Cytology: Diagnostic Principles and Clinical Correlates [5 ed.] 0323636365, 9780323636360

Table of contents :
Cytology
Copyright
Dedication
Contributors
Preface
Acknowledgments
1 - Cervical and Vaginal Cytology
The History of the Pap Test and Its Current Practice
Screening Guidelines
Guidelines for Managing Women with Abnormal Pap Results
HPV Vaccination
Sampling and Preparation Methods
Smears
Liquid-Based Cytologic Study
ThinPrep Pap Test
SurePath Pap Test
Automated Screening
Historical Overview
ThinPrep Imaging System
BD FocalPoint Slide Profiler and Guided Screening Imaging System
Accuracy and Reproducibility
Diagnostic Terminology and Reporting Systems
The Bethesda System
Specimen Adequacy
General Categorization
Interpretation and Results
The Normal Pap
Squamous Cells
Endocervical Cells
Exfoliated Endometrial Cells
Abraded Endometrial Cells and Lower Uterine Segment
Trophoblastic Cells and Decidual Cells
Inflammatory Cells
Lactobacilli
Artifacts and Contaminants
Organisms and Infections
Shift in Flora Suggestive of Bacterial Vaginosis
Trichomonas Vaginalis
Candida
Actinomyces
Herpes Simplex Virus
Cytomegalovirus
Chlamydia Trachomatis
Rare Infections
Benign and Reactive Changes
Benign Squamous Changes
Benign Endocervical Changes
Repair
Radiation Changes
Cellular Changes Associated with Intrauterine Devices
Glandular Cells Status Post Hysterectomy
Other Benign Changes
Vaginal Specimens in “Des Daughters”
Squamous Abnormalities
Squamous Intraepithelial Lesions
Grading Squamous Intraepithelial Lesions
Low-Grade Squamous Intraepithelial Lesion
Management
High-Grade Squamous Intraepithelial Lesion
Management
Problems in the Diagnosis of Squamous Intraepithelial Lesions
Avoiding Overdiagnosis of LSIL
Distinguishing LSIL from HSIL
Distinguishing HSIL from Invasive Carcinoma
Squamous Cell Carcinoma
Atypical Squamous Cells
Atypical Squamous Cells of Undetermined Significance
Management
Atypical Squamous Cells, Cannot Exclude Hsil
Management
Glandular Abnormalities
Endocervical Adenocarcinoma in Situ
Adenocarcinoma
Endocervical Adenocarcinoma
Endometrial Adenocarcinoma
Differential Diagnosis of Adenocarcinoma
Atypical Glandular Cells
Atypical Endocervical Cells
Atypical Endometrial Cells
Other Malignant Neoplasms
Small Cell Neuroendocrine Carcinoma
Malignant Melanoma
Malignant Lymphoma
Carcinosarcoma
Metastatic Tumors
Endometrial Cells in Women ≥45 Years of Age
References
2 - Respiratory Tract and Mediastinum
Normal Anatomy, Histology, and Cytology of the Respiratory Tract
Sampling Techniques, Preparation Methods, Reporting Terminology, and Accuracy
Sputum
Bronchial Specimens
Bronchial aspirations and washings
Bronchial brushings
Bronchoalveolar lavage
Transbronchial FNA (“Wang Needle”)
Endobronchial Ultrasound-Guided (EBUS) FNA
Transesophageal FNA
Percutaneous FNA
Benign Cellular Changes
Reactive Squamous Cell Changes
Reactive Bronchial Cell Changes
Bronchial Reserve Cell Hyperplasia
Repair
Type II Pneumocyte Hyperplasia
Noncellular Elements and Specimen Contaminants
Infections
Viral Infections
Herpes Simplex
Cytomegalovirus
Measles Virus and Respiratory Syncytial Virus
Adenovirus
Bacterial Pneumonias
Tuberculosis
Pulmonary Fungal Infections
Cryptococcosis
Histoplasmosis
Blastomycosis
Coccidioidomycosis
Paracoccidioidomycosis
Sporotrichosis
Invasive Fungi
Aspergillosis
Zygomycosis
Candidiasis
Pneumocystis Jirovecii
Parasitic Infections
Strongyloidiasis
Dirofilariasis
Echinococcosis (Hydatid Disease)
Nonneoplastic, Noninfectious Pulmonary Diseases
Sarcoidosis
Granulomatosis with Polyangiitis
Pulmonary Amyloidosis
Pulmonary Alveolar Proteinosis
Common Inflammatory Processes, Including Organizing Pneumonia
Benign Neoplasms of the Lung
Pulmonary Hamartoma
Langerhans Cell Histiocytosis
Sclerosing Pneumocytoma
Papillomas and Related Lesions
Inflammatory Myofibroblastic Tumor
Endobronchial Granular Cell Tumor
Precursor Lesions of the Respiratory Epithelium
Lung Cancer
Molecular Testing of Lung Cancers
EGFR
MET
ERBB-2 (HER2)
ALK
ROS1
RET
BRAF
PIK3CA
KRAS
PD-L1
Squamous Cell Carcinoma
Adenocarcinoma
Large Cell Carcinoma
Sarcomatoid Carcinoma
Neuroendocrine Tumors
Typical Carcinoid Tumor
Atypical Carcinoid Tumor
Large Cell Neuroendocrine Carcinoma
Small Cell Carcinoma
Uncommon Pulmonary Tumors
Lymphoepithelioma-Like Carcinoma
Adenoid Cystic Carcinoma and Other Bronchial Gland Tumors
PEComa (“Clear Cell Tumor,” “Sugar Tumor”)
Sarcomas
Lymphomas and Leukemias
Metastatic Cancers to the Lung
Tumors of the Mediastinum
Thymoma
Thymic Carcinoma
Mediastinal Lymphomas
Germ Cell Tumors
NUT Carcinoma
References
3 - Urine and Bladder Washings
Indications
Specimen Collection
Voided Urine
Catheterized Urine
Bladder Washings
Upper Tract Washings and Brushings
Ileal Conduits
Sample Processing
Adequacy Criteria
Reporting Terminology
Accuracy
Normal Elements
Benign Lesions
Infections
Noninfectious Findings and Conditions
Crystals
Casts
Nonspecific reactive urothelial cell changes
Effects of radiation and chemotherapy
Urothelial atypia associated with urinary calculi
Other Benign Lesions
Nephrogenic adenoma
Müllerianosis
Urothelial Neoplasms
High-Grade Urothelial Carcinoma and Carcinoma in Situ
Atypical Urothelial Cells
Other Malignant Lesions
Other Primary Cancers of the Urinary Tract
Squamous cell carcinoma
Adenocarcinoma
Clear cell carcinoma
Small cell carcinoma
Melanoma
Paraganglioma
Sarcoma
Metastatic Cancers
Renal cell carcinoma
Prostatic carcinoma
Colonic carcinoma
Other metastatic tumors
Ancillary Techniques
Summary
References
4 - Pleural, Pericardial, and Peritoneal Fluids
Specimen Collection, Preparation, and Reporting Terminology
Accuracy
Benign Elements
Nonneoplastic Conditions
Acute Serositis
Eosinophilic Effusions
Lymphocytic Effusions
Rheumatoid Pleuritis
Lupus Pleuritis
Other Nonneoplastic Conditions
Malignant Effusions
Primary Tumors
Diffuse Malignant Mesothelioma
Mesothelioma versus Reactive Mesothelial Cells
Mesothelioma versus Adenocarcinoma
Mesothelioma versus Squamous Cell Carcinoma
Mesothelioma versus Vascular Tumors
Primary Effusion Lymphoma
Metastatic Tumors
Adenocarcinoma
Immunohistochemistry for ER, PR, and HER2
Molecular Testing of Effusions with Metastatic Lung Cancer
Squamous Cell Carcinoma
Small Cell Carcinoma
Melanoma
Non-Hodgkin Lymphoma
Hodgkin Lymphoma
Multiple Myeloma
Acute and Chronic Leukemias
Myeloproliferative Neoplasms
Sarcomas
Germ Cell Tumors
References
5 - Peritoneal Washings
Specimen Collection, Preparation, and Reporting Terminology
Accuracy
The Normal Peritoneal Washing
Benign Conditions
Endosalpingiosis and Similar Benign Proliferations
Endometriosis
Malignant Tumors
Ovarian and Fallopian Tube Cancers
Endometrial Cancer
Cervical Cancer
Pancreatic and Gastrointestinal Malignancies
Pancreatic Cancer
Gastric Cancer
Colorectal Cancer
Monitoring Response to Treatment (“Second-Look Procedures”)
References
6 - Cerebrospinal Fluid
Anatomy and Physiology
Obtaining and Preparing the Specimen
Reporting Terminology
Accuracy
Normal Elements
Abnormal Inflammatory Cells
Nonneoplastic Disorders
Acute Bacterial Meningitis
Aseptic Meningitis
Cryptococcal Meningitis
Toxoplasmosis
Cysticercosis
Angiostrongyliasis
Primary Amebic Meningoencephalitis
Neoplasms
Metastatic Solid Tumors
Carcinoma of the lung
Carcinoma of the breast
Melanoma
Leukemia
Acute lymphoblastic leukemia
Acute myeloid leukemia
Chronic lymphocytic leukemia
Myeloproliferative Neoplasms
Malignant Lymphoma
Primary CNS Tumors
Primary central nervous system lymphoma
Medulloblastoma
Anchor 513
Astrocytomas and oligodendroglial tumors
Ependymoma
Atypical teratoid/rhabdoid tumor
Choroid plexus tumors
Pineal tumors
Germ cell tumors
Other tumors of the central nervous system
References
7 - Gastrointestinal Tract
Clinical Indications
Sampling a Wider Area and Reaching Deep Organs
Better Recognition of Lymphoid Cells
Less Invasive
Shorter Turnaround Time
Sample Collection and Processing
Sample Collection
Processing the Sample
Accuracy
Review of Morphologic Findings
Esophagus
Infections
Epithelial Repair and Reactive Changes
Barrett’s Esophagus
Dysplasia in Barrett’s Esophagus
Adenocarcinoma
Squamous cell carcinoma
Uncommon Tumors of the Esophagus
Stomach
Infections
Epithelial Repair
Dysplasia and Gastric Adenomas
Adenocarcinoma
Neuroendocrine Neoplasms
Non-Hodgkin Lymphoma
Gastrointestinal Stromal Tumor
Duodenum
Infections
Adenoma and Adenocarcinoma
Colon
The Anal Pap Test
References
8 - Fine Needle Aspiration Biopsy Technique and Specimen Handling
Introduction
Materials and Supplies
Procedure for Performing FNA of a Palpable Mass
Determining Whether FNA Is Warranted
Obtaining Patient Consent
Sample Explanation of the Procedure
Readying the Equipment
Positioning the Patient and Immobilizing the Lesion
Sampling the Lesion
“Feeling With the Needle”
Preparing the Sample
Making Smears
The “one-smear” method (Fig. 8.2A–D)
The “two-smear” method (Fig. 8.3A–C)
Splitting Material for Multiple Smears
Method 1 (Fig. 8.4A–F)
Method 2
Fixing the Smears
Handling Cystic Masses
Retrieving Material from the Needle Hub
Rinsing the Needle and Reserving Material for Ancillary Studies
Making a Cell Block from a Smear
Postprocedure Information for the Patient
Variations on Biopsy Technique
Ultrasound-Guided Techniques
Perpendicular Technique
Parallel Technique
Complications
Management of Adverse and Unexpected Events
Acknowledgments
References
9 - Breast
Specimen Types
Fine-Needle Aspiration
Nipple Discharge
Sample Preparation
Reporting Terminology
Evaluation of the Specimen
The Normal Breast
Benign Conditions
Cysts
Fibrocystic Changes
Nonproliferative Fibrocystic Changes
Proliferative Fibrocystic Changes
Fibroadenoma
Pregnancy and Lactational Changes
Fat Necrosis
Radiation Change
Mastitis
Subareolar Abscess
Gynecomastia
Papillary Neoplasms
Phyllodes Tumor
Breast Cancer
Invasive Ductal Carcinoma
Invasive Lobular Carcinoma
Medullary Carcinoma
Mucinous (Colloid) Carcinoma
Tubular Carcinoma
Metaplastic Carcinoma
Uncommon Breast Tumors
Microglandular Adenosis
Apocrine Carcinoma
Adenoid Cystic Carcinoma
Non-Hodgkin Lymphoma
Peri-implant Seroma and Anaplastic Large Cell Lymphoma
Sarcoma
Metastatic Tumors
References
10 - Thyroid
Aspiration Technique and Slide Preparation
Terminology for Reporting Results
Nondiagnostic (ND) or Unsatisfactory
Benign
The “Indeterminate” Categories
Malignant
Accuracy
Molecular Testing
Evaluation of the Specimen
Benign Conditions
Benign Follicular Nodules
Chronic Lymphocytic (Hashimoto) Thyroiditis
Subacute (de Quervain) Thyroiditis
Riedel Disease
Amyloid Goiter
Black Thyroid
Radiation Changes
Suspicious for a Follicular Neoplasm or Follicular Neoplasm
Suspicious for a Follicular Neoplasm, Hürthle Cell Type
Malignant
Papillary Thyroid Carcinoma
Management
Poorly Differentiated Thyroid Carcinoma
Anaplastic Thyroid Carcinoma
Squamous Cell Carcinoma
Medullary Thyroid Carcinoma
Management
Primary Thyroid Lymphoma
Rare Primary Thyroid Tumors
Metastatic Tumors
Atypia of Undetermined Significance or Follicular Lesion of Undetermined Significance
Parathyroid Tumors
References
11 - Salivary Gland
Rationale, Indications, and Technical Considerations
Classification
Diagnostic Overview
The Normal Aspirate
Nonneoplastic Conditions
Acute and Chronic Sialadenitis
Granulomatous Sialadenitis
Sialadenosis
Lymphoepithelial Sialadenitis
Non-neoplastic Cysts
Squamous-Lined Cysts
Mucin-Containing Cysts
Amyloidosis
Benign Neoplasms
Pleomorphic Adenoma
Pitfalls Associated with Pleomorphic Adenomas
Sparse or Absent Matrix
Adenoid Cystic-Like Matrix
Cytologic Atypia
Metaplastic Changes
Myoepithelioma
Basal Cell Adenoma
Warthin tumor
Oncocytoma
Carcinomas of Salivary Gland Origin
Mucoepidermoid Carcinoma
Acinic Cell Carcinoma
Adenoid Cystic Carcinoma
Carcinoma Ex Pleomorphic Adenoma
Salivary Duct Carcinoma
Secretory Carcinoma
Polymorphous Adenocarcinoma
Rare Malignant Neoplasms
Basal Cell Adenocarcinoma
Epithelial-Myoepithelial Carcinoma
Clear Cell Carcinoma
Primary Small Cell Carcinoma
Lymphoepithelial Carcinoma
Adenocarcinoma, Not Otherwise Specified
Other Malignancies
Squamous Cell Carcinoma
Lymphoma Involving the Salivary Gland
Miscellaneous
Summary of Salivary Gland FNA
References
12 - Lymph Nodes
Technical Aspects
Reporting Terminology and Accuracy
Ancillary Studies
Flow Cytometry
Immunocytochemistry
Molecular Genetic Studies
Nonneoplastic Lesions
Reactive Hyperplasia (Without Specific Etiology)
Benign Lymphadenopathies with Specific Etiology but without Characteristic Fine-Needle Aspiration Findings
Inflammatory/Infectious Conditions with Characteristic Fine-Needle Aspiration Findings
Sarcoidosis
Bacterial and Fungal Lymphadenitis
Cat Scratch Disease
Mycobacterial Lymphadenitis
Rosai–Dorfman Disease (Sinus Histiocytosis with Massive Lymphadenopathy)
Kikuchi Lymphadenitis
Infectious Mononucleosis
HIV-Associated Lymphadenopathy
Dermatopathic Lymphadenitis
Silicone Lymphadenitis
Neoplasms
Hodgkin Lymphoma
Non-Hodgkin Lymphoma
Lymphomas of Small Cells
Follicular Lymphoma
Marginal Zone Lymphoma
Small Lymphocytic Lymphoma
Mantle Cell Lymphoma
Differential Diagnosis: Small Cell Lymphomas
Lymphomas of Large Cells
Diffuse Large B-cell Lymphoma
Variants of Diffuse Large B-Cell Lymphoma
High-Grade B-Cell Lymphoma
Burkitt Lymphoma
Plasmablastic Lymphoma
T-Cell Lymphomas
. Peripheral T-cell lymphoma of unspecified type (PTCL, NOS) is much more common in Asia than Europe or North America. In the Un...
. Anaplastic large cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma that was recognized after the discovery of the CD30...
. AITL is a typically an advanced stage lymphoma of T cells with a T follicular helper immunophenotype (e.g., expressing PD1, CD...
. Mycosis fungoides is a primary cutaneous T-cell lymphoma, but in advanced stages lymph nodes and viscera may be involved. Such...
. Adult T-cell leukemia/lymphoma is a disease of adult patients in endemic regions (including the Caribbean islands in the Weste...
. Lymphoblastic lymphoma is an aggressive lymphoma that comprises almost one-half of childhood non-Hodgkin lymphoma, is more com...
Posttransplant Lymphoproliferative Disorders
Differential Diagnosis: Large Cell Lymphomas
Nonlymphoid Neoplasms
Carcinomas
Malignant Melanoma
Seminoma/Germinoma
Sarcomas
References
13 - Liver
Normal Liver
Infections
Hepatic Abscess
Echinococcal Cyst (Hydatid Cyst)
Other Infections
Benign Lesions
Solitary Cysts
Cirrhosis
Focal Nodular Hyperplasia
Hepatic Adenoma
Bile Duct Hamartoma and Adenoma
Hemangioma
Angiomyolipoma
Malignant Tumors
Hepatocellular Carcinoma
Cholangiocarcinoma
Hepatoblastoma
Angiosarcoma
Epithelioid Hemangioendothelioma
Metastatic Tumors
References
14 - Pancreas and Biliary Tree
Indications
Sampling Techniques
Complications
Rapid On-Site Evaluation
Sample Preparation and Cyst Fluid Analysis
Accuracy and Limitations
Reporting Terminology
Normal Pancreas and Bile Duct
Pancreatitis and Reactive Changes
Pancreatic Intraepithelial Neoplasia
Ductal Adenocarcinoma
Variants of Ductal Adenocarcinoma
Neuroendocrine Neoplasms
Acinar Cell Carcinoma
Solid-Pseudopapillary Neoplasm
Pancreatoblastoma
Pancreatic Cysts
Pseudocyst
Serous Cystadenoma
Lymphoepithelial Cyst
Mucinous Cysts: Mucinous Cystic Neoplasm and Intraductal Papillary Mucinous Neoplasm
Secondary Pancreatic Neoplasms and Ectopic Splenic Tissue
References
15 - Kidney and Adrenal Gland
The Kidney
Indications and Sampling Methods
Specimen Collection and Preparation
Accuracy
Adequacy
Normal Elements
Glomeruli and Tubular Cells
Benign Lesions
Oncocytoma
Papillary Adenoma
Angiomyolipoma
Metanephric Adenoma
Mixed Epithelial and Stromal Tumor
Renal Abscess
Tuberculosis
Xanthogranulomatous Pyelonephritis
Renal Infarct
Renal Cysts
Other Benign Lesions
Renal Cell Carcinoma
Clear Cell Renal Cell Carcinoma
Papillary Renal Cell Carcinoma
Oncocytic Papillary Renal Cell Carcinoma
Chromophobe Renal Cell Carcinoma
Clear Cell Papillary Renal Cell Carcinoma
Tubulocystic Renal Cell Carcinoma
Renal Medullary Carcinoma
Collecting Duct Carcinoma (Bellini Tumor)
MiT Family Translocation Renal Cell Carcinoma
Mucinous Tubular and Spindle Cell Carcinoma
Urothelial Carcinoma
Lymphoma
Metastatic Tumors
Pediatric Tumors
Rare Entities
The Adrenal Gland
Specimen Collection, Preparation, and Accuracy
Myelolipoma
Adrenal Cortical Neoplasms
Pheochromocytoma
Metastatic Tumors
References
16 - Ovary
Obtaining the Specimen
Preparing the Specimen and Reporting Results
Accuracy
Benign Tumor-Like Lesions of the Ovary
Nonneoplastic Cysts
Cystic Follicle and Follicle Cyst
Corpus Luteum Cyst
Endometriotic Cyst
Simple Ovarian, Paraovarian, and Paratubal Cysts
Hydrosalpinx
Tuboovarian Abscess
Benign Surface Epithelial–Stromal Tumors
Benign Serous Tumors
Benign Mucinous Tumors
Benign Brenner Tumor
Malignant Surface Epithelial-Stromal Tumors
Serous Borderline Tumor and Serous Carcinoma
Mucinous Borderline Tumor and Mucinous Carcinoma
Endometrioid Carcinoma
Clear Cell Carcinoma
Germ Cell Tumors
Teratoma
Mature Teratoma
Immature Teratoma
Carcinoid Tumor
Dysgerminoma
Embryonal Carcinoma and Other Malignant Germ Cell Tumors
Sex Cord–Stromal Tumors
Granulosa Cell Tumors
Adult Granulosa Cell Tumor
Juvenile Granulosa Cell Tumor
Thecoma
Fibroma
Uncommon Primary Ovarian Tumors
Metastatic Tumors
References
17 - Soft Tissue
Specimen Collection and Preparation
Ancillary Studies
Reporting Terminology
Adipocytic and Lipogenic Neoplasms
Lipoma
Hibernoma
Spindle Cell/Pleomorphic Lipoma
Atypical Lipomatous Tumor/Well-Differentiated Liposarcoma
Pleomorphic Liposarcoma
Myxoid Neoplasms
Intramuscular Myxoma
Soft Tissue Perineurioma
Myxofibrosarcoma
Low-Grade Fibromyxoid Sarcoma
Myxoid Liposarcoma
Lipoblastoma
Myxofibrosarcoma-Like Dedifferentiated Liposarcoma
Myxoinflammatory Fibroblastic Sarcoma
Extraskeletal Myxoid Chondrosarcoma
Spindle Cell Neoplasms
Leiomyosarcoma
Schwannoma
Malignant Peripheral Nerve Sheath Tumor
Synovial Sarcoma
Solitary Fibrous Tumor
Desmoid (Deep) Fibromatosis
Nodular Fasciitis
Dermatofibrosarcoma Protuberans
Inflammatory Myofibroblastic Tumor
Adult Fibrosarcoma
Fibrohistiocytoid Neoplasms
Tenosynovial Giant Cell Tumor, Localized and Diffuse Types
Giant Cell Tumor of Soft Tissue
Angiomatoid Fibrous Histiocytoma
Round Cell Neoplasms
Neuroblastoma
Ewing Sarcoma
Desmoplastic Small Round Cell Tumor
Embryonal Rhabdomyosarcoma
Alveolar Rhabdomyosarcoma
Undifferentiated Round Cell Sarcomas, including CIC-Rearranged Sarcoma
Epithelioid Neoplasms
Epithelioid Sarcoma
Clear Cell Sarcoma of Soft Tissue
Alveolar Soft Part Sarcoma
Epithelioid Hemangioendothelioma
Pseudomyogenic Hemangioendothelioma
Epithelioid Angiosarcoma
Granular Cell Tumor
Pleomorphic Neoplasms
Undifferentiated Pleomorphic Sarcoma
Pleomorphic Rhabdomyosarcoma
Dedifferentiated Sarcomas
Nonneoplastic Soft Tissue Lesions
Idiopathic Retroperitoneal Fibrosis
Elastofibroma
Amyloidoma (Tumoral Amyloidosis)
References
18 - Bone
Introduction
Cartilaginous Tumors
Chondroblastoma
Chondromyxoid Fibroma
Enchondroma
Chondrosarcoma and Chondrosarcoma Variants
Giant Cell–Rich Tumors
Giant Cell Tumor of Bone
Aneurysmal Bone Cyst
Osteogenic Tumors
Osteoblastoma
Conventional Osteosarcoma and Osteosarcoma Variants
Low-Grade Osteosarsarcoma
Notochordal Tumors
Benign Notochodal Cell Tumor
Chordoma
Tumors of Other Lineages
Adamantinoma
Vascular Tumors
Hematopoietic and Histiocytic Neoplasms
Plasmacytoma/Myeloma and Non-Hodgkin Lymphoma
Langerhans Cell Histiocytosis
Nonneoplastic Bone Lesions
Metastasis
References
19 - Laboratory Management
Agencies and Organizations
Centers for Medicare and Medicaid Services
The Joint Commission
College of American Pathologists
Commission on Accreditation of Allied Health Education Programs
Occupational Safety and Health Administration
National Fire Protection Association
Regulations
Clinical Laboratory Improvement Amendments of 1988
Health Insurance Portability and Accountability Act of 1996 (HIPAA)
Laboratory Personnel
Laboratory Director
Technical Supervisor
General Supervisor
Cytotechnologist
Policy and Procedure Manuals
Workflow
Billing
Procedure Codes
. This is the most widely used in pathology. It denotes that only the physician professional component of the service is being b...
. This modifier denotes a reduced service from the customary procedure. In cytology, a good example is the manual review of a sl...
. Modifier 59 denotes a “separate procedure,” such as a different specimen (e.g., washing vs brushing) or anatomic site. Payers ...
. Teaching physicians must append modifier GC to CPT and HCPCS codes on Medicare claims when a resident or fellow actively parti...
. These modifiers are applied to Pap test HCPCS codes when billing Medicare. They clarify the laboratory’s right (or lack thereo...
. This modifier denotes the facility technical component of the service being billed, and thus is the counterpart of the CPT 26 ...
International Classification of Diseases (ICD)-10-CM Codes
Coding Pap Tests
The Screening (Routine) Pap Test
The Screening (High-Risk) Pap Test
The Diagnostic Pap Test
Coding Nongynecologic, Non-Fine-Needle Aspiration Cases
Coding Fine-Needle Aspirates
Coding Consultation Cases
Quality Control and Quality Assurance
Prospective 10% Rescreen
Retrospective Rescreen (“5-Year Lookback”)
Cytologic-Histologic Correlation
Annual Statistics
Workload Records
Competency Assessment
Proficiency Testing
General Description of Cytology PT
Scoring
Results
Performance Evaluation
Measures of Cytotechnologist Performance
Screening Skills
. Inevitably, when enough cases interpreted as negative by one CT are rescreened by another, abnormal cells are identified in so...
. The abnormal rate is the percentage of abnormal cases (ASC, AGC, SIL, and carcinoma) diagnosed by a CT divided by the total nu...
Interpretive Skills
. CLIA 88 requires that the performance of a CT be based, in part, on an evaluation of the cases submitted to the pathologist fo...
. The unsatisfactory rate is the proportion of all Pap test results that are interpreted as unsatisfactory by a CT. A low unsati...
Measures of Cytopathologist Performance
Atypical Squamous Cells-to-Squamous Intraepithelial Lesion Ratio
High-Risk Human Papillomavirus Positivity Rates for Atypical Squamous Cells of Undetermined Significance
Cytology/Biopsy Correlation
Safety
OSHA Bloodborne Pathogens Standard
Exposure Control
Hepatitis B Vaccination
Communication of Hazards to Employees
Recordkeeping
OSHA Laboratory Standard
National Fire Protection Association Standard for Health Care Facilities
NFPA Standard on Fire Protection for Laboratories Using Chemicals
References
content

