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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7

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

230

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 th