnicity; Asians OR 1.38, Caucasians OR 1.04 Chinese only

961

Chapter 24: Epidemiological and clinical aspects of lung cancer Table 4 (cont.)

Author

Number of studies (cases/ controls)

Meta or pooled analysis

OR (95% CI)

Risk genotype

Comments

10 (2686/ 3325) 12 (4285/ 4656)

Meta

0.86 (0.31–2.32)

G-463A

Meta

0.81 (0.64–1.02)

G-463A

Results influenced by a single large study Multiple ethnic groups

3 (741/846) 3 (499/959)

Meta Meta

1.12 (0.96–1.47) 0.70 (0.56–0.88)

C609T ¼ Pro187Ser C609T

Caucasians Japanese

7 (2078/3081)

Meta

1.54 (0.77–3.07)

High-activity alleles

8 (986/1633)

Pooled

1.18 (0.92–1.52)

High-activity alleles

8 (1944/2670) 6 (815/1286)

Pooled Pooled

0.75 (0.53–1.07) 0.72 (0.43–1.22)

Low-activity alleles Low-activity alleles

mEH Tyrl 13His (decreased activity) and Hisl 38 Arg (increased activity) mEH Tyrl 13His (decreased activity) and Hisl 38 Arg (increased activity) Nine ethnic groups Caucasians

16 (3865/ 6077)

Meta

1.04 (0.96–1.14)

Slow acetylator phenotype

C481T, G590A, G857A; 2 or more mutant alleles ¼ slow acetylator phenotype

Methylenetetrahydrofolate reductase Mao et al.642 8 (5111/6415)

Meta

1.12 (0.97–1.28)

C677T

7 (5087/6232)

Meta

1.00 (0.92–1.08)

A1298C

10 (5274/ 7435) 7 (5098/6243)

Meta

1.22 (0.95–1.55)

C677T

Statistical bias not for lung cancer, possibly for colorectal cancer, gastric, leukemia Both polymorphisms Hom/het not significant

Meta

1.07 (0.83–1.38)

A198C

25 (9387/ 9922) 7 (6424/6612) 13 (3209/ 3301) 23 (7495/ 8362) 6(3610/5293)

Meta

1.08 (1.00–1.17)

G12139C ¼ Arg72Pro

Meta Meta

1.14 (1.06–1.22) 1.05 (0.96–1.15)

G12139C G12139C

Meta

1.22 (1.05–1.42)

G12139C

Pooled

1.20 (1.02–1.42)

G12139C

Risk associated with smoking and with SCC

H-ras-1 Weston and Godbold648

3 (643/402)

Meta

1.9 (1.3–2.8)

VTR variants 6 bp

MSPI/HpaII, Rare allele (3.7%)

ERCC1 Kiyohara and Yoshimasu649

5 (2531/3073)

Meta

0.72 (0.46–1.11)

T19007C

Similar data Li et al.650

15 (5004/ 6478) 11 (4110/ 53050) 6(2286/3085) 9 (6463/6603)

Meta

1.30 (1.13–1.49)

A35931C ¼ Lys751Gln

2007 In Chinese, similar data651,652

Meta

0.95 (0.84–1.07)

G23592A ¼ Asp312Asn Asian, Caucasian

Meta Pooled

1.27 (1.04–1.56) 1.19 (1.02–1.39)

G23592A A35931C

Myeloperoxidase Feyler et al.637 Kiyohara et al.638 NQO1 Kiyohara et al.638 Epoxide hydrolase (EH) Lee et al.639

Kiyohara et al.640 N-Acetyltransferase 2 (NAT2) Borlak et al.641

Boccia et al.643

TP53 Yan et al.644

Li et al.646 Hung et al.647

ERCC2/ XPD Kiyohara and Yoshimasu649 Hu et al.652 Hung et al.647

962

Different ethnic groups, some hospital-based selection, folate intake in future studies may be useful Statistical bias; association of 12139 C allele and adenocarcinoma Asians hom and hets; Pro allele 32% Caucasians, similar data;645 Pro allele 27% Statistical bias

2004 2008

Chapter 24: Epidemiological and clinical aspects of lung cancer Table 4 (cont.)

Author

XRCC1 Wang et al.653 Zheng et al.654 Kiyohara et al.655

XRCC3 Manuguerra et al.651 Hung et al.647 OGG1 Li et al.656 Hung et al.647 Mdm2 Bai et al.657 XPA/XP1 Kiyohara and Yoshimasu649 XPC/ RAD4 Qiu et al.659

Number of studies (cases/ controls)

Meta or pooled analysis

OR (95% CI)

Risk genotype

Comments

16 (4848/ 6592) 8 (2861/2783) 18 (7385/ 9380) 6 (1702/2010)

Meta

0.88 (0.79–0.97)

Arg194Trp

Meta Meta

1.06 (0.89–1.27) 0.99 (0.93–1.06)

Arg194Trp Arg399Gln

Mixed ethnicity; Codon 399, no association Only Chinese All

Meta

1.34 (1.16–1.54)

Arg399Gln

Asian

6 (1486/1850) 8 (3467/5021)

Meta Pooled

1.25 (0.97–1.60) 0.84 (0.71–1.00)

Thr241Met C/T Thr241Met

Other allele combinations OR ~1 Risk associated with smoking and small cell carcinoma

17 (6375/ 6406) 4 (2569/4178)

Meta

1.15 (0.94–1.41)

Ser326Cys

All ethnicities, similar data552

Pooled

1.34 (1.01–1.79)

Ser326Cys

Caucasian

8 (6063/6678)

Meta

1.16 (1.01–1.34)

Thr309Gly

Enhanced in never smokers (1.36), similar data658

7 (1913/1882)

Meta

0.75 (0.59–0.95)

Gly23Ala

GG protective effect in all, but not significant in Caucasians only

16 (6797/ 9018)

Meta

1.28 (1.07–1.53)

Lys939Gln

Similar data660

Data are compiled from reviews on polymorphisms using meta-analysis (Meta) or pooled analysis (Pooled). If the 95% confidence interval (CI) does not include the odds ratio of 1.0, the OR is significantly linked to lung cancer risk as either decreased risk (protective: OR 95% CI < 1) or increased risk (OR 95% CI > 1).

The biological interpretation of the higher risk in neversmokers is not straightforward. It is possible the effects of some genetic susceptibility profiles are more easily observed in people with lower levels of exposure, pointing to an internal effect at the gene regulatory level. As this finding apparently violates the dose-response exposure expectation, as yet unknown factors in the surrounding environment may have an impact. These observations suggest the existence of genegene interactions in lung carcinogenesis. It is likely that larger prospective studies in progress will yield clearer insights.563 People with rare combinations of common gene variants have a high risk of cancer and can be compared to people with highly penetrant mutations. In theory, a future application may be genetic screening for high-risk alleles as a basis for lung cancer detection.

Genome-wide studies and lung cancer risk Genome-wide association studies have been conducted in five of the commonest cancer types: breast, prostate, colorectal, lung and melanoma.564 Twenty novel disease loci have been identified. The risks conferred by the susceptibility alleles are

low, generally 1.3-fold or less, indicating susceptibility to these diseases is polygenic.564 For lung cancer, the genome-wide approach revealed higher-risk loci in chromosomes 5q15.33,543,565 6p21.33,543 and 15q24–25.566–568 The 5p15.33 locus (single nucleotide polymorphism, SNP, rs4975616569) contains a probable lung cancer-related locus within the 60 kb region of linkage disequilibrium, comprising at least two genes, the “Cleft lip and palate transmembrane protein 1-like protein” (CLPTM1L) gene and human telomerase reverse transcriptase (HTERT) gene. 5p15.33 is frequently amplified in early stages of non-small cell lung cancer.570 Moreover, rs402710 SNP in 5p15.33 is associated with higher DNA adduct levels in “normal” lung tissue.571 Recently, the rs2736100 SNP in the HTERT chromosome on 5p15.33 was linked to adenocarcinoma.569,572 The 5p15.33 region (rs 401681 SNP in intron CLPTM1L) in addition to being associated with lung cancer risk (OR ¼ 1.15, 95% CI 1.10–1.22) is also associated with a higher risk of basal cell carcinoma (OR ¼ 1.25, 95% CI 1.18–1.34) and cancer of the urinary bladder (OR ¼ 1.12, 95% CI 1.06–1.18), prostate (OR ¼ 1.07, 95% CI 1.03–1.11) and cervix (OR ¼1.31, 95% CI 1.03–1.32).573

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Chapter 24: Epidemiological and clinical aspects of lung cancer

The 6p21.33 region was confirmed (SNP 3117582)569 and contains probably a single locus in a 630 kb region between “large proline-rich protein BAT3” (BAT3) and MutS protein homolog 5 (MSH5). BAT3 is involved in apoptosis and the protein complexes with E1A-binding protein p300, required for acetylation of p53 in response to DNA damage.574 MSH5 is involved in DNA mismatch repair (MMR)575 and meiotic recombination. Deficiency of MMR plays a role in lung cancer development.576 The chromosome 15q association between smoking and lung cancer is specific for lung cancer, as no evidence for an elevated risk was found in never-smoking patients with lung cancer, nor for other smoking-related tumors (e.g. bladder and kidney).577 The 15q24–25 region has been divided in two SNP haplotype maps.578 One region encompasses “proteasome subunit alpha type-4” (PSMA4), “iron-responsive element-binding protein 2” (IREB2) and a hypothetical gene LOC123688. In the other region, three neuronal acetylcholine receptor subunit genes, α5 (CHRNA5), α3 (CHRNA3) and β4 (CHRNAB4), are

present. Which of the six candidate genes account(s) for the increased lung cancer risk is unknown. The N-acetylcholine receptor has a role in nicotine addiction.577,579 A mechanistic link seen in early nicotine exposure (before the age of 17 years), common CHRNA5-A3-B4 haplotypes and adult nicotine addiction has been identified in three independent European populations. In cancer cells, activation of the N-acetylcholine receptor involves an autocrine loop, and leads to increased proliferation, inhibition of apoptosis, increased angiogenesis, and reduced cellular adhesion (Figure 9). In lung cancer patients continuation of smoking leads to decreased survival.580 PSMA4 has been suggested as a candidate gene, since expression of this gene is upregulated in lung and other cancer(s) and modulates cell proliferation.578 Downregulation of PSMA4 expression increases apoptosis and decreases proteasome activity. Detailed analysis of one 15q single nucleotide polymorphism (SNP, rs16969968,581 rs12914385569) identified an association with an increased risk of lung cancer (OR ¼ 1.30). In addition, for the variant allele, an association with smoking and an earlier age of lung cancer onset was demonstrated.581

Figure 9. The acetylcholine receptor (AChR) pathway. Nicotine activation leads to cellular proliferation, inhibition of apoptosis and angiogenesis.

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Chapter 24: Epidemiological and clinical aspects of lung cancer

The CHRNA3 gene contains SNP rs1051730, which in a European population is strongly associated with smoking, lung cancer and peripheral arterial disease.582 In Eastern populations, the frequency of the susceptible haplotype, on 15q25, is much lower (~1%) than in European and American populations (30–40%). Nevertheless, the increased lung cancer risk of this haplotype was confirmed in a Japanese583 but not in a Chinese population.584 In the Chinese study, novel SNPs were detected with an increased risk of lung cancer, one of which (rs6495309T>C, OR ¼1.4) affected the OCT-1 binding ability in the CHRNA3 promoter region. It was characterized as a functional variant, leading to increased CHRNA3 expression.584 Carriers of CHRNA3 variants extract a greater amount of nicotine from tobacco smoke and are exposed to a higher internal dose of 4-(methylnitrosamino)-1-(3-pyridyl)1-butanone per cigarette than non-carriers.585

Genome-wide linkage studies and familial lung cancer Genome-wide linkage studies revealed two loci at 6q23 and and 15q24–25. The locus at chromosome 6q23 was disclosed in a study of 52 extended pedigrees affected by lung cancer.586 The genomic area on chromosome 6 extends from C6S1848 at 146 centimorgan to 164 centimorgan near D6S1035. This region is frequently deleted in sporadic lung cancer and in many other cancer types (breast,587–590 ovary,587,591 mesothelioma,592 pancreas,593,594 oral SqCC,595 melanoma596 and Hodgkin lymphoma).597 This area probably contains at least one tumor suppressor gene. Recently, fine mapping of chromosome 6q23 revealed “regulator of G-protein 17” (RGS17) as a likely candidate gene.598 The RGS17 protein inhibits signal transduction by increasing the GTPase activity of G-protein alpha subunits. This drives them into their inactive GDP-bound form and negatively regulates µ-opioid receptor-mediated activation of the G-proteins. Overexpressed RGS17, via induction of cAMP signaling and CREB phosphorylation, plays a role in the maintenance of tumor cell proliferation. For the locus on chromosome 15q24–25, the presence of two high-risk alleles and a family history of lung cancer resulted in an OR of 7.567 This recent progress with genome-wide analysis and the subsequent detection of RGS17 as a likely candidate gene for hereditary lung cancer is promising. However, this field has large hurdles to overcome since documented relative risks are very low, implying a negligible prognostic relevance.599 The distribution of the probes on the array is approximately 80% of the whole genome. The question remains whether the haplotype map of one population can be extrapolated to another population.

Lung cancer screening Lung cancer screening studies are undertaken based on the premise that screening a population with a high risk of lung cancer may reduce the lung cancer-associated death rate.

The possible errors, also called biases, are well recognized in epidemiological science.600 In the late 1970s and early 1980s, chest X-ray (CXR) and sputum cytology were studied. More carcinomas were detected with CXR than with cytology. The complementary role of cytology was minimal, as only a few, mainly centrally located SqCC were detected.601 The ultimate conclusion of these studies was that CXR screening of high-risk individuals did not lower the lung cancer-associated death rate.602–610 High-resolution low-dose computer tomography (LDCT) has a higher analytical sensitivity than a CXR and for the past 15 years a variety of single arm (observational) and randomized controlled trials (RCT) have revisited the issue. Japanese researchers were the frontrunners of HRCT screening, followed by the International Early Lung Cancer Action Project (I-ELCAP) collaboration with a single-arm, lung cancer screening study. The results of the ELCAP study suggested lung cancer screening was very effective, conferring a probability of increased survival. An overview of the singlearm studies is shown in Table 5. Whether lung cancer screening with LDCT will reduce mortality truly depends on the results of ongoing RCTs. Recently, the American “National Lung Cancer Screening Trial” (NLST) reported a 20% mortality reduction.611 This seems a promising outcome for patients with lung cancer. In this study the control arm consisted of chest X-ray. As pathology review has not been undertaken and in situ adenocarcinomas (formerly bronchioloalveolar carcinomas) are included in the analysis, the mortality reduction may be on the optimistic side. In Europe the RCT design of some studies is different: the control arm has no intervention except for smoking cessation efforts. Initial results from the other RCT undertaken in Denmark and Holland/Belgium are not expected before 2016.612,613 Results may be reported sooner for smaller studies undertaken in Italy.614,615 Study design information from current RCT lung cancer screening trials with low-dose computed tomography (LDCT) is shown in Table 6 and baseline screening results in Table 7. The first screening round (baseline) detects the prevalence of lung cancer, while the following round detects incident cases. More than half of screened patients had nodules. However, most of these were small, with round edges, calcified (i.e. intensity similar to the rib) and considered benign or too small to further qualify (< 5 mm, 41%).613 The non-calcified nodules ranged from 7% to 47% in incidence. Only a small fraction of the screen-positive cases (range 0.36–2.7%) were diagnosed as lung cancer.616 The chance of detection of lung cancer is size-dependent: for nodules < 10 mm the figure is 5%, for 10 to 20 mm 21%, and > 20 mm 35%.617 The prevalence rate of lung cancer in RCT studies varies from 0.8% to 2.4% (Table 7). After a few years of screening, a reduction in the number of advanced lung cancer cases (stages III and IV) may be expected. However, this has not yet occurred.618,619 To establish a possible reduction in mortality, at least 6–8 years of follow-up is required.

965

Table 5 An overview of the single-arm CT lung cancer screening studies

Study /reference

Number of patients

High-risk group

Age

Gender male (%)

Selection

Incidence screen rounds

True positive rate baseline

Cancers baseline

Cancers incidence (% rate/ year)

Sone et al.621,662

5483

Smokers ~ 50%, pky > 1

40–74

54%

þSputum

N/A

396 (7.2%)a

19(0.4%) (incl 1 sputum only)

(0.4%)

ELCAP616

1000

Smokers, pky > 10, asbestos 14%

> 59

54%

Antibiotics; < 63 m FU 6–10 mm individual; >10 Bx/ Cx

Annually

233 (23%)

27 (2.7%)

N/A

Swensen et al.663,664

1520

Smokers, pky > 20

> 50

52%

Sputum

4 annual

713 (47%)

31 (2%) (incl. 1 sputum only)

32 (0.5%) (incl. 1 sputum only)

Sobue et al.665

1611

Smokers ~ 85%, pky > 50%

40–79

88%

X-ray, 3 day pooled sputum

11 annual

186 (11.5%)

14 (0.9%) (incl. 1 sputum only)

22 (0.3%) (incl. 3 sputum only)

Tiitola et al.666

602

Smokers > 95%, pky > 10, asbestos 100%

38–81

98%

Asbestos-related occupational disease, FNA

N/A

111 (18%)

5 (0.83%)

N/A

Nawa et al.667

7956

Smokers > 60%, pky ¼ 50%

50–69

79%

Hitachi

N/A

2099 (26%)

40 (0.44%) b

4 (0.07%)

Diederich et al.668,669

817

Smokers 100%, pky > 20

>40

72%

Media

2 annual

350 (43%)

11 (1.3%)

10 (0.6%)

MacRedmond et al.670,671

449

Smokers 100%, pky > 10

50–74

50%

Media, FNA for NCN

2 annual

93 (21%)

2 (0.46%)

6 (1.3%)

6406

Smokers 100%, pky > 20

> 45

86%

N/A

N/A

2255 (35%)

23 (0.36%)

N/A

30449

Smokers, pky 15–40

> 40

NS

FNA

N/A

4197 (14%)

404 (1.3%)

74 (0.27%)

Chong et al.672 673,674

Henschke et al.

b

FNA, fine-needle aspirate; NCN, non-calcified nodule; Bx, biopsy; Cx, cytology; 3 m FU, 3 months follow up HR-CT, no growth was considered benign; pky, pack years; N/A, not available; ELCAP, Early Lung Cancer Action Program; international ELCAP, includes ELCAP data; Hitachi, Hitachi employee health insurance group; NS, gender not specified.673,674 a Excluding 1047 abnormalities of little clinical importance (19%). b One case synchronous double primary cancer.

Table 6 Randomized lung cancer screening trials with low-dose computed tomography (LDCT)

Country/name

Patients in LDCT arm (n)

Patients in control arm (n)

Study design

High-risk group

Age

Gender Male (%)

Selection

Incidence screen rounds

Begin / end intervention / end follow-up

Netherlands, Belgium/NELSON675

7557

7557

LDCT vs. no intervention

(ex-)Smokers > 30 pky

50–75

N/A

PB

Yr 2, yr 4 and yr 5.5

2003/2009/2015

Denmark613

2052

2052

LDCT vs. no intervention

(ex-)Smokers > 30 pky

50–70

55%

JA

Yr 2, 3, 4, 5

2004/2010/2016

Italy/ITALUNG615,676

1593

1613

LDCT vs. no intervention

(ex-)Smokers > 30 pky

55–69

65%

HC

Yr 2, 3, 4

2004/2015 / N/A

Italy/DANTE677

1276

1198

LDCT vs. chest X-ray

(ex-)Smokers > 20 pky

60–74

100%

JA

Yr 2, 3, 4

Germany678/LUSI

1254

1251

(ex-)Smokers > 30 pky

50–69

N/A

PB

Yr 2, 3, 4, 5

France (pilot)/ Depiscan679

375

390

LDCT vs. chest X-ray

>30 pky Smokers 64%, former smokers 36%

50–75

71%

HC

N/A

US NLST611/LSS617

26722

26732

LDCT vs. chest X-ray

Heavy smokers and ex-smokers 15 mm or growth þ Bx/VATS

>500 mm3 or growtha in follow-up CT

Malignant tumors screen arm (prevalence %, % pos screening)

30 (1.9%, 9.%)

8 (2.4%)

28 (2.2%)

21(1.5%)

17 (0.8%, 9.4%)

70 (0.9%, 4.4% or 36%)1b

Cancer detection rate control arm

0.45%

N/A

0.7%

N/A

N/A

N/A

Invasive procedures benign lesion (n) (% screened, % positive screen)

N/A

N/A

6 (0.47%, 3%)

N/A

8 (0.4%, 4.4%)

N/A

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Chapter 24: Epidemiological and clinical aspects of lung cancer

746. Anderson NE, Rosenblum MK, Graus F, Wiley RG, Posner JB. Autoantibodies in paraneoplastic syndromes associated with small-cell lung cancer. Neurology 1988;38:1391–8.

758. Stavrianeas NG, Katoulis AC, Neofotistou O, Stratigeas NP, Neamonitos C. Tripe palms preceding squamous cell carcinoma of the lung by 11 months. Dermatology 1999; 198:173–4.

747. Grunwald GB, Klein R, Simmonds MA, Kornguth SE. Autoimmune basis for visual paraneoplastic syndrome in patients with small-cell lung carcinoma. Lancet 1985;1:658–61.

759. Thiers BH, Sahn RE, Callen JP. Cutaneous manifestations of internal malignancy. CA Cancer J Clin 2009;59:73–98.

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761. Wakata N, Kurihara T, Saito E, Kinoshita M. Polymyositis and dermatomyositis associated with malignancy: a 30-year retrospective study. Int J Dermatol 2002;41:729–34.

750. Payne M, Bradbury P, Lang B, et al. Prospective study into the incidence of Lambert Eaton myasthenic syndrome in small cell lung cancer. J Thorac Oncol 2010;5:34–8. 751. Cheran SK, Herndon JE 2nd, Patz EF Jr. Comparison of whole-body FDG-PET to bone scan for detection of bone metastases in patients with a new diagnosis of lung cancer. Lung Cancer 2004;44:317–25. 752. Gavrilovic IT, Posner JB. Brain metastases: epidemiology and pathophysiology. J Neurooncol 2005;75:5–14. 753. Oliver TW Jr, Bernardino ME, Miller JI, et al. Isolated adrenal masses in nonsmall-cell bronchogenic carcinoma. Radiology 1984;153:217–8. 754. Lorusso L, Hart IK, Ferrari D, et al. Autonomic paraneoplastic neurological syndromes. Autoimmun Rev 2007;6:162–8. 755. De Giorgio R, Sarnelli G, Corinaldesi R, Stanghellini V. Advances in our understanding of the pathology of chronic intestinal pseudo-obstruction. Gut 2004;53:1549–52. 756. van Meerbeeck JP, Fennell DA, De Ruysscher DK. Small-cell lung cancer. Lancet 2011;378:1741–55. 757. Cohen PR. Hypertrophic pulmonary osteoarthropathy and tripe palms in a man with squamous cell carcinoma of the larynx and lung. Report of a case and review of cutaneous paraneoplastic syndromes associated with laryngeal and lung malignancies. Am J Clin Oncol 1993;16:268–76.

762. Sparsa A, Liozon E, Herrmann F, et al. Routine vs extensive malignancy search for adult dermatomyositis and polymyositis: a study of 40 patients. Arch Dermatol 2002;138:885–90. 763. Andras C, Ponyi A, Constantin T, et al. Dermatomyositis and polymyositis associated with malignancy: a 21-year retrospective study. J Rheumatol 2008;35:438–44. 764. Kissel JT, Halterman RK, Rammohan KW, Mendell JR. The relationship of complement-mediated microvasculopathy to the histologic features and clinical duration of disease in dermatomyositis. Arch Neurol 1991;48:26–30. 765. Ascensao JL, Oken MM, Ewing SL, Goldberg RJ, Kaplan ME. Leukocytosis and large cell lung cancer. A frequent association. Cancer 1987;60:903–5. 766. Gastl G, Plante M, Finstad CL, et al. High IL-6 levels in ascitic fluid correlate with reactive thrombocytosis in patients with epithelial ovarian cancer. Br J Haematol 1993;83:433–41. 767. Estrov Z, Talpaz M, Mavligit G, et al. Elevated plasma thrombopoietic activity in patients with metastatic cancer-related thrombocytosis. Am J Med 1995;98:551–8. 768. Monreal M, Prandoni P. Venous thromboembolism as first manifestation of cancer. Semin Thromb Hemost 1999;25:131–6. 769. Henschke CI, Yankelevitz DF, Libby DM, et al. Survival of patients with stage I lung cancer detected on CT

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operable non-small cell lung cancer. J Thorac Cardiovasc Surg 2003;126:1943–51. 780. Maziak DE, Darling GE, Inculet RI, et al. Positron emission tomography in staging early lung cancer: a randomized trial. Ann Intern Med 2009;151:221–8, W-48. 781. Keidar Z, Haim N, Guralnik L, et al. PET/CT using 18F-FDG in suspected lung cancer recurrence: diagnostic value and impact on patient management. J Nucl Med 2004;45:1640–6.