Citation preview

Cytology Diagnostic Principles and Clinical Correlates

Fifth Edition

Edmund S. Cibas, MD Professor of Pathology Harvard Medical School; Director of Cytopathology Department of Pathology Brigham and Women’s Hospital Boston, Massachusetts

Barbara S. Ducatman, MD Chief, Pathology and Laboratory Medicine Service Line Physician Executive, Beaumont Medical Group Beaumont Health; Chair of Pathology Beaumont Hospital-Royal Oak Royal Oak, Michigan; Professor and Chair of Pathology Oakland University William Beaumont School of Medicine Rochester, Michigan

Elsevier 1600 John F. Kennedy Blvd. Ste 1800 Philadelphia, PA 19103-2899

CYTOLOGY: DIAGNOSTIC PRINCIPLES AND CLINICAL CORRELATES, FIFTH EDITION Copyright © 2021 by Elsevier Inc. All rights reserved. Previous edition copyrighted 2014.

ISBN: 978-0-323-63636-0

No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein).

Notice Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds or experiments described herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made. To the fullest extent of the law, no responsibility is assumed by Elsevier, authors, editors or ­contributors for any injury and/or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. Library of Congress Control Number: 2019943432

Content Strategist: Michael Houston Content Development Specialist: Laura Klein Publishing Services Manager: Shereen Jameel Senior Project Manager: Kamatchi Madhavan Design Direction: Amy Buxton Printed in Canada Last digit is the print number:  9 8 7 6 5 4 3 2 1

To Todd Bryant Stewart and Alan M. Ducatman

v

Contributors

Gamze Ayata, MD Assistant Professor of Pathology Harvard Medical School; Pathologist Beth Israel Deaconess Medical Center Boston, Massachusetts Kurt D. Bernacki, MD System Medical Director, Surgical Pathology Beaumont Health; Medical Director, Surgical Pathology Beaumont Hospital- Royal Oak Royal Oak, Michigan; Assistant Professor of Pathology Oakland University William Beaumont School of Medicine Rochester, Michigan

Christopher A. French, MD Associate Professor of Pathology Harvard Medical School Brigham and Women’s Hospital Boston, Massachusetts Vickie Y. Jo, MD Assistant Professor of Pathology Brigham and Women’s Hospital Harvard Medical School Boston, Massachusetts Jeffrey F. Krane, MD, PhD Professor of Pathology David Geffen School of Medicine at UCLA Los Angeles, California

Edmund S. Cibas, MD Professor of Pathology Harvard Medical School; Director of Cytopathology Brigham and Women’s Hospital Boston, Massachusetts

Amy Ly, MD Assistant Professor of Pathology Harvard Medical School; Director of Fine Needle Aspiration Biopsy Service, Pathology Massachusetts General Hospital Boston, Massachusetts

Barbara S. Ducatman, MD Chief, Pathology and Laboratory Medicine Service Line Physician Executive, Beaumont Medical Group Beaumont Health; Chair of Pathology Beaumont Hospital-Royal Oak Royal Oak, Michigan; Professor and Chair of Pathology Oakland University William Beaumont School of Medicine Rochester, Michigan

Vaishali Pansare, MD Medical Director, Outreach Beaumont Health; Chief of Pathology Beaumont Hospital-Grosse Pointe and Beaumont Hospital-Troy Grosse Pointe and Troy, Michigan; Associate Professor of Pathology Oakland University William Beaumont School of Medicine Rochester, Michigan

William C. Faquin, MD, PhD Professor of Pathology Harvard Medical School Massachusetts General Hospital; Director, Head and Neck Pathology Massachusetts Eye and Ear Infirmary Boston, Massachusetts

Martha Bishop Pitman, MD Professor of Pathology Harvard Medical School; Director of Cytopathology Massachusetts General Hospital Boston, Massachusetts

vi

Contributors

Xiaohua Qian, MD, PhD Assistant Professor of Pathology Harvard Medical School; Director, Fine-Needle Aspiration Biopsy Service Brigham and Women’s Hospital Boston, Massachusetts Andrew A. Renshaw, MD Pathologist Baptist Hospital and Miami Cancer Institute Miami, Florida Marina Vivero, MD Assistant Professor of Pathology Harvard Medical School; Associate Pathologist Brigham and Women’s Hospital Boston, Massachusetts

Paul E. Wakely, Jr., MD Professor of Pathology The Ohio State University College of Medicine Wexner Medical Center Columbus, Ohio Helen H. Wang, MD, DrPH Professor of Pathology Harvard Medical School; Pathologist Beth Israel Deaconess Medical Center Boston, Massachusetts Tad J. Wieczorek, MD Instructor in Pathology Harvard Medical School; Pathologist Brigham and Women’s Hospital Boston, Massachusetts

vii

Preface

We hope this book will serve as a useful guide for the pathologist in practice and for the trainee—resident or fellow—who is looking to obtain expertise in the subspecialty of cytopathology. It has been 6 years since the publication of the fourth edition of Cytology: Diagnostic Principles and Clinical Correlates. Since then, cytology has continued to grow and evolve as a discipline devoted to the diagnosis of cellular tissue obtained by minimally invasive methods (e.g., scraping, brushing, aspiration), thus the need for this updated edition. We have retained many of the qualities of the prior editions. This edition again aims to be concise yet comprehensive. We have emphasized brevity and clarity. The text is grounded in an understanding of surgical pathology and current diagnostic terminology. Where relevant, we have illustrated the value of established ancillary studies. Although the book is multiauthored, the chapters follow a similar format: indications, sample collection and preparation methods, terminology for reporting results, accuracy (including common pitfalls that lead to false-negative and false-positive diagnoses), a description of normal elements, and, finally, a how-to guide for the diagnosis of benign and malignant lesions, with an emphasis on differential

viii

diagnosis. We have retained the bulleted “capsule summaries,” particularly for summarizing cytomorphologic features and differential diagnoses. We have continued to emphasize clinical correlation (hence, the title) because good cytologists are those who understand the clinical implications of their interpretations. A major enhancement of the prior edition was the inclusion of a dedicated chapter on fine-needle aspiration technique and specimen handling, accompanied by a video demonstration. This fifth edition is enhanced by a new chapter on bone cytology that we trust readers will find useful. Once again, we hope we have conveyed the beauty, strength, and challenge of cytology. With this book we have strived to take some of the mystery out of cytology, but mysteries remain, their solutions still obscure. If this text inspires the reader to explore and even solve some of them, we will consider ourselves doubly rewarded. Edmund S. Cibas, MD Barbara S. Ducatman, MD 2019

Acknowledgments

We owe a great debt to many individuals for their help with this book. To Bill Schmitt, Michael Houston, Lauren Klein, Anne Snyder, Humayra Rahman Khan, and Kamatchi Madhavan at Elsevier, who shepherded this book gently to completion: a thousand thank-yous. You exemplified the spirit of teamwork, and we enjoyed working with each of you. Maryann Kennedy’s administrative skills and hard work at the Brigham and Women’s Hospital contributed immeasurably to this edition. Thanks also to Sandy George and Deanna Reynolds at West Virginia University, who were invaluable in providing their assistance. We extend our thanks to Olga Pozdnyakova, MD, PhD, for her contributions to the video that accompanies Chapter 8. We also thank Jessica L. Wang, MD, and Bonnie Choy, MD, for their assistance with the visual material for this chapter. Mark Rublee and David Sewell (Motion Video, Philadelphia, Pa.), who shot and edited the video, were indispensable, and we thank them for the high standards and professionalism they brought to the project. We are grateful to Dr. David Wilbur for his review, with helpful suggestions, of the Automated Screening section of Chapter 1. Ryan Callaghan, Global Product Manager for Women’s Health & Cancer at BD Life Sciences, kindly contributed the BD FocalPoint images in Chapter 1. We express our deep appreciation to Brenda Cox of Cox Consulting for her help with the complexities of billing in Chapter 19. Ms. Cox is editor of the American Pathology Foundation’s Pathology Service Coding Handbook, which

served as a resource for information set forth in this chapter. Readers who want more information on pathology coding can contact Ms. Cox (Cox Consulting) at brenda@cox consultant.onmicrosoft.com for information. Subscription information for the Pathology Service Coding Handbook can be obtained at www.apf-connect.org. We are indebted to many members of the staff of the Brigham and Women’s Hospital and West Virginia University School of Medicine and Hospital—the cytotechnologists, cytopathologists, and trainees—who inspire us with their devotion to cytopathology and who continue to challenge us. In particular, we acknowledge Kristen DeRobbio, CT (ASCP), Dorothy Nappi, CT (ASCP), and Grace Goffi, CT, MIAC, who have helped us train so many pathology residents and fellows over the years. Without their help we would not have our extraordinary collections of cytology teaching cases from which so many of the images in this book are derived. Finally, to our friends, families, and loved ones, especially Todd Stewart and Alan Ducatman, who tolerated the long evening and weekend hours that deprived them (temporarily!) of a large share of our time. This book would not exist without their love, support, and strength. Edmund S. Cibas Barbara S. Ducatman

ix

1

Cervical and Vaginal Cytology EDMUND S. CIBAS

T

he 20th century witnessed a remarkable decline in the mortality from cervical cancer in developed countries, an achievement attributable to the implementation of the Papanicolaou (Pap) test. In the 1930s, before Pap test screening was introduced, cervical cancer was the most common cause of cancer deaths in women in the United States.1 Today it is not even in the top 10, but it has not been eradicated; there are still approximately 13,000 new cases of cervical cancer in the United States each year, with 4000 deaths.2 Worldwide, cervical cancer incidence (over 500,000 cases annually) and mortality (over 300,000 deaths per year) rank fourth, after breast, lung, and colorectal cancers.3,4 Indeed, in some countries (e.g., those in sub-Saharan Africa), cervical cancer remains the most common type of cancer in women. Unfortunately, screening programs are rudimentary or nonexistent in many parts of the world: fewer than 5% of women in developing countries have ever had a Pap test.5 In contrast, 89% of women in the United States report having had a Pap test in the preceding 5 years.6 Around the world, Pap test screening is implemented in two different ways, commonly referred to as opportunistic versus organized. An organized screening program is planned at the national or regional level. It specifies a target population and screening intervals and has a mechanism for inviting women to attend screening services, informing them of their result, and referring them for treatment. Opportunistic screening, the system in place in the United States, for example, is done independently of an organized or population-based program, often for women who are visiting health services for other reasons. Screening is recommended during a consultation or requested by the woman. Opportunistic screening tends to reach younger, lower-risk women who are attending family planning and antenatal services. Organized screening is more cost-effective than opportunistic screening, making better use of available resources and ensuring that the greatest number of women benefit.

The History of the Pap Test and Its Current Practice The Pap test is the most successful cancer reduction program ever devised.1,7 Credit for its conception and development

goes to George N. Papanicolaou, an anatomist and Greek immigrant to the United States. In 1928 he reported that malignant cells from the cervix can be identified in vaginal smears.8 Later, in collaboration with the gynecologist Herbert Traut, who provided him with a large number of clinical samples, Papanicolaou published detailed descriptions of preinvasive cervical lesions.9,10 Pathologists and clinicians initially greeted this technique with skepticism, but by the late 1940s Papanicolaou’s observations had been confirmed by others. The Canadian gynecologist J. Ernest Ayre suggested taking samples directly from the cervix with a wooden spatula rather than from the vagina with a pipette as originally described by Papanicolaou.11 Eventually, cytologic smears were embraced as an ideal screening test for preinvasive lesions, which, if treated, would be prevented from developing into invasive cancer. The first cervical cancer screening clinics in the United States were established in the 1940s.12 Although it was not evaluated in a controlled, prospective study, several pieces of evidence link the Pap test to the prevention of cervical cancer. First, the mortality rate from cervical cancer fell dramatically after screening was introduced, by 72% in British Columbia13 and 70% in Kentucky.14 Second, there was a direct correlation between the intensity of screening and the decrease in mortality. Among Scandinavian countries, the mortality rate fell by 80% in Iceland, where screening was greatest; in Norway, where screening was lowest, the mortality rate fell by only 10%.15 A similar correlation was observed in high and low screening regions of Scotland16 and Canada.17 In the United States, the decrease in deaths from cervical cancer was proportional to the screening rates in various states.18 Finally, women who do not develop invasive cancer are more likely to have had a Pap test than women with cancer. In a Canadian study the relative risk for women who had not had a Pap test for 5 years was 2.7,19 and screening history was a highly significant risk factor independent of other factors such as age, income, education, sexual history, and smoking. In Denmark a woman’s risk of developing cervical cancer decreased in proportion to the number of negative smears she had had: by 48% with just one negative smear, 69% with two to four negative smears, and 100% with five or more smears.20 More recently, prospective studies from Japan and Sweden have provided 1

2 CHA P T ER 1    Cervical and Vaginal Cytology

additional evidence that the Pap test significantly reduces the incidence of and mortality from cervical cancer.21,22

Screening Guidelines Cervical cancer screening guidelines differ around the world. In the United States, recommendations are issued by the American College of Obstetricians and Gynecologists,23 the US Preventive Services Task Force,24 and a consortium of the American Cancer Society, the American Society for Colposcopy and Cervical Pathology (ASCCP), and the American Society for Clinical Pathology.25 Their guidelines differ in minor ways, but there is general agreement on the larger points, including longer screening intervals and a later age to start screening (age 21) than had been recommended in the past (Table 1.1). For women 21 to 29 years of age, they recommend a Pap test every 3 years. For women between ages 30 to 65, there are several options: a Pap test alone every 3 years; a test for high-risk types of the human papillomavirus (HPV) every 5 years; or a “cotest” (a Pap plus an HPV test) every 5 years. The guidelines address women with an average risk for cervical cancer. Women at higher risk—those with a history of cervical cancer, in utero diethylstilbestrol (DES) exposure, and immunocompromise (due to organ transplantation, chemotherapy, long-term corticosteroid treatment, or infection with the human immunodeficiency virus [HIV])—may benefit from more frequent screening. Because women with human immunodeficiency TABLE   Cervical Cancer Screening Guidelines in the 1.1  United States (for Women at Average Risk)

Circumstance

Recommendation

Age to begin screening

Age 21. Women younger than age 21 should not be screened, regardless of the age of sexual initiation

Women aged 21 to 29

Every 3 years with cytology (liquidbased or smears) alone

Women aged 30 to 65

Every 3 years with cytology alone, or every 5 years with HPV, or every 5 years with cytology and HPV (“cotest”)

Discontinuation of screening

Age 65 years if adequate prior screening and no history of CIN 2 or higher*

Screening after total hysterectomy

Not recommended if no history of CIN 2 or higher

*ACOG and ACS/ASCCP/ASCP define “adequate prior screening” as three consecutive negative cytology results or two consecutive negative cotest results within the previous 10 years, with the most recent test performed within the past 5 years. “No history of CIN 2 or higher” defined by ACS/ASCCP/ASCP as within the last 20 years. ACOG=American College of Obstetrics and Gynecology; ACS/ASCCP/ ASCP= American Cancer Society/American Society for Colposcopy and Cervical Pathology/American Society for Clinical Pathology; CIN 2= cervical intraepithelial lesion grade 2

virus/acquired immunodeficiency syndrome have higher rates of cervical cancer than the general population, it is recommended that HIV-seropositive women have a Pap test twice during the first year after diagnosis of HIV infection and, if the results are normal, annually thereafter.26 Adherence to screening guidelines is critical for cervical cancer prevention: in Sweden, for example, women who had not had a Pap test within the recommended screening interval had a higher risk of cervical cancer than those who had been screened (odds ratio 2.52).27 Because of the strong association between HPV and cervical neoplasia (see “Squamous Intraepithelial Lesions” below), a test for high-risk HPV types can be used by itself as an initial screen (so-called “primary HPV screening”). If positive, partial genotyping (for HPVs 16 and 18) and Pap cytologic study are performed; a referral to colposcopy is recommended if genotyping shows HPV 16 or 18, if the Pap cytology result is abnormal, or if a subsequent HPV test shows persistence of virus.28 There is evidence that primary HPV screening is equivalent or superior to screening algorithms based on Pap cytologic study,29-31 and the US Food and Drug Administration (FDA) has approved the Roche cobas HPV Test and the BD Onclarity HPV Assay for primary HPV screening. The International Agency for Research on Cancer and the World Health Organization have endorsed primary HPV testing, and in 2017 the Netherlands was the first country to implement it nationwide. 

Guidelines for Managing Women with Abnormal Pap Results In 2012, the ASCCP revised its guidelines for the management of women with abnormal cervical cytologic study, HPV, and histopathologic results.32 These guidelines, mentioned throughout this chapter in the relevant sections, apply only to women whose abnormalities are detected during screening. Management is individualized for women with postcoital or unexplained abnormal vaginal bleeding, pelvic pain, abnormal discharge, or a visible cervical lesion. 

HPV Vaccination The development of prophylactic HPV vaccines has opened a new opportunity for cervical cancer prevention. The vaccines consist of empty protein shells called virus-like particles that are made up of the major HPV capsid protein L1; they contain no DNA and are not infectious. Two of the vaccines are Gardasil and Gardasil 9 (Merck & Co., Inc.). Gardasil is a quadrivalent vaccine against HPV types 6, 11, 16, and 18, and Gardasil 9 is a nine-valent vaccine against HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58. A third vaccine—Cervarix (GlaxoSmithKline)—withdrew from the US market in 2016 due to low market demand. They have all shown extraordinary efficacy in preventing type-specific histologic CIN 2,3 lesions, with no difference in serious adverse effects compared with placebo.33,34 The American Cancer Society recommends routine HPV vaccination for girls and boys ages 11 and 12

CHAPTER 1  Cervical and Vaginal Cytology

years. It also recommends the vaccine to males and females ages 13 to 26 to “catch up,” having missed the opportunity to be vaccinated at the recommended age.35 In 2016, it was estimated that 65% of girls and 56% of boys aged 13 to 17 years in the United States had initiated HPV vaccination.36 Of note, screening guidelines are no different for the vaccinated population as compared with those not vaccinated. Continued screening, even for the vaccinated population, remains important because (1) these vaccines do not protect against all cervical cancers; (2) the duration of protection is unknown; (3) they are not effective in treating prevalent HPV infections; and (4) the cost of the vaccines might limit their use in some populations. 