792. Thiberville L, Salaun M, Lachkar S, et al. Human in vivo fluorescence microimaging of the alveolar ducts and sacs during bronchoscopy. Eur Respir J 2009;33:974–85. 793. Lam S, Kennedy T, Unger M, et al. Localization of bronchial intraepithelial neoplastic lesions by fluorescence bronchoscopy. Chest 1998;113:696–702.

782. Ohno Y, Koyama H, Nogami M, et al. Whole-body MR imaging vs. FDGPET: comparison of accuracy of Mstage diagnosis for lung cancer patients. J Magn Reson Imaging 2007; 26:498–509.

794. Edell E, Lam S, Pass H, et al. Detection and localization of intraepithelial neoplasia and invasive carcinoma using fluorescence-reflectance bronchoscopy: an international, multicenter clinical trial. J Thorac Oncol 2009;4:49–54.

783. Schreiber G, McCrory DC. Performance characteristics of different modalities for diagnosis of suspected lung cancer: summary of published evidence. Chest 2003;123:115S–28S.

795. Hirsch FR, Prindiville SA, Miller YE, et al. Fluorescence versus white-light bronchoscopy for detection of preneoplastic lesions: a randomized study. J Natl Cancer Inst 2001; 93:1385–91.

784. Rivera MP, Detterbeck F, Mehta AC. Diagnosis of lung cancer: the guidelines. Chest 2003;123:129S–36S. 785. Hermens FH, Van Engelenburg TC, Visser FJ, et al. Diagnostic yield of transbronchial histology needle aspiration in patients with mediastinal lymph node enlargement. Respiration 2003;70:631–5. 786. Yung RC. Tissue diagnosis of suspected lung cancer: selecting between bronchoscopy, transthoracic needle aspiration, and resectional biopsy. Respir Care Clin N Am 2003;9:51–76. 787. Yasufuku K, Chiyo M, Koh E, et al. Endobronchial ultrasound guided transbronchial needle aspiration for staging of lung cancer. Lung Cancer 2005;50(3):347–54. 788. Detterbeck FC, DeCamp MM Jr, Kohman LJ, Silvestri GA. Lung cancer. Invasive staging: the guidelines. Chest 2003;123:167S–75S. 789. Ouellette DR. The safety of bronchoscopy in a pulmonary fellowship program. Chest 2006;130:1185–90. 790. Hwangbo B, Kim SK, Lee HS, et al. Application of endobronchial ultrasound-guided transbronchial needle aspiration following integrated PET/CT in mediastinal staging of potentially operable non-small cell lung cancer. Chest 2009;135:1280–7.

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791. Wallace MB, Pascual JM, Raimondo M, et al. Minimally invasive endoscopic staging of suspected lung cancer. JAMA 2008;299:540–6.

796. Lee P, van den Berg RM, Lam S, et al. Color fluorescence ratio for detection of bronchial dysplasia and carcinoma in situ. Clin Cancer Res 2009;15:4700–5. 797. Detterbeck F, Puchalski J, Rubinowitz A, Cheng D. Classification of the thoroughness of mediastinal staging of lung cancer. Chest 2010;137:436–42. 798. de Cabanyes Candela S, Detterbeck FC. A systematic review of restaging after induction therapy for stage IIIa lung cancer: prediction of pathologic stage. J Thorac Oncol 2010;5:389–98. 799. Maskell NA, Gleeson FV, Davies RJ. Standard pleural biopsy versus CTguided cutting-needle biopsy for diagnosis of malignant disease in pleural effusions: a randomised controlled trial. Lancet 2003; 361:1326–30. 800. Nance KV, Shermer RW, Askin FB. Diagnostic efficacy of pleural biopsy as compared with that of pleural fluid examination. Mod Pathol 1991;4:320–4. 801. Ong KC, Indumathi V, Poh WT, Ong YY. The diagnostic yield of pleural fluid cytology in malignant pleural effusions. Singapore Med J 2000; 41:19–23. 802. Prakash UB, Reiman HM. Comparison of needle biopsy with cytologic analysis

for the evaluation of pleural effusion: analysis of 414 cases. Mayo Clin Proc 1985;60:158–64. 803. Delgado PI, Jorda M, Ganjei-Azar P. Small cell carcinoma versus other lung malignancies: diagnosis by fine-needle aspiration cytology. Cancer 2000;90:279–85. 804. Jones AM, Hanson IM, Armstrong GR, O’Driscoll BR. Value and accuracy of cytology in addition to histology in the diagnosis of lung cancer at flexible bronchoscopy. Respir Med 2001;95:374–8. 805. Marchevsky AM, Changsri C, Gupta I, et al. Frozen section diagnoses of small pulmonary nodules: accuracy and clinical implications. Ann Thorac Surg 2004;78:1755–9. 806. Novis DA, Zarbo RJ. Interinstitutional comparison of frozen section turnaround time. A College of American Pathologists Q-Probes study of 32868 frozen sections in 700 hospitals. Arch Pathol Lab Med 1997;121:559–67. 807. Gephardt GN, Zarbo RJ. Interinstitutional comparison of frozen section consultations. A college of American Pathologists Q-Probes study of 90,538 cases in 461 institutions. Arch Pathol Lab Med 1996;120:804–9. 808. Gupta R, Dastane A, McKenna RJ Jr, Marchevsky AM. What can we learn from the errors in the frozen section diagnosis of pulmonary carcinoid tumors? An evidence-based approach. Hum Pathol 2009;40:1–9. 809. Soga J, Yakuwa Y. Bronchopulmonary carcinoids: An analysis of 1,875 reported cases with special reference to a comparison between typical carcinoids and atypical varieties. Ann Thorac Cardiovasc Surg 1999; 5:211–9. 810. Suda T, Mizoguchi Y, Hasegawa S, Negi K, Hattori Y. Frozen-section diagnosis of small adenocarcinoma of the lung for intentional limited surgery. Surg Today 2006;36:676–9. 811. Goldstraw P. Lung. In Edge S, Byrd D, Compton C, eds. AJCC Cancer Staging Manual, 7th ed. New York: SpringerVerlag, 2010. pp. 252–70. 812. Goldstraw P, Crowley J, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the

Chapter 24: Epidemiological and clinical aspects of lung cancer

TNM Classification of malignant tumours. J Thorac Oncol 2007;2:706–14. 813. Kato Y, Ferguson TB, Bennett DE, Burford TH. Oat cell carcinoma of the lung. A review of 138 cases. Cancer 1969;23:517–24. 814. Nicholson SA, Beasley MB, Brambilla E, et al. Small cell lung carcinoma (SCLC): a clinicopathologic study of 100 cases with surgical specimens. Am J Surg Pathol 2002;26:1184–97. 815. Lally BE, Urbanic JJ, Blackstock AW, Miller AA, Perry MC. Small cell lung

cancer: have we made any progress over the last 25 years? The Oncologist 2007;12:1096–104. 816. Turrisi AT 3rd, Kim K, Blum R, et al. Twice-daily compared with once-daily thoracic radiotherapy in limited smallcell lung cancer treated concurrently with cisplatin and etoposide. N Engl J Med 1999;340:265–71. 817. Sundstrom S, Bremnes RM, Kaasa S, Aasebo U, Aamdal S. Second-line chemotherapy in recurrent small cell lung cancer. Results from a crossover schedule after primary

treatment with cisplatin and etoposide (EP-regimen) or cyclophosphamide, epirubicin, and vincristin (CEVregimen). Lung Cancer 2005; 48:251–61. 818. Colby TV, Koss MN, Travis WD. Carcinoma of the lung: clinical and radiographic aspects, spread, staging, management, and prognosis. In Colby TV, Koss MN, Travis WD, eds. Tumors of the Lower Respiratory Tract, 3rd ed. Washington DC: Armed Forces Institute of Pathoogy, 1995. pp. 107–34.

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25

Lung cancer staging Eric Lim and Peter Goldstraw

Introduction Cancer staging refers to the classification of patients based on the anatomic extent of disease. The Tumor Node Metastasis (TNM) classification evolved from the work of Pierre Denoix in the 1940s and has since been adopted as the reference standard by the International Union Against Cancer (Union Internationale Contre le Cancer – UICC) and the American Joint Committee on Cancer (AJCC). These two bodies collaborate to ensure a uniform TNM classification. The most recent classification, the seventh edition, was published in 2009.1 This revision contains the first changes since the 1997 fifth edition. The changes were based upon proposals submitted by the International Association for the Study of Lung Cancer (IASLC)*.2 The seventh edition will be used throughout this chapter, with reference to the sixth edition where necessary to highlight important changes. The TNM Classification fulfills a number of related objectives, namely: 1. To aid the clinician in the planning of treatment. 2. To give some indication of prognosis. 3. To assist in evaluation of the results of treatment. 4. To facilitate the exchange of information between treatment centers. 5. To contribute to the continuing investigation of human cancer.

*

When generating the dataset for the seventh edition of the TNM classification, the difficulties with retrograde compatibility were recognized, and staging information was collected by category. Therefore for the future, emphasis is placed on collection of the primary measurements and anatomical descriptions (e.g. 2.5 cm in addition to T1b) which require more detail than that currently specified by the 2007 proforma recommended by the Royal College of Pathologists.23 In the United States the College of American Pathologists maintains updated cancer protocols, which can be accessed at www.cap.org.

The TNM classification The TNM classification requires the primary site be defined and the diagnosis confirmed microscopically before stage is assigned. Such requires that primary lung tumors must be differentiated from metastatic lesions (see Chapter 26). The system is based on three principal components that describe the anatomic extent of disease. The T category describes the extent of the primary tumor, the N category describes the absence/ presence and extent of regional lymph node metastasis, and the M category describes the absence/presence of distant metastases. The T, N and M categories are further classified by numerals, which indicate progressively advanced disease. The resultant TNM categories are amalgamated into stage groupings for convenience and practicality (Table 1){. Staging information may be acquired by clinical examination, imaging (e.g. chest-film, computed tomography (CT), positron emission tomography (PET), magnetic resonance imaging (MRI)), endoscopy (e.g. bronchoscopy, mediastinoscopy), mediastinotomy, video-assisted thoracoscopic examination, esophageal ultrasound with or without ultrasound-guided biopsies (EUS) and endobronchial ultrasound-guided biopsy (EBUS), other forms of biopsy (e.g. needle biopsies, surgical biopsies) and surgery (e.g. resection of the primary tumor). Two classifications of the TNM system are described for each organ site – clinical (cTNM) and pathological (pTNM). However, the difference between these two is not entirely based upon whether or not tissue confirmation has been obtained but upon the time at which a decision has been made about treatment. Stage, without further clarification, refers to clinical or cTNM and incorporates all the information, including biopsies where appropriate, needed to make a decision regarding treatment. Once this classification has been determined it should {

The information provided in this chapter serves as an overview and to highlight some important changes; however, readers are strongly advised to refer to the complete staging handbook and manual for more detailed information.21 If any further doubts remain, clarification on questions regarding TNM staging can be obtained at the UICC “TNM helpdesk” located at www.uicc.org.

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 25: Lung cancer staging Table 1 Seventh edition stage grouping

Sixth edition T/M descriptor

Seventh N0 edition T/M

N1

N2

N3

T1 (
2 cm.)

T1a

IA

IIA

IIIA

IIIB

T1 (> 2–3 cm)

T1b

IA

IIA

IIIA

IIIB

T2 (> 3–5 cm)

T2a

IB

IIA

IIIA

IIIB

T2 (> 5–7 cm)

T2b

IIA

IIB

IIIA

IIIB

T2 (> 7 cm)

T3

IIB

IIIA

IIIA

IIIB

T3 invasion

IIB

IIIA

IIIA

IIIB

T4 (same lobe nodules)

IIB

IIIA

IIIA

IIIB

IIIA

IIIA

IIIB

IIIB

IIIA

IIIA

IIIB

IIIB

IV

IV

IV

IV

IV

IV

IV

IV

IV

IV

IV

IV

T4 (extension)

T4

M1 (ipsilateral lung) T4 (pleural effusion)

M1a

M1 (contralateral lung) M1 (distant)

M1b

Stages in bold indicate a change from the sixth edition for a particular TNM category.

be entered into the case file and remains unchanged. If treatment includes surgical resection, then the additional pathological information is the basis for the pathological stage, pTNM. The pathological assessment of the primary tumor (pT) entails resection of the primary tumor or a biopsy adequate to evaluate the highest pT category. Removal of nodes adequate to validate the absence of regional lymph node metastasis is required for pN0. The pathological assessment of distant metastasis (pM) entails microscopic examination. A pathological stage may be assigned if the anatomic extent of disease has been proven, whether or not the primary lesion has been completely removed. If a biopsied primary tumor technically cannot be removed, or when it is unreasonable to remove it, the criteria for pathological classification and staging are satisfied without total removal of the primary cancer if (a) biopsy has confirmed a pT category and there is microscopic confirmation of nodal disease at any level (pN1–3), (b) there is microscopic confirmation of the highest N category (pN3) or (c) there is microscopic confirmation of pM1. When information is obtained for staging after induction treatment (in lung cancer, this usually implies initial treatment with chemotherapy or radiotherapy prior to surgery) the prefix y is used (e.g. ycTNM, ypTNM). For the staging of recurrent tumour after a disease free interval, the prefix “r” is used (e.g. rTNM).

T classification The T descriptors are assigned based on tumor size, anatomic relation/invasion or the state of the lung distal to the primary tumor. In the seventh edition, new T size cut points (at 2 cm, 5 cm and 7 cm) were introduced, in addition to the established

cut point of 3 cm that traditionally separated T1 and T2 tumors. T1 tumors are now sub-divided into T1a (
2 cm) and T1b (> 2 cm but
3 cm), T2 tumors have been subdivided into T2a (> 3cm but
5 cm) and T2b (> 5 cm but
7 cm), and tumors > 7 cm are now classified as T3.3 In addition, in the seventh edition, the classification of additional tumor nodules in certain locations has changed (see below). It is unclear whether clinical tumor size is determined with mediastinal or lung windows on CT scan. The IASLC manual simply recommends settings as chosen by the radiologist. This recommendation is problematic for semisolid and groundglass opacities, since prognosis may be related to the size of the solid component only.4 Although lung carcinomas are often measured and sectioned after formalin fixation, it is important that the pathologist measure the tumor in the unfixed state. A recent study noted that 20% of tumors > 3.0 cm shrank by an average of more than 1.0 cm following formalin fixation. This may result in tumor downstaging from T2 to T1. In addition, formalin fixation-induced tumor shrinkage caused stage shift from stage Ib to stage Ia in 10% of the study cohort.5 Not surprisingly, patients with “downstaged” tumors had 5-year survival rates expected for their prefixation tumor size. The T descriptors are listed in Table 2 and illustrated in Figures 1–4. In the seventh edition, for the first time, an agreed definition of visceral pleural invasion has been given, i.e. invasion through the elastic lamina or onto the surface of the visceral pleura (Figure 5). A more detailed classification, a PL category, has been proposed.6 The definitions for the degree of visceral pleural invasion are: PL0 if the tumor is either within the subpleural lung parenchyma or invading superficially into the pleural connective tissue beneath the elastic layer, PL1 if a tumor invades beyond the elastic layer, PL2 if the tumor invades onto the pleural surface and PL3 if a tumor invades into any component of the parietal pleura. In the seventh edition, PL0 is not regarded as a T descriptor and the T category should be assigned on other features. PL1 or PL2 indicates “visceral pleural invasion”, i.e. a T2 descriptor. PL3 indicates invasion of the parietal pleura, i.e. T3. Histological sampling of the pleura in the lung cancer resection specimen should be obtained in the area where there is the greatest macroscopic concern for invasion on gross examination. One can turn down the microscope condenser to visualize the elastic fibers. However, in a substantial percentage of cases the elastic layer is imperceptible on hematoxylin and eosin-stained tissue sections. The use of elastic stains is recommended if the presence or absence of visceral pleural invasion or the PL category is not clear on routine sections.7 Carcinoma must invade the thick elastic layer. This layer may be near the lung parenchyma, near the pleural surface or in the middle. In some cases there is a connective tissue layer between the lung parenchyma and the thick elastic layer. Invasion of this connective tissue is regarded as PL0.7 In some

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Chapter 25: Lung cancer staging

cases elastic stains are difficult to interpret due to pleural inflammation and/or fibrosis. These processes result in alteration of the elastic layers, often with reduplication. If one is not certain about visceral pleural invasion, then one should follow the general TNM rules and assign the lower category T.7 The seventh edition also dramatically revised the TNM approach to multiple tumor nodules. First, the confusing term

“satellite nodules” has been replaced with the more reasonable descriptor “additional tumor nodules”. Yet the pathologist must be aware that these lesions must be recognized grossly and that incidental microscopic additional tumor nodules do not impact the TNM designation. Grossly identified “additional tumor nodules” in the major tumor-bearing lobe require a T3 assignment. Separate tumor nodule(s) in a different ipsilateral

Table 2 T descriptors for lung cancer

a b c

TX

Primary tumor cannot be assessed, or tumor proven by the presence of malignant cells in sputum or bronchial washings but not visualized by imaging or bronchoscopy

T0

No evidence of primary tumor

Tis

Carcinoma in situ

T1

Tumor 3 cm or less in greatest dimension, surrounded by lung or visceral pleura, without bronchoscopic evidence of invasion more proximal than the lobar bronchus (i.e., not in the main bronchus) T1a Tumor 2 cm or less in greatest dimensiona T1b Tumor more than 2 cm but not more than 3 cm in greatest dimension

T2

Tumor more than 3 cm but not more than 7 cm; or tumor with any of the following features:b  involves main bronchus, 2 cm or more distal to the carina  invades visceral pleura  associated with atelectasis or obstructive pneumonitis that extends to the hilar region but does not involve the entire lung T2a Tumor more than 3 cm but not more than 5 cm in greatest dimension T2b Tumor more than 5 cm but not more than 7 cm in greatest dimension

T3

Tumor more than 7 cm or one that directly invades any of the following: chest wall (including superior sulcus tumors), diaphragm, phrenic nerve, mediastinal pleura, parietal pericardium; or tumor in the main bronchus less than 2 cm distal to the carinaa but without involvement of the carina; or associated atelectasis or obstructive pneumonitis of the entire lung or separate tumor nodule(s) in the same lobe as the primary

T4

Tumor of any size that invades any of the following: mediastinum, heart, great vessels,c trachea, recurrent laryngeal nerve, esophagus, vertebral body, carina; separate tumor nodule(s) in a different ipsilateral lobe to that of the primary

The uncommon superficial spreading tumor of any size with its invasive component limited to the bronchial wall, which may extend proximal to the main bronchus, is also classified as T1a. T2 tumors with these features are classified T2a if 5 cm or less or if size cannot be determined, and T2b if greater than 5 cm but not larger than 7 cm. Great vessels are defined as aorta, superior vena cava, inferior vena cava, main pulmonary artery, the intrapericardial segments of the trunks of pulmonary arteries or veins. Figures 1–4 T descriptors for lung carcinomas according to the 7th edition AJCC/UICC staging system. (Images courtesy of A. Frazier, MD, Baltimore, Maryland, USA. Reprinted with permission courtesy of the International Association for the Study of Lung Cancer. Copyright © 2008 Aletta Ann Frazier, MD.)

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Chapter 25: Lung cancer staging

Figures 1–4 (cont.)

lobe are given a T4 assignment. Separate tumor nodule(s) in a contralateral lobe are staged M1a. Discerning intrapulmonary metastases from synchronous primary carcinomas may be impossible (see below) (see Chapter 27). Furthermore, data supporting these designations are lacking. Compliance with the designations will generate a useful database.

N classification The N descriptors are based on the absence/presence and extent of metastasis in regional lymph nodes. Direct invasion into regional lymph nodes is considered a metastasis. In lung cancer, the anatomic locations of the lymph nodes are divided into distinct stations, as defined by the IASLC and illustrated

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Chapter 25: Lung cancer staging Figures 1–4 (cont.)

Table 3 N descriptors for lung cancer

Figure 5. Visceral pleural invasion. PL0 is not regarded as a T descriptor. PL1 and PL2 indicate visceral pleural invasion, i.e. T2a. PL3 indicates parietal pleural invasion, i.e. T3. (Image courtesy of A. Frazier, MD, Baltimore, Maryland, USA. Reprinted with permission courtesy of the International Association for the Study of Lung Cancer. Copyright © 2008 Aletta Ann Frazier, MD.)

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NX

Regional lymph nodes cannot be assessed

N0

No regional lymph node metastasis

N1

Metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodes, including involvement by direct extension

N2

Metastasis in ipsilateral mediastinal and/or subcarinal lymph node(s)

N3

Metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s)

in the new IASLC nodal map (Figure 6).8 This station assignment reconciles differences between the Japanese map9 and that of Mountain/Dresler.10 In addition, the concept of “nodal zones” has been proposed with an aim of making the classification relevant to radiologists and oncologists dealing with larger nodal masses that transgress nodal stations. The N descriptors are listed in Table 3 and illustrated in Figures 7–9. It is essential for physicians, radiologists, surgeons and pathologists to classify the lymph nodes in a standardized manner as management, prognosis and treatment hinge on the correct

Chapter 25: Lung cancer staging

Figure 6. International Association for the Study of Lung Cancer Nodal Chart with stations and zones. (Image courtesy of A. Frazier, MD, Baltimore, Maryland, USA. Reprinted with permission courtesy of the International Association for the Study of Lung Cancer. Copyright © 2008 Aletta Ann Frazier, MD.)

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Chapter 25: Lung cancer staging

Figures 7–9. N descriptors for lung carcinomas according to the 7th edition AJCC/UICC staging system. (Images courtesy of A. Frazier, MD, Baltimore, Maryland, USA. Reprinted with permission courtesy of the International Association for the Study of Lung Cancer. Copyright © 2008 Aletta Ann Frazier, MD.)

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Chapter 25: Lung cancer staging Table 4 M Descriptors for lung cancer

a

M0

No distant metastasis

M1

Distant metastasis 1a Separate tumor nodule(s) in a contralateral lobe; tumor with pleural nodules or malignant pleural or pericardial effusiona 1b Distant metastasis

Most pleural (pericardial) effusions with lung cancer are due to tumor. In a few patients, however, multiple microscopical examinations of pleural (pericardial) fluid are negative for tumor, and the fluid is non-bloody and is not an exudate. Where these elements and clinical judgment dictate that the effusion is not related to the tumor, the effusion should be excluded as a staging element and the patient should be classified as M0.

classification of the extent of lymph node metastases. A detailed description of the individual stations is not within the scope of this chapter, but can be obtained from the primary reference.8 The extent of nodal evaluation at thoracotomy can influence survival.11,12 Adequate N staging is generally considered to include sampling or dissection of lymph nodes from stations 2R, 4R, 7, 10R and 11R for right-sided tumors, and stations 5, 6, 7, 10L and 11L for left-sided neoplasms. Station 9 lymph nodes should also be evaluated for lower lobe tumors. The more peripheral lymph nodes at stations 12–14 are usually evaluated by the pathologist in lobectomy or pneumonectomy specimens but may be separately removed when sublobar resections (e.g. segmentectomy) are performed. The UICC recommends that at least six lymph nodes/ stations should be removed/sampled and confirmed on histology to be free of disease to confer pN0 status. Three of these nodes/stations should be mediastinal, including the sub-carinal nodes (station 7), and three from N1 nodes/stations. If all resected/sampled lymph nodes are negative, but the recommended number is not met, the nodal status should nevertheless be classified as pN0. If resection has been performed, and otherwise fulfills the requirements for complete resection, it should be classified as R0. Prognosis is influenced by the presence or absence of nodal disease, but also by the extent, pattern and bulk of any nodal disease. The IASLC found three groups with differing prognoses; patients who had N1 single zone disease, those who had either multiple N1 or single N2 zone metastases, and those who had multiple N2 lymph node zones involved.13 These findings underscore the importance of careful dissection and assessment of the lymph nodes in resected lung specimens. However, it is uncertain whether the anatomic location of lymph node metastases is more meaningful than the number of metastatic lymph nodes.14 The use of the nodal zone concept is recommended for evaluation in the seventh edition of TNM.

M classification In the seventh edition, important amendments have been made to the M descriptor (Table 4) (Figures 10 and 11). MX is no longer a valid designation, as the presence of distant metastases can be assessed on clinical examination. The presence of a

Figures 10 and 11. M descriptors for lung carcinomas according to the 7th edition AJCC/UICC staging system. (Images courtesy of A. Frazier, MD, Baltimore, Maryland, USA. Reprinted with permission courtesy of the International Association for the Study of Lung Cancer. Copyright © 2008 Aletta Ann Frazier, MD.)

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Chapter 25: Lung cancer staging

separate tumor nodule in the contralateral lung, tumor with pleural nodules or the presence of a metastatic pleural (or pericardial) effusion has been assigned M1a, differentiating these conditions from distant disease (M1b).15 It is important to appreciate that malignant cells within the pleural or pericardial effusion rather than the presence of an effusion per se constitutes the designation of M1a. In the presence of distant disease, it is important to document the number and sites of metastases.

Optional descriptors In the seventh edition of the TNM system, a number of optional descriptors are detailed. These include the cLy category for the classification of lymphangitis carcinomatosis, determined on radiological studies. Such a finding is usually a contraindication to surgical treatment. Unlike other tumor sites, in lung cancer the “V” classification refers not only to venous invasion but also to arteriolar invasion, a not uncommon feature in lung cancer. Vascular invasion (venous or arteriolar) is classified as VX, V0, V1 (microscopic invasion) and V2 (macroscopic invasion).

Specific situations Isolated tumor cells (ITC) are single or small clusters of tumor cells not more than 0.2 mm in greatest dimension that are detected by routine (H&E) stains or immunohistochemistry. ITCs do not typically induce a stromal reaction or penetrate vascular or lymphatic sinus walls. Cases with ITC in lymph nodes or at distant sites should be classified as N0 or M0, respectively. Immunohistochemical studies do not apply. The same applies to cases with findings suggestive of tumor cells or their components by non-morphological techniques, such as flow cytometry or DNA analysis. These cases should be analyzed separately. Direct invasion of a tumor into an adjacent lobe, across the fissure or by direct extension at a point where the fissure is deficient should be classified as T2a unless other criteria assign a higher T category. As mentioned above, in the seventh edition of the TNM system there has been a paradigm shift in the classification of separate tumor nodules, with emphasis placed on the opinion of the pathologist in making such distinctions. Determining whether multiple carcinomas represent synchronous primaries or intrapulmonary metastases is a difficult topic.16 Pathologists should record the nodule number, size and the relative distance between the lesions, and in particular the distance from the dominant lesion, as well as the relationship to the pleura and bronchial margin. When a pathologist encounters multiple lesions, it is useful to speak to the surgeon or radiologist to review CTs to correlate the pathology with the clinical and/or radiological findings. Because the resected lung tissue is collapsed, pathologists may have more difficulty than radiologists or surgeons identifying small tumors. This is particularly the case with adenocarcinomas with a predominant lepidic component.7 In most situations where additional tumor nodules are found in association with a lung primary, these are metastatic nodules, with identical histological appearance to that of the

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primary tumor. Multiple tumors are considered synchronous primaries if they are of different histological cell types. Multiple tumors of similar histological appearance should only be considered to be synchronous primary tumors if in the opinion of the pathologist, based on features such as differences in morphology, immunohistochemistry and/or molecular studies, or, in the case of squamous cancers, being associated with carcinoma in situ, they represent differing subtypes of the same histopathological cell type. Such cases should also have no evidence of mediastinal nodal metastases or of nodal metastases within a common nodal drainage. These circumstances are most commonly encountered when dealing with lepidic-pattern adenocarcinomas (see Chapter 27). Multiple synchronous primary tumors should be staged separately. The highest T category and stage of disease should be assigned and the multiplicity or the number of tumors should be indicated in parenthesis, e.g. T2 (m) or T2 (5).9 This distinction may require histopathological confirmation of cell type from more than one tumor nodule, where clinically appropriate. Careful palpation during surgery and by the pathologist of the resected specimen is therefore required to screen for the presence of additional tumor nodules. Only nodules discovered for the first time at pathological examination are classified as additional nodules. Small cell lung cancer (SCLC) has always been included within the TNM classification. However, oncologists have not found it useful, preferring to continue with the “limited” versus “extensive” classification. The IASLC, in accumulating data to inform revisions to the sixth edition, collected 13 000 cases of SCLC, the largest database ever in this disease. The analysis confirms that the TNM classification is of prognostic value, especially in limited disease (LD) SCLC.17 In the context of “limited disease,” survival for clinical stages I and II was significantly different from stage III with N2 or N3 involvement.17 Moreover, after surgical resection, survival was noted to correlate more strongly with the extent of lymph node disease rather than T stage.18 As a consequence there is greater emphasis on the use of TNM in SCLC in the seventh edition. In addition, analysis of 600 carcinoid tumor cases within the IASLC database confirmed the validity of the TNM classification for this tumor type with several caveats.19 Travis et al. showed the combined stage groupings (I versus II versus III/IV) had significant differences in survival, but the subcategories of the groupings (IA versus IB and IIA versus IIB) did not. However, many issues remain unresolved including the validity of this system for typical versus atypical carcinoid tumors, the validity of staging multicentric carcinoid tumors, the validity of size cut-offs in the determination of T value, and the prognostic significance of thoracic lymph node metastases.6,20 Additional staging definitions have been introduced with regard to the completeness of resection.21 RX is assigned when the presence of residual tumor cannot be assessed, R0 (complete resection) when resection margins are free of tumor on microscopy with a minimum of six nodes/nodal stations removed/sampled for histological examination. As stated

Chapter 25: Lung cancer staging

Figure 12. Survival rates for clinically staged lung cancer. Data modified from reference 2.

Figure 13. Survival rates for pathologically staged lung cancer. Data modified from reference 2.

above, these lymph node stations should include three regions from the mediastinum, one of which should be sub-carinal node 7, and three nodes from the hilum or other N1 locations. The designation R1 (microscopic incomplete resection) is assigned when there is microscopic evidence of residual disease at either the resection margins or lymph node margins feature extracapsular extension. When pleural lavage cytology (PLC) collected at thoracotomy is positive for malignant cells, the patient is also staged R1(cyþ).22 This finding has an adverse and independent prognostic impact following complete resection, but is not a standard staging procedure.22 R1(is) is assigned when the requirements for R0 have been met, but in situ carcinoma is found at the bronchial resection margin. The designation R2 (macroscopic incomplete resection) is assigned if macroscopic evidence of residual disease is identified at the resection margins, at the edge of lymph nodes with extracapsular extension or if either involved lymph nodes, pleural or pericardial nodules were not resected at surgery. Since concerns have been raised that the definition of complete resection is too imprecise, the seventh edition of TNM includes a new category,23 “R0 (un)” to study the cases of “uncertain resection”. Cases where there is no macroscopic or microscopic evidence of residual disease but nodal assessment has been

based on less than the number of nodes/stations recommended for complete resection and/or the highest mediastinal node removed/sampled is positive qualify for this designation.

References 1. Goldstraw P, for the IASLC International Staging Committee. Lung. In Edge SB, Byrd DR, Compton CC, et al, eds. AJCC Cancer Staging Manual, 7th edition. New York: Springer-Verlag, 2010. pp. 253–70. 2. Goldstraw P, Crowley J, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the TNM stage groupings in the forthcoming (seventh) edition of the TNM Classification of malignant tumours. J Thorac Oncol 2007; 2(8):706–14. 3. Rami-Porta R, Ball D, Crowley J, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of

Overall survival The prognostic information from TNM staging is used to create stage groupings (Table 1).2 Corresponding survival plots for the clinical and pathological classifications are illustrated in Figures 12 and 13. For clinically staged lung cancer patients, the 5-year survival rate for stage IA is 50%, IB 43%, IIA 36%, IIB 25%, IIIA 19%, IIIB 7% and IV 2%. For pathologically staged lung cancer patients the 5-year survival rate for stage IA is 73%, IB 58%, IIA 46%, IIB 36%, IIIA 24%, IIIB 9% and IV 13%.21

Conclusion The pathologist has a key role in pre-treatment clinical staging and especially post-surgical pathological staging of patients with lung cancer. Meticulous attention to the gross specimens, dissections and microscopic examinations within the context of the revised TNM descriptors is necessary to ensure complete and accurate pathological staging.

the T descriptors in the forthcoming (seventh) edition of the TNM classification for lung cancer. J Thorac Oncol 2007;2(7):593–602. 4. Detterbeck FC, Boffa DJ, Tanoue LT, Wilson LD. Details and difficulties regarding the new lung cancer staging system. Chest 2010; 137(5):1172–80. 5. Hsu PK, Huang HC, Hsieh CC, et al. Effect of formalin fixation on tumor size determination in stage I non-small cell lung cancer. Ann Thorac Surg 2007;84 (6):1825–9. 6. Travis WD, Brambilla E, Rami-Porta R, et al. Visceral pleural invasion: pathologic criteria and use of elastic stains: proposal for the 7th edition of the TNM classification for lung

cancer. J Thorac Oncol 2008;3(12): 1384–90. 7. Travis WD. Reporting lung cancer pathology specimens. Impact of the anticipated 7th Edition TNM classification based on recommendations of the IASLC Staging Committee. Histopathology 2009; 54(1):3–11. 8. Rusch VW, Asamura H, Watanabe H, et al. The IASLC lung cancer staging project: a proposal for a new international lymph node map in the forthcoming seventh edition of the TNM classification for lung cancer. J Thorac Oncol 2009;4(5):568–77. 9. Naruke T, Suemasu K, Ishikawa S. Lymph node mapping and curability at

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various levels of metastasis in resected lung cancer. J Thorac Cardiovasc Surg 1978;76(6):832–9. 10. Mountain CF, Dresler CM. Regional lymph node classification for lung cancer staging. Chest 1997;111(6): 1718–23. 11. Gajra A, Newman N, Gamble GP, Kohman LJ, Graziano SL. Effect of number of lymph nodes sampled on outcome in patients with stage I non-small-cell lung cancer. J Clin Oncol 2003;21(6):1029–34. 12. Wu YC, Lin CF, Hsu WH, et al. Long-term results of pathological stage I non-small cell lung cancer: validation of using the number of totally removed lymph nodes as a staging control. Eur J Cardiothorac Surg 2003; 24(6):994–1001. 13. Rusch VW, Crowley J, Giroux DJ, et al. The IASLC Lung Cancer Staging Project: proposals for the revision of the N descriptors in the forthcoming seventh edition of the TNM classification for lung cancer. J Thorac Oncol 2007;2(7):603–12. 14. Wei S, Asamura H, Kawachi R, Sakurai H, Watanabe S. Which is the better prognostic factor for resected non-small cell lung cancer: the number of metastatic lymph nodes or the currently

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used nodal stage classification? J Thorac Oncol 2011;6(2):310–8. 15. Postmus PE, Brambilla E, Chansky K, et al. The IASLC Lung Cancer Staging Project: proposals for revision of the M descriptors in the forthcoming (seventh) edition of the TNM classification of lung cancer. J Thorac Oncol 2007;2(8):686–93. 16. Martini N, Melamed MR. Multiple primary lung cancers. J Thorac Cardiovasc Surg 1975;70(4):606–12. 17. Shepherd FA, Crowley J, Van Houtte P, et al. The International Association for the Study of Lung Cancer lung cancer staging project: proposals regarding the clinical staging of small cell lung cancer in the forthcoming (seventh) edition of the tumor, node, metastasis classification for lung cancer. J Thorac Oncol 2007;2(12):1067–77. 18. Vallieres E, Shepherd FA, Crowley J, et al. The IASLC Lung Cancer Staging Project: proposals regarding the relevance of TNM in the pathologic staging of small cell lung cancer in the forthcoming (seventh) edition of the TNM classification for lung cancer. J Thorac Oncol 2009;4(9): 1049–59. 19. Travis WD, Giroux DJ, Chansky K, et al. The IASLC Lung Cancer Staging

Project: proposals for the inclusion of broncho-pulmonary carcinoid tumors in the forthcoming (seventh) edition of the TNM Classification for Lung Cancer. J Thorac Oncol 2008; 3(11):1213–23. 20. Lim E, Yap YK, De Stavola BL, Nicholson AG, Goldstraw P. The impact of stage and cell type on the prognosis of pulmonary neuroendocrine tumors. J Thorac Cardiovasc Surg 2005;130(4): 969–72. 21. Goldstraw P. International Association for the Study of Lung Cancer Staging Handbook in Thoracic Oncology. Florida: Editorial Rx Press, 2009. 22. Lim E, Clough R, Goldstraw P, et al. Impact of positive pleural lavage cytology on survival in patients having lung resection for non-small-cell lung cancer: an international individual patient data meta-analysis. J Thorac Cardiovasc Surg 2010;139(6):1441–6. 23. Gibbs AR, Nicholson AG. Dataset for Lung Cancer Histopathology Reports, 2nd ed. London: The Royal College of Pathologists. 2007. [Available from: http://www.rcpath.org/resources/ pdfG048LungDataset-Sept07-AR.pdf. Accessed 15 June 2009.]

Chapter

26

Immunohistochemistry in the diagnosis of pulmonary tumors Paul William Bishop

Introduction Immunohistochemistry is an indispensible diagnostic tool in the study of lung tumors. It may be helpful as a supplement to morphology in classifying primary lung tumours. This subclassification is increasingly important, as emerging therapeutic options demand increasing diagnostic exactitude. The technique is also invaluable in deciding whether a tumor, particularly an adenocarcinoma, is a pulmonary primary or arises from an extra-pulmonary site. If metastatic, immunohistochemical stains can also often determine the primary site. However, immunohistochemistry has limitations. No marker is absolutely specific or absolutely sensitive. Since the exact conditions of tissue fixation, antigen retrieval and staining vary between laboratories, diagnostic laboratories do not exactly reproduce the conditions of the published studies. Deciding whether a tumor shows positive staining also has an element of subjectivity. Cut-off levels vary between studies and often involve a combination of staining intensity and proportion of cells stained, for which there is no universally agreed scoring system. Some antibodies, when applied to some tumors, have given rise to hundreds of reported cases with consistent results. Published results with other antibodies and tumors have given varied results for reasons that may or may not be apparent. Particularly for less common pulmonary or extra-pulmonary tumors, the published data may be sparse in the extreme. The corpus of published data is immense and ever growing, particularly as tissue microarrays have the potential to examine hundreds of tumors within the scope of one study. Mastering this body of knowledge is an all but impossible task. Sensitivities of antibodies can be derived from the published literature. Deriving specificities is more problematic. A numerical value for specificity is only applicable to binary classifications. So it is useful when differentiating, for example, mesothelioma from adenocarcinoma (see Chapter 36) or metastatic colonic adenocarcinoma from a lung adenocarcinoma. However, when multiple possible primary sites exist, the relative probabilities of tumors metastisizing from each possible primary site are unknown. Even with the large numbers of tumors that can be examined using tissue microarrays, many

tumors that may enter a differential diagnosis have never been tested in sufficient numbers for any of the antibodies in use. All one can do is be aware of the publications which show that all antibodies are less than absolutely specific. Lastly, there is the challenge for pathologists to communicate findings to clinicians. Reports with probabilistic conclusions must be clearly articulated. This falls within the realm of quality assurance monitoring.

TTF-1 Thyroid transcription factor (TTF-1, thyroid-specific enhancerbinding protein) is the single most important target for immunohistochemistry in the study of pulmonary pathology. As a transcription factor, there is a large body of publications elucidating its biological role. The protein product is a 38 kilodalton (kDa) homeodomain-containing tissue-specific nuclear transcription protein of the Nkx2 gene family. The human TTF-1 polypeptide of 371 amino acids is highly conserved, sharing 98% homology with the rat TTF-1 polypeptide.1 The expression of TTF-1 is under the control of several genes. These include the homeobox gene HOXB3, which is itself expressed in early mammalian embryogenesis in the anterior neuroectoderm, branchial arches and their derivatives, including the area of the thyroid primordia and thyroid gland.2 Within the thyroid, TTF-1 activates the transcription of thyroid-specific gene promoters by binding to them. Thus, it is crucial in the maintenance of the thyroid differentiation phenotype.3 In the lung, it acts as a master regulator gene, binding to the promoters for surfactant apoproteins A, B, C and D, Clara cell antigen and T1a. This binding is itself modulated by various cofactors.4–20

Role of TTF-1 in embryology and non-neoplastic conditions Normally, expression of TTF-1 occurs in type II pneumocytes, epithelial cells of the thyroid, in certain regions of the brain, the anterior pituitary and the parathyroid glands. A group of transcription factors are active in the endodermally derived

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors

cells of the developing lung tubules and are required for normal lung morphogenesis and function. These include TTF-1, beta-catenin, Forkhead orthologs (FOX), GATA, SOX and ETS family members.21 In contrast, a second group of distinct proteins, including FOXF1, POD1, GLI, and HOX family members, play important roles in the developing lung mesenchyme, from which pulmonary vessels and bronchial smooth muscle develop.21 It is likely that TTF-1, in particular, plays an important role in distal lung formation.22 TTF-1 protein has been detected by immunohistochemistry in human fetal lung as early as 11 weeks gestation, being localized in the nuclei of epithelial cells of the developing airways. Staining is observed in both ciliated and non-ciliated cells of the bronchial and bronchiolar epithelia and in cells lining the distal airspaces. Budding tips of terminal airways have prominently labelled nuclei. TTF-1 expression decreases in bronchial, bronchiolar and alveolar epithelia with advancing gestational age and cytodifferentiation. By mid-gestation, proximal bronchial cells are negative for TTF-1.23,24 At term, TTF-1 expression persists in a few bronchial and bronchiolar basal cells and in all alveolar type II cells, whereas type I cells are negative. Expression of TTF-1 is essential for morphogenesis of the thyroid, lung and ventral forebrain, as TTF-1 knockout mice lack these organs. Partial deficiency of TTF-1 produces predominantly neurological defects in humans and mice.25 Brain-thyroid-lung syndrome has been described as a severe multi-system disorder of infants characterized by cerebral dysgenesis, thyroid dysfunction and respiratory failure. This syndrome is due to a de novo mutation or heterozygous deletions in the thyroid transcription factor 1 gene.26–28 A patient with thyroglobulin deficiency attributed to decreased TTF-1 expression has been described.29 In the light of these studies, it seems probable that mutations in TTF-1 underlie certain abnormalities of organogenesis or of cellular function within these organs. It is reasonable to infer from its embryological expression that a normal TTF-1 expression pattern is crucial in the control of distal lung development. Failure to selectively switch off expression of TTF-1 after a gestational age of 24 weeks is proposed as a final common pathway leading to pulmonary hypoplasia.30 Downregulation of thyroid transcription factor-1 gene expression occurs in fetal rat lung hypoplasia and is restored by glucocorticoids.31 However, a study in humans of various forms of pulmonary hypoplasia, including term infants who had undergone extracorporeal membrane oxygenation, showed no modulation of TTF-1 expression.32 In hyaline membrane disease with alveolar hemorrhage, edema or airway collapse, little or no TTF-1 is present except in open terminal airways. In bronchopulmonary dysplasia, TTF-1 is absent in areas of alveolar collapse or infection, and is only present in regenerating open airways.23 In congenital cystic adenomatoid malformation (CCAM), TTF-1 is detected in the nuclei of epithelial cells lining the cysts. TTF-1 was expressed in a majority of the bronchiolar-like epithelial cells of the cysts in CCAM types 1, 2 and 3, almost all of the cells being positive.

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By contrast, TTF-1 expression in the alveolar-like epithelium of CCAM type 4 cysts is restricted to type II cells, with only 30–60% of the lining cells being TTF-1-positive. These results support the hypothesis that CCAM types 1, 2 and 3 reflect abnormalities in lung morphogenesis and differentiation, distinct from that for CCAM type 4. However, they do not establish that TTF-1 plays a causative role in the development of CCAM.24 In human neonatal esophageal atresia with tracheoesophageal fistula, the epithelium of the fistula tract expresses TTF-1, indicating it is derived from a respiratory cell lineage. This respiratory origin of the human fistula may explain the poor esophageal motility (and subsequent serious respiratory complications) of the distal segment after standard repair.33 In medicolegal investigations, TTF-1 and PE-10 (a monoclonal antibody that recognizes surfactant apoproteins) have been used to aid visualization of peripheral airways in term infants in order to assess whether the infant was stillborn or live-born.34

TTF-1 expression in lung tumors TTF-1 has been applied extensively as an immunohistochemical marker for epithelial neoplasms of both lung and thyroid, whether benign or malignant, orthotopic or heterotopic, primary or metastatic. The rate of positivity for TTF-1 varies across the range of lung tumors. Broadly, 84% of small cell carcinomas, 77% of adenocarcinomas but only 8% of squamous cell carcinomas are positive. Rates of positivity in various lung tumors were collated by Ordonez in two reviews.35,36 Adenocarcinomas of the lung have been studied by a large number of researchers (see Chapter 27). Results are summarized in Table 1. With few exceptions, these papers give similar proportions of positive cases, with an overall positivity rate of 77.0% (95% confidence interval 75.4% to 78.6%). TTF-1 is the superior immunohistochemical marker for pulmonary adenocarcinomas and large cell carcinomas compared to surfactant proteins A and B.37 Small cell carcinoma is positive for TTF-1 in a marginally higher percentage of cases than adenocarcinoma. Based on the data in Table 2, tumors are positive in 84.2% of cases (95% confidence interval 81.4% to 86.9%) (see Chapter 31). Squamous cell carcinoma is positive for TTF-1 in only a small percentage of cases. This is in keeping with the general failure of immunohistochemistry to demonstrate the organ of origin of squamous cell carcinomas. Based on the data in Table 3, tumors are positive in 8.5% of cases (95% confidence interval 6.7% to 10.3%) One paper with a large number of cases has an unusually high rate of positivity of 25%.38 If this paper is excluded, the rate is 6.1% of cases (95% confidence interval 4.4% to 7.7%). Basaloid squamous cell carcinomas are consistently negative.39 The values for large cell carcinoma are more variable but overall 30% of cases are positive (95% confidence interval 24% to 35%) (see Table 4). Since many large cell carcinomas are poorly differentiated adenocarcinomas, these may account for a proportion of the positive cases (see Chapters 27 and 29).