Sampling and Preparation Methods To obtain an ideal Pap specimen, the American Cancer Society recommends the following patient instructions7: PATIENT INSTRUCTIONS • T  ry not to schedule an appointment for a time during your menstrual period. The best time is at least 5 days after your menstrual period stops. • Don’t use tampons, birth-control foams, jellies, or vaginal creams, lubricants, or medications for 2 to 3 days before the test. • Don’t douche for 2 to 3 days before the test. • Don’t have vaginal sex for 2 days before the test.   

Once the patient is positioned, a bivalve speculum of appropriate size is gently inserted into the vagina.37 SPECIMEN COLLECTION • T  he speculum can be lubricated with warm water or sparingly applied water-soluble lubricant. • Excess mucus or other discharge should be removed gently with a cotton swab. • The sample should be obtained before the application of acetic acid or Lugol’s iodine. • An optimal sample includes cells from the ectocervix and endocervix.   

Water-soluble gel lubricant, if used, should be applied sparingly to the posterior blade of the speculum, avoiding the tip: excessive lubricant can result in an unsatisfactory specimen.37-41 When visible, different lubricants have different effects and different appearances on cytologic preparations.41-43 It can be helpful to check any guidelines issued by the manufacturers of liquid-based cytology instruments with regard to recommended lubricants. In particular, lubricants containing carbomers or Carbopol polymers have a deleterious effect on ThinPrep (Hologic, Malborough, MA) slides and should be avoided.44 There are no clinically important differences between smears and liquid-based cytology methods, and therefore either is considered acceptable for cytologic screening.24,25

3

Smears Smears are often prepared using the combination of a spatula and brush. The spatula is used first. Although a wooden or plastic spatula is acceptable, the plastic spatula is recommended because wooden fibers trap diagnostic material.37 The spatula is rotated at least 360 degrees. The sample can be smeared on half of a slide and spray fixed (the other half should be covered to avoid coating it with fixative before the endocervical sample is applied). Alternatively, one may set aside the spatula sample momentarily while the endocervical brush sample is obtained. After the brush is inserted in the endocervical canal, some bristles should still be visible. If inserted too far, there may be inadvertent sampling of the lower uterine segment (LUS), which causes diagnostic difficulties because its epithelium resembles a high-grade squamous intraepithelial lesion (HSIL) and adenocarcinoma in situ (AIS). The brush should be rotated gently only one-quarter turn. A larger rotation is unnecessary because the circumferential bristles are in contact with the entire surface the moment the brush is inserted. The spatula sample, if not already applied and fixed, should be applied to the slide, then the brush sample rolled over the slide, followed by immediate fixation. The two samples can be placed in quick succession on two separate halves of the slide, or the endocervical sample can be rolled directly over the spatula sample, both covering the entire slide. Immediate fixation (within seconds) is critical to prevent air-drying artifact, which distorts the cells and hinders interpretation. The broomlike brush (“broom”) has a flat array of plastic strips contoured to conform to the cervix, with longer strips in the middle. This design allows simultaneous sampling of the endocervix and ectocervix. The long middle strips are inserted into the os until the shorter outer strips bend against the ectocervix. The broom is rotated three to five times. To transfer the material, each side of the broom is stroked once across the slide in a painting motion. The cotton swab moistened with saline solution is no longer recommended because its fibers trap cells, reducing the efficiency of cell transfer onto slides. There are two options for smear fixation. Coating fixatives contain alcohol and polyethylene glycol and are applied by pump sprays, by droppers from dropper bottles, or by pouring from an individual envelope included as part of a slide preparation kit. Alternatively, the smear can be immersed directly into a container filled with 95% ethanol. Samples for liquid-based cytologic study are obtained as described above except that, instead of smearing the cells on a slide, the collection device is rinsed in a vial containing a liquid fixative. In the United States, the liquid-based Pap test is more common than the smear. 

Liquid-Based Cytologic Study In 1996, the US (FDA) approved the ThinPrep as an alternative to the cervicovaginal smear. This was followed 3 years later by approval of the AutoCyte Prep (now SurePath;

4 CHA P T ER 1    Cervical and Vaginal Cytology

BD TriPath, Burlington, NC). Liquid-based cytologic study (LBC) was an important step in the development of automated Pap screening devices—an improved preparation was needed to minimize cell overlap so that automated instruments would perform better in identifying abnormal cells. But LBC performed so well in clinical trials against smears that it found a market independent of automated screening. Although a number of studies showed an increased detection of cytologic low-grade squamous intraepithelial lesions (LSIL) or HSIL with LBC,45 subsequent meta-analyses and prospective randomized trials failed to demonstrate a significant difference between smears and LBC in the detection of histologic CIN2,3.46,47 Nevertheless, LBC offers several advantages over smears: the opportunity to prepare duplicate slides and even cell block preparations from the residual sample48,49; the option

1. Dispersion

of “out-of-vial” aliquoting for HPV, chlamydia, and gonorrhea testing; an improved substrate for automated screening devices; and a thinner cell preparation that most pathologists and cytotechnologists find less tiring to review than smears.

ThinPrep Pap Test The practitioner obtains the ThinPrep Pap sample with either a broom-type device or a plastic spatula/endocervical brush combination. The sampling device is swirled/rinsed in a methanol-based preservative solution (PreservCyt) for transport to the cytology laboratory and then discarded. Red blood cells are lysed by the solution. The vials are placed one at a time in the ThinPrep 2000 instrument or in batches in the ThinPrep 5000 (Fig. 1.1A). This results in a thin deposit of cells in a circle 20 mm in diameter (contrast

2. Cell collection

3. Cell transfer

2. Disaggregation

3. Transfer to sedimentation tube

A

1. Vortexing

4. Sedimentation 2

5. Cell deposition and staining

B • Fig. 1.1  Liquid-Based Slide Preparation Methods.  (A) ThinPrep method. 1. The sample vial sits on a

stage, and a hollow plastic cylinder with a 20 mm diameter polycarbonate filter bonded to its lower surface is inserted into the vial. A rotor spins the cylinder for a few seconds, dispersing the cells. 2. A vacuum is applied to the cylinder, trapping cells on the filter. The instrument monitors cell density on the filter. 3. With continued application of vacuum, the cylinder (with cells attached to the filter) is inverted 180 degrees, and the filter is pressed against a glass slide. The slide is immediately dropped into an alcohol bath. (B) SurePath method. 1. The sample is vortexed. 2. Cell clusters are disaggregated by syringing the sample through a small orifice. 3. The sample is poured into a centrifuge tube filled with a density gradient reagent. 4. Sedimentation is performed in a centrifuge. A pellet is obtained, resuspended, and the sedimentation is repeated. 5. The tubes are transferred to the PrepStain instrument, where a robotic arm transfers the fluid into a cylinder. Cells settle by gravity onto a cationic polyelectrolyte-coated slide. The same robotic arm also dispenses sequential stains to individual cylinders.

CHAPTER 1  Cervical and Vaginal Cytology

with Cytospin: diameter = 6 mm). In most cases, only a fraction of the sample is used to prepare the slide used for diagnosis. If needed, the residual sample is available for additional ThinPrep slide preparation, cell block preparation, or molecular diagnostic testing (e.g., high-risk HPV, Chlamydia, gonorrhea). A multicenter, split-sample study found that the ThinPrep detected 18% more cytologic cases of LSIL and more serious lesions as compared with smears, with no significant difference in the detection of organisms.50 A number of studies have shown significant increases in the detection of cytologic HSIL after the implementation of the ThinPrep.45,51-55 Subsequent meta-analyses and a prospective randomized trial, however, failed to demonstrate a significant difference between smears and ThinPrep in the detection of histologic CIN2,3.46,47 Data suggest that the ThinPrep is equivalent to the smear in the detection of endocervical adenocarcinoma in situ and endometrial disease.56,57 The ThinPrep collection vial has been approved by the FDA for testing for HPV, useful for primary screening alongside the pap (“cotesting”) and for managing women whose Pap test results show atypical squamous cells (ASC).32,58 

SurePath Pap Test TriPath Imaging (acquired by Becton Dickinson in 2006) developed the SurePath Pap test (formerly AutoCyte Prep) for samples collected in a mostly ethanol-based transport medium (BD SurePath Preservative Fluid). The process is shown in Fig. 1.1B. Unlike the ThinPrep method, the practitioner snips off the tip of the collection device and includes it in the sample vial. Red blood cells and some leukocytes are eliminated by density centrifugation. In addition to preparing an evenly distributed deposit of cells in a circle 13 mm in diameter, the method incorporates a final staining step that discretely stains each individual slide. A multicenter, split-sample clinical trial showed a 7.2% increase in the detection of cytologic LSIL and more serious lesions, as well as a significant decrease in the percentage of unsatisfactory specimens.59 As with ThinPrep, however, subsequent meta-analyses failed to demonstrate a significant difference between smears and SurePath in the detection of histologic CIN2,3.47 

Automated Screening Historical Overview Automated cytology screening devices have been under development since the 1950s. The first computerized screening system was developed in the United States by Airborne Instruments Inc. and was called the Cytoanalyzer.60 In preclinical trials it did not perform as well as expected, and the project was discontinued. The difficulty of the task was soon appreciated, especially the inherent problems with analyzing smears prepared in the conventional manner. Despite setbacks, research into cervical cytology screening continued throughout the following decades, with the development of

5

the TI-CAS,61 Quantimet,62 BIOPEPR,63 CERVIFIP,64 CYBEST,65 DIASCANNER,66,67 FAZYTAN,68 and LEYTAS.69 Some of these instruments are now in museums, but others have served as prototypes for systems that are now commercially available. In the 1990s, researchers in the United States and Canada established private enterprises supported by venture capital to develop a commercial automated screening instrument. Foremost in the field were AutoCyte (formerly Roche Image Analysis Systems), Cytyc, Neopath, and Neuromedical Systems. A three-way merger took place in 1999, when AutoCyte, after purchasing the intellectual property of Neuromedical Systems, merged with Neopath to form a new company called TriPath Imaging, acquired in 2006 by Becton Dickinson. In 2007, Cytyc Corporation, developer of the ThinPrep Pap Test and ThinPrep Imaging System (TIS), merged with Hologic Inc. and became a whollyowned subsidiary of Hologic. In 1998, the FDA approved the AutoPap System (now called the FocalPoint Slide Profiler; BD TriPath Imaging, Burlington, NC) as a primary screener for cervicovaginal smears, followed by approval in 2002 for use with SurePath slides. In 2003, the FDA approved the TIS (Hologic, Marlborough, MA) as a primary screener for ThinPrep Pap slides, and in 2008 it approved the FocalPoint Guided Screening (GS) Imaging System. Neither is approved in the United States for automated screening of nongynecologic cytology specimens. 

ThinPrep Imaging System The TIS uses the principle of location-guided screening to aid the cytotechnologist in reviewing a ThinPrep Pap slide. The TIS consists of two components, the Image Processor (”imager”) and the Review Scope (Fig. 1.2A–B). Stained and coverslipped ThinPrep slides are placed in a cartridge (each cartridge holding 25 slides), and up to 10 cartridges are loaded onto the bench-top imager. The imager has the capacity to screen more than 300 slides per day. It scans the slides and identifies 22 fields of view (FOV) on each slide that, based on optical density measurements and other features, are the most likely to harbor abnormal cells. The xand y-coordinates of the 22 FOVs are stored in a database and retrieved at a later time. The server is electronically linked to one or more Review Scopes in the laboratory. A Review Scope resembles a standard microscope but is augmented with an automated stage, a pod that controls the stage and objectives, and a keypad. The scope also has a camera that reads the slide identifier when the slide is loaded onto the stage. When a valid slide identifier is recognized, the server sends its coordinate information to the scope, permitting the cytotechnologist to navigate to the 22 FOVs using the pod. Navigation to each FOV is done geographically, that is, using the shortest distance from one FOV to the next. The cytotechnologist uses the pod to advance forward or return back through the FOVs, changing objectives as needed. If no abnormal cells are found

6 CHA P T ER 1    Cervical and Vaginal Cytology

A

B

D

C

• Fig. 1.2  Automated Pap Screening Devices.  (A) ThinPrep Imaging System: the Imager. The Imager con-

sists of (left to right): the Imaging Station, an Image Processor and Server, and a user interface consisting of a monitor, keyboard, and mouse. (B) ThinPrep Imaging System—the Review Scope. Imaging data are electronically linked to a customized microscope called the Review Scope. After the ThinPrep slides have been imaged on the Imager, they are brought to the microscope for location-guided review. In addition to a microscope, there is a console (with display and keypad) and a navigator pod. (C) BD FocalPoint System. (D) BD FocalPoint Guided Screening Review Scope. Imaging data are electronically linked to a customized microscope (the Review Scope). After the SurePath slides have been imaged, they are brought to the microscope for location-guided review.

in any of the FOVs, the case has been completed and can be reported as negative. If any abnormal cells are found in any of the FOVs, a review of the entire slide must be performed. This can be done using the “Autoscan” function on the Review Scope, with preset, customized user screening preferences. The Review Scope has both electronic and physical slide dotting capabilities. The accuracy of the TIS was evaluated in a clinical trial at four laboratories. ThinPrep slides were first screened manually and the results recorded. They were then rescreened using the TIS. Truth adjudication was performed by expert review of all abnormal cases and a proportion of negative slides. The TIS detected significantly more abnormal slides (ASC-US or greater) than manual review (82% versus 76%).70 A later split-sample study comparing smears versus the TIS for ThinPrep slides showed a significantly higher detection rate of histologic HSIL (CIN2,3) with the TIS.71

Because 22 FOVs represent approximately 25% of the ThinPrep cell spot,72 implementation of the TIS enhances productivity.70,73,74 Implementing the TIS requires adopting the proprietary ThinPrep Pap stain, to which some adjustment is necessary because it yields darker nuclear staining of metaplastic and endocervical cell clusters than most traditional Pap stains. The TIS does not eliminate false-negative results, which are still encountered, albeit less frequently than in the absence of imaging.70 A number of postapproval studies have shown significant increases in the detection of cytologic LSIL and HSIL after implementation of the TIS.75-77

BD FocalPoint Slide Profiler and Guided Screening Imaging System The FocalPoint Slide Profiler (FPSP) uses programmed algorithms to measure cellular features such as nuclear size,

CHAPTER 1  Cervical and Vaginal Cytology

integrated optical density, nuclear-to-cytoplasmic ratio, and nuclear contour—morphologic features established using planimetry and ocular micrometry for the diagnosis of squamous and glandular lesions.78 The device assigns a score to each slide that conveys the risk of an abnormality. AutoPap, the predecessor of FPSP, was originally intended as a primary screening device that would eliminate the need to manually screen as many as one-half of all smears. It was temporarily redesigned as a quality control rescreening device called the AutoPap 300 QC System and obtained FDA approval for this function in 1995. The AutoPap 300 QC System did not find a wide audience, however, and became obsolete in the year 2000. A redesign resulted in a new instrument, the AutoPap System-Primary Screener, later renamed FPSP, which obtained FDA approval as a primary screening device in 1998. In this mode, the device is used in the initial screening of smears: it identifies up to 25% of slides as requiring “no further review” based on a low slide score. Of the remaining (higher scoring) slides that require manual review, it also identifies at least 15% (the highest scoring slides) for a second manual review, which may be used as a substitute for the 10% review of negative Pap test results required of all US laboratories (see Chapter 19). A barcode is applied to each slide and slides are loaded into slide trays. Up to 288 slides can be loaded at a time (eight slides per tray, 36 trays). Each slide is analyzed using preset algorithms at 4× magnification for a visual map of the entire slide, then 1000 fields are captured at 20× magnification. After analysis, the device assigns a score (from 0 to 1.0) to each slide according to the likelihood of an abnormality. Slides below a cutoff are considered “no further review,” and those above the cutoff are triaged for full manual review. Any slide deemed unsuitable for analysis because of preparation or coverslipping problems requires manual review. The accuracy of the FPSP was evaluated in a clinical trial at five laboratories.79 Each slide was first evaluated in the conventional manner. The same slides were then processed by the AutoPap System, which detected significantly more abnormal slides—ASCs of undetermined significance (ASC-US) or greater—than conventional practice (86% versus 79%). Importantly, FPSP is not approved for women at high risk for cervical cancer. Thus a laboratory that uses FPSP for primary screening must set aside all Pap test results from high-risk women for manual screening. It is up to the laboratory to define what constitutes a Pap test result from a high-risk patient. False-negative results are occasionally encountered with the FPSP. In the clinical trial, there were 10 false-negative results (5 ASC-US, 4 LSIL, and 1 HSIL) in the 1182 cases considered “no further review” by FPSP, and another study found 9 false-negative results (5 ASC-US and 4 LSIL) in the 296 cases considered “no further review” by FPSP.80 False-negative cases were at least equally identified in the manual screening arm of each study, and overall results between the manual and automated screening methods were at least equivalent. The productivity gain with FPSP is modest because in practice the FPSP archives only about 16% to 17% of Pap test results without full manual review.79,81

7

At this moment, the FPSP in “no further review” mode is used by very few laboratories in the United States. The most recent phase in FocalPoint development occurred in 2008 with FDA approval of the BD FocalPoint GS Imaging System (FIS) (Fig. 1.2C–D). The FIS consists of the FPSP plus a BD FocalPoint GS Review Station and, like the TIS, uses the principle of location-guided screening to aid the cytotechnologist in reviewing a slide. A SurePath slide is first examined by the FPSP, which uses its algorithms to identify the 10 FOVs most likely to harbor abnormal cells. These FOVs are presented hierarchically based on slide score to a cytotechnologist for review at the microscopic Review Station; if no abnormality is detected in the FOVs, the slide is reported as negative without any further review. But if any abnormality is seen in any of the FOVs, or if specimen adequacy cannot be confirmed, the slide is triaged for full manual review. The accuracy of the FIS was evaluated in a clinical trial at four laboratories. The detection of cytologic HSIL+ increased by 20% and for cytologic LSIL+ by 10% in the computer-assisted arm, with small but statistically significant decreases in specificity. For cytologic ASC-US+ sensitivity and specificity, the study arms were not statistically different.82 As with the TIS, implementation of the FIS enhances productivity.83 FocalPoint instrumentation is in use outside the United States, predominantly in Europe, Asia, and Canada, but with slightly different methods. Cervical smears or LBC are scanned, and combinations of guided screening with “slide profiling” are in use. In this method, a low-scoring “no further review” population of slides is archived without further manual screening, whereas the higher scoring “review” population of slides undergoes guided screening as described above. 

Accuracy and Reproducibility The sensitivity of cytology for detecting preinvasive squamous and glandular lesions is difficult to establish, but it is clearly far from perfect. Most studies of preinvasive lesions suffer from verification bias (i.e., cases are referred for biopsy on the basis of an abnormal smear, and women with negative Pap test results are not biopsied). The few relatively unbiased studies show that the mean sensitivity of the Pap test is 47% (range 30% to 80%), and the mean specificity is 95% (range 86% to 100%).84 The sensitivity of cytologic study is less than ideal for invasive cancers as well, and estimates range widely (16% to 82%). Many women with cervical cancer have a history of one or more negative smears.85-97 The majority (56%) of women who develop invasive cervical cancer in the United States have not had a Pap test in the preceding 3 years.98 The relative contributions of sampling and laboratory error vary from one study to another and likely depend on how carefully retrospective rescreening is performed. False-positive diagnoses of cervical cancer occur in 10% to 15% of cases.99,100 The chief culprits are the atrophic smear

8 CHA P T ER 1    Cervical and Vaginal Cytology

with benign squamous atypia in a granular, pseudonecrotic background; reparative changes; and keratinizing HSILs. The interobserver reproducibility of cytologic interpretations is also less than perfect. In a large study of women, most of whom had mild cytologic abnormalities, the unweighted kappa statistic for four categories of diagnosis—negative, atypical, LSIL, and HSIL—was 0.46, indicating moderate reproducibility.101 (Roughly, a κ of 0 or less represents poor agreement, 0–0.2 slight agreement, 0.2–0.4 fair agreement, 0.4–0.6 moderate agreement, 0.6–0.8 very good agreement, and 0.8–1.0 almost perfect agreement.) In the same study, the reproducibility of histologic interpretations of cervical biopsies, also for four categories of diagnosis, was identical (0.46). The greatest disagreement with Pap test results involved those originally interpreted as showing ASC-US; the second reviewer agreed with only 43% of cases. The greatest disagreement with biopsy specimens involved those originally interpreted as CIN1; the second reviewer concurred in only 43% of cases.101 

Diagnostic Terminology and Reporting Systems Papanicolaou devised a numerical system for reporting cervical smears, which was originally intended to convey his degree of suspicion that the patient had cancer: • Class I, absence of atypical or abnormal cells • Class II, atypical but no evidence of malignancy • Class III, suggestive of but not conclusive for malignancy • Class IV, strongly suggestive of malignancy • Class V, conclusive for malignancy Over time the Papanicolaou class system underwent many modifications and was not used in a uniform fashion,102 but it persisted in many laboratories well into the 1980s. In other laboratories it was replaced (or supplemented) by descriptive terms borrowed from histologic classifications of squamous lesions. Squamous cancer precursors were originally divided into carcinoma in situ, a high-risk lesion of immature, undifferentiated atypical cells, and dysplasia (subdivided into mild, moderate, and severe), the latter a lower-risk lesion of more mature squamous cells. In the 1960s, Richart challenged the duality of dysplasia/carcinoma in situ and proposed a new term, cervical intraepithelial neoplasia (CIN). CIN was graded from 1 to 3, but Richart and Barron103 believed that CIN 1 (mild dysplasia) had a strong propensity to progress to CIN 3 and cancer. The high rate of progression found in their study most likely related to stringent entry criteria: for inclusion, CIN 1 had to be confirmed on three consecutive Pap tests.103 Richart’s data showed a higher progression rate for mild dysplasia than most other natural history studies.104 The CIN concept was highly influential, however, and for many years squamous precursors were treated as much on the basis of their size and location as on their grade. In 1989 the Bethesda System was introduced to standardize the reporting of cervical cytology results and incorporate

new insights gained from the discovery of HPV.105 The name for a squamous cancer precursor was changed to squamous intraepithelial lesion (SIL), subdivided into only two grades (low and high) based on the evolving understanding of the biology of HPV. In this system, LSIL encompasses CIN 1, and HSIL encompasses CIN 2 and 3. This was a shift away from the CIN concept, one based on a reevaluation of the existing evidence, which demonstrated that most LSILs are, in fact, transient HPV infections that carry little risk for oncogenesis, whereas most HSILs are associated with viral persistence and a significant potential for progression to invasive cancer. Since 2013, a two-grade reporting terminology has been recommended for the histopathologic reporting of genital and anal HPV-related lesions.106 The first Bethesda System workshop, in 1988, was followed by three others, in 1991, 2001, and 2014, which made modifications to the original framework, terminology, or both. The 2014 Bethesda System, like its predecessors, recommends a specific format for the cytology report, including an explicit statement on the adequacy of the specimen, a general categorization, and an interpretation/result.107,108 