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 1 TTF-1 in adenocarcinoma of the lung

Rate of positivity

Comments

3/16 (19%)154

140

70/97 (72%)

155

23/26 (88%)39 42/47 (89%)69

158/208 (76%)153

8/8 primary and 34/39 metastatic tumors, using cell blocks from fine needle aspirates

12/15 (80%)141

Study of micropapillary carcinoma of lung

14/17 (82%)119

Signet ring cell carcinoma of lung

27/34 (79%)

Using cell blocks from tumor in serous effusions

10/11 (91%)156

Metastases from lung to cervical lymph nodes

157

8/9 (89%)

158

21/39 (54%)

37/43 (86%)142

31/42 (74%)159

24/35 (69%)92

31/42 (74%)160

11/11 (100%)143

Brain metastases from lung primary

30/40 (75%) 37/50 (74%)

In 8 cases > 75% of cells stained, in 15 cases 50–75% of cells, in 10 cases 25–50% of cells, in 4 cases 1–25% of cells)

46/64 (72%)146 110/128 (86%)147

96/128 pulmonary adenocarcinomas showed high levels of TTF-1 expression; 14 showed only weak expression. This study also broke down the results by tumor subtype

37/50 (74%)63

41/50 (82%)162

Using a cell transfer technique on serous effusion specimens

12/22 (55%)163

Metastatic from lung to brain

131

42/46 (91%)

22/30 (73%)164 11/11 (100%)165 19/33 (58%)166 67/98 (68%)37

Using tissue microarray

5/6 (83%)167

27/30 (90%)123

Strong in 14/15 well-differentiated, 7/8 moderately differentiated (the negative case was a mucinous cystadenocarcinoma) and 6/7 poorly differentiated

46/55 (84%)168

169/176 solitary adenocarcinomas, 34/34 multifocal carcinomas, 1/1 signet ring cell carcinoma, 16/20 mucinous carcinomas: using monoclonal 8G7G3/1

13/14 (93%)171

42/50 (84%)148 Cell blocks from cases with metastatic adenocarcinoma to serous cavities, using clone 8G7G3/1

15/17 (88%)150

Cell blocks from cases with metastatic adenocarcinoma to serous cavities, using clone 8G7G3/1

8/13 (62%)151

4/10 (40%)170 5/8 (63%)172 20/28 (71%)173 69/95(73%)85

38/47 well-differentiated, 24/32 moderately differentiated, 7/16 poorly differentiated: 2 cases were positive for TTF-1 but negative for napsin A

160/229 (70%)38

Including 29 bronchoalveolar carcinomas

14/14 (100%)175

16/21 (76%)

Using cytological cell blocks and clone 1–2.A5.9

12/18 (67%)152

Lung adenocarcinoma metastatic to brain

35/46 (76%)90

4/15 (27%)169

18/21 (86%)174

13/16 (81%)149

67

Using cytospin preparations from body cavity fluids

46/55 (84%)130

51/75 (68%)61

220/231 (95%)40

Metastases from lung to CNS

29/40 (73%)161

144 145

On cell blocks, using clone 8G7G3/1

Oncocytic carcinomas

176

30/52 (58%) 127/158 (80%)128

Using cell blocks from serous effusions

Total: 2025/2631 (77.0%: 95% CI 75.4% to 78.6%)

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 2 TTF-1 in small cell carcinoma of the lung

Table 3 TTF-1 in squamous cell carcinoma of the lung

Rate of positivity

Rate of positivity

Comments

Comments

43/52 (83%)53

6/119 (5%)140

1/4 (25%)69

0/12 (0%)39

11/12 (92%)146

1/7 (14%)69

143

6/7 (86%)

Brain metastases from lung primary

0/3 (0%)

143

177

0/10 (0%)

123

24/30 (80%)

0/29 (0%)147

3/5 (60%)178

9/43 21%)61

179

123

20/21 (95%)

1/30 (3%)

19/36 (53%)40

4/99 (4%)40

20/22 (91%)180

3/8 (38%)67

10/12 (83%)

Using cytological cell blocks and clone 1–2.A5.9

154

6/7 (86%)

Metastases from lung to cervical lymph nodes

160

23/28 (82%)

181

27/30 (90%) 3/3 (100%)

131

Using tissue microarray 2% of nuclei stained in one case Using cytological cell blocks and clone 1–2.A5.9

153

0/101 (0%)

0/8 (0%)154

2/5 (40%)

156

Brain metastases

168

47/55 (85%)

67

Using cell blocks from FNA

156

3/8 (38%) 160

0%

On cell blocks Metastases from lung to cervical lymph nodes Of ?34 cases

131

4/7 (57%)

0/12 (0%)165 0/39 (0%)170

13/13 (100%)165 49

27/28 (96%)

53

43/52 (83%)

37

30/37 (81%)

23/28 (82%)160 181

27/30 (90%) 10/10 (100%)169

0/5 (0%)171 1/9 (11%)172 13/60 (22%)182 3/13 (23%)

Using a polyclonal antibody

169

0/20 (0%)37 0/10 (0%)90 0/4885 (0%)

35/36 (97%)52

30/122 (25%)38 This study involved two cancer centers; at one, only 2 of 35 tumors were positive while at the other 28 of 87 were positive

28/33 (85%)55

0/39(0%)128

30/37 (81%)50

1/3586 (3%)

1/385 (33%)

Total: 79/930 (8.5%: 95% CI 6.7% to 10.3%)

38/41 (93%)172

Total: 570/677 (84.2%: 95% CI 81.4% to 86.9%)

Not all reports of large cell carcinoma make it clear whether there was evidence of neuroendocrine differentiation. In those cases where the tumors were classified as large cell neuroendocrine carcinomas, the rate of positivity for TTF-1 is higher at 51% (95% confidence interval 43% to 58%) (see Table 5). The reported rate of positivity in carcinoids is variable, such that aggregating the data is a dubious exercise. However, if one does so, 35% of typical carcinoids are positive (95% confidence interval 29% to 42%) (see Table 6). Of atypical

1018

carcinoids, 37% are positive (95% confidence interval 25% to 49% (see Table 7). Extra-pulmonary carcinoid tumors are rarely positive for TTF-1, so while negativity is uninformative, positivity is useful evidence of a primary pulmonary origin. There are few studies of sarcomatoid carcinoma (see Table 8). One paper reports negativity in all 23 pleomorphic tumors studied.40 A second study of 75 cases found positivity in 55% of pure pleomorphic carcinomas and in 63% of those that included an epithelial component. The determining factor in the latter cases appeared to be the nature of the epithelial

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 4 TTF-1 in large cell carcinoma of the lung

Table 5 TTF-1 in large cell neuroendocrine carcinoma of the lung

Rate of positivity

Rate of positivity

Comments

35

6/62 (10%)

2/4 (50%)50

0/2 (0%)140

2/4 (50%)37

15/19 (79%)143

Brain metastases from lung primary

6/8 (75%)35

3/10 (30%)168

18/44 (41%)39

0/1 (0%)147

2/2 (100%)147

0/2 (0%)61

Using tissue microarray

31/64 (48%)177

4/25 (16%)123

6/10 (60%)123

16/61 (26%)153

6/8 (75%)178

1/6 (17%)154

Using cell blocks

6/8 (75%)179

5/9 (56%)165

6/16 (38%)183

8/20 (40%)37

3/5 (60%)38

0/1 (0%)169

Total: 88/173 (51%: 95% CI 43% to 58%)

85

4/9 (44%)

16/37 (43%)38

Table 7 TTF-1 in atypical carcinoid of the lung

Total: 78/264 (30%: 95% CI 24% to 35%)

Rate of positivity 2/3 (67%)50

Table 6 TTF-1 in typical carcinoid of the lung

2/3 (67%)37

Rate of positivity

Comments

3/3 (100%)184

16/17 (94%)184

Also positive in 8/10 metastatic tumors

0/23 (0%)177

11/16 (69%)185

9/9 (100%)179

1/1 (100%)147

0/3 (0%)40

0/27 (0%)177

5/17 (29%)183

6/23 (26%)123

2/3 (67%)186

18/51 (35%)179

1/1 (100%)85

0/8 (0%)40

Total: 24/65 (37%: 95% CI 25% to 49%) 183

10/36 (28%) 0/8 (0%)

Most positive cases were peripheral and had a spindle cell morphology

169

6/12 (50%)186 6/12 (50%)37 1/2 (50%)85 Total: 75/213 (35%: 95% CI 29% to 42%)

component. Where the epithelial component was squamous, both the squamous and the sarcomatoid components were negative. Where the epithelial component was glandular or large cell, both the epithelial and the sarcomatoid components were commonly positive.41 Concordance between components for cytokeratin 7 was shown in the same study. Among other primary pulmonary tumors investigated, only sclerosing hemangioma has been shown to be consistently positive, with

both the surface epithelial cells and the stromal cells staining (see Table 9) (see Chapter 22).

TTF-1 expression in non-pulmonary tumors Most thyroid neoplasms are positive for both TTF-1 and thyroglobulin. A TTF-1-positive but thyroglobulin-negative anaplastic thyroid carcinoma has been reported.42 This author has seen a thyroid carcinoma that progressively lost its immunoreactivity for thyroglobulin in sequential metastases, while retaining positivity for TTF-1. Although an extremely useful marker for neoplasms of pulmonary and thyroid origin, there are reports of positivity in tumors arising from other organs (see Tables 10 to 14). Many of these are neuroendocrine tumors, with lower rates of positivity reported in other epithelial tumors.43 One paper that documented positivity for TTF-1 in both primaries and metastases of four colorectal adenocarcinomas noted that positivity was only seen in tumors that had

1019

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 8 TTF-1 and cytokeratin 7 in sarcomatoid carcinomas of the lung

Components

TTF-1 Epithelial

Sarcomatoid

Epithelial

41

Sarcomatoid 7/1041

Pure spindle cell carcinoma

4/10

Pure giant cell carcinoma

2/3,41 0/3187

2/3,41 2/3187

Pure pleomorphic carcinoma (giant cell carcinoma/ spindle cell carcinoma)

5/7,41 1/3187

5/7,41 1/3187

Pleomorphic carcinoma with adenocarcinoma

12/14,41 3/4187

11/14,41 1/4187

14/14,41 3/4187

12/14,41 2/4187

Pleomorphic carcinoma with squamous cell carcinoma

0/12,41 0/3187

0/12,41 0/2187

5/1241

2/12,41 1/3187

Pleomorphic carcinoma with large cell carcinoma

13/18,41 2/8187

7/18,41 0/6187

14/18,41 6/8187

13/18,41 6/8187

Pleomorphic carcinoma with adenocarcinoma and large cell carcinoma

4/541

3/541

5/541

4/541

Pleomorphic carcinoma with squamous cell carcinoma and adenocarcinoma

1/241

1/241

1/241

1/241

Carcinosarcoma

1/341

1/341

1/341

1/341

Pulmonary blastoma

1/1,41 1/1188

0/1,41 0/1188

1/141

0/141

Pulmonary blastoma

4/4189

Table 9 Other pulmonary tumors which have been investigated for TTF-1 expression

Rate of positivity Lymphoepithelioma-like carcinoma

0/2540

Sclerosing hemangioma

36/37 (97%),190 16/16 (100%),191 39/44 (89%)

Pulmonary papillary adenoma

1/1192

Inflammatory myofibroblastic tumor

0/9193

Malignant mesothelioma

0/95,153 0/24,166 0/14,69 0/41,92 0/60,145 0/50,144 0/95,153 0/12,154 0/6,131 0/15,164 0/37,174 0/3885

manifested metastatic behavior. This led to speculation that positivity may be associated with the propensity to metastasis.44 Small cell carcinomas, arising as primary tumors at extrapulmonary sites, are not uncommonly positive for TTF-1 (see Table 11). As a result, TTF-1 is of little value in determining whether a small cell carcinoma has arisen from a pulmonary primary or from elsewhere. The number of cases at any one site is too low to determine whether rates differ across anatomical sites. At some sites, other markers are helpful. Salivary gland small cell carcinoma is commonly positive for cytokeratin 20.45–46 A minority of prostatic small cell carcinomas retain immunoreactivity for prostate-specific antigen, prostatic acid phosphatase and other prostate-specific

1020

Cytokeratin 7

markers.47,48 At any site, a search should be made for other tumor components that may be morphologically or immunohistochemically organ-specific. Merkel cell carcinoma is the one type of carcinoma of small cell morphology that is almost always negative for TTF-1, while positive for cytokeratin 20 (see Table 12).49–56 This author is aware of only one case in a series57 and one case report58 of Merkel cell carcinoma positive for TTF-1. Positivity is uncommon in extra-pulmonary neuroendocrine tumors not of small cell morphology (see Table 13). Among the non-epithelial extra-pulmonary tumors examined, TTF-1 is negative, with rare exceptions (see Table 14). The consistent negativity in mesothelioma makes TTF-1 a useful member of a panel of antibodies for the differentiation of mesothelioma from primary lung carcinoma, particularly adenocarcinoma. There is a case report of misleading positivity in pleural cytology, due to release of TTF-1-positive alveolar epithelial cells into the pleura, secondary to severe pleuropulmonary damage.59

Prognostic value of TTF-1 in lung carcinoma Although the prognostic use of TTF-1 expression has not yet found its way into general diagnostic practice, TTF-1 positivity is generally associated with a better prognosis for patients with non-small cell carcinomas.60–62 This is specifically for those with adenocarcinoma.38,63,64 Publications pre-dating August 2005 have been subject to meta-analysis.65 Interestingly, expression of TTF-1 by ovarian carcinomas is also associated with an earlier stage and a superior prognosis66 but TTF-1positive colorectal tumors may be associated with metastatic behavior.44

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 10 TTF-1 in non-pulmonary adenocarcinomas

References Breast

0/546

35,40,43,67,69,85,90,119,123,128, 131–144,148,149,151,152,154–156,161–163,194

Salivary gland

0/3

143

Gastrointestinal tract NOS

0/48

155

Esophagus

0/24

69,131 143 40,

Stomach

3/177 (1.7%)

69,90,119,131,147–149,162,163,194

See comparison of monoclonal antibodies

Colon

4/474 (1%)

37,40,44,71,85,90,119,123,131,143,144, 147–152,154,162,163,167,194,195

There are also case reports of positivity (see Table 15)

Liver

0/32

40,131,151,156,162–163

Gallbladder

0/21

69,131,154,194

Pancreas

0/108

69,85,131,147,194

Ampulla of Vater

0/6

131

Genitourinary tract NOS

0/34

158

Kidney

0/86

35,90,123,131,143,148,151,152,154

Bladder

0/2

154

Ovary

41/640 (6.4%)

35,59,66,69,123,131,144,147,149,151,155, 156,162,163,196–198

Positive cases include endometrioid, serous, clear cell and mucinous carcinomas and a malignant mixed Müllerian tumor. 35 of the positive cases are to be found in just two papers66,198 and one abstract (see Table 15)196

Endometrium

16/219 (7%)

40,66,69,90,123,131,147,151,154,199,200

Positive cases include endometrioid and serous carcinomas; see also a case report (see Table 15)201

Cervix uteri

3/100 (3%)

66,199,200

Peritoneal serous carcinoma

1/13 (8%)

202

Prostate

4/104 (4%)

35,69,90,131,144,151,152,156,194,203,204

Paranasal sinus

0/1

152

Thyroid-like nasopharyngeal papillary adenocarcinoma

2/2,1/1,3/3 (100%)

205–207

Total

46/1749 (2.6%)

In two cases, positivity was seen in mucinous areas There was negativity for thyroglobulin207

Assessing the rate of positivity requires that case reports of positivity be excluded, since the denominator, the number of cases examined by the reporting pathologists, is not known.

Comparison of monoclonal antibodies and practical considerations The commercially available antibodies can be used on formalinfixed, paraffin-embedded tissues. Early immunohistochemical studies of TTF-1 used polyclonal antibodies, which probably had lower sensitivity than the monoclonal antibodies now in

routine use. 8G7G3/1 is the anti-TTF-1 antibody most studied, and can be assumed to be the default antibody in this chapter, except where stated otherwise. A second monoclonal antibody, SPT24, appears to have greater sensitivity, but with less specificity (see Table 15). Most, but not all, published papers state which antibody was used but some multicenter studies have used the two

1021

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 11 TTF-1 in non-pulmonary small cell carcinomas

Orbit

0/153

Sinonasal cavities

0/8,49 1/1a,49 0/453

Thyroid

0/2,49 1/150

Salivary gland

0/2,49 0/1,50 0/1,53 3/15,46 1/3b,178

Breast

1/1,50 1/2,53 1/3b,178 2/10,208 2/2,209 0/1,210 1/1,211 0/1,210 0/1,212 1,213 1214

Thymus

0/353

Larynx

1/153

Gastrointestinal tract, NOS

2/12 49

Esophagus

5/7,

53,215

15/21,

215

9/15

216

Reference

Merkel cell carcinoma of skin

Small cell carcinoma of lung

52

0/21

35/36 (97%)

49

0/18

27/28 (96%)

53

0/23

43/52 (83%)

54

0/13

11/13 (85%)

55

0/21

28/33 (85%)

226

0/22

9/9 (100%)

57

1/30

43/59 (73%)

Total

Total: 1/148 (0.7%)

196/230 (85%)

53

Stomach

2/4

Small intestine

0/1,53 1/2b178 217

Appendix

0/1

Colorectum

0/2,53 1/7b178

Liver

1/1b,178 0/1,218 1/1219

Pancreas

0/1,49 1/153

Renal pelvis

Table 13 TTF-1 in other extra-pulmonary neuroendocrine tumors

Carcinoid, gastrointestinal

0/49,51 1/46,185 0/50,184 0/63,186 0/103227

Large cell neuroendocrine carcinoma of the paranasal sinus

0/150

0/1,51 1/153

Large cell neuroendocrine carcinoma of the larynx

0/150

Urinary bladder

1/5,49 2/4,51 2/3,50 1/3,53 2/10b,178 2/10,220 17/44c,221 11/44d,222

Carcinoid tumor of breast

0/543

Prostate

0/3,49 4/4,51 5/5, 1/3,53 5/5b,178 23/44,47 15/1848

Breast carcinoma with neuroendocrine features

5/5,209 0/543

Ovary, hypercalcemic type

0/7,180 0/15223

Type B breast carcinoma with colloid features

0/443

Ovary, pulmonary type

1/2,180 1/1e224

Large cell neuroendocrine tumor of bladder

2/2222

Uterine cervix

1/3,49 1/7,51 0/1,50 7/16,53 2/3b,178 3/8180

Large cell neuroendocrine carcinoma of the prostate

1/150

Vagina

0/1,53 1/3225

0/150

Endometrium

0/153

Large cell neuroendocrine carcinoma of the ovary

Adrenal

1/153

Large cell neuroendocrine carcinoma of the uterine cervix

1/1228

Pancreatic endocrine tumors

0/12,185 0/10184

Parathyroid adenoma

0/10184

Pituitary adenoma

0/10184

Phaeochromocytoma

0/5184

Paraganglioma

0/49,51 0/1229

Brain metastasis from a paranasal sinus tumor. Described as high-grade neuroendocrine tumor but not well characterized in this paper. c A urothelial component present in 29 of the cases was consistently negative for TTF-1. d In addition 2/2 large cell neuroendocrine tumors were positive for TTF-1. e This small cell carcinoma arose within a microinvasive mucinous cystadenocarcinoma of the ovary; the small cell carcinoma showed moderate to strong positivity of most nuclei, the borderline epithelium moderate positivity of a few nuclei. a b

antibodies indiscriminately. Other clones have been marketed. This author is aware of one study of each of the clones 1–2. A5.967 and BGX-397A.68 The staining methodology impacts the rate of staining, at least in gynecological tumors. The rates are least for the combination of 8G7G3/1 and automated Envisionþ HRP detection system with the Dako autostainer. They are greatest for the

1022

Table 12 Studies comparing TTF-1 in Merkel cell carcinoma with small cell carcinoma of the lung

combination of clone SPT24 with the automated Bond Max system from Vision Systems.68 The impact of such details of methodology has not been systematically studied for pulmonary tumors. It is my practice to use the clone 8G7G3/1 in the first instance. If, in combination with other selected antibodies, this is not decisive and the staining with 8G7G3/1 is negative, equivocal or obscured by cytoplasmic staining, this author uses SPT24.

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 14 Other extra-pulmonary tumors which have been examined for TTF-1 expression

Nephroblastoma

8/4843

Metanephric adenoma

0/543

Cystic nephroma

0/143

Leiomyosarcoma

0/3166,193

Synovial sarcoma

1/5193

Malignant melanoma

0/1,69 0/1,152 0/4,193 1/70 (primary tumors),137 1/70 (metastases)137

Glioblastoma multiforme

0/50 (see Table 15)230,231

Ependymomas

2/27232

Astrocytic tumors

0/32232

Other brain tumors

0/46232

Thymoma

1/1233

In the experience arising from this practice, SPT24 has a sensitivity for pulmonary adenocarcinomas some 5 percentage points higher than that of 8G7G3/1. The report will state that “positivity for SPT24 supports a lung primary but this clone has superior sensitivity at the expense of lower specificity” (Figures 1 and 2). The diagnostically informative staining for TTF-1 is nuclear. Cases can be regarded as positive even if the nuclear staining is only focal in the tumor (i.e. 1% to 10% of the tumor cells). For example, in up to 10% of TTF-1-positive lung adenocarcinomas, staining is present in fewer than 10% of the neoplastic cells. Such focal staining is not commonly encountered in adenocarcinomas of non-pulmonary and non-thyroid origin. Course granular cytoplasmic staining occurs as a confounding factor in some lung adenocarcinomas with clone 8G7G3/169,70 and is not of diagnostic significance (Figure 2a). It has also been reported in squamous cell carcinoma of the head and neck and of the cervix,37,50 oncocytic thyroid tumours,42 gastrointestinal and pancreatic carcinoids,51 colonic adenocarcinomas70 and in decalcified bone.70 It is particularly common in hepatocytes70 and hepatocellular carcinoma,71–73 such that it has been proposed as diagnostically useful in the differentiation from other adenocarcinomas. Cytoplasmic staining varies not only with the clone of TTF-1, but also with the supplier of the clone. Cytoplasmic staining is also more common when EDTA is used in the buffer rather than DTRS.74 A rapid immunohistochemical technique has been developed for use with intra-operative frozen sections.75,76 This technique may inform a decision whether to perform a wedge resection for a metastasis or a lobectomy for a primary carcinoma of the lung. Although positivity would indicate a lung primary and direct the surgeon towards a lobectomy, negativity is not conclusive. It would support a decision for a wedge resection, in a patient in whom the probability of a lung tumor being metastatic is high, on the grounds of a known

prior extra-pulmonary primary. Extending this rapid technique to other antigens, such as CDX-2, which yield complementary information, would greatly enhance the value of such rapid staining.

Napsin Napsin A is an aspartic protease with a molecular weight of approximately 38 kDa.77 Aspartic proteases form a widely distributed protein superfamily, which includes cathepsins, pepsins and rennin.78 Napsin A, along with cathepsin H, is required for the processing of the proforms of SP-B and SP-C and hence for the synthesis of surfactant.79,80 Napsin is expressed in type II pneumocytes, where it is involved in the N- and C-terminal processing of pro-surfactant protein-B.79,80 TAO2 has been shown to be identical to napsin A.81 There is also a napsin B gene, which is transcribed exclusively in cells related to the immune system. The gene lacks a stop codon and may represent a transcribed pseudogene.82 In the lung, antigenicity for napsin A has been demonstrated in type II pneumocytes. In these cells it co-localizes with Sp-B multivesicular bodies, composite bodies and lamellar bodies. Napsin A is also detected in alveolar macrophages, perhaps related to phagocytosis. It is also expressed in normal kidney but only in a minority of renal tumors. When its expression is experimentally re-established in renal tumor cell lines, this has a suppressive influence on the tumor, including colony formation in vitro and slower establishment of tumors in mouse xenografts.83 Immunohistochemical staining for napsin is cytoplasmic. It is positive in 82.4% (95% confidence interval 79.2% to 85.6%) of primary lung adenocarcinomas by immunohistochemistry, a somewhat higher percentage than that seen with TTF-1 (see Table 16). The potential for this marker to join TTF-1 in daily practice is confirmed by the report of occasional adenocarcinomas of the lung positive for napsin A but negative for TTF-1.84 Poorly differentiated cancers do not stain as frequently as well-differentiated lesions.85 Squamous cell carcinomas, small cell carcinomas and pulmonary carcinoids are considered by most authors to be negative for napsin A. One exception is a recent paper which reports a rate of positivity of 26% in pulmonary squamous cell carcinoma, along with positivity in a minority of extrapulmonary squamous cell carcinomas.86 A minority of renal cell carcinomas are positive, although it is claimed that expression of napsin A in these tumors is a false-positive, probably because of intrinsic biotin.87 Less than 5% of a range of other adenocarcinomas, including thyroid, breast, pancreas, biliary tract and colon, stain with napsin A. Expression, when present in breast and colonic adenocarcinomas, appears to be granular. This is said to be unlike the staining pattern in the lung,87 although others have described the staining in pulmonary adenocarcinoma as granular.88 Despite its promise as a marker complementary to TTF-1, there have been relatively few publications on the diagnostic application of napsin A.

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 15 Comparisons of TTF-1 clones SPT24 and 8G7G3/1

SPT24

a b

1024

234

8G7G3/1 235

16/19

7/8,234 14/19235

Pulmonary small cell carcinoma

7/8,

Pulmonary atypical carcinoid

5/5,234 2/10235

2/5,234 0/10235

Pulmonary typical carcinoid

5/11,23415/41235

1/11,234 4/41235

Pulmonary carcinoid, NOS

14/23236

4/23236

Primary pulmonary adenocarcinoma

72/86,237 12/17,234 134/185236 (76%)

56/86,237 11/17,234 121/185236 (65%)

Pulmonary large cell carcinoma

6/12,234 22/47236

5/12,234 17/47236

Pulmonary large cell neuroendocrine carcinoma

10/13235

4/13235

Pulmonary squamous cell carcinoma

2/14,234 14/97236

1/14,234 1/97236

Pulmonary tumor unclassified

10/22,236 10/22236

7/22,236 7/22236

Head and neck squamous cell carcinoma

0/38236

0/38236

Salivary gland tumor

1/56236

1/56236

Gastric adenocarcinoma

1/12,234 1/110236

0/12,234 1/110236

Colorectal carcinoma

4/90,237 3/6,238 1/17,234 3/120236

0/90,237 0/6,238 0/17,234 3/120236

Lung metastases from primary colorectal adenocarcinoma

4/41237

0/41237

Lung metastases from renal carcinoma

0/6237

0/6237

Gastric carcinoid

0/6235

0/6235

Intestinal carcinoid

0/28,234 0/54235

0/28,234 0/54235

Pancreatic carcinoid

0/26235

0/26235

Non-pulmonary poorly differentiated neuroendocrine carcinoma, various sites

8/45235

5/45235

Pancreatic adenocarcinoma

0/5,234 0/110236

0/5,234 0/110236

Breast adenocarcinoma

0/2,237 0/14,234 0/34236

0/2,237 0/14,234 0/34236

Bladder urothelial carcinoma

5/98236

5/98236

Prostatic adenocarcinoma

0/8,234 2/160236

0/8,234 2/160236

Ovarian serous carcinoma

1/6,234 1/20a,68 6/36b68

1/6,234 0/20a,68 2/36b68

Ovarian clear cell carcinoma

0/5,234 2/20a,68 1/7b68

0/5,234 0/20a,68 1/7b68

Ovarian endometrioid carcinoma

0/5,234 2/20a,68 0/7b68

0/5,234 1/20a,68 1/7b68

Ovarian mucinous carcinoma

1/20a,68 1/3b68

0/20a,68 0/3b68

Ovarian poorly differentiated carcinoma

0/20a68

0/20a68

Ovarian serous cystadenoma

0/4a68

0/4a68

Ovarian mucinous cystadenoma

1/3a68, 0/2b68

0/3a68, 0/2b68

Ovarian malignant mixed Mullerian tumor

1/2b68

0/2b68

Uterine serous carcinoma

4/18b68

1/18b68

Uterine endometrioid carcinoma

4/18b68

1/18b68

Uterine malignant mixed Mullerian tumour

9/11b68

0/11b68

Glioblastoma multiforme

14/28231

0/28231

Gliosarcoma

0/1231

0/1231

Using Tissue Microarray (TMA). Using whole tissue sections.

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors

(a)

(b)

Figure 1. Pulmonary adenocarcinoma stained with anti-TTF-1 clones 8G7G3/1 and SPT24, respectively. (a) There is only equivocal staining of the tumor with 8G7G3/1. (b) SPT24 demonstrates strong nuclear staining.

(a)

(b)

Figure 2. Pulmonary adenocarcinoma stained with anti-TTF-1 clones 8G7G3/1 and SPT24, respectively. (a) Clone 8G7G3/1 has produced granular cytoplasmic staining which is not diagnostically significant. (b) SPT24 produced nuclear staining without confounding cytoplasmic staining.

Surfactant proteins Pulmonary surfactant contains a number of proteins, including the 10 kDa Clara cell protein and surfactant proteins (SP) A, B, C and D. Surfactant apoprotein A appears to be a major constituent of giant lamellar bodies. Since surfactant is exclusively produced by the lung, one might expect surfactant proteins to be lung-specific marker. This does not appear to be the case. Antibodies to surfactants used for immunohistochemistry have been shown in a number of studies to lack both sensitivity and specificity. According to one author, only 63% of primary lung carcinomas stain with both surfactant protein A and B, while 46% of metastatic carcinomas,

including primary breast cancers, stain with surfactant A and B antibodies.87 As markers of primary pulmonary adenocarcinoma, surfactant proteins are inferior to TTF-1 and napsin. The combination of TTF-1 with SP-A does not surpass the diagnostic utility of TTF-1 alone.89 Breast tumor cells metastatic to the lung incorporate pulmonary surfactant proteins, which are therefore of little value in differentiating metastatic breast carcinoma from primary pulmonary adenocarcinoma.90 Papillary and lepidic subtypes of lung adenocarcinoma may show nuclear positivity, attributable to the presence of nuclear inclusions containing material with the antigenicity of

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors

surfactant A: such nuclear inclusions co-express TTF-1.91 In pleomorphic carcinomas, positivity for SP-A is usually restricted to the epithelial component.41 The pattern of staining for surfactant A is usually cytoplasmic in primary pulmonary adenocarcinoma but membranous in mesothelioma.92 PE-10 is a monoclonal antibody that recognizes 34–37 kDa and 62 kDa human surfactant apoproteins. Staining may detect Table 16 Expression of napsin by tumors

Lung

Adenocarcinoma

Adenocarcinoma with enteric differentiation Large cell carcinoma Squamous cell carcinoma Small cell carcinoma Carcinoid Other tumors

Mesothelioma Melanoma Breast carcinoma Gastric carcinoma Colonic carcinoma Pancreatic carcinoma Renal cell carcinoma Urothelial carcinoma of bladder Prostatic carcinoma Ovarian carcinoma Endometrial carcinoma Salivary gland carcinoma Thyroid carcinoma Sarcoma Various adenocarcinomas

33/39,239 47/58,81 39/43,88 70/83,240 17/21 (nodal metastases),240 10/12,84 27/30,241 79/95,85 122/158128 (overall 82%) 0/7241 2/11,239 2/7,81 2/7,88 3/985 0/31,239 0/26,240 0/46,85 0/39,128 9/3586 0/15,239 0/6,240 0/385 0/6,239 0/385 0/5,88 0/5,84 0/3885 0/4239 0/25,240 0/8,84 0/17,85 0/115128 0/1,239 0/284 0/1,239 0/38,240 0/1,84 0/14,241 0/585 0/1,84 0/3185 1/1,239 49/11885 0/284 0/1239 0/784 0/8,240 0/184 0/1,239 0/1240 0/21,240 2/8185 0/5239 0/3388

a combination of cytoplasmic and nuclear inclusions.93 It has superior specificity, compared with polyclonal antibodies against surfactants A or B.94 Table 17 lists comparative results for PE-10, surfactants A and B and TTF-1.

Neuroendocrine markers There are a number of neuroendocrine markers which are effective on paraffin-embedded tissue. These are useful in the identification of neuroendocrine differentiation in lung tumors (see Table 18) (see Chapter 31). Chromogranins are members of a family of acidic glycoproteins located in neurosecretory granules. The most abundant is chromogranin A (molecular weight 68 kDa). The two other major members are chromogranin B (secretogranin I) and chromogranin C (secretogranin II), with other granins including secretogranins III, IV and V5, VGF and NESP-55 recognized. Chromogranins can be detected in nearly all neuroendocrine tumors. Positivity depends on the number of dense core granules; there may be a lack of immunoreactivity in small cell carcinoma and Merkel cell carcinoma, where secretory granules are sparse.95 Greater sensitivity in small cell carcinomas may be achieved with antigen retrieval.96 Synaptophysin is a 38 kDa transmembrane glycoprotein located in presynaptic vesicles and is both chemically and topographically different from chromogranin. Other proteins in pre-synaptic vesicles include synaptobrevin, synaptotagmin, SNAP-25. SNAP receptor (SNARE), syntaxin and Rab3A.95 In nerves, it is co-expressed with neurofilament, whereas in solitary bronchial neuroendocrine cells, it is co-expressed with cytokeratins.97 Antigenicity may be lost with prolonged fixation.98 CD56 (neural cell adhesion molecule, NCAM, Leu19) is a membrane glycoprotein homophilic cell adhesion molecule, originally isolated on the basis of its role in neural cell adhesion. There have been reports that NCAM-positive carcinomas are more aggressive than NCAM-negative carcinomas.99 Monoclonal antibodies 123C3 and MOC-1 recognize non-carbohydrate epitopes. JLP5B9 recognizes the polysialated epitopes of embryonic NCAM (eNCAM).100 PGP9.5 is an additional broad-spectrum neuroendocrine marker, which is now little used in diagnostic practice.

Table 17 Expression of surfactants by tumors

PE-10 Primary lung tumors

Adenocarcinoma Squamous cell carcinoma Adenosquamous carcinoma Small cell carcinoma Large cell carcinoma Pleomorphic carcinoma

Malignant mesothelioma

154

6/16, 13/21 0/8154 0/5154 0/6154

90

Breast carcinoma metastatic to lung

1/6154

Various carcinomas metastatic to lung

1/14154

Anti-SP-B 128

25/46, 71/158 2/1090 1/190

19/5541

0/9242

Primary breast carcinoma

1026

Anti-SP-A 242

90

TTF-1 159

153

29/46, 22/42, 119/208, 0/10,90 0/101,153 0/593 1/190

93

18/75

35/4690 0/1090 1/190

0/193 12/61,153 0/893 0/95153

4/5190

0/5190

0/5190

6/1390

6/1390

0/1390

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 18 Expression of neuroendocrine markers by pulmonary tumors

CD56

Chromogranin 243

8/10

244

(overall 97%)

243

96

7/66, 32/38, 8/18, 8/3597 (overall 37%)

Synaptophysin 245

19/43,

244

27/66,243 21/36,96 9/10,244 23/3597 (overall 54%)

Small cell carcinoma

64/66,

Carcinoid tumor

9/11,246 7/10244 (overall 76%)

15/17,246 10/10244 (overall 93%)

11/11,246 8/10244 (overall 91%)

Large cell carcinoma (non-neuroendocrine)

2/3,243 2/35100 (overall 11%)

0/3,243 1/9,96 0/35100 (overall 2%)

0/3,243 1/9,96 3/35100 (overall 9%)

Large cell neuroendocrine

2/2,243 40/44,39 2/2244 (overall 92%)

0/2,243 30/44,39 5/6,96 2/2244 (overall 69%)

2/2,243 37/44,39 6/6,96 2/2244 (overall 87%)

Adenocarcinoma

1/38,243 0/25,110 1/10,244 11/209100 (overall 5%)

0/38,243 3/67,245 2/33,96 0/10,244 1/209100 (overall 2%)

1/38,243 2/33,96 23/209100 (overall 9%)

Squamous and basaloid cell carcinoma

4/52,243 0/25,110 3/21,244 29/242,100 3/28110 (overall 11%)

1/52,243 2/37,96 1/21,244 1/242,100 0/28110 (overall 1%)

1/52,243 0/35,96 1/21,244 10/242,100 1/28110 (overall 3%)

Table 19 Antibodies helpful in the differentiation of squamous from adenocarcinoma of the lung

Squamous cell carcinoma

Adenocarcinoma

TTF-1

5% of cases: see Table 3

78% of cases: see Table 1

Napsin

0%: see Table 16 123

170

82%: see Table 16 107

0/30,123 2/10170 (overall 5%)

Cytokeratins 5/6

30/30,

Cytokeratin7

8/37,96 0/12,247 7/30,123 4/12,165 0/5171 (overall 20%)

31/33,96 45/54,247 70/74,248 80/80,121 30/30,123 11/11,165 15/15171 (overall 95%)

Cytokeratin14

35/37,96 13/15281 (overall 92%)

7/33,96 2/10281 (overall 21%)

p63

13/13,282 28/28,101 13/13,283 4/4,284 32/39,170 21/23107 (overall 93%)

0/12,106 0/4,284 0/10170 (overall 0%)

Desmocollin-3

20/20115

1/20115

31/39,

19/23

(overall 87%)

Neuron-specific enolase (NSE) is now rarely used, owing to its utter nonspecificity. Positivity for neuroendocrine markers is not uncommon in non-neuroendocrine tumors of the lung (see Chapter 27). The diagnosis of large cell neuroendocrine carcinoma requires morphological features of neuroendocrine differentiation, namely organoid nesting, trabecular growth, rosette formation or perilobular palisading, in addition to immunophenotypic neuroendocrine differentiation (see Chapter 31). CD56 is the most useful neuroendocrine marker in small cell carcinomas, while chromogranin and synaptophysin are better markers in carcinoid tumors, typical and atypical. CD56 stains a number of extra-pulmonary tumors, even some that were not considered to manifest neuroendocrine differentiation, which may metastazise to the lung.

p16 and p63 p16 acts as a tumor suppressor gene. It binds to cyclin 4/6 complexes, to control the cell cycle at G1/S. In the absence of p16, CDK4 phosphorylates the retinoblastoma gene, which in turn releases E2F, inducing DNA synthesis. Aberrant p16 methylation is an early event in pulmonary non-small cell carcinogenesis, particularly adenocarcinomas, leading to loss of both p16 activity and expression (see Chapter 22).101–102

Since p16 expression is almost never abnormal in small cell carcinoma of the lung,103 immunoreactivity is retained by small cell carcinoma, as well as some squamous cell carcinomas.101 Staining for p16 is both nuclear and cytoplasmic. p63 is a member of the p53 tumor suppressor gene family. The gene is located on chromosome 3q27–29. It encodes at least six different transcripts with transactivation (TAp63) or negative effects (DNp63) on the p53 reporter genes, resulting in tumor suppressor and oncogenic effects respectively.104 The DNp63 isoforms lack the N-terminal transactivation domain and inhibit p53.105 p63 is expressed predominantly in basal cell and squamous cell carcinomas, as well as transitional cell carcinomas, but not in adenocarcinomas (primary or metastatic),106 large cell carcinomas106 or small cell carcinomas (see Table 19). Other non-pulmonary tumours, possibly metastatic to lung, may be positive with p63. Low levels of nuclear staining with p63 may be detected in non-squamous carcinomas. A cutoff value of 10% tumor cell positivity has been proposed.107

Cytokeratins Cytokeratins are a family of 20 intermediate filaments present in all epithelial cells and in a number of non-epithelial cells. Cytokeratin-positivity has been reported in almost every

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors

tumor type, including uterine smooth muscle tumors, most soft tissue sarcomas, melanomas, gliomas, plasmacytomas and occasionally lymphomas. The coordinate expression of cytokeratins 7 and 20 is particularly useful in ascertaining the site of origin of adenocarcinomas. Adenocarcinomas of the lung are characteristically positive for CK7 but negative for CK20, a combination shared with adenocarcinomas of many other sites (see Table 20) (Figure 3). However, adenocarcinomas of a number of other major organs usually show different results for CK7 and 20 and therefore these two cytokeratins in combination may be useful to narrow down possible primary sites for an adenocarcinoma (Figure 3). Cytokeratins 5/6 and 14 are characteristic of squamous cell differentiation. Table 20 Rates of coordinate expression cytokeratins 7 and 20 by pulmonary adenocarcinomas

References

67,119,142,154,159,161,247–250

CK7þ

CK7

CK20þ

14%

0%

CK20

76%

10%

Other markers A number of markers that are usually negative in lung carcinomas are useful in differentiating primary pulmonary tumors from those arising at extra-pulmonary sites. The intelligent selection of such markers depends on the patient’s gender, a clinical history, radiological evidence and the morphological assessment of the tumor. The use of immunohistochemistry to evaluate an unknown primary cancer has recently been reviewed (Figure 4).108 As a routine and for economy, TTF-1 (clone 8G7G3/1) may be applied to all adenocarcinomas within the lung. Only if it proves negative need one consider whether a panel of further antibodies should be examined. While this discussion is beyond the scope of this chapter, one should be aware of the growing number of useful antibodies (see Table 21).

Problems of differential diagnosis Small cell carcinoma versus lymphoid infiltrates When working with small or crushed bronchial biopsies, interpretation needs to be judicious. It is unwise to rely on immunohistochemistry alone, without assessing the

Figure 3. Cytokeratin 7 and 20 positivity rates in various neoplasms. Note that most tumors show near-constancy (positive or negative) for one or both cytokeratins. Gastric carcinoma is the exception, with unpredictable immunoreactivity for both cytokeratins. Data from references 42,67,119,142,154,159,161,185,248–250,304–315.

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors

(a)

(b)

(c)

Figure 4. Metastatic adenocarcinoma with a papillary architecture in soft tissues of the neck. (a) Nuclear positivity for TTF-1. (b) Cytoplasmic positivity for napsin A. (c) Negativity for thyroglobulin. These findings indicate that the carcinoma is metastatic from the lung rather than the thyroid.

morphology of at least a few intact cells, either in the biopsy or in accompanying cytology. The supporting immunohistochemistry may allow one to make a diagnosis, where on morphological grounds alone it would not be prudent. In such biopsies, the differentiation of small cell carcinoma both from non-small cell carcinoma and from lymphoid infiltrates may be difficult. Lymphoid infiltrates are positive for CD45 but negative for cytokeratins, TTF-1 and neuroendocrine markers. Small cell carcinomas are likely to be positive for CD56, TTF-1 and cytokeratins. In areas of crush, immunoreactivity for CD56 has been shown to be retained, but staining for TTF-1 and the anti-cytokeratin MNF116 is lost.109 Unfortunately, immunohistochemical markers do not distinguish crushed small cell carcinoma from crushed carcinoid tumours (see Chapter 31).

Small cell carcinoma versus small cell variant of squamous carcinoma The small cell variant of squamous carcinoma may closely mimic small cell carcinoma morphologically (see Chapter 28). This differential is critical in making the clinical decision between surgical management and chemotherapy. Cytokeratins that are positive in small cell carcinoma, such as CAM5.2 and MNF116, tend to produce punctate staining. The range of neuroendocrine markers are positive in small cell carcinoma, with CD56 having a greater sensitivity than chromogranin and synaptophysin. TTF-1 is usually positive in small cell carcinoma. p16 is also usually positive in small cell carcinoma but is of lesser value; it is not uncommonly positive in squamous cell carcinomas, although usually to a lesser degree (see Table 22). Squamous cell carcinoma is likely to be positive for CK5/6 and 34βE12 (an anti-cytokeratin which reacts with cytokeratins 1, 5, 10 and 14).110–111 Cytokeratin 14 should be positive in squamous cell carcinoma, but my personal experience is that in poorly differentiated squamous cell carcinoma, it is often negative. Nuclear staining for p63 is seen in squamous carcinoma. p63, like TTF-1, being a nuclear stain, avoids the problem of limited staining of the scant cytoplasm in both small cell carcinoma and the small cell variant of squamous

cell carcinoma. Combined small cell carcinomas (tumors with both small cell and non-small cell components) typically show a concordant small cell immunophenotype in both components with positivity for CD56 and synaptophysin (see Chapter 31).112

Squamous cell carcinoma versus adenocarcinoma With the development of specific chemotherapeutic regimes, the differentiation of squamous from non-squamous, particularly adenocarcinoma, is increasingly required by clinicians. Unfortunately it is often unclear whether the pathological classification in the course of drug trials was made on morphology alone or in addition whether immunohistochemistry, including mucin stains, was applied (see Table 19). Of these, an appropriate routine panel might comprise CK5/6, p63, TTF-1, napsin A and a mucin stain.113–114 Desmocollin-3 is a constituent of desmosomes and therefore expression in squamous cell carcinoma is not surprising. To date there is only a single report of its use in the subclassification of lung tumors. It also has the potential to reassign some large cell carcinomas as poorly differentiated squamous cell carcinomas.115 A recent paper examined the use of a panel of five antibodies, not currently in routine use (TRIM29, CEACAM5, SLC7A5, MUC1 AND CK5/6), in a weighed algorithm. The group demonstrated this panel’s superiority to the combination of TTF-1 and p63. Furthermore, the five-antibody panel was able to assign 59% of morphological large cell carcinomas as either squamous or adenocarcinomas.116

Differential diagnosis of clear cell carcinoma A variety of lung tumors may be dominated by clear cell morphology. These include clear cell variants of pulmonary adenocarcinoma, squamous cell carcinoma and neuroendocrine carcinoma, benign clear cell tumor of the lung and clear cell carcinomas metastatic from other sites. These are most commonly the kidney but also liver and ovary. Table 23 lists markers which may be useful in the identification of these metastatic tumors.

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 21 Markers commonly positive in extra-pulmonary tumors but usually negative in primary lung tumors

a b

1030

Antibody

Tumors commonly positive for marker

Positivity in lung tumors

CDX-2

Gastrointestinal adenocarcinomas and carcinoid tumors, ovary (mucinous)

Adenocarcinoma NOS: 9.7% (0/11,251 29/212,118 0/35,252 0/22,253 7/55,163 1/46131) Goblet cell mucinous carcinoma: 11/11117 Mucinous cystic carcinoma: 3/3254 Mucinous bronchioloalveolar carcinoma: 0/10,117 0/30,125 4/40126 Non-mucinous bronchioloalveolar carcinoma of lung: 0/32125 Solid adenocarcinoma of lung with mucin: 0/20126 Signet ring cell carcinoma of lung: 0/2,117 0/40126 Squamous cell carcinoma: 0/10,142 0/11,251 3/41,118 0/13,252 0/7131 Large cell carcinoma: 0/10,142 0/6,118 0/1252 Small cell carcinoma: 0/10,142 0/4,251 3/16,118 0/2,252 1/8,255 0/3131 Large cell neuroendocrine carcinoma: 3/8,118 5/8255 Carcinoid: 0/5,142 1/8,118 2/7,252 0/30,142 0/20227

NESP-55 (Neuroendocrine Secretory Protein-55)

Pancreatic well differentiated neuroendocrine tumors

1/20227

PDX-1 (Pancreatic Duodenal Homeobox)

Gastrointestinal carcinoids

0/20227

CD10

Conventional renal cell carcinoma, canalicular pattern in hepatocellular carcinoma

Stromal cells of lung carcinomas256

Renal cell carcinoma marker

Conventional and papillary renal cell carcinoma,

0/23257

Hep Par 1 (Hepatocyte Paraffin 1)

Hepatocellular carcinoma

0/5,258 1/10,259 3/55,260 0/6,261 5/21262

Prostate-specific antigen

Prostatic adenocarcinoma

0/40,263 0/56131

Prostatic acid phosphatase

Prostatic adenocarcinoma

Rarely, pulmonary small cell carcinoma may produce ectopic prostatespecific antigen and prostatic acid phosphatase.264 Weak positivity for prostatic acid phosphatase has been reported in a pulmonary carcinosarcoma265 and a pulmonary carcinoid266

Prostate-specific membrane antigen

Prostatic adenocarcinoma

Estrogen receptora

Breast and female genital tract carcinomas

0/33,267 0/35,133 3/42,268 0/248,269 0/111,270 3/49,129 62/64,271 0/23,272 10/41,132 10/55,130 4/46131

Progesterone receptor

Breast and female genital tract carcinomas

No data

Gross cystic disease fluid proteinb (GCDFP)

Breast carcinomas

11/211,134 2/35,133 2/46131

Mammaglobin

Breast carcinomas

0/106,135 0/45,273 0/197128

CD5

Thymic carcinomas

0/15,274 0/6,275 0/14,276 1/20168

CD70

Thymic carcinomas

0/17277

Thyroglobulin

Thyroid neoplasms

No data

Inhibin

Sex cord stromal tumors, adrenocortical carcinomas

9/48,278 7/56279

Estrogen receptor: the clone TE111 has a lower sensitivity than either 1D5 or 6F11, but has the advantage that it does not stain any tumors other than those of breast or ovary: in particular, bronchogenic carcinomas are reliably negative.132 A subset of primary adenocarcinomas of lung show positivity for GCDF-15; these tumors occur in both sexes and have a distinctive morphology with mucin production and co-expression of TTF-1.134

Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 22 Markers useful in the differentiation of small cell from squamous cell carcinoma of the lung

Small cell carcinoma 96

244

97

9/35, 7/56,

Squamous cell carcinoma 111

0/10,

280

0/28

101

(overall 10%)

41/42,96 12/12,39 15/16,244 22/23,280 5/5,171 28/28101 (overall 98%)

34βE12

1/37, 3/43,

Cytokeratin 5/6

0%,95 1/30, 2/38170 (overall 1%)

30/30,123 31/39170 (overall 88%)

Cytokeratin 14

2/37,96 1/7,281 5%95 (overall 7%)

35/37,96 13/15281 (overall 92%)

p16

28/28101 (100%)

15/28101 (54%)

p63

0/23,282 0/28,101 0/13,283 0/16,284 0/28170 (0%)

13/13,282 28/28,101 13/13,283 4/4,284 32/39170 (overall 91%)

CD56

64/66,243 8/10244 (overall 95%)

4/52,243 0/25,110 2/16,244 29/242,100 0/5171 (overall 10%)

TTF-1

20/23,282 26/28,101 12/13283 (overall 91%)

0/13,282 1/128,101 0/13283 (overall 1%)

Table 23 Markers useful in the differential diagnosis of clear cell tumor of the lung

Clear cell adenocarcinoma of lung

Usually positive for TTF-1147,285

Clear cell squamous carcinoma of lung

Positive for cytokeratins 5/6123,170 and 14.96,281 Immunohistochemistry is of little value in distinguishing the site of origin of squamous cell carcinoma

Clear cell neuroendocrine carcinoma

Positive for neuroendocrine markers

Clear cell carcinoma of kidney

Usually negative for both cytokeratins 7 and 20,250 commonly negative for BerEP4,286 positive for CD10,286–289 renal cell carcinoma marker257,290 and vimentin286,291–292

Clear cell hepatocellular carcinoma

Usually negative for cytokeratins 7 and 20293–296 and for BerEP4.293 Positive for Hep Par 1.258,262,294,297 CD10262,298 and polyclonal CEA262,293,296,298 show canalicular staining

Clear cell carcinoma of ovary

Usually positive for CA-125 and negative for CEA.285 May be positive for OCT-4285

Benign clear cell tumor of lung

Positive for HMB45,299–301 negative for cytokeratins300–302

Adenocarcinoma of the lung versus metastatic adenocarcinoma of the colorectum

Adenocarcinoma of the lung versus metastatic adenocarcinoma of the breast

It is increasingly common for patients with prior colorectal adenocarcinomas to have subsequent secondary lung tumors resected. In distinguishing between a metastasis from the colorectum and a lung primary, TTF-1, CDX-2, cytokeratin 7 and cytokeratin 20 are the most useful markers. One should use the more specific TTF-1 clone, 8G7G3/1 (see Table 24). However, if the tumor is mucinous, immunohistochemistry becomes unreliable, as mucinous primary adenocarcinomas of the lung acquire an intestinal immunophenotype. There is limited published information addressing this difficult area. The diversity of descriptive names given to mucinous adenocarcinomas of the lung (including colloid carcinoma,117 goblet cell mucinous,118 signet ring cell,119 mucinous bronchioloalveolar120–125 and solid adenocarcinoma with mucin126) make it difficult to know which studies are applicable to any specific case (see Chapter 27). Table 24 therefore does not include mucinous primary lung tumours.