The Bethesda System Specimen Adequacy One of the most important advances of the Bethesda System is its recommendation that each Pap report include an explicit statement of adequacy. In 1988, the Bethesda System proposed three categories for specimen adequacy: “satisfactory,” “less than optimal” (renamed “satisfactory but limited by ….” in 1991), and “unsatisfactory.” The 2001 Bethesda System eliminated the middle category because it was confusing to clinicians and prompted unnecessary repeat Pap tests. Nevertheless, the 2014 Bethesda System advocates mentioning the presence or absence of a transformation zone component and permits comments on obscuring elements. It is easy to determine whether a specimen is adequate or unsatisfactory in most cases. Slides received without patient identification or broken beyond repair should be rejected as unsatisfactory. An appropriately labeled smear with an adequate complement of well-preserved squamous and endocervical cells is clearly satisfactory. About 1% or less of Pap test results are interpreted as unsatisfactory.109,110 Unsatisfactory Pap test results can be finalized by a cytotechnologist and need not be reviewed by a cytopathologist (see Chapter 19). One of the components of an adequate Pap test result is an adequate squamous component, defined as a minimum estimated number of well-preserved, well-visualized squamous cells, the minimum being different for smears versus liquid-based preparations: The minimum number of squamous cells for adequacy depends on the preparation method: • Liquid-based: 5000 • Smear: 8000–12,000   

CHAPTER 1  Cervical and Vaginal Cytology

The minimum number of 5000 squamous cells for an adequate LBC Pap test result was based on correlations made between the false-negative rate and squamous cell cellularity.111 Because LBCs likely represent a more homogeneous representation of the material obtained by the collection device,112 a more stringent squamous cellularity requirement was imposed on smears. The cellularity of the squamous cell component is estimated; laboratories are not expected to count individual cells. With experience, an adequate squamous cell component is apparent in most cases. In borderline cases, techniques are available for estimating adequacy: reference images for smears and a spot-counting procedure for liquidbased preparations.108 A spot-counting method is used to evaluate LBCs with borderline squamous cellularity. A minimum of ten fields are counted along a diameter that includes the center of the slide (Fig. 1.3A). If the cell circle has blank spots, these should be represented in the fields counted (Fig. 1.3B). The average number of squamous cells is then compared against tables that take into account the objective, the eyepiece field number, and the diameter of the circle that contains cellular material.108 For example, with an Field Number 20 (FN 20)) eyepiece and a ×40 objective, the sample is adequate if the average number of squamous cells counted is greater than 3.1 for a ThinPrep slide. An additional slide can be prepared when the initial slide has insufficient cellularity, in the hopes that the new slide will be more cellular. The addition of a washing step with 10% glacial acetic acid increases the percentage of satisfactory ThinPrep Pap test results, uncovering occasional cases of SIL and invasive cancer.113,114 Adequacy should be assessed individually, however, not cumulatively; two unsatisfactory slides do not add up to a satisfactory slide.108

9

Squamous cellularity is sometimes difficult to estimate, for example, when there is marked cell clustering or cytolysis. In certain clinical settings, particularly in women with atrophy and after radiation therapy to the pelvis, a lower number may be adequate. In these situations, cytologists are expected to use their judgment when evaluating adequacy rather than rigidly applying the numerical criteria.108 For women with an unsatisfactory Pap test result, repeat cytologic study in 2 to 4 months is recommended. In women with an unsatisfactory Pap test result and a positive HPV test result, either a repeat Pap test in 2 to 4 months or colposcopy is acceptable.32 In the 2014 Bethesda System, the presence or absence of an endocervical/transformation zone component is noted on the report. An endocervical component is considered present if 10 or more endocervical or squamous metaplastic cells, either isolated or in groups, are present. The data on the endocervical component as a measure of sample adequacy are contradictory.115 The importance of endocervical cells was first suggested by cross-sectional studies, which showed that smears are more likely to contain SIL when endocervical cells are present.116-118 Data from retrospective case–control studies, however, do not support this: investigators have found no association between false-negative Pap test results and the absence of endocervical cells.119,120 Retrospective cohort studies have shown that women whose initial smears lack endocervical cells do not develop more lesions on follow-up than women whose smears do have an endocervical component,121-123 implying that an endocervical component is not essential. Currently, a smear without endocervical cells is not considered unsatisfactory, although the absence of an endocervical/transformation zone component is mentioned as a “quality indicator.” A repeat Pap test is not necessary.32 

General Categorization The general categorization is an optional component of the Bethesda System. A

THREE GENERAL CLASSIFICATION CATEGORIES • N  egative for intraepithelial lesion or malignancy • Other: See Interpretation/Result • Epithelial cell abnormality: See Interpretation/Result   

B • Fig. 1.3  Method

for Estimating the Adequacy of the Squamous Component of Liquid-Based Preparations.  (A) At magnification ×40, 10 fields are counted starting at the edge (horizontal or vertical) and including the center of the preparation. (B) An attempt is made to include “holes” in proportion to their size, making sure that the fields counted cover both cellular and sparsely cellular areas in proportion to their size.

“Other” includes benign-appearing endometrial cells in women over 45 years of age. Specimens are categorized according to the most significant abnormality identified. 

Interpretation and Results Recommended terminology for reporting findings is listed in Table 1.2. Mentioning nonneoplastic findings, other than organisms, is optional, given that many clinicians desire the Pap test report to be as concise as possible. Findings of no

10 CHA P T ER 1    Cervical and Vaginal Cytology

clinical consequence, if mentioned, may result in confusion and even unnecessary repeat testing. Nevertheless, many cytologists believe it is important to document that certain findings were interpreted as benign, particularly those that can mimic a neoplasm. 

The Normal Pap A normal Pap test result is reported as “Negative for Intraepithelial Lesion or Malignancy” (NILM). Additional findings (e.g., reactive changes, infectious organisms) are listed subsequently. Approximately 90% of Pap test results are interpreted as NILM.124 Pap test results, with the exception of those cases that show reactive/reparative changes, can be finalized by a cytotechnologist in the United States and need not be reviewed by a pathologist (see Chapter 19). In

the United States, a pathologist is required to review cases that show reactive/reparative changes and any abnormality at the level of ASC-US or higher. This represents about 10% to 20% of the total Pap volume in most laboratories.

Squamous Cells The ectocervix is lined by a stratified squamous epithelium that matures under the influence of estrogen. The most mature squamous cells are called superficial cells. They have a small, pyknotic nucleus that is 5 to 6 μm in diameter. Intermediate cells have a larger nucleus measuring 8 μm in diameter, which is not pyknotic but instead has a finely granular texture. Intermediate cells are occasionally binucleated and even multinucleated. Both superficial and intermediate cells are large polygonal cells with transparent pink or green

TABLE 1.2    The 2014 Bethesda System for Reporting Cervical Cytology

SPECIMEN ADEQUACY

OTHER

Satisfactory for evaluation

Endometrial cells in a woman ≥45 years of age

Unsatisfactory for evaluation

EPITHELIAL CELL ABNORMALITIES SQUAMOUS CELL

GENERAL CATEGORIZATION (Optional) Negative for Intraepithelial Lesion or Malignancy (NILM)

Atypical squamous cells (ASC)

Other

• Of undetermined significance (ASC-US)

Epithelial Cell Abnormality

• Cannot exclude HSIL (ASC-H)

INTERPRETATION/RESULT NEGATIVE FOR INTRAEPITHELIAL LESION OR MALIGNANCY NONNEOPLASTIC FINDINGS (optional to report)

Low-grade squamous intraepithelial lesion (LSIL)

Squamous metaplasia Keratotic changes Tubal metaplasia Atrophy Pregnancy-associated changes Reactive cellular changes associated with: • Inflammation (includes typical repair) • Radiation • Intrauterine contraceptive device (IUD) Glandular cells status post hysterectomy

ORGANISMS Trichomonas vaginalis Fungal organisms morphologically consistent with Candida species Shift in flora suggestive of bacterial vaginosis Bacteria morphologically consistent with Actinomyces species Cellular changes consistent with herpes simplex virus Cellular changes consistent with cytomegalovirus NOS=not otherwise specified.

High-grade squamous intraepithelial lesion (HSIL) Squamous cell carcinoma

GLANDULAR CELL Atypical endocervical cells, NOS Atypical endometrial cells, NOS Atypical glandular cells, NOS Atypical endocervical cells, favor neoplastic Atypical glandular cells, favor neoplastic Endocervical adenocarcinoma in situ (AIS) Adenocarcinoma • Endocervical • Endometrial • Extrauterine • Not otherwise specified

OTHER MALIGNANT NEOPLASMS (specify) ADJUNCTIVE TESTING COMPUTER-ASSISTED INTERPRETATION EDUCATIONAL NOTES AND COMMENTS (optional)

CHAPTER 1  Cervical and Vaginal Cytology

cytoplasm (Fig. 1.4). Superficial and intermediate cells are the predominant cells in cytologic samples from women of reproductive age. Immature squamous cells are called parabasal cells and basal cells. Because a Pap test does not usually scrape off the entire thickness of the epithelium but only the upper few layers, immature cells near the base of a mature epithelium are not usually sampled. An immature epithelium, however, is composed throughout its thickness by parabasal-type or basal-type cells. Immature epithelium is common at the transformation zone, where it is called squamous metaplasia, and whenever there is squamous epithelial atrophy due to a low estrogen state. Thus so-called “parabasal” and “basal” cells are typically obtained from squamous metaplasia or atrophic epithelium. Squamous atrophy is encountered in a variety of clinical settings associated with a low estrogen state. LOW ESTROGEN STATES INCLUDE: • • • • •

 remenarche P Postpartum Postmenopause Turner syndrome Status post bilateral oophorectomy   

Parabasal cells are round or oval rather than polygonal and have a variably sized nucleus that is usually larger than that of an intermediate cell. Basal cells are even smaller and have very scant cytoplasm (Fig. 1.5). Basal and parabasal cells are the hallmark of atrophy. With a deeply atrophic cervical epithelium, no superficial or intermediate cells are seen, only parabasal and basal cells. In addition, atrophic epithelium, particularly in postmenopausal women, is prone to injury and inflammation and often shows a spectrum of changes that should be recognized as normal and not confused with a significant lesion. Sheets of immature squamous cells are crowded and

• Fig. 1.4  Superficial and Intermediate Squamous Cells.  The mature squamous epithelium of the ectocervix in women of reproductive age is composed throughout most of Its thickness by superficial (arrowhead) and intermediate (arrow) cells.

11

syncytium-like, mimicking the crowded cells of an HSIL (Fig. 1.6A). Nevertheless, the chromatin texture in atrophy is finely granular and evenly distributed, nuclear contours remain mostly smooth and thin, and mitoses are generally absent. A curious variant, transitional cell metaplasia, is notable for prominent longitudinal nuclear grooves (“coffeebean nuclei”), wrinkled nuclei, and small perinuclear halos (Fig. 1.6B).125 Cellular degeneration is seen in some cases of atrophy (Fig. 1.7A). Dark blue, rounded, amorphous masses known as “blue blobs,” thought to represent either condensed mucus or degenerated bare nuclei, are sometimes seen (Fig. 1.7B), as is a granular background (see Fig. 1.7A) that resembles the necrosis associated with invasive cancers. Parabasal-type cells are also the constituents of squamous metaplasia of the endocervix. Squamous metaplasia is a common morphologic alteration of the endocervical epithelium usually limited to the transformation zone in women who otherwise have good squamous maturation. It is identified cytologically as flat sheets of immature squamous cells, often arranged in an interlocking fashion like paving stones (Fig. 1.8). They may show mild variation in nuclear size, with slightly irregular contours and slight hyperchromasia. Squamous metaplasia, as defined cytologically, is always composed of immature squamous cells. So-called mature squamous metaplasia, a histologic term describing mature squamous epithelium overlying endocervical glands, is not recognized as such on cytologic preparations. Other normal changes of squamous cells are hyperkeratosis and parakeratosis. Hyperkeratosis is a benign response of stratified squamous epithelium due to chronic mucosal irritation, as in uterine prolapse. Anucleate, mature, polygonal squamous cells appear as isolated cells or plaques of tightly adherent cells (Fig. 1.9A). Such cells are benign and should not be considered abnormal. This cytologic picture is mimicked by contamination of the slide by squamous cells of the vulva or skin from the fingers of the persons handling the slide.

• Fig. 1.5  Parabasal and Basal Cells (postpartum smear). Parabasal cells (large arrow) are oval and typically have dense cytoplasm. Basal cells (small arrow) are similar but have less cytoplasm. Many cells have abundant pale-yellow staining glycogen, a characteristic but nonspecific feature of squamous cells of pregnancy and the postpartum period.

12 CHA P T ER 1    Cervical and Vaginal Cytology

A

B

• Fig. 1.6  Parabasal Cells (postmenopausal smear).  (A) Atrophic epithelium is composed almost exclu-

sively of parabasal cells, often arranged in broad, flowing sheets. (B) Transitional cell metaplasia. In this uncommon condition, the atrophic epithelium resembles transitional cell epithelium by virtue of its longitudinal nuclear grooves. Nuclear membrane irregularities raise the possibility of a high-grade squamous intraepithelial lesion, but the chromatin is pale and finely textured.

B

A

• Fig. 1.7  Parabasal Cells (postmenopausal smear).  (A) Degenerated parabasal cells in atrophic Pap test results have hypereosinophilic cytoplasm and a pyknotic nucleus. Note the granular background, which is commonly seen. (B) Dark blue blobs are seen in some atrophic smears. These featureless structures should not be interpreted as a significant abnormality.

• Fig. 1.8  Squamous Metaplasia.  Interlocking parabasal-type cells, as seen here, represent squamous metaplasia.

CHAPTER 1  Cervical and Vaginal Cytology

13

B

A

• Fig. 1.9  Keratosis.  (A) Hyperkeratosis. Anucleate squames are a protective response of the squamous epithelium. (B) Parakeratosis. Parakeratosis appears as plaques, as seen here, or isolated cells.

B

A

• Fig. 1.10  Endocervical Cells.  (A) Normal endocervical cells are often arranged in cohesive sheets. Note the even spacing of the nuclei, their pale, finely granular chromatin, and the honeycomb appearance imparted by the sharp cell membranes. (B) Sometimes they appear as strips or isolated cells. Abundant intracytoplasmic mucin results in a cup-shaped nucleus.

Parakeratosis, a benign reactive change also caused by chronic irritation, is characterized by small, heavily keratinized squamous cells with dense orangeophilic cytoplasm and small, pyknotic nuclei (Fig. 1.9B). When such densely keratinized cells show nuclear atypia in the form of enlargement and membrane irregularity, they are called “atypical parakeratosis” and should be categorized as an epithelial cell abnormality (see further on). 

Endocervical Cells The endocervix is lined by mucin-producing columnar cells that have abundant vacuolated cytoplasm and a basally placed nucleus with finely granular chromatin. Nucleoli are inconspicuous but become prominent in reactive conditions such as cervicitis. Endocervical cells are often identified in strips or sheets rather than as isolated cells (Fig. 1.10). When arranged as strips, the cells have the appearance of a picket fence. When in sheets, they resemble a honeycomb because of the well-defined cell borders and uniform cell arrangement. Rarely, mitoses are identified. They should not raise suspicion of a neoplasm if the cells are otherwise normal in

appearance. Tubal metaplasia is a benign alteration of the endocervical epithelium found in about 30% of cone biopsy and hysterectomy specimens (Fig. 1.11).126 

Exfoliated Endometrial Cells Spontaneously exfoliated, menstrual endometrial cells are seen if the Pap test specimen is taken during the first 12 days of the menstrual cycle.127 CYTOMORPHOLOGY OF EXFOLIATED ENDOMETRIAL CELLS • • • • • •

 alls of small cells B Isolated small cells Scant cytoplasm Dark nucleus Nuclear molding Nuclear fragmentation   

Exfoliated endometrial cells are most easily recognized when they are arranged in spherical clusters (Fig. 1.12A). They are small, with a dark nucleus and (usually) scant

14 CHA P T ER 1    Cervical and Vaginal Cytology

• Fig. 1.11  Tubal Metaplasia.  Ciliated endocervical cells are occasionally seen.

B

A

• Fig. 1.12  Endometrial Cells.  (A) Spontaneously exfoliated endometrial cells, as in menses, are small cells

arranged in balls. Cytoplasm is scant. Nuclei around the perimeter appear to be wrapping around adjacent cells (arrow), a characteristic but nonspecific feature. (B) Some exfoliated endometrial cells have a doublecontoured appearance and resemble a sombrero. The inner, dense core consists of endometrial stromal cells and the outer rim of endometrial glandular cells.

cytoplasm; occasional cells may have more abundant, clear cytoplasm. Clusters have a scalloped contour due to the slight protrusion of individual cells. Apoptosis is common. Isolated endometrial cells are also seen, but they are less conspicuous because of their small size. Occasionally, endometrial cell clusters consist of an obvious dual cell population, with small, dark stromal cells (in the center) and larger glandular cells (around the edges) (Fig. 1.12B). Most endometrial cell clusters, however, do not have this dual population; clusters like that in Fig. 1.12A might be of endometrial glandular or stromal origin or a mix of both and are best referred to simply as “exfoliated endometrial cells.”128 Shedding endometrial cells after day 12 (“out of phase”) is associated with endometritis, endometrial polyps, and intrauterine devices (IUDs). In a young woman, abnormal shedding is almost never due to endometrial adenocarcinoma.129,130 For this reason, endometrial cells need not be mentioned in the report for women under 45 years of age. Sometimes a pathologist might do so anyway, to document that the cells were identified and interpreted as benign endometrial cells. Endometrial cells are notorious for their

ability to cause diagnostic difficulty because a variety of neoplastic cells resemble endometrial cells. In a woman 45 years of age or older, benign-appearing endometrial cells are reported because of the small risk of endometrial neoplasia. DIFFERENTIAL DIAGNOSIS OF EXFOLIATED ENDOMETRIAL CELLS • • • •

 SIL H Squamous cell carcinoma Adenocarcinoma in situ Small cell carcinoma   

The differential diagnosis includes a number of very significant lesions that mimic endometrial cells and thus are sometimes mistakenly interpreted as normal, particularly if the woman is in the first 12 days of her menstrual cycle. Attention to certain cytologic details can help avoid some if not all of these misattributions. A minority of HSILs are comprised of relatively small cells. Like endometrial cells, their nuclei are dark, and they have scant cytoplasm (Fig. 1.13A). HSIL cells, even when small, are usually bigger

CHAPTER 1  Cervical and Vaginal Cytology

A

B

C

D

15

• Fig. 1.13  Mimics of Exfoliated Endometrial Cells.  (A) High-grade squamous intraepithelial lesion (HSIL).

The cells of some HSILs are small, but still larger than endometrial cells and usually arranged in flatter aggregates rather than spheres. (B) Squamous cell carcinoma (SQC). Some poorly differentiated SQCs are indistinguishable from endometrial cells. The granular debris (“tumor diathesis”) is more prominent than in most normal menstrual Pap test results. (C) Adenocarcinoma in situ (AIS). Some cases of AIS have an endometrioid appearance, but mitoses (arrows) are distinctly uncommon in exfoliated endometrial cells. (D) Small cell carcinoma. The cells resemble endometrial cells but are even darker. There is nuclear smearing, which is not characteristic of benign endometrial cells.

than endometrial cells, vary more in size, and have denser cytoplasm. HSIL clusters are usually less well circumscribed and not as spherical as endometrial cell balls. Some poorly differentiated squamous cell carcinomas are comprised of small dark cells that mimic endometrial cells to perfection (Fig. 1.13B). In such cases, suspicious clinical findings (e.g., postcoital bleeding) might be the only clue to the correct interpretation. Most AIS have a columnar cell structure, but a minority are made up of smaller and rounder cells (Fig. 1.13C), particularly with liquid-based preparations. Careful examination for focal columnar differentiation and mitoses can be very helpful. The rare small cell carcinoma of the cervix may display crush artifact (Fig. 1.13D), which is rarely seen with endometrial cells. 

Abraded Endometrial Cells and Lower Uterine Segment The endocervical sampling device occasionally inadvertently samples the LUS or endometrium.131 This is especially likely when the endocervical canal is abnormally shortened, such as after a cone biopsy or trachelectomy.132,133

CYTOMORPHOLOGY OF ABRADED ENDOMETRIUM AND LOWER UTERINE SEGMENT • • • •

 arge and small tissue fragments L Glands and stroma Stromal cells • Uniform • Oval or spindle-shaped • Finely granular chromatin • Occasional mitoses • Capillaries traverse larger fragments Glands • Tubular • Straight or branching • Mitoses (some cases) • Extreme nuclear crowding • Scant cytoplasm   

The characteristic feature is the combination of glands and stroma, often in large fragments (Fig. 1.14A–C), either together or separated. Glandular cells of the LUS resemble endocervical cells, but have a higher nuclear-to-cytoplasmic

A

B

C • Fig. 1.14  Endometrial Cells, Directly Sampled.  (A) An intact endometrial tubule is surrounded by well-

preserved endometrial stromal cells. (B) Benign stromal cells are elongated and mitotically active (arrow) and might suggest a high-grade squamous intraepithelial lesion or malignancy. The pale, finely granular chromatin and the association with intact endometrial glands are clues to a benign interpretation. (C) The glandular cells are crowded and mitotically active (arrow), but evenly spaced.

CHAPTER 1  Cervical and Vaginal Cytology

ratio, are more hyperchromatic, and can be mitotically active. Because of their very high nuclear-to-cytoplasmic ratio, there’s a risk of confusion with a significant squamous or glandular lesion.131 

Trophoblastic Cells and Decidual Cells Syncytiotrophoblastic cells from placental tissue are seen very rarely, perhaps in about 0.1% of Pap test results from pregnant women.134 The cells are large, with abundant blue or pink cytoplasm. They have multiple nuclei that have a granular chromatin texture and slightly irregular contours. Trophoblastic cells can be distinguished from multinucleated histiocytes because their nuclei are darker and more irregular in contour (Fig. 1.15). They do not show the prominent molding and ground-glass appearance of nuclei of herpes simplex infection. Immunostains for human chorionic gonadotropin and human placental lactogen can be used to confirm their identity as trophoblastic cells. The presence of syncytiotrophoblastic cells is not a reliable predictor of an impending abortion.134

17

Decidual cells may be seen during pregnancy and the postpartum period, but they are often difficult to recognize and identify as such. They are usually isolated cells with abundant granular cytoplasm, a large vesicular nucleus, and a prominent nucleolus. They often show degenerative changes. 