Lung is a common metastatic site for breast carcinomas. Women who have had a breast carcinoma are at a 30% increased risk of a second extra-mammary primary tumor, particularly a primary lung carcinoma. Thus 4% to 9% of women with a history of breast carcinoma can be expected to develop a subsequent primary lung carcinoma. A solitary pulmonary nodule in a woman with such a history has a 52% probability of being a primary lung tumor, a 43% probability of being a metastasis from a breast primary and a 5% probability of being a benign lesion, such as a hamartoma.127 Although primary squamous carcinoma of the breast is rare, breast carcinomas may show focal squamous metaplasia which may lead to an erroneous diagnosis of a primary squamous carcinoma of the lung. Comparison with the morphology of the primary tumor is invaluable in this and virtually every other circumstance. Among primary pulmonary adenocarcinomas, one can expect 84% of tumours to be positive for TTF-1 and/or napsin A.128 Breast carcinomas are reliably negative for both (see Tables 10 and 16). Estrogen

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors Table 24 Markers useful in the differential diagnosis of primary adenocarcinoma of the lung and metastatic colorectal adenocarcinoma

Pulmonary adenocarcinoma

Colorectal adenocarcinoma

TTF-1

77% (2025/2631, see Table 1)

1% (4/474, see Table 10)

CDX-2

10% (37/381, see Table 21)

97% (320/331118,131,142,251–253,303)

CK7þ/CK20þ

11% (36/31667,142,154,159,161,247–250)

9% (48/510142,159,161,247,248,250,304–307)

CK7þ/CK20-

80% (254/316)

2% (11/510)

CK7/CK20þ

0% (0/316)

76% (386/510)

CK7/CK20-

8% (26/316)

13% (65/510)

and progesterone receptors are useful markers for primary breast carcinoma, thus knowing the steroid receptor status of the primary is essential. It should be remembered, however, that primary lung tumors may be positive for estrogen receptors.128–131 There is a trade-off between the sensitivity and the specificity of clones: one paper that detected positivity in pulmonary adenocarcinomas using clones 1D5 or 6F11 was negative with clone TE111.132 Other useful markers for breast carcinomas are GATA-3, Mammaglobin and Gross Cystic Disease Fluid Protein-15 (GCDFP-15).128,133–135 Although the former two are reliably negative in lung adenocarcinomas, a small proportion stain for GCDFP-15.128,131,133–134

Diagnosis of metastatic melanoma Diagnosis of metastatic melanoma can be problematic. Pulmonary metastatectomy is performed in patients thought to have malignant melanoma metastatic to the lung. They have

References 1. Ikeda K, Clark JC, Shaw-White JR, et al. Gene structure and expression of human thyroid transcription factor-1 in respiratory epithelial cells. J Biol Chem 1995;270(14):8108–14. 2. Guazzi S, Lonigro R, Pintonello L, et al. The thyroid transcription factor-1 gene is a candidate target for regulation by Hox proteins. Embo J 1994;13(14):3339–47. 3. Saiardi A, Tassi V, De Filippis V, Civitareale D. Cloning and sequence analysis of human thyroid transcription factor 1. Biochim Biophys Acta 1995; 1261(2):307–10. 4. Boggaram V. Regulation of lung surfactant protein gene expression. Front Biosci 2003;8:d751–64. 5. Alcorn JL, Islam KN, Young PP, Mendelson CR. Glucocorticoid inhibition of SP-A gene expression in lung type II cells is mediated via the TTF-1-binding element. Am J Physiol Lung Cell Mol Physiol 2004;286(4):L767–76. 6. Li J, Gao E, Mendelson CR. Cyclic AMPresponsive expression of the surfactant

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often had one or more local or regional recurrences before developing pulmonary metastases. The difficulty arises because melanomas have a propensity to progressively dedifferentiate with successive metastases. By the time lung metastases occur, the tumor is often amelanotic. It may also often have lost its immunoreactivity for melanoma antigens.136 S100, the most sensitive but least specific melanoma marker, is the most likely to be retained.136 Some melanomas acquire focal expression of cytokeratins,136,137–139 EMA137–138 or CEA,137–139 which may be diagnostically confusing. One should be aware that TTF-1 positivity has been reported in metastatic melanomas.137 It may be helpful to review the previous specimens and assess their immunophenotype. If they have shown progressive loss of melanocytic markers, the negativity of the lung metastases is not surprising. Much of the information in this chapter, along with subsequent updates, may be found at: http://www.e-immunohistochemistry.info.

protein-A gene is mediated by increased DNA binding and transcriptional activity of thyroid transcription factor1. J Biol Chem 1998;273(8):4592–600. 7. Bruno MD, Bohinski RJ, Huelsman KM, Whitsett JA, Korfhagen TR. Lung cell-specific expression of the murine surfactant protein A (SP-A) gene is mediated by interactions between the SP-A promoter and thyroid transcription factor-1. J Biol Chem 1995;270(12):6531–6. 8. Kelly SE, Bachurski CJ, Burhans MS, Glasser SW. Transcription of the lungspecific surfactant protein C gene is mediated by thyroid transcription factor 1. J Biol Chem 1996;271(12): 6881–8. 9. Yan C, Whitsett JA. Protein kinase A activation of the surfactant protein B gene is mediated by phosphorylation of thyroid transcription factor 1. J Biol Chem 1997;272(28):17327–32. 10. Kumar AS, Venkatesh VC, Planer BC, Feinstein SI, Ballard PL. Phorbol ester down-regulation of lung surfactant protein B gene expression by

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Chapter 26: Immunohistochemistry in the diagnosis of pulmonary tumors

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297. Wu PC, Fang JW, Lau VK, et al. Classification of hepatocellular carcinoma according to hepatocellular and biliary differentiation markers. Clinical and biological implications. Am J Pathol 1996;149(4):1167–75.

300. Gal AA, Koss MN, Hochholzer L, Chejfec G. An immunohistochemical study of benign clear cell (‘sugar’) tumor of the lung. Arch Pathol Lab Med 1991;115 (10):1034–8. 301. Lantuejoul S, Isaac S, Pinel N, et al. Clear cell tumor of the lung: an immunohistochemical and ultrastructural study supporting a pericytic differentiation. Mod Pathol 1997;10(10):1001–8. 302. Gaffey MJ, Mills SE, Askin FB, et al. Clear cell tumor of the lung. A clinicopathologic, immunohistochemical, and ultrastructural study of eight cases. Am J Surg Pathol 1990; 14(3):248–59. 303. Hinoi T, Tani M, Lucas PC, et al. Loss of CDX2 expression and microsatellite instability are prominent features of large cell minimally differentiated carcinomas of the colon. Am J Pathol 2001;159(6):2239–48. 304. Kende AI, Carr NJ, Sobin LH. Expression of cytokeratins 7 and 20 in carcinomas of the gastrointestinal tract. Histopathology 2003;42(2):137–40. 305. Park SY, Kim HS, Hong EK, Kim WH. Expression of cytokeratins 7 and 20 in primary carcinomas of the stomach and colorectum and their value in the differential diagnosis of metastatic carcinomas to the ovary. Hum Pathol 2002;33(11):1078–85. 306. Chen ZM, Wang HL. Alteration of cytokeratin 7 and cytokeratin 20 expression profile is uniquely associated with tumorigenesis of

primary adenocarcinoma of the small intestine. Am J Surg Pathol 2004; 28(10):1352–9. 307. Vang R, Gown AM, Barry TS, et al. Cytokeratins 7 and 20 in primary and secondary mucinous tumors of the ovary: analysis of coordinate immunohistochemical expression profiles and staining distribution in 179 cases. Am J Surg Pathol 2006; 30(9):1130–9. 308. Ormsby AH, Goldblum JR, Rice TW, Richter JE, Gramlich TL. The utility of cytokeratin subsets in distinguishing Barrett’s-related oesophageal adenocarcinoma from gastric adenocarcinoma. Histopathology 2001;38(4):307–11. 309. Jiang J, Ulbright TM, Younger C, et al. Cytokeratin 7 and cytokeratin 20 in primary urinary bladder carcinoma and matched lymph node metastasis. Arch Pathol Lab Med 2001;125(7):921–3. 310. Bassily NH, Vallorosi CJ, Akdas G, Montie JE, Rubin MA. Coordinate expression of cytokeratins 7 and 20 in prostate adenocarcinoma and bladder urothelial carcinoma. Am J Clin Pathol 2000;113(3):383–8. 311. Rullier A, Le Bail B, Fawaz R, et al. Cytokeratin 7 and 20 expression in cholangiocarcinomas varies along the biliary tract but still differs from that in colorectal carcinoma metastasis. Am J Surg Pathol 2000; 24(6):870–6. 312. Kuo T. Cytokeratin profiles of the thymus and thymomas: histogenetic correlations and proposal for a histological classification of thymomas. Histopathology 2000;36 (5):403–14. 313. Meer S, Altini M. CK7þ/CK20immunoexpression profile is typical of salivary gland neoplasia. Histopathology 2007;51(1):26–32. 314. Nikitakis NG, Tosios KI, Papanikolaou VS, Rivera H, Papanicolaou SI, Ioffe OB. Immunohistochemical expression of cytokeratins 7 and 20 in malignant salivary gland tumors. Mod Pathol 2004;17(4):407–15. 315. Khunamornpong S, Siriaunkgul S, Suprasert P, et al. Intrahepatic cholangiocarcinoma metastatic to the ovary: a report of 16 cases of an underemphasized form of secondary tumor in the ovary that may mimic primary neoplasia. Am J Surg Pathol 2007;31(12):1788–99.

Chapter

27

Adenocarcinoma of the lung Douglas B. Flieder, Alain C. Borczuk and Masayuki Noguchi

Introduction Adenocarcinoma is the commonest histological subtype of lung cancer in most of the world and accounts for almost half of all lung cancers.1 Over the past 20 years it has become clear that this general category of carcinoma is not nearly as monolithic as once thought. Major advances in epidemiological, radiological, histological, immunohistochemical and molecular research paint a very complex picture. These suggest a wide spectrum of different entities, when viewed from different perspectives. All agree, however, that adenocarcinoma is a malignant epithelial tumor with glandular differentiation and/or mucin production. In light of recent therapeutic advances affecting particular subgroups of adenocarcinoma, histopathologists must be aware of subtle morphological and molecular distinctions. This chapter aims to present the

current level of understanding within our traditional morphological framework.

Classification and cell of origin Tumor classifications dating back to the first World Health Organization in 1967 and progressing through to the 2004 document were conceived by and for pathologists to ensure uniform tumor reporting and to aid clinical trials. Adenocarcinoma subclassification was expanded from three entities in 1967 to five major subtypes and five variants in 2004 (Table 1).2–5 This development illustrates increased interest in this tumor and the deluge of information shaping our understanding of the carcinoma. The 2004 classification was the first to include relevant clinical and genetic information.

Table 1 Historical WHO classifications of pulmonary adenocarcinoma

1967

1981

1999

Bronchogenic

2004 Adenocarcinoma, mixed subtype

Acinar

Acinar adenocarcinoma

Acinar adenocarcinoma

Acinar adenocarcinoma

Papillary

Papillary adenocarcinoma

Papillary adenocarcinoma

Papillary adenocarcinoma

Bronchioloalveolar carcinoma

Bronchioloalveolar carcinoma Non-mucinous Mucinous Mixed Solid with mucin Variants Well-differentiated fetal adenocarcinoma Mucinous (colloid) adenocarcinoma Mucinous cystadenocarcinoma Signet-ring adenocarcinoma Clear-cell adenocarcinoma

Bronchioloalveolar carcinoma Non-mucinous Mucinous Mixed or indeterminate Solid with mucin production Variants Fetal adenocarcinoma

Bronchioloalveolar carcinoma

Solid with mucus formation

Mucinous (colloid) adenocarcinoma Mucinous cystadenocarcinoma Signet-ring adenocarcinoma Clear-cell adenocarcinoma

Sources: World Health Organization Histological Typing of Lung Tumours.2–5

Spencer’s Pathology of the Lung, Sixth Edition, ed. Philip Hasleton and Douglas B. Flieder. Published by Cambridge University Press. © Cambridge University Press 2013.

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The current 2004 classification, not unlike the previous 1999 version, is notable in that it requires a bronchioloalveolar carcinoma (BAC) to have pure lepidic growth without stromal, pleural or vascular invasion. The 2004 classification also added a mixed subtype category of adenocarcinoma, as an acknowledgement that most adenocarcinomas feature at least two if not more morphological patterns.5 While the definition of BAC is reasonable, the term “BAC” has become cumbersome and confusing for pathologists and clinicians. Some use it as a diagnosis, others as a descriptive term. The mixed subtype is also problematic since at least 80% of lung adenocarcinomas belong in this category. Thus, the classification may not separate possibly distinct subgroups and does not allow for morphological-radiological-molecular studies on particular architectural patterns. These problems interfere with targeted therapy choices for patients with advanced carcinomas. These issues along with the growing need for greater understanding between pathologists, radiologists and medical oncologists spurred a collaborative attempt to focus clinically relevant issues and create a multidisciplinary classification scheme. This effort resulted in the 2011 International Association for the Study of Lung Cancer (IASLC)/American Thoracic Society (ATS)/European Respiratory Society (ERS) International Multidisciplinary classification (Table 2).6 This scheme proposes presumptive improvements including radical changes in nomenclature and subtype definitions, and to diagnostic algorithms utilizing immunohistochemical studies to potentially refine diagnoses. While multidisciplinary in nature, the backbone of the classification is histopathology. Radiological and molecular profile results support this proposed classification scheme (Table 3). Although the classification is unproven, those involved in this work hope it is an improvement over the 2004 WHO system and will readily support clinical practice, clinical trials and research investigations. The IASLC/ATS/ERS classification marks the death of the term/diagnosis bronchioloalveolar carcinoma. The rationale is simple, as the term has come to define many different lesions over the years. These include solitary small invasive peripheral lung tumors with 100% 5-year survival, mixed-type adenocarcinomas, invasive mucinous tumors and advanced disease.7–14 Its removal should be met with enthusiasm from pathologists, radiologists and oncologists. This proposed classification represents a true paradigm shift. The scheme recognizes that a proportion of, but certainly not all, lung adenocarcinomas develop from dysplasia and progress through in situ carcinoma to invasive carcinoma with the ability to metastasize. Thus, atypical adenomatous hyperplasia remains a preinvasive lesion, i.e. a dysplastic lesion, while bronchioloalveolar carcinoma measuring 3.0 cm or less is renamed adenocarcinoma in situ (AIS). Predominant bronchioloalveolar carcinomas with only minute (0.5 cm or smaller) foci of invasion are renamed minimally invasive adenocarcinomas (MIA).

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Table 2 IASLC/ATS/ERS classification of lung adenocarcinoma in resection specimens

Pre-invasive lesions Atypical adenomatous hyperplasia Adenocarcinoma in situ (
3 cm formerly BAC) Non-mucinous Mucinous Mixed mucinous/non-mucinous Minimally invasive adenocarcinoma (
3 cm lepidic predominant tumor with
0.5 cm invasion) Non-mucinous Mucinous Mixed mucinous/non-mucinous Invasive adenocarcinoma Lepidic predominant (formerly non-mucinous BAC pattern, with >0.5 cm. invasion) Acinar predominant Papillary predominant Micropapillary predominant Solid predominant with mucin production Variants of invasive adenocarcinoma Invasive mucinous adenocarcinoma (formerly mucinous BAC) Colloid Fetal (low and high grade) Enteric BAC, bronchioloalveolar carcinoma; IASLC, International Association for the Study of Lung Cancer; ATS, American Thoracic Society; ERS, European Respiratory Society. Source: reference6.

In addition, the mixed adenocarcinoma subtype is replaced with a system that classifies carcinomas according to the predominant architectural pattern. A micropapillary subtype is added to the lepidic, acinar, papillary and solid predominant patterns. Carcinomas featuring predominant lepidic growth with more than 0.5 cm of invasion are named lepidic predominant adenocarcinoma (LPA). The system also encourages one to report the percentages of all recognized patterns in 5% increments. Such is also helpful when attempting to distinguish separate synchronous tumors from intrapulmonary metastases. Mucinous lung adenocarcinomas are also separated from the more common non-mucinous lesions on the strength of epidemiological, morphological, radiological and molecular findings. Though not proven, it is suggested that a similar progression from mucinous AIS through mucinous MIA to invasive mucinous adenocarcinoma occurs. Finally, the IASLC/ATS/ERS classification revises the 2004 WHO classification grouping of invasive adenocarcinoma variants. Mucinous cystadenocarcinoma is added to the colloid adenocarcinoma group as an acknowledgement that this very rare lesion is probably a member of the same group. Clear cell and signet ring adenocarcinomas are not recognized as architectural subtypes but rather as cytological features noted in many of the histological patterns. Enteric adenocarcinoma is

Chapter 27: Adenocarcinoma of the lung Table 3 Adenocarcinoma histological subtypes with radiological and molecular associations

Predominant histological subtype

CT scan appearance

Molecular features

Non-mucinous AIS and MIA

GGN, part solid nodule

TTF-1 positive 100%

Non-mucinous lepidic

Part solid nodule GGN or solid nodule

TTF-1 positive 100%

EGFR mutation neversmokers 10–30% KRAS mutation smokers 10–30%

EGFR mutation neversmokers 10–30% KRAS mutation smokers 10% BRAF mutation 5%

Papillary

Solid nodule

TTF-1 positive 90–100% EGFR mutation 10–30% KRAS mutation 3% ERBB2 mutation 3% p53 mutation 30% BRAF mutation 5%

Acinar

Solid nodule

TTF-1 positive or negative KRAS mutation smokers 20% EGFR mutation nonsmokers 5% p53 mutation 40%

Micropapillary

Unknown

KRAS mutation 33% EGFR mutation 20% BRAF mutation 20%

Solid

Solid

TTF-1 positive 70% KRAS mutation smokers 10–30% EGFR mutation never smokers 10–30% EML4/ALK translocation >5% p53 mutation 50%

Invasive mucinous

Consolidation

TTF-1 positive 0–30% KRAS mutation 80–100% EGFR mutation 0%

Air bronchograms Less often GGO

AIS, adenocarcinoma in situ; MIA, minimally invasive adenocarcinoma; GGN, ground-glass nodule; GGO, ground-glass opacity. Source: modified from reference 6.

added as a variant type simply to call attention to its existence. The added visibility may prevent misdiagnoses as colorectal metastases and raise research interest. Given their morphological heterogeneity, it is highly unlikely that pulmonary adenocarcinomas arise from a single cell type; however, it is quite likely that “normal” epithelial cells transform into malignant cells.15–17 Expression profile studies suggest two separate histogenetic origins of lung adenocarcinoma.18 Clara cells, non-ciliated bronchiolar cells and type II pneumocytes in the peripheral lung represent one group of progenitor cells. Basal cells and mucous cells from the central conducting airways are probably the other tumor progenitor cells. These terminal respiratory unit (TRU) and non-TRU clusters, respectively, appear to represent the two major subtypes of adenocarcinoma.18,19

Genetics Less than 20 years ago, karyotypic studies were the standard method of evaluating tumor cytogenetics. However, molecular methods including multicolor fluorescence in situ hybridization (FISH) and comparative genomic hybridization (CGH) analysis have almost replaced cell culture-based research.20 Lung adenocarcinomas feature extensive karyotypic changes and genomic imbalances (Figure 1). Hyper- and hypodiploidy are almost always present while the mean chromosome number is nearly triploid.21 Chromosome 1q gain is the most frequent chromosomal imbalance. Other frequently observed imbalances include, but are not limited to, deletions on chromosomes 3p, 4q, 5q, 6q, 8p, 9, 13q and 14q and gains on 5p, 8q, 16p and 20q.21–25 Expression profiles generate much information and several different investigators have used microarray analyses to subdivide lung adenocarcinomas into several groups.18,26–30 These studies may not demonstrate complete agreement with each other, making reconciliation of findings or definitive conclusions

Figure 1. Comparative genomic hybridization sum karyogram of a lung adenocarcinoma from a woman. Significant chromosomal anomalies are apparent. The red color indicates DNA loss, green indicates DNA gain, and blue indicates no imbalance. Artificial overrepresentation of the X chromosome and loss of the Y chromosome are due to technical reasons. (Figure courtesy of I. Petersen, MD, Jena, Germany.)

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difficult. However, all identify a subset of adenocarcinomas with high-level expression of peripheral lung epithelial cell genes. This group can be considered the TRU subclass of adenocarcinomas, while those carcinomas featuring more cell cycle and proliferation-related features form the non-TRU subclass (Table 4).31,32 Interestingly, the TRU subclass contains a large number of female non- or light smokers with well-differentiated LPA and a high frequency of epidermal growth factor receptor (EGFR) mutations. These findings exemplify how recent technologies facilitate pathogenetic, clinicopathological, therapeutic and prognostic separations as well as the ability to distinguish primary lung adenocarcinomas from metastatic adenocarcinomas (see Molecular findings and Prognosis and natural history below).

Table 4 Expression profile-defined adenocarcinoma subtypes

Biological processes

TRU type

Non-TRU type

Regulation of lipid surface tension Sex differences Lipoprotein metabolism Innate immune response

Nucleotide biosynthesis Cell cycle Circulation

Mesoderm development Phosphate transport Respiratory gaseous exchange Lipid metabolism Fertilization Molecular functions

Antigen binding Oxidoreductase activity Oxygen binding Phospholipidtranslocating ATPase activity Nonspecific monooxygenase activity Steroid binding

Cellular components

Extracellular region Microsome

TRU, terminal respiratory unit. Source: modified from reference 18.

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Chromatin establishment/ maintenance Mitoses Pregnancy Cell surface receptor linked signal transduction Epidermis development

Special epidemiological features Although adenocarcinoma of the lung represented no more than 5% of lung cancers in the 1950’s, this subtype is now the commonest form in at least the United States, Canada, Japan, Korea and China, and constitutes between 35 and 50% of recorded pulmonary cancers.33–38 Although its ascent to the top of the incidence list has taken several decades, this rise is a remarkable event for a major cancer subtype. In Connecticut, USA, adenocarcinoma rates increased nearly 17-fold in women and nearly 10-fold in men from 1959 through to 1991.39 This change in incidence is probably due to changes in cigarette design and smoking behavior, rather than diagnostic advances or changes in histological interpretation.40–45 Increased smoking cessation, at least in parts of the Western world, may also contribute since an ex-smoker’s risk of developing a pulmonary squamous cell carcinoma drops faster than the risk of developing an adenocarcinoma.46,47 While adenocarcinoma is the most frequent lung cancer type in men and women, smokers and non-smokers, Asians and Caucasians alike, virtually all attention now focuses on the unique subgroup of non-smokers who develop lung cancer. An estimated 25% of all lung cancers worldwide are not attributable to tobacco use, making this disease the sixth or seventh leading cause of cancer death for both sexes.48,49 The estimated lung cancer death rate among non-smoking Americans is reported as 17.1 per 100 000 for men and 14.7 per 100 000 for women.49 Sixty-four to 70% of neversmokers who develop lung cancer have adenocarcinomas while “only” 30 to 42% of smokers have this type of cancer (Figure 2).50

Polypeptide N-acetylgalactosaminyl transferase adenosine triphosphate binding

Soluble fraction Chromosome, pericentric region Kinetochore Nucleolus Figure 2. A stunning percentage of adenocarcinoma patients are nonsmokers. Data from reference 50.