Inflammatory Cells Neutrophils are seen in all Pap samples and do not necessarily indicate infection, but they are present in increased numbers after injury or infection. Lymphocytes and plasma cells are rare, but occasionally—most often in older women—they are numerous (Fig. 1.16A–B). This pattern is called follicular cervicitis because biopsy specimens show lymphoid follicle formation. The lymphocytes of follicular cervicitis can be confused with HSIL cells, endometrial cells, and lymphoma. Histiocytes are associated with a myriad of conditions (e.g., menses, pregnancy, foreign bodies, radiotherapy, and endometrial hyperplasia and carcinoma) (Fig. 1.17), but by themselves are a nonspecific finding of no clinical significance. 

Lactobacilli

•

Fig. 1.15  Syncytiotrophoblast.  The nuclei of these multinucleated cells are dark and coarsely granular, unlike those of histiocytes.

A

The vagina is colonized by Gram-positive rod-shaped bacteria of the genus Lactobacillus. They are beneficial because they produce lactic acid, which reduces the ambient pH and possibly protects from infection by Candida and other pathogens. Lactobacilli metabolize the glycogen contained within exfoliated squamous cells. The resulting cellular pattern, commonly seen during the second (luteal) phase of the menstrual cycle, is known as cytolysis—bare intermediate cell nuclei, fragments of squamous cytoplasm, and abundant bacterial rods (Fig. 1.18). Cytolysis can interfere with one’s ability to evaluate the nuclear-to-cytoplasmic ratio, an important criterion in grading SILs. 

B •

Fig. 1.16  Follicular Cervicitis.  (A) This smear from a 61-year-old woman contains numerous lymphocytes in various stages of maturation, including an occasional plasma cell (arrow). Most normal lymphocytes have a round nuclear contour, unlike the cells of a high-grade squamous intraepithelial lesion, to which they bear a superficial resemblance. (B) Lymphocytes are also a mimic of exfoliated endometrial cells. They are roughly the same size or a bit smaller, more heterogeneous in size, and less tightly clustered than most endometrial cells.

18 CHA P T ER 1    Cervical and Vaginal Cytology

“String of pearls” is the descriptive name given to an uncommon but striking artifact seen with ThinPrep Pap tests: a long string with bulbous excrescences (Fig. 1.19C). The cause is unclear, but it might represent a tight coil of mucus. Although originally described in a case report from a postmenopausal woman,137 they can be seen at any age. 

Organisms and Infections Shift in Flora Suggestive of Bacterial Vaginosis

•

Fig. 1.17  Histiocytes. Histiocytes have abundant multivacuolated cytoplasm and an oval, occasionally folded nucleus.

A steep reduction in the proportion of Lactobacilli, with a concomitant predominance of coccobacilli, is associated with bacterial vaginosis, a disorder characterized by a thin, milky vaginal discharge and a foul, fishy odor. It is not considered a sexually transmitted disease. At one time attributed solely to Gardnerella vaginalis, it is now clear that bacterial vaginosis can be caused by other bacteria as well.138 The diagnosis is made by correlating morphologic findings on a Pap or wet prep with other test results (vaginal pH and the amine-odor “whiff” test after adding potassium hydroxide).139 CYTOMORPHOLOGY OF A SHIFT IN FLORA • S  hort bacilli (coccobacilli), curved bacilli, or mixed bacteria • No Lactobacilli • “Filmy” appearance • “Clue cells”   

• Fig. 1.18  Lactobacilli.  These bacteria are part of the normal flora of

the vagina. Note the bare nuclei of the intermediate cells, which are subject to cytolysis by these organisms.

Artifacts and Contaminants Artifacts and specimen contaminants such as trichomes135 are illustrated in Fig. 1.19. For laboratories that use the ThinPrep method, one of the most commonly encountered artifacts happens when the sample is contaminated by a lubricant, gel, or vaginal medication containing carbomers or Carbopol polymers (Fig. 1.19B). These interfere with cellular deposition during slide preparation, resulting in an unsatisfactory slide.136 A note in the report can be helpful, for example: NOTE: This sample appears to be unsatisfactory due to the presence of an obscuring material, possibly lubricant. Nonapproved lubricants, and some gels or medications, may contain ingredients that interfere with Pap slide preparation (notably carbomers and Carbopol polymers). Of note, disposable specula may be precoated with interfering lubricants. In all cases, lubricant should be used sparingly when obtaining a Pap. For more information, please contact the Cytology Laboratory.

The cytologic hallmark is the replacement of the normal Lactobacilli by shorter bacilli (coccobacilli), curved bacilli, and mixed bacteria (Fig. 1.20). These small organisms are numerous and give a filmy appearance to the preparation. They frequently adhere to squamous cells, completely covering them like a shag carpet (“clue cells”). Clue cells are not specific for the diagnosis, especially in small numbers, and exactly how much “shift” is required for this diagnosis is an unresolved question. Data suggest that requiring at least 20% clue cells may increase the specificity of the diagnosis.140 Neutrophils are often scarce. This pattern is common and seen in about 50% of patients referred to a dysplasia clinic.138 Clinical correlation is required for a definite diagnosis of bacterial vaginosis because the cytologic pattern is neither sufficient nor necessary for the diagnosis. Women who are symptomatic are treated with metronidazole or clindamycin.141 

Trichomonas Vaginalis Trichomonas vaginalis is a primitive eukaryotic organism, a parasitic protozoan that causes trichomoniasis, a sexually transmitted disease. Patients may experience burning, itching, and a malodorous vaginal discharge, but up to 50% are symptom free.142 Although primarily a disease of women, it also occurs in men, most of whom are symptom free.

CHAPTER 1  Cervical and Vaginal Cytology

B

A

C

E

D

F • Fig. 1.19  Artifacts and Contaminants.  (A) “Cornflaking.” This refractile brown artifact results from bub-

bles of air trapped on superficial squamous cells, resulting in obscuring of the nuclei. It can be reversed by returning the slide through xylene and alcohol to water, then restaining and recoverslipping. (B) Lubricant artifact. In this example, the lubricant is purple and has a granular, thread-like appearance. Depending on the formulation, it may be purple or red, and sometimes it has a denser, sheet-like appearance. (C) “String of pearls.” (D) “Cockleburs.” This is the name given to radiating arrays of club-shaped orange bodies composed of lipid, glycoprotein, and calcium, surrounded by histiocytes. They are most commonly associated with, but not limited to, pregnant patients. They have no clinical significance. (E) Trichome. These large star-shaped structures are derived from plants. They stain a pale yellow and have from three to eight legs. Trichomes are produced by many different plants and vary in color, size, and shape. (F) Carpet beetle parts. These arrow-shaped structures are contaminants from sources such as gauze pads and tampons.

19

20 CHA P T ER 1    Cervical and Vaginal Cytology

Trichomonas-related halos are smaller and accompanied by only minimal nuclear atypia. Patients and their sexual partners are treated with metronidazole or tinidazole.144 

CYTOMORPHOLOGY OF TRICHOMONAS VAGINALIS • • • • •

 5 to 30 μm long 1 Pear-shaped Pale, eccentrically placed nucleus Red cytoplasmic granules “Trich parties”

Candida Candida albicans and Candida glabrata are fungal species that infect the vulva, vagina, and cervix. Patients may be symptom free, or they may complain of burning, itching, and a thick, cheesy discharge.

  

The organism is a 15- to 30-μm pear-shaped protozoon that has a small, very pale, eccentrically placed nucleus (Fig. 1.21A). The cytoplasm often contains tiny red granules. Clusters of the organisms are colloquially called “Trich parties,”143 “Trich picnics” (or “Trich-nics”), “Trich hotels,” or “Trich conferences” (Fig. 1.21B). Trichomonas is sometimes accompanied by Leptothrix, a nonpathogenic, long, filamentous bacterium. Some squamous cells have a small, narrow, indistinct perinuclear halo that calls to mind the cytopathic changes of HPV, but

CYTOMORPHOLOGY OF CANDIDA • • • •

 ink P Yeast forms (3 to 7 μm diameter) Long pseudohyphae and true hyphae Tangles of pseudohyphae and yeast forms (“spaghetti and meatballs”) • Skewers of squamous cells around pseudohyphae (“shish kebabs”)   

These fungi are eosinophilic and often interspersed among squamous cells (Fig. 1.22). In many cases, some squamous cells appear in linear arrays, as if skewered by the pseudohyphae. Tangles of pseudohyphae (“spaghetti”) admixed with yeast forms (“meatballs”) are common. Thin mucus strands are a common mimic of Candida pseudohyphae, but they are pale blue rather than pink like Candida. Not all women with this finding are symptomatic, and usually only symptomatic women are treated. Treatment is a short course of an over-the-counter topical (intravaginal) azole drug (e.g., Clotrimazole, Miconazole, others).145 

Actinomyces

• Fig. 1.20  Shift in Flora Suggestive of Bacterial Vaginosis. Numerous small bacteria cover large portions of the slide. In some but not all cases, these bacteria adhere to squamous cells (“clue cells”), giving them the appearance of a shag rug, as seen here. Lactobacilli are absent.

A

Actinomyces organisms are Gram-positive anaerobic bacteria that are normal inhabitants of the mouth and bowel.

B • Fig. 1.21  Trichomonas Vaginalis.  (A) This organism is pear-shaped, with a pale oval nucleus and red cytoplasmic granules, often inconspicuous. The flagellum, barely seen here, is usually inapparent (SurePath preparation, image courtesy Keith V. Nance, MD, UNC Rex Healthcare, Raleigh, NC). (B) “Trich picnic.” Numerous faintly visible organisms are aggregated, in this example on a platform of squamous cells.

CHAPTER 1  Cervical and Vaginal Cytology

They are uncommon in the cervix and vagina (found in 10% sequence diversity in the code for the L1 protein.) Only a minority (12 to 15) cause cervical cancer.183,184 The genital HPVs are divided into low-risk and high-risk types based on the frequency of their association with invasive cervical cancer. By definition, an HPV is low risk if it has never (or virtually never) been isolated from a cervical carcinoma, and high risk if it has been. Persistent infection with any one of about 15 high risk (carcinogenic) types accounts for virtually all cervical cancers.185

•

Fig. 1.34  Human Papillomavirus (HPV) Detection By in Situ Hybridization.  The dark brown signal is centered around the nuclei of infected cells. (Courtesy Miu-Fun Chau, Dakocytomation, Carpinteria, CA.)

EXAMPLES OF LOW-RISK AND HIGH-RISK HUMAN PAPILLOMAVIRUSES • L  ow-risk: 6, 11, 42, 43, 44, 53, 54, 57, 66 • High-risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68   

HPV 16 is the prototype of the high-risk viruses: it is the most prevalent of the high-risk HPVs and the one most commonly detected in cervical cancers, responsible for 50% of all cervical cancers.186 Although they are not (or only rarely) carcinogenic, low-risk HPVs are nevertheless important because they cause genital warts. The receptor for HPV entry into the cell has not been definitively established, but uptake seems to occur by clathrin-coated endocytosis, caveolae, or both, after which viral DNA is transported to the nucleus.186 The small HPV genome consists of about 8000 base pairs of circular, ­double-stranded DNA. It codes for only eight genes, which are classified as “early” (E) or “late” (L) depending on the timing of their expression in the epithelium. HPV infection is established at the transformation zone and in the basal layers of the epithelium, where the HPV genome is maintained, with expression of the E genes (E1, E2, E4, E5, E6, and E7). As the epithelium matures toward the surface, gene amplification and viral assembly occur, with expression of L1 and L2, with eventual viral release. L1 is the major viral capsid protein and is the principal component of HPV vaccines. The carcinogenicity of HPV results primarily from the activity of the oncogenes E6 and E7. They have a number of cellular targets, with a multitude of effects that lead to malignant transformation.185,186 Important ones appear to be (1) the binding of E6 to the tumor suppressor protein p53, which results in the degradation of p53 and the blocking of apoptosis; (2) the binding of E7 to the retinoblastoma tumor suppressor protein pRb, which abolishes cell-cycle arrest and leads to increased cellular proliferation185,187; (3) the induction of centrosomal abnormalities by E7 (mechanism not clear), which may cause genomic instability by the accumulation of mitotic errors. These effects alone are probably insufficient for cancer development: HPV viral proteins cannot fully transform human keratinocytes in culture.186 Because p53 mediates DNA damage repair, p53 degradation predisposes the host cell to secondary events such as point mutations. Such secondary changes may take years to accumulate, and this may explain the relatively long clinical interval between preinvasive lesions and cervical cancer. Given that smoking and long-term oral contraceptive use are known risk factors for cervical cancer, cofactors likely enhance the role of HPV. Cellular immune responses play an important role in clearing most HPV infections, but how they work is still poorly understood. For reasons that are not well understood, E6 and E7 proteins encoded by low-risk HPVs have a much more limited effect on cells—one that does not stimulate carcinogenesis—as compared with E6 and E7 proteins encoded by high-risk types.

CHAPTER 1  Cervical and Vaginal Cytology

The risk of HPV infection per sexual contact is not known but is probably fairly high. Most sexually active women are infected with one or more HPV types at some point in their lives. For unclear reasons, the virus has a strong predilection for the transformation zone. Serologic study is not an accurate measure of infection because only 50% to 60% of infected women have circulating antibodies to HPV.188 Clearly, only a minority of HPV infections persist and lead to cancer. Clinical HPV testing is generally restricted to high-risk types, and in this context “HPV testing” refers to testing for high-risk types. Testing for low-risk types has no role in screening or the management of women with abnormal cytology.32 A variety of molecular techniques—hybrid capture, target amplification, invader chemistry—can be used to detect HPV. The Hybrid Capture 2 test (Qiagen), which was evaluated in the multicenter ASCUS/LSIL Triage Study trial, uses a cocktail of probes to the 13 high-risk HPV types listed above, which account for nearly 90% of HPVs detected in HSIL and invasive cancers.58 Other FDA-approved HPV tests include Cervista HPV HR (Hologic), the cobas HPV test (Roche Molecular Diagnostics), and the Aptima HPV assays (Hologic Gen-Probe).

Grading Squamous Intraepithelial Lesions The Bethesda System recommends a low-grade/high-grade approach to grading SIL. This is based on the evidence that most LSILs are transient infections that carry little risk for oncogenesis, whereas most HSILs are associated with viral persistence and a significant potential for progression to invasive cancer. HPV typing plays no role in the grading of SIL. Although low-risk viruses are more common in LSIL than HSIL, high-risk viruses predominate in both.58,189 Morphologic assessment by conventional light microscopy is still the gold standard for grading SILs. 

Low-Grade Squamous Intraepithelial Lesion LSIL accounts for approximately 2.5% of all Pap test results.124 It is caused by a large number of different HPVs, including low-risk and high-risk types. Many LSILs regress spontaneously, but some persist for long periods of time. Approximately 21% progress to HSIL, but it is possible that at least some of these may have been HSILs from the beginning and were initially misclassified as LSIL. In fact, 18% of women with an LSIL Pap result prove to have HSIL

(CIN2,3) on biopsy.190 Less than 1% of untreated LSILs progress to invasive cancer.104 LSIL is a lesion of intermediate or superficial cells that show nuclear enlargement accompanied by moderate variation in nuclear size and slight irregularities in nuclear shape and contour. Hyperchromasia is present and can take the form of either a uniformly granular increase in chromatin or the smudgy hyperchromasia seen in some koilocytes. Nucleoli are inconspicuous. Classic koilocytes have large, sharply defined perinuclear cytoplasmic cavities surrounded by a dense rim of cytoplasm. Their nuclei are usually enlarged, but they are diagnostic of LSIL even in the absence of nuclear enlargement (Fig. 1.35). Some LSILs show prominent keratinization manifested by deeply orangeophilic cytoplasm and squamous pearls (Fig. 1.36). DIFFERENTIAL DIAGNOSIS OF LOW-GRADE SQUAMOUS INTRAEPITHELIAL LESION • • • •

 eactive squamous cells R Squamous cells with nonspecific halos Reactive endocervical cells ASC-US   

Nuclear enlargement by itself is not diagnostic of LSIL. It is common with benign squamous cells, particularly those seen in perimenopausal women (PM cells, see Fig. 1.25A). Similarly, small, nonspecific halos mimic the cavities of koilocytes. They are seen in association with Trichomonas and other infections, and they can be an artifact of slide preparation. Nonspecific halos are often smaller than koilocyte cavities and unassociated with nuclear atypia (see Fig. 1.26 A–B). Some markedly enlarged reactive endocervical cells have the size and polygonal shape of a squamous cell. With their enlarged nucleus they mimic an LSIL (see Fig. 1.27B). They are recognized by the company they keep (arranged alongside smaller, more recognizable endocervical cells) and by their granular rather than smooth cytoplasm. Mild but noticeable nuclear changes and larger cytoplasmic cavities raise the possibility of LSIL but sometimes fall short qualitatively or quantitatively. Squamous cells that are suspicious but not conclusive for LSIL are reported as ASC-US. Management

CYTOMORPHOLOGY OF LOW-GRADE SQUAMOUS INTRAEPITHELIAL LESION • L  arge, mature cells (equal in size to a normal superficial or intermediate squamous cell) • Nuclear atypia: • Enlargement • Irregular contour (slight) • Hyperchromasia • Slight chromatin coarseness • Cytoplasmic cavities (koilocytes) • Keratinizing variant   

29

The management of a woman with an LSIL Pap depends on her particular circumstances. HPV testing to triage women with LSIL Pap test results is not generally recommended because the high rate of positivity (77%) limits its usefulness.32 Colposcopy is recommended in most cases.32 If the patient is pregnant, it is acceptable but not necessary to defer colposcopy until 6 weeks postpartum. Postmenopausal women with LSIL cytology can be managed by immediate colposcopy, but repeat Pap testing at 6 and 12 months or HPV testing are also acceptable.32 With the latter two options, the postmenopausal woman is referred to

30 CHA P T ER 1    Cervical and Vaginal Cytology

A

B •

Fig. 1.35  Low-Grade Squamous Intraepithelial Lesions (LSIL).  (A) LSIL. Classic koilocytes, as seen here, have a large cytoplasmic cavity with a sharply defined inner edge and are frequently binucleated. Nuclear enlargement may not be as marked as in the nonkoilocytic LSILs. (B) Nonkoilocytic LSIL. Nuclei are significantly enlarged and show hyperchromasia and nuclear contour irregularity. No definite koilocytes are seen. This pattern was once called mild dysplasia or CIN 1.

colposcopy only if the HPV test result is positive or any one of the Pap test results is ASC-US or greater. After two consecutive negative Pap test results, return to routine screening is recommended. 

High-Grade Squamous Intraepithelial Lesion HSIL accounts for about 0.5% of all Pap test results. Virtually all women (97%) with an HSIL Pap result test positive for high-risk HPV.58 If left untreated, it carries a significant risk of progression to cervical cancer.168,170

CYTOMORPHOLOGY OF HIGH-GRADE SQUAMOUS INTRAEPITHELIAL LESION • U  sually parabasal-sized cells • Discrete cells or syncytium-like groups (hyperchromatic crowded groups) • Nuclear atypia • Enlargement • Marked irregularity in contour • Usually marked hyperchromasia • Marked chromatin coarseness • Keratinizing variant   

CHAPTER 1  Cervical and Vaginal Cytology

• Fig. 1.36  Low-Grade Squamous Intraepithelial Lesion, Keratinizing Type.  A squamous pearl is being formed.

31

HSIL is usually a lesion of immature squamous cells. Patten divided HSILs into three categories based on cell size (frequencies in parentheses): large cell (20%), intermediate (70%), and small cell (10%).191 These subtypes have no biologic significance but are helpful to keep in mind. Nuclear enlargement is generally in the same range as in LSILs, but the nuclear-to-cytoplasmic ratio is higher because the cells are smaller (Fig. 1.37). In general, hyperchromasia, irregular chromatin distribution, and membrane contour irregularity are all more severe than in LSIL. In any given HSIL, one or more of the characteristic nuclear changes predominate. Thus some HSILs have very irregular nuclear contours but only mild to moderate hyperchromasia. Architecturally, the cells of HSIL are arranged in two main patterns: as distinct individual cells

A

B • Fig. 1.37  High-Grade Squamous Intraepithelial Lesion (HSIL).  (A) These cells have scant cytoplasm

and a markedly hyperchromatic nucleus with highly irregular nuclear contours. (B) Cells with a moderate amount of cytoplasm, formerly called moderate dysplasia or CIN 2, are included in the HSIL category.

32 CHA P T ER 1    Cervical and Vaginal Cytology

(Fig. 1.37) or as cohesive groups of cells with indistinct cell borders (syncytium-like clusters; Fig. 1.38). They may have dense, squamoid cytoplasm, but HSIL cells are often completely undifferentiated in appearance and lack any defining squamous features. In fact, cytoplasmic transparency and vacuoles (Fig. 1.39) and an elongated configuration (Fig. 1.40) can cause them to be mistaken for cells of glandular origin. Although usually a lesion of small, immature squamous cells, mature keratinizing cells with marked nuclear atypia are classified as HSIL (Fig. 1.41).

DIFFERENTIAL DIAGNOSIS OF HIGH-GRADE SQUAMOUS INTRAEPITHELIAL LESION • • • • • • • • • • • •

 quamous metaplasia S Atrophy Transitional cell metaplasia Exfoliated endometrial cells Follicular cervicitis Histiocytes IUD effect Endocervical polyp atypia Adenocarcinoma in situ Squamous cell carcinoma ASC-H ASC-US associated with atrophy   

• Fig. 1.38  High-Grade Squamous Intraepithelial Lesion (HSIL).  The cells of an HSIL are often arranged in dark, three-­dimensional groups in which individual cell borders are indistinct (syncytium-like).

• Fig. 1.40  High-Grade Squamous Intraepithelial Lesion (HSIL).  The

cells of some HSILs have an elongated configuration that makes them look columnar. In the absence of strips, rosettes, or feathering, this should not be taken for evidence of glandular differentiation (i.e., an adenocarcinoma in situ).

• Fig. 1.39  High-Grade Squamous Intraepithelial Lesion (HSIL). Some

HSILs are comprised of very small, dispersed, highly atypical cells. The nucleus of these small cells is not much larger than that of normal intermediate cells. They are nevertheless identified as abnormal because of their hyperchromasia, markedly irregular nuclear outline, or both. Some HSIL cells have cytoplasmic vacuoles. These do not indicate a glandular lesion.

•

Fig. 1.41  High-Grade Squamous Intraepithelial Lesion (HSIL), Keratinizing Type.  Although the cells show differentiation by keratinizing, they are classified as HSIL if the nuclei are sufficiently abnormal.

CHAPTER 1  Cervical and Vaginal Cytology

Distinguishing HSIL from its many mimics is an important skill of the cytologist. As with histologic specimens, the most frequent cytologic mimics are squamous metaplasia with reactive changes and atrophy. Squamous metaplastic cells commonly show mild degrees of nuclear enlargement, nuclear membrane irregularity, and even chromatin coarsening. These changes rarely rise to the level of atypia seen in HSIL. In postmenopausal women, sheets of atrophic squamous epithelium mimic the syncytium-like clusters of HSIL (see Fig. 1.6A). Although atrophic squamous cells have a high nuclear-to-cytoplasmic ratio, their nuclei are usually very regular, with finely textured chromatin. Transitional cell metaplasia, associated with Pap test results from older women, is likely to raise the possibility of HSIL because of the irregularity of the nuclear outlines and prominence of nuclear grooves (see Fig. 1.6B). The minimal hyperchromasia and the abundance of coffee-bean–shaped nuclei is a clue to the benign metaplastic nature of these cells. HSIL cells, even those of the small-cell type,191 are usually bigger than endometrial cells (compare Figs. 1.12 and 1.13A), vary more in size, and may have denser cytoplasm. HSIL clusters are usually less well circumscribed and not as spherical as endometrial cell clusters. Lymphoid cells, commonly seen in postmenopausal women, are smaller than HSIL cells, their chromatin is even more coarsely textured, and there are often admixed plasma cells, dendritic cells (with a larger, pale nucleus) and tingible-body macrophages (see Fig. 1.16). Histiocytes are roughly the same size as HSIL cells, and many have irregular nuclear contours, but their chromatin is finely textured and cytoplasm is often abundant and fluffy (see Fig. 1.17). The small cells of IUD effect are usually few in number and have a more prominent nucleolus than is commonly seen with HSIL (see Fig. 1.31). Occasional inflamed endocervical polyps are lined by a single layer of highly atypical, hyperchromatic endocervical cells that are easily overinterpreted as HSIL. Their true nature is often clarified only after histologic correlation (Fig. 1.42). The neoplastic cells of AIS share many of the nuclear features of HSIL. Clusters of neoplastic cells are more

A

33

likely to represent HSIL rather than AIS, unless there is clear columnar differentiation in the form of feathering or rosette formation. Squamous cell carcinoma should be considered whenever the cytologic criteria for HSIL are fulfilled but in addition one finds prominent nucleoli, irregular chromatin distribution, or necrotic debris. Cell block preparations from a residual liquid-based sample can help by providing a “histologic” look at hyperchromatic cell clusters (Fig. 1.43A–B).48 Immunohistochemistry for p16 and Ki-67 (MIB1) helps distinguish benign from neoplastic cells192; p63, which highlights squamous but not glandular lesions, helps distinguish squamous from glandular lesions.193 In some cases, uncertainty remains regarding the true nature of the cells examined. Cells with the features of squamous metaplasia sometimes show a degree of nuclear atypia that makes it impossible to exclude an HSIL. These cases of atypical squamous metaplasia are reported as “ASC, cannot exclude HSIL.” Another diagnostically difficult pattern is the marked squamous atypia associated with a deeply atrophic Pap test result. Atrophic cervical epithelium sometimes displays a marked squamous atypia that is impossible to distinguish from HSIL. The recommended approach is to call such cases ASC-US. Management

Management is more aggressive for cytologic HSIL than LSIL because HSIL has a higher risk of progression to invasive cancer. With the exception of younger women (ages 21 to 24) and those who are pregnant, an immediate loop electrosurgical excision (the “see-and-treat” approach) is acceptable as the initial treatment if the woman has an HSIL Pap, but not LSIL. An alternative to loop is colposcopy with endocervical assessment (evaluating the canal using the colposcope or tissue sampling).32 In younger and pregnant women, colposcopy is recommended. Histologic CIN2+ is found in 60% of women with cytologic HSIL, and cervical cancer in about 2%.32 

B • Fig. 1.42  Endocervical Polyp Atypia Mimicking HSIL.  (A) The slide contains scattered isolated cells with dark nuclei. (B) The surface of the endocervical polyp reveals a single layer of reactive endocervical cells.

34 CHA P T ER 1    Cervical and Vaginal Cytology

Problems in the Diagnosis of Squamous Intraepithelial Lesions Avoiding Overdiagnosis of LSIL

Care must be taken not to overinterpret nonspecific halos (see Fig. 1.26A–B) and the minimal nuclear changes of benign cells like PM cells (Fig. 1.25A).157 Without hyperchromasia or nuclear membrane irregularity, such cells are best called NILM. Cellular changes that include some hyperchromasia or nuclear membrane irregularity are suggestive of LSIL and should be categorized as ASC-US. 

are less numerous than the LSIL cells. In a small percentage of cases, morphologic features intermediate between typical LSIL and HSIL make grading difficult.194 Although there are generally fewer abnormal cells in an LSIL than in an HSIL, the quantity of cells is an unreliable discriminator. CYTOMORPHOLOGIC PATTERNS OF “SIL, GRADE CANNOT BE DETERMINED” • • • •

Distinguishing LSIL from HSIL

The distinction between cytologic LSIL and HSIL is an important one, with significantly different implications for clinical management. Proficiency in this distinction is an important skill of the cytology practitioner. As mentioned above, HSIL is usually a lesion of immature squamous cells, and nuclear atypia (hyperchromasia, irregular chromatin distribution, and membrane contour irregularity) is more severe than in LSIL. If a specimen is composed of both LSIL and HSIL, it should be reported as an HSIL even if the HSIL cells

A

 ew dysplastic cells F Extensive cytolysis LSIL, with a small number of equivocal HSIL cells Extensively keratinized SILs, without definite HSIL   

Grading is difficult when dysplastic cells are few in number, when the cytoplasm of the dysplastic cells is obviously affected by cytolysis, or when an LSIL is accompanied by a small number of cells suggestive of but not conclusive for HSIL. Extensively keratinized SILs without definite HSIL are especially difficult to grade195 (Fig. 1.44). In all such cases, a diagnosis of “SIL, grade cannot be determined” (or

B • Fig. 1.43  Utility of Cell Blocks in Evaluating Hyperchromatic Cell Clusters.  (A) The distinction between

a squamous and glandular lesion can be problematic, especially when individual cells in dark cell clusters are poorly visualized. (B) A cell block prepared from the residual liquid-based sample in this case clarified the findings as HSIL.

B

A •

Fig. 1.44  Squamous Intraepithelial Lesion, Cannot Determine Grade. When a lesion is extensively keratinized and there is no definite high-grade squamous intraepithelial lesion, it is difficult to grade. Colposcopically directed biopsies showed (A) CIN1 and (B) CIN2,3.

CHAPTER 1  Cervical and Vaginal Cytology

“LSIL, cannot exclude HSIL”) is appropriate,196 and about one-half of surveyed laboratories report using such a category.124 This interpretation accounts for 3% to 12% of all cytologic SILs.190,197-199 Patients with this diagnosis have an intermediate risk (between that of cytologic LSIL and HSIL) of harboring histologic HSIL (CIN 2,3).196-198  Distinguishing HSIL from Invasive Carcinoma

The criteria used to distinguish HSIL from invasive carcinoma are by no means perfect. Occasionally, a cytologically typical HSIL proves to be invasive squamous cancer on biopsy. Conversely, the possibility of invasive cancer is often raised in cases of HSIL when the cells have marked nuclear abnormalities associated with abundant, heavily keratinized cytoplasm and unusual cell shapes, but the lesion turns out to be just a keratinizing HSIL on biopsy.100 Clinicians understand that no diagnosis of HSIL on cytologic material excludes the possibility of invasive cancer, and that colposcopy and biopsy are necessary for confirmation. HSILs with features worrisome for invasive cancer can be reported as “HSIL, with features suggestive of invasive cancer.” 

Squamous Cell Carcinoma Squamous cell carcinoma (SQC) is the most common malignant tumor of the cervix, accounting for about 75% of cervical cancers.200 Although most patients are between the ages of 35 and 55 years, cervical SQC does occur in younger patients, including those under 30 years of age.87 HPV 16 accounts for about 50% to 60% of cervical SQCs worldwide, and HPV 18 for an additional 10% to 15%.201 Early invasive tumors can be asymptomatic, but as the tumor

35

grows patients can develop abnormal vaginal bleeding, vaginal discharge, and pain during intercourse (dyspareunia). With more advanced tumors there can be back pain, sciatica, tenesmus, and hematuria. Histologically and cytologically, SQCs range from welldifferentiated, keratinizing tumors to poorly differentiated, non-keratinizing tumors.202 Some SQCs cannot be distinguished cytologically from HSIL, particularly the smaller, less deeply invasive tumors.203 CYTOMORPHOLOGY OF SQUAMOUS CELL CARCINOMA • •

HSIL features, plus • Macronucleolus • Irregular chromatin distribution • Tumor diathesis “Tadpoles” and “fiber cells” (keratinizing type)   

The classic pattern of SQC shows abundant necrotic debris: a granular, amorphous precipitate with nuclear debris and red blood cells called “tumor diathesis” (Fig. 1.45). It is not specific for invasive cancers; a similar pattern is seen in some atrophic smears and even during heavy menstrual bleeding. When associated with hyperchromatic crowded groups of atypical cells or abundant atypical keratinized cells with unusual shapes (“tadpoles,” “fiber cells”), the pattern is diagnostic. The cells of a non-keratinizing SQC look like modified HSIL cells (Figs. 1.46 and 1.47). Like HSIL, they are hyperchromatic and have scant cytoplasm, but they have a prominent nucleolus and a highly irregular pattern of

• Fig. 1.45  Squamous Cell Carcinoma.  Slides from deeply invasive tumors show abundant tumor dia-

thesis, a granular precipitate of lysed blood and cell fragments. In some cases, the malignant cells can be hard to identify.

36 CHA P T ER 1    Cervical and Vaginal Cytology

chromatin distribution. The cells of a keratinizing SQC are often bizarrely elongated (Fig. 1.48). Some are long and spindle-shaped, with small condensed nuclei (“fiber cells”). Others have a larger cytoplasmic body with a long tail (“tadpole cells”). Such cells are uncommon in keratinizing HSILs. Most SQCs are associated with an adjacent or overlying HSIL, and therefore cytologic preparations from SQCs often contain a population of HSIL cells as well. DIFFERENTIAL DIAGNOSIS OF SQUAMOUS CELL CARCINOMA • • • • • •

• Fig. 1.46  Squamous Cell Carcinoma (SQC), Non-Keratinizing.  The

malignant cells have irregularly distributed chromatin and a prominent nucleolus, characteristic features of invasive SQCs.

•

Fig. 1.47  Squamous Cell Carcinoma (SQC), Nonkeratinizing.  The sheet-like arrangement of poorly differentiated squamous carcinoma cells with nucleoli and mitoses mimics the appearance of reparative epithelium, but the crowding and haphazard arrangement of the cells are not typical of repair.

A

 SIL H Atypia of atrophy Atypia of repair Benign endometrial cells Behçet disease Pemphigus vulgaris   

The differential diagnosis of a SQC includes HSIL. Prominent nucleoli and tumor diathesis are the principal cytologic features that help distinguish SQC from HSIL, but these features are not present in all samples from patients with SQC. A significant number of women with a SQC are diagnosed as having HSIL because prominent nucleoli and tumor diathesises are absent.203 Conversely, a granular, tumor diathesis-like background is not specific for invasive cancers and is seen in women with atrophic vaginitis99 (see Fig. 1.7), severe cervicitis, and rare cases of HSIL.100 In postmenopausal women, marked atrophy atypia is one of the most common benign mimics of a keratinizing SQC. The benign atypia of atrophy contains scattered cells with large, dark nuclei and eosinophilic or orangeophilic cytoplasm. Their large, dark nuclei are alarming, but chromatin is usually smudgy. Such cells, if seen in a deeply atrophic squamous background, should be interpreted as ASC-US and not HSIL or invasive cancer.

B • Fig. 1.48  Squamous Cell Carcinoma, Keratinizing.  (A) In keratinizing carcinomas, the cells have mark-

edly aberrant shapes, as seen here. “Fiber cells” are numerous. (B) Tumor diathesis and a tadpole cell are seen in this tumor.

CHAPTER 1  Cervical and Vaginal Cytology

Marked repair atypia is another good mimic of non-­ keratinizing SQC. Both repair and SQC contain large cells with prominent nucleoli, and mitoses are seen in both. Repair cells are recognized by their finely textured chromatin pattern, the flatness and cohesion of the sheets. If the nuclei have coarsely textured chromatin, show marked crowding, or demonstrate significant dyshesion, a SQC should be considered. A minority of non-keratinizing SQCs are comprised of small cells that are indistinguishable from endometrial cells (see Fig. 1.13B). The blood that accompanies menstrual endometrial cells resembles the granular necrosis that is tumor diathesis, adding to the similarity. Mitoses, if identified, should raise the suspicion of SQC. In some cases, knowledge that the patient has a suspicious cervical mass or suspicious clinical symptoms (e.g., dyspareunia) may be the only clue to the correct interpretation. Behçet disease, a chronic disease of uncertain cause that is characterized by oral and genital ulcers, can mimic SQC. Slides may show numerous isolated, keratinized cells with dark, pleomorphic nuclei and large nucleoli. A history of this disorder may be critical for correct diagnosis.204 Samples from patients with pemphigus vulgaris, a blistering disorder that involves mucosal surfaces, may mimic a poorly differentiated SQC. A complete history may be important to avoid making an overcall, although cases of coexisting SQC and pemphigus vulgaris have been reported.205 Management Treatment choices for women with cervical cancer depend primarily on tumor stage.206 The primary treatment of early stage cervical cancer is either surgery or radiation therapy. Chemoradiation is the primary treatment of choice for women with higher stage cancers (IB2 to IVA). 

Atypical Squamous Cells Since the days of Papanicolaou, cytology laboratories have used a borderline category to report squamous and glandular findings of uncertain significance. Historically, terminology was inconsistent and often confusing. In the Bethesda System, the preferred term for borderline squamous cases is atypical squamous cells (ASC). ASC is defined as cytologic changes suggestive of SIL. Most cytologists agree that this category is essential. Eliminating ASC would result in increased reporting of LSIL (which probably contributes little to cancer prevention) and decreased recognition of CIN2,3.207 It is risky to eliminate an equivocal category because of the large number of women with underlying HSIL who are discovered through a workup for an equivocal cytology reading. In fact, histologic HSIL is found in 10% to 20% of women with ASC Pap test results,58,190 and, ironically, ASC Pap test results, because they are more common, detect more cases of HSIL than HSIL Pap test results.208 Finally, the elimination of an equivocal category seems imprudent given the expectations in the United States for a very sensitive Pap test.

37

ASC diagnoses should be kept to a minimum. There is no correct rate of ASC, but expert consensus suggests that it be kept to less than 5% of all Pap cases. For laboratories that serve high-risk populations, a better gauge is the ASC/SIL ratio, which should not exceed 3:1.108 The ASC rate can be kept low through education, optimal sample preparation, and the monitoring (with feedback) of individual ASC/SIL ratios.209,210 According to a College of American Pathologists survey, most US laboratories are complying with the above recommendations: ASC accounts for 4.6% of all Pap test results, and the median ASC/SIL ratio is 1.5.124 In the Bethesda System, ASC is qualified as either “of undetermined significance” (ASC-US) or “cannot exclude HSIL” (ASC-H). The higher risk of the latter category is well recognized.211,212

Atypical Squamous Cells of Undetermined Significance The cases described in this section are daily dilemmas for cytologists. ASC-US, the most common nonnegative interpretation, constitutes 4.3% of all Pap test interpretations.124 The decision to categorize a Pap test result as negative (NILM), ASC-US, ASC-H, LSIL, or HSIL rests on the quantity of the altered squamous cells, the severity of the abnormalities, the state of preservation of the specimen, and the clinical setting. If the changes are suspicious but not conclusive for SIL, the findings are reported as ASC-US. CYTOMORPHOLOGIC PATTERNS OF ASC-US • A  typical cells with mature, intermediate-type cytoplasm, including cells suggestive of koilocytes • Atypical squamous cells in atrophy • Atypical parakeratosis • Atypical repair • “Atypia” due to a compromised specimen   

Atypical mature squamous cells with features suspicious for a SIL are classified as ASC-US (Fig. 1.49A). Some cases have incomplete features of koilocytosis (Fig. 1.49B). Atypical squamous cells associated with atrophy are diagnosed as ASC-US when there is nuclear enlargement with hyperchromasia, when nuclei are irregular in contour and chromatin distribution, and when there is cellular pleomorphism with unusual shapes. In extreme cases, the changes seen in atrophy with inflammation are difficult to distinguish from a SIL or invasive cancer (Fig. 1.50). Historically, management options included a course of intravaginal estrogen cream (e.g., 1 g estrogen cream three times a week for several months), followed by a repeat Pap test a week after completing the regimen.213 A significant squamous lesion is more easily detected among the mature cells, whereas a benign alteration due to atrophy is transformed into normal epithelium. This option has been omitted from more recent management guidelines.32 Parakeratosis with mild nuclear enlargement and mild to moderate nuclear membrane irregularity (atypical parakeratosis) is suggestive of SIL (Fig. 1.51). In some cases such cells are

38 CHA P T ER 1    Cervical and Vaginal Cytology

B

A

• Fig. 1.49  Atypical Squamous Cells of Undetermined Significance (ASC-US).  (A) The nucleus of this

mature squamous cell is significantly enlarged and there is moderate hyperchromasia. Cells like this, particularly if few in number, are suggestive but not diagnostic of a squamous intraepithelial lesion. (B) Some cells have large cytoplasmic cavities but minimal nuclear atypia. It is preferable to diagnose such cases as ASC-US when abnormal cells are few and the changes minimal.

B

A •

Fig. 1.50  Atypical Squamous Cells of Undetermined Significance, Associated with Atrophy. (A) Histologic section of benign atrophy-associated atypia. (B) A cytologic smear shows occasional markedly enlarged, hyperchromatic nuclei.

accompanied by other changes diagnostic of a SIL, but when the changes are mild, such cases are best classified as ASC-US. Highly exuberant atypical repair reactions can demonstrate cellular crowding and overlap (in contrast with typical repair, which is in flat sheets), marked variation in nuclear size, prominent and irregular nucleoli, and irregular chromatin distribution (Fig. 1.52). Such cases are difficult to distinguish from invasive carcinoma. Carcinomas often have a tumor diathesis and many isolated atypical cells, features that are usually absent in repair reactions. Management

Reflex HPV testing is the preferred approach for managing most women with ASC-US.32 Women who test positive for HPV should be referred for colposcopy with directed biopsies. Women who test negative for HPV return for cotesting in 3 years. Repeat cytologic study in 1 year is an acceptable option, but reflex HPV testing is preferred for managing a

• Fig. 1.51  Atypical Squamous Cells of Undetermined Significance, with Features of Atypical Parakeratosis. Small, keratinized squamous cells with mild variation in nuclear size and contour may represent either a reactive process or a significant squamous lesion.

CHAPTER 1  Cervical and Vaginal Cytology

woman with ASC-US because it is more sensitive than a single repeat Pap test.58 If repeat Pap testing is selected, colposcopy is recommended if the result is ASC-US or worse; if the result is negative, return to testing every 3 years is recommended. There are minor variations in the recommendations for younger (ages 21 to 24) and pregnant women. In younger women with an ASC-US Pap, cytology at 12 months is preferred, but reflex HPV testing is acceptable. If the cytology option is selected, only women with an ASC-H or HSIL Pap (or worse) at the 12 month follow-up should be referred to colposcopy. The recommendations for pregnant women are the same as the general recommendations, except that it is acceptable to defer colposcopy until 6 weeks postpartum. Endocervical curettage in pregnant women is unacceptable. 

•

Fig. 1.52  Atypical Squamous Cells of Undetermined Significance, Atypical Repair Reaction.  In some cases of repair there is such striking nuclear atypia that an invasive cancer cannot be excluded. The flat, sheet-like arrangement with nuclear separation, the finely textured chromatin, and the prominent streaming appearance are more typical of benign repair rather than cancer. (Contrast with Fig. 1.47.)

A

Atypical Squamous Cells, Cannot Exclude HSIL ASC-H is the second, by far less common subtype of ASC, representing only 0.3% of all Pap test interpretations.124 This category is reserved for Pap test results that are specifically suspicious for HSIL. The most common pattern is that of immature (small) squamous cells with mild to moderate nuclear atypia (enlargement, hyperchromasia, membrane irregularity), commonly called atypical squamous metaplasia (Figs. 1.53 and 1.54). Management

ASC-H cytology has a higher risk for histologic CIN 2,3 than ASC-US (50% versus 17%).214 For this reason, women with an ASC-H Pap should be referred for colposcopy, regardless of HPV result.32 

•

Fig. 1.53  Atypical Squamous Cells, Cannot Exclude High-Grade Squamous Intraepithelial Lesion (HSIL).  These metaplastic-like cells show significant nuclear membrane irregularity. There is no hyperchromasia or nuclear size variation, however, which makes the diagnosis of HSIL uncertain.

B •

39

Fig. 1.54  Atypical Squamous Cells, Cannot Exclude High-Grade Squamous Intraepithelial Lesion (HSIL).  (A) Immature squamous metaplastic cells sometimes show some nuclear atypia, which raises the possibility of HSIL, but the degree of nuclear enlargement, hyperchromasia, and membrane irregularity is insufficient for a definite diagnosis. (B) Subsequent colposcopy revealed benign immature squamous metaplasia, and a human papillomavirus (HPV) test was negative for high-risk HPV.