Chapter 27: Adenocarcinoma of the lung

Almost 53% of lung cancers in women and 15% in men are not tobacco-related. Research suggests that never-smoking women are at higher risk than never-smoking men of developing lung adenocarcinoma yet women are probably not more susceptible than men to the carcinogenic effects of cigarette smoke in the lung.51 Asian countries also report an earlier age at diagnosis among never-smokers compared with smokers.52 Observational pathology studies also report that from 58 to 72% of small adenocarcinomas considered AIS, MIA, PLA or invasive mucinous adenocarcinoma are diagnosed in women, and more than half of these women are non-smokers.6,7 Molecular findings, discussed below, follow this thread and such evidence serves as the framework for the IASLC/ATS/ ERS classification. Most lung adenocarcinomas are diagnosed in the sixth and seventh decades of life, while octogenarians are a distinct subset.53,54 No more than 10% of adenocarcinomas are diagnosed in those younger than 50 years and less than 5% in individuals below 40 years of age.55–59 Two distinct groups of pediatric adenocarcinomas are recognized. Most are found in conjunction with congenital cystic adenomatoid malformations of the lung. The second group comprises the rare primary lung adenocarcinomas in children and adolescents treated for assorted malignancies, including osteosarcoma, chondrosarcoma, neuroblastoma and Wilms tumor.60–67 Previously referred to as “pulmonary nodules resembling bronchioloalveolar carcinoma”, these mostly subcentimeter lesions can feature stromal invasion as well as EGFR or KRAS mutations.68,69

Special clinical features Since more than three-quarters of lung adenocarcinomas are peripheral tumors, clinical manifestations are as likely to be due to metastatic disease as to the primary tumor (Table 5). Pain, cough, dyspnea and recurrent pneumonia are common complaints attributable to a peripheral lesion. Chest pain is poorly understood and, aside from Pancoast syndrome patients (lower brachial plexopathy, Horner syndrome and shoulder pain), is not secondary to cancer invasion of chest wall nerves. Cough, hemoptysis, wheezing, dyspnea and pneumonia are frequent symptoms in patients with central tumors.70,71 Atelectasis often develops but massive bleeding is rare. Intrathoracic spread of lung adenocarcinoma can lead to shortness of breath due to pleural effusion, while subcarinal or posterior mediastinal nodal involvement may cause dysphagia. Left recurrent laryngeal and phrenic nerve involvements cause

hoarseness and an elevated hemidiaphragm, with or without dyspnea, respectively. Pericardial or cardiac involvement can present with tamponade. Superior vena cava obstruction manifests as superior vena cava syndrome. Extrathoracic manifestations of lung adenocarcinoma secondary to lymphangitic and/or hematogenous spread produce initial symptoms in up to 25% of patients (see Chapter 24). Constitutional symptoms are common but poorly understood. Malaise, fever, anorexia and weight loss are likely to have a biochemical basis, with cytokines and tumor-derived factors probably playing large roles.72–75 While paraneoplastic syndromes, mostly neurological, metabolic, hematological and dermatological, affect up to 10% of lung adenocarcinoma patients, the clinical literature reports mostly individual cases and rarely separates the processes into adeno- or squamous cell carcinoma-specific groups (see Chapter 24). Given the popularity of screening studies in developed nations, it is not surprising that 5–20% of lung cancer patients are asymptomatic and only diagnosed during workup of other medical complaints.70,76,77 This percentage is likely to change depending on the outcome of CT screening studies (see Chapter 24).

Radiographic findings Compiled data from chest radiographs present a virtual macroscopic picture of non-mucinous lung adenocarcinoma. Carcinomas are most frequent in the right upper lobe and the anterior segment is most often affected.78 Apical carcinomas are as likely to be adenocarcinomas as squamous cell carcinomas.79–82 More than half of adenocarcinomas are peripheral and the remainder are either peripheral masses with hilar lymphadenopathy, or central lesions (Figure 3).80,83,84 Not surprisingly, obstructive pneumonitis or atelectasis is seen in no more than 30% of adenocarcinomas.80,85 Calcification or cavitation is rare but visceral pleural puckering, the so-called “tail sign” or “pleural tag”, is seen in up to 80% of adenocarcinomas.86–91 The “tail sign” is not specific for lung adenocarcinoma, as it is also associated with granulomas and metastatic lesions.92,93 Air bronchograms are also common.91 Pleural, chest wall and bone destruction, mostly osteolytic, is not uncommon. Hemidiaphragmatic elevation may be secondary to phrenic nerve involvement, infra-pulmonary pleural effusion or atelectasis.94 While chest radiography recognizes parenchymal abnormalities and nodules larger than 1.0 cm, high-resolution CT scans practically represent lung adenocarcinoma imaging biomarkers (Table 3).95 Since radiographic images may suggest

Table 5 Clinical manifestations of pulmonary adenocarcinoma

Adenocarcinoma

Effects of primary tumor

Intrathoracic spread

Distant metastases

Paraneoplastic syndromes

10–25%

< 10%

10–25%

< 10%

Source: modified from reference 409.

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Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

Figure 3. Chest radiograph of a lung adenocarcinoma. (a) Posterior-anterior view. A 2.0 cm right middle lobe ill-defined nodule is noted. (b) One easily appreciates the right middle lobe nodule in this inverted image. (c) The corresponding computed tomogram demonstrates the spiculated solid nodule. (Images courtesy of K. Edwards, MD, Philadelphia, Pennsylvania, USA.)

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Figure 4. Computed tomogram of a ground-glass nodule. The right middle lobe ground-glass nodule features preserved lobular architecture. This lesion was resected and diagnosed as non-mucinous adenocarcinoma in situ. (Image courtesy of M. Yu, MD, Philadelphia, Pennsylvania, USA.)

Figure 5. Computed tomogram of a solid mass. This almost 5.0 cm left lower lobe spiculated solid mass obliterates lung parenchyma. (Image courtesy of M. Yu, MD, Philadelphia, Pennsylvania, USA.)

morphological diagnoses, one must be aware of particular terms describing these morphological lesions. Nodules refer to lesions measuring up to 3.0 cm, while masses are larger than 3.0 cm.96,97 Pure ground-glass nodules (GGN) are focal areas of increased lung attenuation within which the margins or normal structures, e.g. vessels, remain outlined (Figure 4). Solid nodules are focal areas of increased attenuation that are dense enough to obscure normal structures (Figure 5). Part-solid nodules contain both solid

and ground-glass components (Figure 6). This lesion should be evaluated with thin-section CT scans (
3.0 mm thickness).96,98 Observational studies suggest GGNs represent either AAH or AIS, part-solid nodules represent MIA, while solid nodules are invasive carcinomas.7,91,99–109 These correlates are not foolproof, as the differential diagnosis includes infections and inflammatory processes for each of these radiographic findings. In addition, there is overlap among the imaging features.

Chapter 27: Adenocarcinoma of the lung

Figure 6. Computed tomogram of a part solid nodule. A coronal view demonstrates a right upper lobe part solid nodule with a central 5.5 mm solid component. Vessels course through the lesion. If the entire solid area represents invasive carcinoma, then this tumor would be a lepidic predominant carcinoma rather than a minimally invasive adenocarcinoma. The left upper lobe contains a 4.0 cm ground-glass opacity as well. (Image courtesy of B. Milestone, MD, Philadelphia, Pennsylvania, USA.)

AIS can be part solid or bubble-like, while invasive adenocarcinoma might be part-solid or manifest as a GGN.91,105,110 Computed tomography studies also allow accurate size and growth rate assessments. Given the radiologist’s newfound ability to independently measure the solid and ground-glass components of a lung cancer, the pathology community must begin to address the issue of tumor size. It is uncertain whether the size of the invasive component or the size of the entire lesion holds accurate prognostic information. Another issue relates to the optimal frequency and duration of CT follow-ups for GGNs and part-solid nodules.107,108,111–114 GGNs less than 0.5 cm are rarely followed up, while larger lesions are usually rescanned at least annually. An increase in size or attenuation suggests progression to invasive disease.96 Little is known about part-solid nodules.115 Small solid nodules, suspicious for lung cancer, should be managed according to the Fleischner Society guidelines.116,117 These remarkable advances in describing and classifying lung adenocarcinoma images practically mirror the radiological prognostic power associated with other organ malignancies. Correlative radiological-morphological studies suggest that for solid nodules smaller than 2.0 cm, the smaller the tumor, the lesser the likelihood of vascular invasion.118 Tumors greater than 2.0 cm with coarse, lesional spiculations are associated with vascular invasion, lymph node metastases and decreased survival following resection.99,119 The

Figure 7. 18F-fluorodeoxyglucose-positron emission tomography of metastatic adenocarcinoma. Left hilar and subcarinal lymphadenopathy features intense 18F-fluorodeoxyglucose avidity. (Image courtesy of K. Edwards, MD, Philadelphia, Pennsylvania, USA.)

proportion of ground-glass opacity versus solid pattern in patients with clinical T1 adenocarcinoma also appears to predict lymph node metastases and outcome.120–122 Imaging characteristics are not associated with EGFR or KRAS mutations.123 Positron emission tomography (PET) imaging combined with CT scans might one day differentiate adenocarcinoma subtypes or prognosticate outcomes. At the moment, clinicians rely on this modality to aid in mediastinal lymph node evaluation, detection of recurrences and metastases, as well as possibly evaluate the clinical response to chemo-radiation therapy (Figure 7).124–128 18F-fluorodeoxyglucose (FDG)-PET has a high sensitivity for cancer, but a low specificity. False-positive PET images may be due to infection or granulomatous diseases such as sarcoidosis while false-negative images are seen in lowmetabolism lung adenocarcinomas such as AIS.129 For adenocarcinomas larger than 0.7 cm, standard uptake values (SUV) tend to be lower than for other histological types and may inversely correlate with survival.124,126,128 Standard uptake values do not differ between non-mucinous and mucinous adenocarcinomas and correlate with Ki-67 proliferation indices.130–132

Macroscopic pathology Pre-invasive lesions are either atypical adenomatous hyperplasia (AAH) or adenocarcinoma in situ (AIS). The former is discussed in Chapter 23. According to the IASLC/ATS/ERS, AIS measures less than 3.0 cm. At least two-thirds are of the

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Chapter 27: Adenocarcinoma of the lung

Figure 9. Adenocarcinoma in situ. Lesions may be associated with emphysematous lung. Such cases may impart a part solid CT appearance. Note the significant anthracosis.

Figure 8. Adenocarcinoma in situ. This peripheral tan lesion distorts but does not destroy underlying lung parenchyma.

Figure 11. Invasive adenocarcinoma. This carcinoma appears to respect underlying lung; however, widespread microscopic invasion was noted. Parenchymal consolidation, although historically associated with lepidic growth, is a rare presentation for even invasive adenocarcinoma.

Figure 10. Invasive adenocarcinoma. Peripheral adenocarcinomas often pucker overlying visceral pleura. While the center of the carcinoma is solid, one appreciates a feathered edge representing a lepidic component.

non-mucinous type and most are solitary lesions, although up to 25% of cases feature multiple nodules. These non-invasive carcinomas are usually irregular, tan, semi-firm peripheral nodules with circumscribed borders. Since tumor cells grow along intact alveolar walls, airspaces are visible with the naked

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eye (Figure 8). Multiloculated cysts may be seen (Figure 9). Central scars with anthracosis and overlying pleural fibrosis with or without puckering may be noted, while hemorrhage or necrosis is usually absent. Invasive adenocarcinomas are most often peripheral but up to 13% may be central.133–137 While mid-twentieth-century tumors usually measured more than 3.0 cm, these days the great majority are smaller than 4.0 cm. Non-mucinous solitary, firm and gritty, gray-tan lesions feature either lobulated or spiculated edges (Figure 10). Central necrosis or hemorrhage is common but cavitation rare. The periphery may be semi-firm with preserved airspaces, indicating a probable lepidic component. Papillary fronds are rarely observed and one cannot discriminate morphological acinar or solid patterns on gross inspection. Pleural puckering can be striking and consolidation is rare (Figures 10 and 11). Spread along the visceral pleura can

Chapter 27: Adenocarcinoma of the lung

manifest with a thick pleural rind resembling malignant mesothelioma and is referred to as pseudomesotheliomatous carcinoma (Figure 12). Lymphangitic spread may be seen,

especially at autopsy. If multiple nodules are identified, one should consider the possibility of a metastatic process. Central carcinomas may be hilar/perihilar or completely endobronchial (Figure 13). These are grossly indistinguishable from squamous cell carcinomas. Atelectasis and/or postobstructive pneumonia are common findings.

Histopathology

Figure 12. Pseudomesotheliomatous adenocarcinoma. In the absence of a dominant lung mass, obliteration of the pleural space along with tumor extension along a fissure suggests a diagnosis of malignant mesothelioma.

(a)

Adenocarcinoma in situ (AIS), formerly known as BAC, is a morphologically striking lesion unique to the lung. At low magnification tumor cells appear to replace normal pneumocytes, hence the popularity of the term “replacement pattern adenocarcinoma” in many parts of the world (Figure 14).12 These carcinomas represent no more than 10% of worldwide lung adenocarcinomas, perhaps 15% in screening studies. Most are non-mucinous.138,139 Central areas are notably cellular, compared with the periphery, which is poorly defined owing to decreased cell density, height and cytological atypia. Thus, tumor cells blend with reactive type II pneumocytes at the tumor’s edge. These carcinomas feature crowded, uniform, cuboidal to columnar epithelial cells with mild to moderate cytological atypia sitting on alveolar walls (Figure 15). Cellular tufts may be seen but complex papillary structures indicate a papillary carcinoma. Single or clusters of tumor cells often float in alveolar ducts and sacs along with macrophages. Tumor cells resemble either Clara cells or type II pneumocytes. The former feature columnar cells with eosinophilic cytoplasm, apical granules and sometime apical nuclei. Type II-like cells are (b)

Figure 13. Central adenocarcinoma. (a) This carcinoma invades through visceral pleura into hilar adipose tissue and crosses the left major fissure. (b) Endobronchial adenocarcinoma arising from a lower lobe segmental bronchus virtually occludes the entire airway. Not surprisingly, the patient presented with cough. (Image courtesy of G. Ishii, MD, PhD, Chiba, Japan.)

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Chapter 27: Adenocarcinoma of the lung

(a)

(b)

Figure 14. Non-mucinous adenocarcinoma in situ. (a) Alveolar walls are lined by neoplastic cells. Parenchymal destruction is not seen. (b) Alveolar septa are often thickened in these lesions, yet inflammatory cell infiltrates are not usually striking.

Figure 16. Non-mucinous adenocarcinoma in situ. Although most lesions feature relatively bland cytological features, nuclear atypia can be striking.

Figure 15. Non-mucinous adenocarcinoma in situ. Malignant epithelial cells line alveolar walls and normal pneumocytes are not seen. Moderately atypical cells range from cuboidal to columnar and papillary fronds are not present.

cuboidal with clear to foamy cytoplasm and eosinophilic or clear intranuclear pseudoinclusions.140,141 Some tumors feature a combination of Clara cell and type II pneumocyte cell types. Nucleomegaly and prominent nucleoli are frequent and nuclear

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pleomorphism is not uncommon (Figure 16). Cytoplasmic glycogen or mucin can be seen in either cell type. Alveolar septa may be expanded with lymphoplasmacytic infiltrates or germinal centers. T-lymphocytes predominate.142 Septa may be fibrotic but are not markedly distorted (Figure 14b). Central scars may contain entrapped benign or neoplastic epithelium in angular arrangements (see below) (Figure 17). Surrounding lung often features a desquamative interstitial pneumonia (DIP)-like pattern and scattered non-necrotizing granulomas.

Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

Figure 17. Non-mucinous adenocarcinoma in situ. (a) This pure lepidic growth carcinoma is associated with parenchymal fibrosis and distortion. (b) Tumor cells line entrapped and distorted airspaces. (c) Entrapped glands may be difficult to distinguish from invasive carcinoma. The absence of desmoplasia and presence of intra-alveolar macrophages suggest a noninvasive process.

The new category of MIA is not well studied. This diagnosis probably applies to many part-solid nodules and tumors with large central scars. Since BAC was defined as a non-invasive carcinoma in the 1999 WHO classification, pathologists, including lung experts, have struggled to reproducibly identify small foci of stromal invasion in otherwise non-invasive lesions. Such cases may be classified in this subgroup. By definition MIA is a solitary, discrete and circumscribed 3.0 cm or smaller adenocarcinoma with a predominant lepidic pattern and stromal invasion (Figure 18). Multiple foci of invasion are permitted but no single focus should be larger than 0.5 cm. The invasive component must have a non-lepidic architecture or demonstrate single tumor cells infiltrating myofibroblastic stroma (Figure 19). A diagnosis of MIA cannot be considered in cases with tumor necrosis, or lymphatic, blood vessel or pleural invasion.

Most lung adenocarcinomas are invasive. They feature a mixture of histological subtypes, while smaller numbers have only one growth pattern (Figure 20). Over 80% of resected adenocarcinomas feature at least two and often three individual patterns, while less than 10% lack a lepidic component.143 The use of the WHO term “mixed subtype” does not convey particular information about tumor morphology and it is reasonable to report tumor types according to the most prevalent type observed and the approximate percentage of all subtypes present. Such may have therapeutic and/or prognostic importance.144–149 Recognized major subtypes are lepidic, acinar, papillary, micropapillary and solid predominant with mucin production (Table 1). Regardless of the subtype, several morphological points should be emphasized. Tumor cells range from cuboidal to

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Chapter 27: Adenocarcinoma of the lung

columnar or polygonal with moderate to abundant clear, eosinophilic or mucin-filled basophilic cytoplasm (Figure 21). Oncocytic or clear cell features may be extensive.150 Nuclei are

Figure 18. Microinvasive adenocarcinoma. This 2.2 cm non-mucinous adenocarcinoma features a peripheral lepidic pattern with a 0.4 cm area of parenchymal destruction.

(a)

large and vesicular with prominent nucleoli. Intranuclear pseudo-inclusions, indicative of type II pneumocyte differentiation, may be prominent (Figure 22). Clear cell and signet ring morphology are not exclusive to any subtype. Cilia are exceedingly rare.151 Extracellular mucin can manifest as intraluminal wisps or extensive acellular pools; the latter is a feature of mucinous adenocarcinoma (see below). Aside from the papillary subtype, invasive adenocarcinomas elicit stromal desmoplasia, and varying degrees of host inflammation, including neutrophils, eosinophils, lymphocytes and plasma cells. Germinal centers may be seen. Punctate and geographic necrosis, as well as secondary vasculitis and granulomatous inflammation, are not infrequent findings. Psammoma bodies, calcification and metaplastic bone are sometimes noted.152 The acinar subtype is the commonest pure subtype, representing almost half of all cases.149 Glands are round, oval or angulated and a cribriform pattern is occasionally observed (Figure 23).153 Cells cytologically resemble bronchial gland epithelium, bronchial lining epithelium or Clara cells.134,154,155 Signet ring morphology may be seen. Papillary predominant adenocarcinoma is the second most common lung adenocarcinoma subtype, representing 28% of all cancers in a recent large series.149 This tumor is composed of true papillary structures that replace alveolar architecture, or on rare occasion presents as an endobronchial mass. Architectural distortion may not be obvious, as the epithelial-lined fibrovascular cores resemble normal alveolar parenchyma (Figure 24a). Stromal invasion is not a prerequisite for (b)

Figure 19. Microinvasive adenocarcinoma. (a) Irregular malignant glands and nests infiltrate lung tissue. Desmoplasia is difficult to distinguish from fibroelastosis. As long as the largest focus of invasive carcinoma measures no more than 0.5 cm, the carcinoma is considered a good-prognosis lesion. (b) Individual cell nests indicate stromal invasion.

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Chapter 27: Adenocarcinoma of the lung

Figure 20. Invasive adenocarcinoma. Architectural and cytological heterogeneity are hallmarks of these carcinomas. This field features acinar (right) and solid (left) growth patterns along with focal clear cell cytology (upper left).

Figure 22. Invasive adenocarcinoma with type II pneumocyte features. Cuboidal cells display prominent intranuclear pseudoinclusions.

diagnosis as complex papillary cores may simply fill airspaces. The complex fibrovascular cores are often fibrotic with lymphoplasmacytic infiltrates. Crowded lining cells may be cudoidal or columnar with either clear or eosinophilic cytoplasm (Figure 24b). Mucinous differentiation is rare. Nuclei are large with vesicular chromatin and at least focally prominent nucleoli. Mitotic figures are apparent. Peculiar spindle cell morules and/or psammoma bodies as well as luminal necrosis may be seen (Figure 24c).156,157 The solid predominant adenocarcinoma is frequently identified in both small and large adenocarcinomas. Sheets, lobules

Figure 21. Invasive adenocarcinoma with Clara cell features. Columnar tumor cells have eosinophilic cytoplasm and occasional apical nuclei.

or small nests of tumor cells are recognizable as epithelial, owing to tumor cell cohesion (Figure 25a). Polygonal cells feature ample cytoplasm and overtly malignant nuclear features, including prominent nucleoli (Figure 25b,c). When it is the only subtype present in a tissue sample, intracellular mucin, confirmed with either a mucicarmine or periodic acid-Schiff stain with diastase, should be found in at least five tumor cells in each of two high-power fields. Lepidic predominant adenocarcinoma is uncommon and features the typically bland epithelial cells growing on alveolar septa, described above for AIS and MIA; however, this lesion also features at least one focus of invasive carcinoma measuring more than 0.5 cm. The invasive carcinoma is usually acinar pattern, but the other major subtypes can be seen. It is also distinguished from MIA by tumor necrosis, or lymphatic, blood vessel or pleural invasion. Micropapillary predominant adenocarcinoma is the rarest morphological pattern but has been elevated to a major subtype in the IASLC/ATS/ERS classification scheme owing to its aggressive behavior.145,149,158–163 This histology is usually seen at the periphery of carcinomas with significant amounts of papillary pattern and is characterized by small papillary tufts of tumor cells, either budding from alveolar septa or floating within alveolar spaces (Figure 26a). Ring-like structures may be noted (Figure 26b). This pattern lacks fibrovascular cores yet stromal and vascular invasion are common. The cells feature high nuclear/cytoplasmic ratios with eosinophilic cytoplasm (Figure 26c). This morphology may comprise a far greater percentage of tumor metastasis as compared with its percentage in the primary lung carcinoma.148,164

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Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

Figure 23. Invasive adenocarcinoma, acinar pattern. (a) Malignant glands feature cuboidal to columnar tumor cells with prominent nucleoli. (b) Intraluminal mucin is often seen in these carcinomas. (c) Clear cell change is not uncommon in gland-forming adenocarcinomas.

No well-established histological or cytological grading system exists for lung adenocarcinomas. The WHO suggests a three-tier system with well, moderate and poorly differentiated categories but strict criteria are lacking.5 No single detailed morphological grading system has met with worldwide approval. Several investigators including those involved in the IASLC/ATS/ERS classification believe architectural patterns hold the key to a prognostic grading scheme. Others believe a system combining particular histological findings, such as vascular invasion, with architectural patterns or pure nuclear criteria predict outcomes.148,149,165–169

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Cytology More than 50 years ago George Papanicolaou wrote that “the use of the cytological method in the diagnosis of malignant lesions of the respiratory tract has been generally acclaimed as one of its most successful applications”.170 While he might not have imagined that pathologists today should not issue a report with a diagnosis of non-small cell carcinoma before attempting to subtype the malignancy as either adeno- or squamous cell carcinoma (see below), the myriad non-invasive and minimally invasive procedures utilized to procure cytological material would not surprise him. Although lung adenocarcinomas may

Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

Figure 24. Invasive adenocarcinoma, papillary pattern. (a) Lung parenchyma is replaced by a complex papillary proliferation. (b) Columnar cells grow along fibrovascular cores. Areas may resemble lepidic growth but normal lung architecture is not seen. Luminal necrosis is obvious. (c) Crowded columnar tumor cells line fibrovascular cores. Intraluminal morules may be seen.

not exfoliate large numbers of diagnostic cells, technological advances, such as fine-needle aspirates (FNA), allow for relatively easy sampling and reliable diagnosis of peripheral pulmonary nodules and masses.171 Regardless of the en vogue classification scheme, one should be able to diagnose adenocarcinoma on most adequate sputum, bronchial washings, brushings, lavage samples, as well as FNA and associated cell block tissue.