40 CHA P T ER 1    Cervical and Vaginal Cytology

Glandular Abnormalities Endocervical Adenocarcinoma in Situ Endocervical AIS is the recognized precursor to endocervical adenocarcinoma. The evidence linking them is similar to that linking SIL to squamous cell carcinoma: women with AIS are an average 13 years younger than those with adenocarcinoma (39 versus 52 years old); AIS resembles adenocarcinoma morphologically and is often found in histologic sections adjacent to invasive carcinoma; AIS has been discovered retrospectively in biopsies originally called negative in women who later develop invasive adenocarcinomas215; and HPV 16 and 18 have been identified in AIS and adenocarcinomas in similar proportions. The concept of endocervical AIS was first introduced in 1953 when its histologic features were convincingly illustrated by Friedell and McKay.216 Widespread recognition of the cytologic characteristics of AIS came only in the late 1970s and 1980s, when a group of Australian investigators published their experience with a large number of histologically confirmed cases.217-219 It was not until 2001 that the cytologic criteria for AIS were considered sufficiently reliable to merit a separate, explicit diagnostic category in the Bethesda System. There has been a steady increase in the incidence of AIS, due in part to better cytologic recognition of AIS, but cytologic diagnosis remains a challenge, mainly because it is still a very uncommon lesion. The incidence of AIS is a mere 1.25/100,000, as compared with 44.4/100,000 for squamous cell carcinoma in situ.200 Therefore, in practice, one is likely to see one case of AIS for every 36 cases of HSIL. CYTOMORPHOLOGY OF ADENOCARCINOMA IN SITU • • • •

 yperchromatic crowded groups H Glandular differentiation • Columnar cells • Strips and rosettes • “Feathering” Neoplastic nucleus • Hyperchromasia • Crowding, stratification • Inconspicuous nucleolus • Apoptosis • Mitoses No tumor diathesis   

In the Bethesda System, AIS is a separate diagnostic category because there is a consensus that the cytologic criteria are accurate and reproducible.219-221 Examination of the slide under low magnification reveals hyperchromatic crowded groups (Fig. 1.55) similar to those of HSIL. Closer inspection reveals evidence of glandular differentiation: columnar cells arranged in strips (Fig. 1.55C), rosettes (Fig. 1.56A), or both. Columnar cells in sheets reveal their glandular nature by “feathering,” a splaying out around the edges (Fig. 1.56B). Nuclei are hyperchromatic and crowded,

and there is scant cytoplasm. Apoptotic bodies are seen in most cases and are a useful clue to the diagnosis.222 Mitoses are seen in some cases and are helpful, but only if accompanied by the typical nuclear changes described above. DIFFERENTIAL DIAGNOSIS OF ADENOCARCINOMA IN SITU • • • • • • •

 xfoliated endometrial cells E Tubal metaplasia Abraded endometrial cells and LUS Reactive endocervical cells Reparative changes HSIL Invasive adenocarcinoma   

A serious (and not uncommon) problem is mistaking AIS for benign cells.223,224 Some cases of AIS, in fact, strongly resemble menstrual endometrial cells (“endometrioid” AIS),225 and apoptosis is a feature of both (see Fig. 1.13C). The cells of AIS are generally better preserved and have a coarser chromatin texture. Feathering, rosettes, and mitoses are virtually never seen in menstrual endometrium. AIS resembles tubal metaplasia (Fig. 1.57A–D), but tubal metaplasia is recognized by the presence of terminal bars and cilia. In addition, tubal metaplasia lacks mitoses and apoptosis. AIS also strongly resembles directly sampled endometrium and LUS, particularly because mitoses can be seen in both (see Fig. 1.14B–C). The intact tubules alongside stroma that are typical of endometrium and LUS are rarely seen in AIS (see Fig. 1.14A), however, and the nuclei of endometrium and LUS, although crowded, are arranged in an orderly rather than haphazard pattern. Reactive endocervical cells and reparative epithelia show a greater range of nuclear size and less hyperchromasia than AIS, which generally has strikingly uniform dark nuclei: the nuclei of reactive endocervical cells typically have prominent nucleoli, a feature seen in only a very small proportion of AIS cases. AIS can resemble HSIL almost to perfection (see Fig. 1.55). Both are characterized by hyperchromatic crowded groups, mitoses, apoptosis, and coarse chromatin. The cells of HSIL, like those of AIS, can have pale, granular cytoplasm. The diagnosis of AIS should be reserved for cases with clear columnar glandular differentiation: strips of columnar cells, rosettes, and feathering. If AIS is suspected, cell block preparations from a residual liquid-based sample can help by providing a “histologic” look at hyperchromatic cell clusters48 (see Fig. 1.43A–B). Immunohistochemistry for p16 helps to identify HPV-related neoplastic cells192; p63, which highlights squamous but not glandular lesions, helps distinguish squamous from glandular lesions.193 The distinction between AIS and invasive adenocarcinoma is problematic. Some cases of adenocarcinoma are clearly invasive because the cells are large, with abundant cytoplasm and prominent nucleoli, and a tumor diathesis is present. These features are absent in some cases of

CHAPTER 1  Cervical and Vaginal Cytology

A

B

C • Fig. 1.55  Adenocarcinoma in Situ (AIS).  (A) At first glance, some groups of neoplastic cells resemble

the hyperchromatic crowded groups of a high-grade squamous intraepithelial lesion. Only slight feathering is seen (arrows). (B) The columnar features are subtle with liquid-based preparations. (C) A picket-fence arrangement is indicative of glandular differentiation.

A

B • Fig. 1.56  Adenocarcinoma in Situ (AIS).  (A) Rosettes are highly characteristic of AIS and virtually never seen with high-grade squamous intraepithelial lesion, benign endocervical cells, or lower uterine segment (LUS) or endometrial epithelium. (B) The endocervical glandular nature of these neoplastic cells is betrayed by “feathering.”

41

42 CHA P T ER 1    Cervical and Vaginal Cytology

A

B

C

D • Fig. 1.57  Adenocarcinoma in Situ (AIS) Compared to Tubal Metaplasia.  (A) Endocervical AIS. Cells are columnar in shape, dark, crowded, and arranged in a curved strip. (B) A cone biopsy revealed AIS. (C) Tubal metaplasia. Atypical glandular cells bear a resemblance to those in (A), except that cilia are identified. (D) Subsequent biopsy specimens showed tubal metaplasia of surface endocervical epithelium.

adenocarcinoma, however, resulting in significant morphologic overlap between AIS and cervical adenocarcinoma. Thus the cytologic diagnosis of AIS does not exclude invasive adenocarcinoma; histologic evaluation is necessary for a definite distinction. Even the histologic distinction between AIS and adenocarcinoma is often a difficult judgment based in part on whether the lesion extends below the normal location of endocervical glands. Management

A woman with a cytologic diagnosis of AIS should undergo colposcopic examination with endocervical sampling.32 For women older than 35 years and younger women with certain clinical indications such as unexplained vaginal bleeding, endometrial sampling should be included. If there is no evidence of invasive disease, she should have a diagnostic excision procedure, one that yields an intact specimen with interpretable margins. 

Adenocarcinoma Adenocarcinomas of the endocervix, endometrium, vagina, and even the ovaries and fallopian tubes are sometimes detected with the Pap test. There is significant overlap in

their morphologic features, so that a precise site of origin often cannot be established. Additional testing (imaging studies, histologic sampling) is usually required for definitive classification and treatment.

Endocervical Adenocarcinoma Adenocarcinoma of the endocervix represents approximately 20% to 25% of cervical cancers in the United States.200 Some patients complain of bleeding or vaginal discharge, but others are symptom free. As with cervical squamous cell carcinomas, HPV is commonly present in endocervical adenocarcinomas; HPV 16 accounts for 40% and HPV 18 for an additional 30%.226 There are many histologic subtypes: “usual” type (the most common, characterized by mucin depletion), mucinous (subdivided into gastric, intestinal, and signet-ring cell types), villoglandular, endometrioid, clear cell, serous, mesonephric, and adenocarcinoma admixed with neuroendocrine carcinoma.202 Minimal deviation adenocarcinoma (syn., adenoma malignum) is an extremely well-­differentiated form of mucinous adenocarcinoma, gastric type and, unlike most endocervical adenocarcinomas, is unassociated with HPV. Adenosquamous carcinoma of the cervix shows a mixture of neoplastic glands and cells with squamous differentiation (intercellular

CHAPTER 1  Cervical and Vaginal Cytology

A

43

B • Fig. 1.58  Endocervical Adenocarcinoma.  (A) The cells are round rather than elongated as in adenocar-

cinoma in situ. They are crowded and hyperchromatic, and a tumor diathesis is present. Tumor diathesis on liquid-based preparations appears as clumps and as a granular ring around groups of malignant cells (“clinging diathesis”). (B) High magnification reveals nuclear crowding and very large nucleoli.

bridges, keratinization). Glassy cell carcinoma is a poorly differentiated variant of adenosquamous carcinoma. The myriad subtypes make cytologic recognition particularly challenging. Some cases of invasive endocervical adenocarcinoma are cytologically indistinguishable from AIS, but in many cases the diagnosis of an invasive adenocarcinoma can be made or at least suggested. CYTOMORPHOLOGY OF ENDOCERVICAL ADENOCARCINOMA • • • • • •

 umor diathesis (50% or fewer of cases) T Abundant large abnormal cells Isolated cells, sheets, crowded clusters Large, round nucleus Prominent nucleolus Abundant cytoplasm   

The cells of most endocervical adenocarcinomas are usually numerous and arranged in crowded sheets and clusters and as isolated cells. Well-differentiated endocervical adenocarcinomas of mucinous type are composed of columnar cells with abundant, foamy cytoplasm and a basally located nucleus (Fig. 1.58A–B). They are sometimes difficult to distinguish from reactive endocervical cells (Fig. 1.59A–D). Nuclei are pale or hyperchromatic, and mitoses are sometimes present. In moderately and poorly differentiated tumors there is greater variation in nuclear size and shape, and nucleoli are prominent (Fig. 1.60). A tumor diathesis is present in only about one-half of cases227 (see Fig. 1.58A–B). Adenosquamous carcinoma is composed of malignant squamous and glandular cells arranged in sheets of large pleomorphic cells with abundant dense cytoplasm and prominent macronucleoli. Clear cell carcinomas of the endocervix and vagina are morphologically identical: both are composed of round cells with pale nuclei, prominent nucleoli, and abundant foamy or finely granular cytoplasm.

The rare, extremely well-differentiated tumor minimal deviation adenocarcinoma (or adenoma malignum) is composed of mucinous glands that show little if any atypia, and yet, if untreated, invade deeply and metastasize. Patients sometimes present with vaginal discharge. In most cases, the neoplastic cells on the Pap test look entirely like normal endocervical cells228 (Fig. 1.61A). Frequently, even cervical biopsy specimens and endocervical curette scrapings are misinterpreted as benign. A correct diagnosis often requires at least a cone biopsy to appreciate the invasive nature of the lesion (Fig. 1.61B). Villoglandular adenocarcinomas are rare low-grade neoplasms. Cytologically, they resemble AIS because the cells are uniform and crowded, with mild to moderate aytpia.229 Like AIS, strips and rosettes are seen, and there may be no tumor diathesis.230 Very few are diagnosed prospectively as an adenocarcinoma by cytology; most are reported as benign or as “atypical glandular cells.”230 The cytologic features of the rare mucoepidermoid carcinoma and adenoid cystic carcinoma of the cervix are similar to their counterparts in the salivary gland and elsewhere. Adenocarcinomas of the cervix are treated in a similar manner to squamous cancers.206 

Endometrial Adenocarcinoma Endometrial adenocarcinoma is predominantly a tumor of postmenopausal women, with a peak incidence in women in their late 50s and early 60s; it is rare in women younger than 40. About 90% present with postmenopausal bleeding, but some are symptom free. Most endometrial adenocarcinomas are of the endometrioid type. Less common types include serous and clear cell carcinomas, which present at a more advanced stage and have a worse prognosis. The mucinous type of endometrial carcinoma, by contrast, behaves like the endometrioid type. The Pap test is a screening test for cervical lesions and is not intended for the detection of endometrial lesions.

44 CHA P T ER 1    Cervical and Vaginal Cytology

A

B

C

D •

Fig. 1.59  Endocervical Adenocarcinoma Compared to Reactive Endocervical Cells. (A) Endocervical adenocarcinoma, well-differentiated. The cells are enlarged and crowded, but the features are not conclusive for malignancy (note the absence of tumor diathesis). A diagnosis of atypical glandular cells was made. (B) Histologic sections showed adenocarcinoma. (C) Reactive endocervical cells. These cells appear similar to those in (A). (D) Biopsy specimens in this patient confirmed reactive changes due to inflammation.

cells”). Cervical Pap cytology is atypical, suspicious, or positive for malignancy in 38% to 90% of endometrial adenocarcinomas.231,232 CYTOMORPHOLOGY OF ENDOMETRIAL ADENOCARCINOMA • • • • • • •

•

Fig. 1.60  Endocervical Adenocarcinoma.  These malignant cells show variation in nuclear size, with very prominent nucleoli and coarsely granular chromatin.

Nevertheless, the Pap test does fortuitously pick up cells from many endometrial cancers. The cells that exfoliate from high-grade endometrial adenocarcinomas, particularly those of serous or clear cell type, are obviously malignant, and such cases can and are reported as adenocarcinomas (or, if there is doubt, as “atypical endometrial

Isolated cells and small, tight clusters Round cells Enlarged nucleus Hyperchromatic Prominent nucleolus Scant or abundant vacuolated cytoplasm Cytoplasmic neutrophils (”bags of polys”)   

In many cases of the endometrioid type of endometrial cancer, the malignant cells are not at all numerous, and only about one-third of cases contain a tumor diathesis.233 The malignant cells are round, isolated or in small groups, and larger and more vacuolated than benign endometrial cells (Fig. 1.62). A very characteristic finding, although not entirely specific for endometrial adenocarcinoma, is the

CHAPTER 1  Cervical and Vaginal Cytology

A

45

B •

Fig. 1.61  Minimal Deviation Adenocarcinoma.  (A) The cells are sometimes impossible to distinguish from normal endocervical cells, as in this case. (B) A cone biopsy revealed deeply invasive, misshapen neoplastic glands.

B

A

• Fig. 1.62  Endometrial Adenocarcinoma Compared to Intrauterine Device (IUD) Effect.  (A) Endometrial

adenocarcinoma, endometrioid type. These malignant cells are large, vacuolated, and associated with neutrophils. (B) IUD effect. Benign cells in women with an IUD are indistinguishable morphologically from those of endometrial adenocarcinomas.

“bag of polys” cell: a neoplastic cell with a large cytoplasmic vacuole filled with neutrophils (Fig. 1.63A–B). Such cells should raise a strong suspicion of endometrial neoplasia. Histiocytes frequently accompany the atypical cells and in some cases outnumber them. Cells from serous adenocarcinoma of the endometrium are typically very large, pleomorphic, and easily recognized as malignant. Numerous bare nuclei in a necrotic background are characteristic. Compared with smears from the endometrioid type, smears from serous carcinomas contain more malignant cells.233 Psammoma bodies are present in only 25% of cases.233 Pap tests are more likely to contain malignant cells in patients with a serous rather than an endometrioid type of endometrial adenocarcinoma.234,235 

Differential Diagnosis of Adenocarcinoma Because there is significant morphologic overlap between adenocarcinomas of the cervix, endometrium, and other sites, they are considered together.

DIFFERENTIAL DIAGNOSIS OF ADENOCARCINOMA • • • • • • • • •

 ndocervical adenocarcinoma E Endometrial adenocarcinoma Adenocarcinoma of other sites: • Vaginal • Ovarian • Tubal • Metastatic Squamous cell carcinoma IUD effect Endocervical polyp atypia Reactive endocervical cells AIS Pemphigus vulgaris   

When adenocarcinoma cells are identified on a Pap slide, the two principal suspects are endocervical and endometrial adenocarcinoma. The age of the patient is helpful: the older the patient, the more likely it is that the tumor has

46 CHA P T ER 1    Cervical and Vaginal Cytology

arisen in the endometrium. Morphologic features are also helpful. Endometrial adenocarcinoma cells are rounder and tend to exfoliate as isolated cells and smaller clusters, often arranged as spheres, whereas the cells of endocervical adenocarcinomas are more columnar and arranged as sheets. Histiocytes commonly accompany endometrial carcinomas and not endocervical carcinomas. Ultimately, the cytologist can usually only suggest the possibilities, favoring one site over another: the final classification rests on histologic examination. Adenocarcinoma of the vagina is very rare and often associated with a maternal history of DES use during pregnancy. Adenocarcinomas from the ovaries and fallopian tubes are more commonly associated with psammoma bodies,236 but this is not entirely reliable because endocervical and endometrial cancers sometimes contain them as well. Nonkeratinizing squamous cell cancers resemble endocervical adenocarcinomas. Unless focal keratinization is

identified, a definite distinction is not possible. The cells of IUD effect are indistinguishable from those of endometrial adenocarcinoma (see Fig. 1.62B). If the woman has an IUD and the cells are few in number, it is likely that they represent IUD effect rather than an adenocarcinoma. “Bag of polys” cells are not entirely specific for endometrial neoplasia. Very similar cells are seen with inflamed endocervical polyps and represent reactive endocervical cells (Fig. 1.63C–D). Morphologic distinction can be impossible, and knowledge that the patient has an endocervical polyp may be the only clue to correct interpretation. Reactive endocervical cells and atypical repair (see Figs. 1.52 and 1.59C) mimic adenocarcinomas and vice versa.91 Reactive cells, paradoxically, often show more marked variation in nuclear size and nucleolar size and shape than adenocarcinomas, which are often deceptively uniform.91 Reactive cells have thin nuclear membranes compared with those of adenocarcinomas, which are often thick and sometimes

A

B

C

D • Fig. 1.63  “Bag of Polys” Cells: Differential Diagnosis.  (A and B): Endometrial adenocarcinoma. (A) “Bag

of polys” cells have a large cytoplasmic vacuole filled with neutrophils, often obscuring the nuclear detail of the neoplastic cell. (B) The histologic correlate is seen in the surface of the endometrial adenocarcinoma. (C and D): Inflamed endocervical polyp. (C) The large vacuolated cells are associated with neutrophils, just like the cells of endometrial adenocarcinoma. (D) Histologic sections reveal an acutely inflamed polyp lined by reactive endocervical cells infiltrated by polys.

CHAPTER 1  Cervical and Vaginal Cytology

irregular in contour. Reactive cells form sheets but rarely balls of cells, as is seen with many adenocarcinomas. There are cases, however, where doubt remains: these are diagnosed as “atypical glandular cells.” The distinction from AIS is problematic and not possible in many cases. If a tumor diathesis is present or the cells are round and have prominent nucleoli, the tumor is more than likely an invasive adenocarcinoma. Pemphigus vulgaris is a rare blistering disorder that involves mucous membranes, including the cervix. The squamous cells of the cervix lose their squamous structure and take on a pseudoglandular appearance, with a pale nucleus and prominent nucleolus. The features resemble those of repair except that isolated cells are prominent.237 

Atypical Endocervical Cells This category includes cases when an endocervical cell atypia raises the possibility of AIS or adenocarcinoma, but a benign reaction like repair, pregnancy-related change (e.g., Arias–Stella), endocervical polyp atypia, or microglandular hyperplasia159 cannot be excluded (see Fig. 1.59C–D). Depending on the severity of the atypia, one can leave the interpretation of “atypical endocervical cells” unqualified (“NOS”) or qualify it as “favor neoplastic” (Fig. 1.64A). DIFFERENTIAL DIAGNOSIS OF ATYPICAL ENDOCERVICAL CELLS • R  eactive endocervical cells • ASC-H • HSIL

Atypical Glandular Cells The category atypical glandular cells (AGC) is reserved for cases in which the cellular changes fall between those of a benign reactive process and an unequivocal AIS or adenocarcinoma. It represents 0.2% of all Pap interpretations124 and should be used only when the atypia raises the suspicion of AIS or adenocarcinoma; any case that is recognized as clearly benign should be reported as NILM. AGC is subclassified as “atypical endocervical,” “atypical endometrial,” or “not otherwise specified (NOS).” About 30% of patients with AGC have a significant lesion. Although some are AIS or invasive adenocarcinoma, many if not most of the lesions turn out to be CINs.32,238 This underscores the resemblance of HSIL and AIS. Cell block preparations from the residual LBC sample can be helpful by providing a “histologic” look at the atypical cells.48 Similarly, immunohistochemistry for p63, which highlights squamous but not glandular lesions, can be helpful in selected AGC cases to distinguish between squamous and glandular lesions.193 

A

47

  

The differential diagnosis of atypical endocervical cells includes reactive endocervical cells and squamous lesions. If nuclei are enlarged but round and regular in contour, with finely textured chromatin and prominent nucleoli, they are most likely reactive (see Fig. 1.27B). To qualify as atypical, endocervical cell nuclei should raise the suspicion of AIS, that is, they should be elongated, hyperchromatic, and crowded, often with an elevated nuclear-to-cytoplasmic ratio. A common error is mistaking squamous lesions, particularly HSILs, for atypical endocervical cells. Many HSILs have thin, pale, and even vacuolated cytoplasm (see Fig. 1.39). Atypical round cells are more likely to be HSIL than AIS, and for such cases ASC-H is a more appropriate interpretation; the cells of AIS are usually recognizably columnar. For this reason, “atypical endocervical cells” should be reserved for cells with a recognizably columnar morphology. If AIS is suspected, cell block preparations from a residual liquid-based sample can help by providing a “histologic”

B • Fig. 1.64  Atypical Glandular Cells.  (A) Atypical endocervical cells. These cells are columnar and clearly of endocervical origin but crowded and hyperchromatic. Although mitoses were not seen, the case was reported as “atypical endocervical cells, favor neoplastic.” (B) Atypical endometrial cells. These cells have enlarged nuclei with slightly irregular contours and some infiltration by neutrophils.

48 CHA P T ER 1    Cervical and Vaginal Cytology

look at hyperchromatic cell clusters48 (see Fig. 1.43A–B). Immunohistochemistry for p16 helps to identify HPVrelated neoplastic cells192; p63, which highlights squamous but not glandular lesions, helps distinguish squamous from glandular lesions.193 Management

Because of the high incidence of significant lesions in women with atypical endocervical cells, colposcopy with endocervical sampling is recommended regardless of HPV result.32 For women older than 35 years and younger women with unexplained vaginal bleeding, endometrial sampling should be included. If, after colposcopy, CIN2+ is not identified for atypical endocervical cells, NOS, cotesting at 12 and 24 months is recommended, and return to repeat cotesting in 3 years is recommended if both cotests are negative. (If any test result is abnormal, however, colposcopy is recommended.) If the Pap was qualified as “favor neoplasia” and invasive disease is not identified during the initial colposcopic workup, a diagnostic excisional procedure is recommended. 

Atypical Endometrial Cells Atypical endometrial cells are isolated cells or rounded clusters of cells with an enlarged nucleus and one or more additional features of nuclear atypia (e.g., membrane irregularity, prominence of nucleoli). Cytoplasm is scant or moderately abundant and vacuolated. Such cells are suspicious for endometrial adenocarcinoma, but the quantity of the altered cells or the degree of atypia is insufficient for a conclusive diagnosis of malignancy (Fig. 1.64B). Similar changes are known to be caused by endometrial polyps, chronic endometritis, IUDs, and endometrial hyperplasia. “Atypical endometrial cells” are not further qualified as “NOS” or “favor neoplasia.” Management

Because atypical endometrial cells carry a significant risk of cancer,239 endometrial and endocervical sampling is recommended.32 If no endometrial pathology is identified, colposcopy is recommended. 