While the literature is replete with information regarding the cytological distinctions between WHO subtypes, including the separation of invasive carcinoma from noninvasive lesions, clinically relevant distinctions cannot be made on cytological samples.172–175 Cytology literature, however, emphasizes the range of cytological atypia encountered in both exfoliated and needle samples.

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Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

Figure 25. Invasive adenocarcinoma, solid pattern. (a) At low magnification this well circumscribed carcinoma appears to fill alveolar spaces. (b) Cohesive tumor cells are monotonous with slightly basophilic cytoplasm and prominent nucleoli. (c) Signet ring cells are obvious in this sample. Intracellular mucin can be identified on routine sections but mucin stains may be required to document the presence of at least five tumor cells with mucin in two high-power (40) fields.

Lower-grade adenocarcinomas with cytology, often conforming to AIS, MIA and LPA, are composed of threedimensional ball-like clusters and papillary fronds with a prominent depth of focus lacking significant nuclear molding (Figure 27). These clusters have been referred to as “flowerpetal” or “cartwheel-like”.176 Septal arrangements and sheets may be seen in aspirates. Single cells are obvious and the slide

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background is usually clear. Single cells often resemble alveolar macrophages, which are often numerous in these samples. Nuclei are uniformly round to oval while slightly irregular nuclear contours may be seen. Chromatin is finely granular to powdery and nucleoli are inconspicuous (Figure 28). Optically clear nuclei, i.e. intranuclear pseudo-inclusions, can be noted. The nuclei may be eccentrically located within basophilic and finely

Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

Figure 26. Invasive adenocarcinoma, micropapillary pattern. (a) Malignant tumor cell clusters appear to bud off from lining cells. Fibrovascular cores are not seen. (b) Ring-like structures are frequently noted. Tumor cells rarely elicit a desmoplastic response. (c) Tumor cells feature high nuclear:cytoplasmic ratios and hyperchromatic nuclei. Cytoplasm is usually eosinophilic.

vacuolated cytoplasm but nuclei are not indented by the vacuoles. Cilia or psammoma bodies are exceedingly rare.177,178 Typically invasive adenocarcinomas also feature threedimensional cell balls, acinar groups, small irregular sheets and single cuboidal, columnar or round cells against a clean or necrotic slide background (Figure 29). Bronchial brushing samples may also contain large irregular sheets. Three-dimensional papillary fronds or syncytial arrangements may also be observed. A spectrum of nuclear and cytoplasmic features is noted as cells in a single sample may resemble macrophages in one field but pleomorphic carcinoma in another. Cells vary in size and shape and even groups may be discohesive. Nuclei

may have subtle to fantastically irregular contours while chromatin ranges from finely granular but clumpy to very coarse and hyperchromatic (Figure 30). Prominent nucleoli and nuclear molding are usual findings. Basophilic cytoplasm may be finely or coarsely vacuolated and nuclei are often eccentric and may be indented by the mucin vacuoles. The cytological differential diagnosis includes chemotherapy and radiation therapy effects, viral pneumonia, infarct and metastatic adenocarcinoma. On account of chemotherapeutic considerations, discerning pulmonary adenocarcinoma from squamous cell carcinoma is now very important. Although the older literature reports accuracy rates for non-small cell

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Figure 27. Adenocarcinoma, bronchoalveolar lavage sample. A threedimensional cluster of uniform tumor cells have a septal growth pattern. Note the clean background.

Figure 28. Adenocarcinoma, fine-needle aspirate sample. This sheet of tumor cells features a single enlarged cell with an intranuclear pseudoinclusion. Other cells have virtually identical nuclei and clear cytoplasm.

Figure 29. Adenocarcinoma, fine-needle aspirate sample. A threedimensional acinar group as well as a separate cluster of tumor cells is seen against the neutrophil-rich background. Such findings suggest an invasive process.

Figure 30. Adenocarcinoma, fine-needle aspirate sample. Tumor cells exhibit variation in cell size, nuclear size and chromatin pattern. Nucleoli are obvious. An intranuclear pseudoinclusion is also seen.

carcinoma subtyping in cytology specimens as low as 64%, current practices allow for a definitive morphological diagnosis in more than 90% of cases.179–183 This improvement is due to two factors. FNA samples are far more cellular than

exfoliative specimens and represent most lung cancer specimens today, and on site adequacy evaluation virtually ensures procurement of diagnostic tissue.184 Although intercellular bridges and keratin pearls are rare in cytological preparations,

Chapter 27: Adenocarcinoma of the lung

the Papanicolaou stain highlights keratin far better than hematoxylin and eosin-stained material. Thus, one should attempt to make a determination on smears. Squamous cell carcinomas feature inflamed and necrotic backgrounds, welldefined cell borders, thick orange to yellow cytoplasm and often pyknotic nuclei with rare nucleoli. Whenever possible, cytology should be evaluated in conjunction with any biopsy material. Finally, cytological samples are technically adequate for ancillary studies. Immunohistochemistry is useful in distinguishing adeno- from squamous cell carcinoma while mutational analysis of both localized and advanced lung carcinomas may influence the choice of chemotherapy regimen.184–190

Immunohistochemistry Virtually all non-mucinous pulmonary adenocarcinomas stain with AE1/AE3, CAM5.2, EMA, CK7, CK8 and CK19. CEA, Ber-EP4 and B72.3 stain up to 90% of lung adenocarcinomas. Napsin A and TTF-1, the most rigorously studied antibody, decorate from 60% to 80% of lung adenocarcinomas, respectively.191,192 Surfactant antibodies A and B stain no more than 60% of the tumors (see Chapter 26).192–195 Of note, from 20 to 40% of these carcinomas also stain with so-called squamous cell carcinoma markers K903, P63 and CK5/6.194,196 Antibodies associated with other organ site adenocarcinomas, such as WT1, calretinin, CD117, CDX2, villin, CA125, RCC, CD10, Hep-Par1, estrogen receptor, mammaglobin and GCDFP-15, may stain up to 20% of lung adenocarcinomas.197–203 These findings are important before deciding whether a lung carcinoma is a lung primary or metastasis. Neuroendocrine markers synaptophysin, chromogranin and CD56 stain up to 20% of non-neuroendocrine-appearing adenocarcinomas. The presence of positive neuroendocrine markers should not alter one’s light microscopic impression of adenocarcinoma (see Chapter 31).204,205 The most pressing issue in lung cancer immunohistochemistry pertains to the sub-classification of non-small-cell carcinomas on biopsy and cytology specimens. Since particular therapeutic agents are sub-type dependent, every effort should be made to correctly diagnose these lesions beyond the general category of non-small-cell carcinoma (see below; Treatment). Recent studies propose slightly different immunohistochemical panels, containing TTF-1, napsin A, p63 and/or CK5/6 and several also suggest using a mucin stain.188,194,206–211 Unlike current practice with extended panels for mesothelioma confirmation, in this instance one should pursue a limited marker panel to preserve small tissue samples for possible molecular studies. A stepwise approach, starting with a TTF-1 stain or TTF-1 and a napsin A double stain is reasonable.212 If either marker decorates tumor cells, one can favor a diagnosis of adenocarcinoma. If negative, then p63, CK5/6 and a mucin (mucicarmine or periodic acid-Schiff with diastase) stain should be performed. Since up to one-third of lung adenocarcinomas

stain with p63, diffuse positivity with this antibody only favors squamous cell carcinoma in the absence of TTF-1, napsin A staining or significant cytoplasmic mucin.196,202 Weak and focal p63 positivity in the absence of adenocarcinoma markers is not diagnostic of squamous cell carcinoma and one should diagnose such cases as non-small cell carcinoma.6,194 CK5/6 is less sensitive than p63 and should be considered a second tier choice.213–215 Lastly, if different tumor cell populations stain with TTF-1 and p63, a diagnosis of adenosquamous carcinoma is possible (see Chapter 29). Following such an algorithm allows one to subtype at least 80% of small biopsy samples lacking obvious morphological differentiation. When combined with cytology, some investigators claim over 95% of small samples can be accurately subtyped.216 When samples are studied with these antibodies, discordant results are reported in no more than 5% of biopsyresection pairs.188,189,194,206

Electron microscopy Ultrastructurally, non-mucinous lung adenocarcinomas display the same heterogeneity seen with the light microscope, as well as histochemical and immunohistochemical stains.217–219 Almost 30% of histologically pure carcinomas feature ultrastructural evidence of a second non-small cell carcinoma subtype.220 More than half of large cell carcinomas diagnosed on the basis of light microscopy feature adenocarcinomatous differentiation by ultrastructural evaluation. More than 15% of adenocarcinomas harbor neurosecretory granules.221,222 All pulmonary adenocarcinomas feature lumina bounded by tight junctions and lined with short microvilli.219,223 Endoplasmic reticulum, Golgi complexes, mitochondria and cytoplasmic secretory granules along with free intracytoplasmic mucin or glycogen are often seen.224 Clara cell differentiation manifests with apical bulbous cytoplasm filled with abundant smooth and rough endoplasmic reticulum and electron-dense finely granular granules. Type II pneumocyte differentiation features cytoplasmic lamellar granules and scattered intranuclear inclusions. The former are electron-dense, with osmiophilic whorls resembling surfactant granules. The latter are usually attached to the nuclear membrane and are immunohistochemically positive for surfactant protein.225–227 Intranuclear inclusions are not lung-specific.228 Lepidic adenocarcinomas appear indistinguishable from invasive adenocarcinomas on electron microscopy.217

Molecular findings The molecular pathology of lung adenocarcinoma has undergone major advances in the last decade. Discovery of specific mutations and translocations has allowed for improvements in classification, insights into pathogenesis and, most notably, development of agents that target activated molecular pathways (Table 6).

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Chapter 27: Adenocarcinoma of the lung Table 6 Molecular findings in lung adenocarcinomas

Gene

Overall frequency in adenocarcinoma

Gender

Smoking association

Histological association

Most common mutation variants

Mutations Mutually exclusive

EGFR

20% (higher in Asia)

F>M

NS/LS>S

Varied AIS, papillary/ micropapillary

L858R Exon 19 deletion (E746-A750)

Yes

KRAS

25% (lower in Asia)

? M>F

S>NS

Varied, mucinous

Transversion G12C G12V G12A Transition G12D

Yes

ALK

About 5%

Approximately F¼M

NS/LS>S

Acinar/cribriform, signet ring

Translocation 2p inversion

Yes

BRAF

M

S>NS

Papillary, micropapillary

V600E (exon 15) G469A, G469V, others (exon 11) Other exon 15

Yes

PIK3CA

stage I Prognosis Died of tumor (mean follow-up) Histopathological Arrangement of glands Necrosis Nuclear size Anisonucleosis Nuclear chromatin Nucleoli Morules Biotin-rich nucleus Stroma Amount Appearance Endocrine cells AFP-positive cells b-Catenin expression

Low-grade

High-grade

34 years Male
female 76% Usually early 10% Good 9.5% (70 months)

64 years Male > female 85% Often advanced 37% Poor 42% (24 months)

Orderly

Disorganized

Spotty if present

Commonly broad Large Obvious Dispersed

Small Mild Usually condensed Inconspicuous Common Common Slight to moderate Loose fibromyxoid Almost always Occasional Nuclear and cytoplasmic

Prominent Absent Uncommon Abundant Desmoplastic Common Always Membranous

Source: data from references 378,381,382,393,402.

primitive phenotype rather than a specialized airway epithelium.379 However, FLAC may also combine with typical lung adenocarcinoma, squamous cell carcinoma and small cell carcinoma. Familial FLAC is not recognized; however, a L-FLAC was reported in a family afflicted by familial adenomatous polyposis (FAP).380 b-Catenin, an oncogenic transcriptional activator for Wnt signaling associated with FAP, probably plays a role (see Chapters 22 and 32).381 Low-grade fetal adenocarcinoma (L-FLAC) represents the classic fetal adenocarcinoma. This carcinoma is slightly more common in women than men, and up to 25% are nonsmokers. Unlike typical adenocarcinomas, the mean age is a startling 34 years and 90% present with stage I disease (Table 8).378,382,383 Cases in children, as young as 10 years old, are reported.384,385 High-grade FLAC (H-FLAC) is more

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common in men and almost all patients smoke or smoked.378,382,383 This subgroup has a mean age of 64 years and no more than 40% of patients present with stage I disease. Patients, especially stage I individuals, are often asymptomatic and diagnosed during routine radiological examinations. Other symptoms include cough, shortness of breath, hemoptysis and chest pain.378,382,383 Radiographically, these carcinomas are most often peripheral well-defined homogeneous masses without calcification.386 Lobulation may be prominent.387,388 H-FLAC might feature heterogeneity due to tumor necrosis. Intense focal FDG-PET uptake has been noted and perhaps reflects significant glucose transporter expression in these tumors.387,389 Carcinomas measure from 1.0 to 10 cm with a mean diameter of 4.5 cm.378,382,383 A small percentage are endobronchial.390,391 Cut surfaces demonstrate an unencapsulated white, tan or brown fleshy tumor. Cystic change and hemorrhage may be seen. Low-grade FLAC and H-FLAC cannot be separated on the basis of gross findings. Morphologically, L-FLAC are usually well circumscribed and composed of glands resembling fetal lung tubules at 10 to 16 weeks gestation (Figure 43a,b,c). Branching tubules are lined with pseudostratified nonciliated columnar cells (Figure 43d). Subnuclear or supranuclear cytoplasmic vacuoles are prominent (Figure 43b,d). The clear cytoplasm represents glycogen and only rare intraluminal mucin can be seen. A cribriform pattern may be prominent while cords and ribbons with peripheral palisading and rosette-like structures are not uncommon. Nuclei are usually small, oval to round with mild anisonucleosis and condensed chromatin. Mitoses are frequent and necrosis may be focal (Figure 43e). Solid cell nests or morules, resembling squamous morules of endometrioid adenocarcinoma, are noted at the base of the glands (Figure 43d,f,g). These clusters of polygonal cells have finely granular eosinophilic cytoplasm with round to oval overlapping nuclei. Nuclear clearing is attributed to biotin accumulation; a phenomenon also seen in gestational endometrium and non-pulmonary neoplasms, including pancreatoblastoma.392–394 There may be little or moderate amounts of intervening myxoid stroma without desmoplasia or hypercellularity. Spindle cells are bland and minimal lymphoplasmacytic infiltrates are seen. Low-grade FLAC may occasionally show atypical glandular features, including poorly differentiated sheet-like growth, more enlarged and variously sized nuclei resembling those of conventional adenocarcinomas, and epithelial overgrowth beyond the stromal envelopment (Figure 44). It is not clear whether these atypical features are correlated with biological behavior of L-FLAC, but currently should be categorized as “L-FLAC with atypical histological features” (Y. Nakatani, personal communication). High-grade fetal adenocarcinoma features sheets and disorganized glands with abundant desmoplastic stroma and significant tumor necrosis (Figure 45). The glandular cells

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are larger than in L-FLAC with variation in size, dispersed nuclear chromatin and prominent nucleoli. Morules are not seen. Cytological features from fine-needle aspirates are welldefined. Moderately cellular aspirates feature flat sheets to three-dimensional cohesive spheres and irregular aggregates of cells with sharply defined borders. Acinar formation or at least nuclear palisading is often noted as well as less frequent true glands. Columnar tumor cells demonstrate distinct mostly subnuclear vacuoles as well as tigroid material on air-dried Romanowsky-stained smears but not with alcohol-fixed Papanicolaou-stained preparations.395,396 Nuclei are uniformly small with round to ovoid contours and finely granular chromatin and inconspicuous nucleoli. Cytoplasm is often scant, yielding a high nuclear:cytoplasmic ratio, and homogeneous, delicate and faintly basophilic. Sometimes a second cell population, i.e. morular cells, may be seen and is characterized by larger cells with denser cytoplasm, larger nuclei and more prominent nucleoli.397 The cytological differential diagnosis includes typical adenocarcinoma, carcinoid tumor, primitive neuroectodermal neoplasms and metastatic endometrial carcinoma. Conventional adenocarcinomas rarely feature subnuclear vacuoles and a second cell population, while the cells from a carcinoid tumor are usually round rather than columnar. Carcinoid nuclei feature granular so-called “salt and pepper” chromatin and samples are often more vascular than one sees with FLAC. Rare pulmonary primitive neuroectodermal neoplasms are perhaps indistinguishable on cytology, given the possible presence of columnar, neuroendocrine and even squamous forms. Immunohistochemical studies may be helpful (see below). Since young women rarely develop endometrial carcinoma, metastatic lesions in the FLAC age group are rare. Nuclei in these metastases are higher grade than one sees in L-FLAC. Immunohistochemical studies demonstrate slightly different staining profiles for low- and high-grade FLAC.378,382 Results derived from a biotin-avidin complex method should be interpreted cautiously as optically clear nuclei filled with biotin may yield false-positive staining. Low- and high-grade FLAC stain with cytokeratins, EMA and CEA while varying percentages of epithelial cells, mostly the morular cells, stain with TTF-1, GATA-6, Clara cell antigen and surfactant apoproteins.383,397–400 Morular cells may also stain with CDX2, suggesting, along with GATA-6 and TTF-1 reactivity, that the structure is simply an immature portion of the developing endodermal branching gland.401 H-FLAC, lacking morules, may not stain with these antibodies. All low-grade and approximately one-third of high-grade carcinomas always feature neuroendocrine differentiation evidenced by scattered chromogranin, synaptophysin and CD56 staining in occasional glandular and morular cells. Morules may also react with antibodies directed against specific amines and polypeptide hormones such as calcitonin, serotonin and somatostatin. High-grade FLAC, unlike most cases of L-FLAC,

Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

(d)

Figure 43. Low-grade fetal adenocarcinoma. (a) This well-circumscribed neoplasm is composed of glands and tubules. (b) Tubules are lined by pseudostratified epithelium with prominent cytoplasm vacuoles. Stroma is relatively inconspicuous. (c) Normal lung parenchyma at 16 weeks gestation. Subnuclear vacuoles are obvious in this sample. (d) Pseudostratified non-ciliated columnar tumor cells feature uniform oval nuclei. A small morule stands out with eosinophilic cytoplasm. (e) Rosettes (upper right) and patchy intraluminal necrosis may be seen. Necrosis alone does not distinguish between low-grade and high-grade lesions. Morules (bottom right) are not seen in high-grade lesions though. (f) Morules are usually located at the base of tumor glands. Eosinophilic cytoplasm and overlapping nuclei stand in contrast to the tubular structures lined by clear cytoplasm-rich columnar cells. (g) Morules may protrude into glandular lumens. Optically clear nuclei represent biotin accumulation. (Image courtesy of Y. Nakatani, MD, Chiba, Japan.)

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(e)

(f)

(g)

Figure 43. (cont.)

shows varying numbers of alpha-fetoprotein-positive cells.378 Vimentin and muscle markers are confined to stromal cells.382,383 Low-grade FLAC also features nuclear and cytoplasmic staining with b-catenin, especially in budding glands and morules, while H-FLAC demonstrates a membranous pattern.381 The L-FLAC pattern resembles the pattern in developing fetal

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lung airways.381 This finding suggests that upregulation of the Wnt signaling pathway driven in part by b-catenin gene mutations underlies low-grade but not high-grade fetal adenocarcinogenesis. Interestingly, mutations are noted in tumors with morules and biotin-rich nuclei such as pancreatoblastoma, the cribriform-morular variant of papillary thyroid carcinoma, and endometrioid adenocarcinomas (see Chapter 32).381,394,402

Chapter 27: Adenocarcinoma of the lung

(a)

(b)

Figure 44. Low-grade fetal adenocarcinoma with atypical histological features. (a) Even focal sheet-like growth is a rare histological finding. (b) Usually well-circumscribed, tumor extension into surrounding lung may be a worrisome finding. (Image courtesy of Y. Nakatani, MD, Chiba, Japan.)

Electron microscopy studies accompanied the original morphological description of this tumor.376 Glandular columnar cells are surrounded with a distinct basal lamina and feature apical junctional complexes and rare luminal microvilli. Osmiophilic lamellar bodies suggest type II pneumocyte differentiation. Scattered dense core granules are noted. Sub- or supranuclear glycogen is apparent. The glandular cells feature little differentiation toward mucous or ciliated cells; mucin-secreting granules are inconspicuous and peculiar round bodies with beaded borders may represent rudimentary intracellular components of primitive developing cilia. Morular cells are cohesive with cytoplasmic interdigitations and desmosomes. The cytoplasm contains abundant rough endoplasmic reticulum, mitochondria, lipid droplets, electrondense membrane-bound granules and neuroendocrine-type granules. Optically clear nuclei represent tightly packed 7- to 10-nm filaments.376,383,400 The histological differential diagnosis includes conventional adenocarcinoma with clear cell morphology, carcinoid tumors, pulmonary blastoma, and metastatic carcinomas with endometrioid features. Nodular endometriosis is a rare consideration. Suffice it to say, an unequivocal diagnosis of FLAC cannot be rendered on biopsy or cytology samples due to the possibility of tissue sampling artifact. For example, one may not adequately sample malignant stroma of pulmonary blastoma. The presence of morules distinguishes the L-FLAC from typical adenocarcinoma and carcinoid tumors, while blastoma and metastases feature high-grade cytology. H-FLAC is unique with its endometrioid appearance and high-grade nuclei. The absence of a malignant stromal component distinguishes H-FLAC from pulmonary blastoma.

The primary treatment modality is surgical excision, even for cases with chest wall or lymph node involvement. Chemotherapy and radiation therapy may be useful as palliative agents.382,403 Individuals with L-FLAC have a decidedly better outcome than patients with H-FLAC and typical lung adenocarcinoma. Although 30% of patients experience local recurrences, less than 10% die of disease.382,383,385 These statistics contrast with those with H-FLAC. This high-grade tumor is very aggressive. Approximately half of patients die of the malignancy regardless of presenting stage and performance status.378,382,383

Enteric adenocarcinoma While many unusual architectural and cytological features are seen in many lung adenocarcinomas, the IASLC/ATS/ERS classification only recognizes the enteric pattern as a variant. Although extremely rare as pure carcinomas, focal enteric morphology is not uncommonly encountered and may be diagnostically troublesome since it is easily confused with metastatic colorectal, small intestinal or sinonasal adenocarcinoma.363,404–408 These carcinomas feature epidemiological and clinical features not unlike those of typical adenocarcinomas. They affect both men and women, with a median age of 67 years.404,406 Patients present with cough, chest pain, hemoptysis or nonresolving pneumonia.363 Solitary spiculated lesions are noted on chest radiographs and CT scans. High FDG avidity on FDG-PET scans is noted.406,407 Tumors range from 1.0 to 7.0 cm, with a median size of 3.3 cm. Well-demarcated white-gray firm masses may feature yellow punctate necrosis.

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Chapter 27: Adenocarcinoma of the lung

(a)

(b)

(c)

Figure 45. High-grade fetal adenocarcinoma. (a) Disorganized glands without morules and necrosis are hallmarks of this rare subtype. (b) Tumor cells vary in size and display dispersed nuclear chromatin and occasionally prominent nucleoli. (Image courtesy of T. Kameya, MD, and H. Kondou, MD, Shizuoka, Japan.)(c) Microscopic areas resemble hepatocellular carcinoma. (Image courtesy of T. Kameya, MD, and H. Kondou, MD, Shizuoka, Japan.)

These non-mucinous carcinomas feature complex cribriform “gland in gland” patterns of varying sizes along with garlands and solid areas (Figure 46a). Tumor cells are pseudostratified columnar or cuboidal with oval to columnar nuclei, eosinophilic cytoplasm and brush borders (Figure 46b). Occasional Paneth and/or goblet cells may be seen. Mitoses are frequent. Stromal desmoplasia is not uncommon and lumina are filled with nuclear debris and necrotic material, i.e. dirty necrosis. These findings are also apparent on cytological specimens.

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The immunohistochemical profile is not entirely clear. Several reports note that these tumors only stain with CK7 and TTF-1, and rarely with napsin A, but more recent studies suggest that some may be only CK20-, MUC2- and CDX-2 positive.203,363,404–408 The IASLC/ATS/ERS scheme requires at least one marker of enteric differentiation to be positive or the tumor should be considered an adenocarcinoma with enteric morphology. One is unlikely to exclude a metastasis during an intraoperative frozen section and the patient may require clinical and

Chapter 27: Adenocarcinoma of the lung

(a)

(b)

Figure 46. Enteric adenocarcinoma. (a) This lung primary is histologically indistinguishable from intestinal or sinonasal carcinomas. (b) Cribriform architecture and luminal necrosis, so-called “dirty necrosis”, are obvious. Columnar cells may react with antibodies directed against CK20 and CDX-2.

radiological procedures to exclude an intestinal or upper aerodigestive tract primary. Although definitive data are lacking,

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