CYTOMORPHOLOGY OF SMALL CELL NEUROENDOCRINE CARCINOMA • • • • • •

 lusters of small cells C Hyperchromatic nucleus Nuclear molding Scant cytoplasm Mitoses Nuclear smearing   

Cytologic preparations show clusters of small cells with hyperchromatic nuclei and finely granular chromatin; cytoplasm is scant (Fig. 1.65). Nuclear molding is present (see Fig. 1.13D), as are mitoses. Like their counterparts in the lung, these cells are fragile and show nuclear smearing. Often poorly preserved, the cells are easily confused with menstrual endometrial cells. Nuclear smearing and mitoses, however, are very uncommon with endometrial cells and provide a good clue to the diagnosis of a small cell neuroendocrine carcinoma. 

Malignant Melanoma Although more common in the vulva, melanomas can arise in the vagina and, even less frequently, the cervix. Vaginal melanomas occur predominantly in the elderly and are aggressive tumors. CYTOMORPHOLOGY OF MELANOMA • • • •

Isolated cells Large cells, epithelioid or spindled Round or oval nucleus Melanin (not all cases)   

The malignant cells are often isolated rather than clustered, and this pattern is helpful in distinguishing them from the more common carcinomas (Fig. 1.66). Tumor cells

Other Malignant Neoplasms Small Cell Neuroendocrine Carcinoma Tumors that resemble small cell carcinomas of the lung arise in the uterine cervix. Some have concomitant evidence of squamous differentiation and are a variant of poorly differentiated squamous cell carcinoma. Small cell neuroendocrine carcinoma (termed “high-grade neuroendocrine carcinoma, small cell type” by the World Health Organization), however, is a distinct entity, commonly associated with HPV type 18.202,240 It is highly aggressive, with a predilection for the early development of distant metastases.

• Fig. 1.65  Small Cell Neuroendocrine Carcinoma of the Cervix.  The malignant cells have dark nuclei and scant cytoplasm.

CHAPTER 1  Cervical and Vaginal Cytology

are large, with a round or oval nucleus and often a solitary macronucleolus. Cytoplasm is scant or abundant and in some cases demonstrates the telltale fine, brown granularity of melanin. Melanophages—histiocytes with abundant coarse ingested pigment—may be present. 

Malignant Lymphoma Non-Hodgkin lymphoma frequently involves the cervix and vagina when the disease is advanced. Rarely, it may arise as a primary tumor at these sites.241 Cytologic samples are negative if the mucosa is not ulcerated. The tumor cells are larger than small, mature lymphocytes, with a nucleus that is irregular in contour and coarsely granular. The differential diagnosis includes follicular cervicitis (see Fig. 1.16A–B), which is composed of a mixed population of lymphocytes in various stages of maturation, in contrast to the uniform population of atypical lymphoid cells characteristic of many lymphomas. 

Carcinosarcoma Carcinosarcoma (malignant mixed Müllerian tumor) arises much more commonly in the endometrium than in the cervix. Many cases in which the cervix is involved represent extension from an endometrial primary. As with endometrial carcinoma, the most common symptom is vaginal bleeding. The tumors are composed of malignant glands admixed with malignant spindle cells; the latter may show features of stromal sarcoma, leiomyosarcoma, rhabdomyosarcoma, chondrosarcoma, or liposarcoma. Much of the tumor is made up of undifferentiated cells. Smears are often highly cellular and contain malignant glandular or undifferentiated cells with scant cytoplasm. Malignant spindle cells may be present but are usually a minor component of the specimen. 

Metastatic Tumors Tumors from many sites can metastasize to the cervix or vagina and be detected with the Pap test. Perhaps the most common are stage III or IV adenocarcinomas of the ovary

and fallopian tube, which make their way to the cervix and vagina via the endometrial cavity.242 They are usually serous and resemble the serous carcinoma of the endometrium described above. Cytologic preparations may have a clean background if large, necrotic tumor implants have not been formed in the cervix. Psammoma bodies are small, concentrically laminated calcifications that stain dark blue with the Papanicolaou stain. They are commonly seen in some tumors of the ovary, fallopian tube, endometrium, and peritoneum, but are extremely rare in routine cervical or vaginal smears.236 Their presence should prompt a search for a neoplasm, especially if they are associated with atypical cells236 (Fig. 1.67). Carcinomas of the colon and rectum can spread directly to the vagina. Tumor cells frequently have a columnar shape with large, very hyperchromatic nuclei and a high nuclearto-cytoplasmic ratio. Isolated cells can have a signet ring cell appearance. Tumor necrosis may be present. Carcinomas of the bladder and urethra can also spread to the vagina. Tumor cells are large, often with hyperchromatic nuclei. Clinical correlation is needed to establish the site of origin. Tumors from distant sites such as the breast, kidney, pancreas, and lung can metastasize to the female genital tract. In general, precisely identifying the primary site is impossible without clinical history and previous biopsy material for comparison. 

Endometrial Cells in Women ≥45 Years of Age Although the Pap test is not a screening test for endometrial cancer, it has been known for decades that benign-appearing endometrial cells in an older woman may be a sign of endometrial neoplasia.243-250 Most women come to medical attention because of vaginal bleeding, but 10% to 25% are symptom free.251,252 It is not known whether the exfoliated endometrial

• Fig. 1.67  Psammoma Bodies.  These calcific spheres are dark blue • Fig. 1.66  Malignant Melanoma of the Vagina.  The malignant spin-

dled and epithelioid cells are noncohesive. There is focal finely granular melanin pigment (arrow).

49

or purple and have concentric laminations. They are often fractured, as seen here. These psammoma bodies originated from a borderline serous tumor of the ovary. When cells from ovarian or tubal neoplasms travel through the endometrial cavity, they can be seen on cervical or vaginal Pap samples.

50 CHA P T ER 1    Cervical and Vaginal Cytology

cells are even neoplastic or whether they represent just glandular and stromal breakdown associated with the neoplasm. Because of the associated risk, the 1991 Bethesda System recommended that benign-appearing endometrial cells in postmenopausal women be reported as an epithelial cell abnormality. The recommended terminology was “endometrial cells, cytologically benign, in a postmenopausal woman.” This presented an unanticipated difficulty because menopausal history is not always provided. If the menopausal status was not given, could this diagnosis be made based on age? If so, how old should a woman be for this interpretation to apply? The median age of final menstrual period is 52 years, but the coefficient of variation is large.253 The 2014 Bethesda System recommends reporting this for women aged 45 and above, irrespective of menstrual status and hormone replacement therapy.254 Pap test results with benign-appearing endometrial cells do identify a small number of symptom-free women on hormone replacement therapy with endometrial adenocarcinoma and hyperplasia.255 It was once believed that histiocytes alone convey an increased risk for endometrial cancer.256 This has been widely refuted.252,257,258 Thus only spontaneously exfoliated endometrial cells (Fig. 1.68) are considered significant. Directly abraded endometrium or lower uterine segment, like histiocytes, should not be reported under this heading. An educational note can be particularly helpful, as in this example: SAMPLE REPORT Satisfactory for evaluation. Endometrial cells present in a woman ≥45 years of age. Negative for squamous intraepithelial lesion. Note: Endometrial cells in women 45 years or older may be associated with benign endometrium, hormonal alterations, and, less commonly, endometrial or uterine abnormalities. Endometrial evaluation is recommended in postmenopausal women.   

•

Fig. 1.68  Endometrial Cells in a Woman Older Than 45 Years of Age.  These cells are indistinguishable from menstrual endometrial cells (see Fig. 1.12A).

In the 2014 Bethesda System, this interpretation is no longer categorized as an epithelial cell abnormality; because of the small but definite risk of endometrial neoplasia (3%), neither is it categorized as NILM.108 This orphan diagnosis falls into the general categorization “Other,” a heading some laboratories simply omit from the report, as in the example above. Because the primary goal of the Pap test is the identification of squamous precursors, the explicit statement “negative for squamous intraepithelial lesion” is included. This Pap interpretation represents 0.5% to 1% of all Pap reports.244-247 DIFFERENTIAL DIAGNOSIS OF ENDOMETRIAL CELLS IN WOMEN ≥45 YEARS OF AGE • C  rushed endocervical cells • Follicular cervicitis • Small blue nuclei   

A common mimic of endometrial cells in older women is the cluster of crushed, atrophic endocervical cells. They are recognized on the basis of some residual columnar shape. The lymphoid cell clusters of follicular cervicitis (see Fig. 1.16A–B) are another mimic. Lymphoid cells are about the same size or smaller than exfoliated endometrial cells and less tightly cohesive. Admixed larger, paler dendritic cell nuclei and tingible-body macrophages are typical of follicular cervicitis. Clusters of naked squamous cell nuclei (Fig. 1.69) are easily mistaken for endometrial cells, but can be identified because they have no cytoplasm. Naked squamous cell nuclei (often called “small blue cells”) are common in postmenopausal women and thus a frequent mimic of endometrial cells. They are seen in 21% of Pap test results from women over the age of 50, and their prevalence is proportional to the woman’s age.259 At one time their presence was associated with tamoxifen, a nonsteroidal antiestrogen used in the treatment and prevention of breast cancer, but the frequency of small blue nuclei is no higher in these patients than in women who are not taking tamoxifen.259

•

Fig. 1.69  Bare Squamous Cell Nuclei.  They are about the size of endometrial cells and sometimes aggregate. Cells that lack cytoplasm should not be interpreted as endometrial cells.

CHAPTER 1  Cervical and Vaginal Cytology

Management

Benign-appearing endometrial cells in women ≥45 years of age are usually not from a cancer or hyperplasia. In most women they are physiologic (the woman is still cycling, either naturally or because of hormone replacement therapy) or a result of benign endometrial pathology (e.g., an endometrial polyp). For this reason, an endometrial sample is not indicated for all women with this diagnosis. The woman’s physician, who knows her menstrual and menopausal status, clinical risk factors for endometrial cancer, and whether she is on hormone replacement therapy, should use his or her clinical judgment in deciding whether to take a histologic endometrial sample. Consensus guidelines recommend endometrial assessment for women who are postmenopausal.32

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CHAPTER 1  Cervical and Vaginal Cytology

226. Castellsague X, Diaz M, de Sanjose S, et al. Worldwide human papillomavirus etiology of cervical adenocarcinoma and its cofactors: implications for screening and prevention. J Natl Cancer Inst. 2006;98(5):303–315. 227. DiTomasso JP, Ramzy I, Mody DR. Glandular lesions of the cervix: validity of cytologic criteria used to differentiate reactive changes, intraepithelial lesions, and adenocarcinoma. Acta Cytol. 1996;40:1127–1135. 228. Granter SR, Lee KL. Cytologic findings in minimal deviation adenocarcinoma (adenoma malignum) of the cervix: a report of seven cases. Am J Clin Pathol. 1996;105:327–333. 229. Hagiwara T, Kaku T, Kobayashi H, Wake N, Saito T. Well-differentiated villoglandular adenocarcinoma of the uterine cervix: assessment of cytological features by histological subtypes. Acta Cytol. 2013;57(1):61–68. 230. Ballo MS, Silverberg SG, Sidawy MK. Cytologic features of well-differentiated villoglandular adenocarcinoma of the cervix. Acta Cytol. 1996;40:536–540. 231. Zhou J, Tomashefski Jr JF, Khiyami A. ThinPrep Pap tests in patients with endometrial cancer: a histo-cytological correlation. Diagn Cytopathol. 2007;35(7):448–453. 232. Thrall M, Kjeldahl K, Gulbahce HE, Pambuccian SE. Liquidbased Papanicolaou test (SurePath) interpretations before histologic diagnosis of endometrial hyperplasias and carcinomas: study of 272 cases classified by the 2001 Bethesda System. Cancer. 2007;111(4):217–223. 233. Wright CA, Leiman G, Burgess SM. The cytomorphology of papillary serous carcinoma of the endometrium in cervical smears. Cancer. 1999;87:12–18. 234. Kuebler D, Nikrui N, Bell D. Cytologic features of endometrial papillary serous carcinoma. Acta Cytol. 1989;33:120–126. 235. Todo Y, Minobe S, Okamoto K, et al. Cytological features of cervical smears in serous adenocarcinoma of the endometrium. Jpn J Clin Oncol. 2003;33(12):636–641. 236. Kern SB. Prevalence of psammoma bodies in Papanicolaoustained cervicovaginal smears. Acta Cytol. 1991;35:81–88. 237. Wright C, Pipingas A, Grayson W, Leiman G. Pemphigus vulgaris of the uterine cervix revisited: case report and review of the literature. Diagn Cytopathol. 2000;22:304–307. 238. Schnatz PF, Guile M, O’Sullivan DM, Sorosky JI. Clinical significance of atypical glandular cells on cervical cytology. Obstet Gynecol. 2006;107(3):701–708. 239. Cherkis RC, Patten SF, Dickinson JC, Dekanich AS. Significance of atypical endometrial cells detected by cervical cytology. Obstet Gynecol. 1987;69:786–789. 240. Stoler MH, Mills SE, Gersell DJ, Walker AN. Small cell neuroendocrine carcinoma of the cervix: a human papillomavirus type 18–associated cancer. Am J Surg Pathol. 1991;15:28–32. 241. Harris NL, Scully RE. Malignant lymphoma and granulocytic sarcoma of the uterus and vagina: a clinicopathologic analysis of 27 cases. Cancer. 1984;53:2530–2545. 242. Sasagawa M, Nishino K, Honma S, Kodama S, Takahashi T. Origin of adenocarcinoma cells observed on cervical cytology. Acta Cytol. 2003;47(3):410–414. 243. Ng ABP, Regan JW, Hawliczek S, Wentz B. Significance of endometrial cells in the detection of endometrial carcinoma and its precursors. Acta Cytol. 1974;18:356–361.

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244. Browne TJ, Genest DR, Cibas ES. The clinical significance of benign-appearing endometrial cells on a Papanicolaou test in women 40 years or older. Am J Clin Pathol. 2005;124(6): 834–837. 245. Thrall MJ, Kjeldahl KS, Savik K, Gulbahce HE, Pambuccian SE. Significance of benign endometrial cells in Papanicolaou tests from women aged >or=40 years. Cancer. 2005;105(4):207–216. 246. Bean SM, Connolly K, Roberson J, Eltoum I, Chhieng DC. Incidence and clinical significance of morphologically benignappearing endometrial cells in patients age 40 years or older: the impact of the 2001 Bethesda System. Cancer. 2006;108(1): 39–44. 247. Kapali M, Agaram NP, Dabbs D, Kanbour A, White S, Austin RM. Routine endometrial sampling of asymptomatic premenopausal women shedding normal endometrial cells in Papanicolaou tests is not cost effective. Cancer. 2007;111(1):26–33. 248. Moroney JW, Zahn CM, Heaton RB, Crothers B, Kendall BS, Elkas JC. Normal endometrial cells in liquid-based cervical cytology specimens in women aged 40 or older. Gynecol Oncol. 2007;105(3):672–676. 249. Li Z, Gilbert C, Yang H, Zhao C. Histologic follow-up in patients with Papanicolaou test findings of endometrial cells: results from a large academic women’s hospital laboratory. Am J Clin Pathol. 2012;138(1):79–84. 250. Moatamed NA, Le LT, Levin MR, Govind R, Apple SK. In Papanicolaou smears, benign appearing endometrial cells bear no significance in predicting uterine endometrial adenocarcinomas. Diagn Cytopathol. 2013;41(4):335–341. 251. Cherkis RC, Patten SF, Andrews TJ, Dickinson JC, Patten FW. Significance of normal endometrial cells detected by cervical cytology. Obstet Gynecol. 1988;71:242–244. 252. Zucker PK, Kasdon EJ, Feldstein ML. The validity of Pap smear parameters as predictors of endometrial pathology in menopausal women. Cancer. 1985;56:2256–2263. 253. Gold EB, Crawford SL, Avis NE, et al. Factors related to age at natural menopause: longitudinal analyses from SWAN. Am J Epidemiol. 2013;178(1):70–83. 254. Nayar R, Wilbur DC. The Pap test and Bethesda 2014. “The reports of my demise have been greatly exaggerated.” (after a quotation from Mark Twain). Acta Cytol. 2015;59(2):121–132. 255. Montz FJ. Significance of “normal” endometrial cells in cervical cytology from asymptomatic postmenopausal women receiving hormone replacement therapy. Gynecol Oncol. 2001;81: 33–39. 256. Koss LG, Durfee GR. Cytologic diagnosis of endometrial carcinoma: results of ten years of experience. Acta Cytol. 1962;6:519– 531. 257. Nguyen TN, Bourdeau JL, Ferenczy A, Franco EL. Clinical significance of histiocytes in the detection of endometrial adenocarcinoma and hyperplasia. Diagn Cytopathol. 1998;19:89–93. 258. Tambouret R, Bell DA, Centeno BA. Significance of histiocytes in cervical smears from peri/postmenopausal women. Diagn Cytopathol. 2001;24:271–275. 259. Opjorden SL, Caudill JL, Humphrey SK, Salomao DR. Small cells in cervical-vaginal smears of patients treated with tamoxifen. Cancer. 2001;93(1):23–28.

2

Respiratory Tract and Mediastinum MARINA VIVERO AND CHRISTOPHER A. FRENCH

E

xfoliative cytology was first used to study cells of the respiratory tract in 1845.1 The ability to diagnose pulmonary diseases cytologically was appreciated as early as 1919,2 but it was not until the 1950s and 1960s that pulmonary cytology came into its own as a diagnostic discipline. Its emergence was bolstered by the introduction of direct sampling methods via bronchoscopy and fine-needle aspiration (FNA),3 resulting in an impressive armamentarium of sampling techniques. Since then, the improved sensitivity (and common application) of thoracic imaging has created an ever-increasing need for the cytologic evaluation of pulmonary lesions.

Normal Anatomy, Histology, and Cytology of the Respiratory Tract The respiratory tract can be categorized into upper and lower compartments. The upper airway extends from the sinonasal region to the larynx. The lower respiratory tract, which is the major focus of diagnostic respiratory cytopathology, extends from the trachea to the lungs. The tracheobronchial tree divides into progressively smaller units: bronchi, bronchioles, and respiratory acini. ANATOMY AND CELLULAR COMPONENTS OF THE RESPIRATORY TRACT Upper respiratory tract: • Ciliated columnar cells • Squamous cells Lower respiratory tract: • Trachea and bronchi • Ciliated columnar cells • Goblet cells • Basal/reserve cells • Neuroendocrine cells • Terminal bronchioles • Nonciliated cuboidal/columnar cells (club [previously “Clara”] cells) • Alveoli • Type I and II pneumocytes • Alveolar macrophages   

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The trachea and bronchi are lined by a pseudostratified epithelium. The predominant cell is the ciliated columnar cell, which has a basally placed nucleus with finely textured chromatin. The luminal surface has a thick terminal bar with cilia (Fig. 2.1). Goblet cells, present in a ratio of approximately one per six ciliated cells, also have a basally located nucleus but lack cilia, and their cytoplasm is distended by mucus. Goblet cells secrete mucus, whereas ciliated cells move the mucus and entrapped contaminants up the airway. Adjacent to the basement membrane are basal or reserve cells: small, undifferentiated cells that are the presumed forerunners of the ciliated and goblet cells. Neuroendocrine cells, or Kulchitsky cells, are also present in the respiratory epithelium, but they are identified only with special stains or ultrastructural examination: they are argyrophil-positive and possess dense-core granules. The terminal bronchioles are lined by nonciliated cuboidal to columnar cells called club cells or respiratory exocrine cells (previously called Clara cells); they are not sufficiently distinctive with routine cytologic preparations and thus not specifically identified. The alveolar lining consists of type I and type II pneumocytes. Type I pneumocytes, which are more numerous, are paper thin and cover the gas exchange portion of the alveolar surface. The type II pneumocyte is more conspicuous: plump and cuboidal rather than flat. It secretes pulmonary surfactant, seen ultrastructurally as osmiophilic lamellar bodies. After lung injury, these cells function as reserve cells for the delicate type I pneumocyte. On cytologic preparations, type II pneumocytes are round and have vacuolated cytoplasm; they can be difficult to distinguish from macrophages. Alveolar (pulmonary) macrophages vary in appearance depending on the amount and type of phagocytosed cytoplasmic material. In general, they have one or more round to oval nuclei and lacy or bubbly cytoplasm, often with small black particles from inhaled pollutants (“dust cells,” Fig. 2.2A). After pulmonary hemorrhage, alveolar macrophages contain hemosiderin pigment, which is goldenbrown rather than black. Numerous alveolar macrophages must be present for a sputum sample to be judged adequate. Under normal circumstances, a few white blood cells, such as neutrophils and lymphocytes, are also found within

CHAPTER 2  Respiratory Tract and Mediastinum

the alveolar compartment. An increased number of inflammatory cells is abnormal: abundant neutrophils indicate an acute pneumonia, and numerous lymphocytes are usually associated with chronic inflammation. 

malignant cells,” “positive for malignant cells,” or “nondiagnostic (unsatisfactory),” each of which is associated with a different risk of malignancy.4 Inconclusive findings are commonly reported as “atypical cells present” (connoting a low degree of suspicion) or “suspicious for malignancy” (connoting a high degree of suspicion). Cancer is confirmed in 54% to 59% of “atypical” respiratory specimens and in 82% to 90% of those reported as “suspicious.”4,5 Atypical/ suspicious cases usually remain inconclusive even after careful retrospective reexamination, which fails to reveal any morphologic features to reliably distinguish benign from malignant specimens.6

Sampling Techniques, Preparation Methods, Reporting Terminology, and Accuracy Familiarity with the variety of sampling and preparation methods is crucial for cytologic interpretation because cytomorphology is different depending on the sampling and preparation method. The accuracy of respiratory cytology also varies depending on the specimen type. As with other nongynecologic cytology specimens, respiratory tract diagnoses are typically reported as “negative for

Sputum Sputum consists of a mixture of cellular and noncellular elements that are cleared by the mucociliary apparatus. It was once the most common respiratory tract specimen because it is relatively easy to obtain, with little discomfort to the patient. Sputum cytology is generally reserved for symptomatic individuals; as a screening test (e.g., in symptomfree smokers), sputum cytology is not effective in decreasing rates of death from lung cancer. With the advent of bronchoscopy and FNA, its use as the mainstay in respiratory cytology has declined significantly. Collecting multiple sputum samples over several days optimizes sensitivity. Early morning, deep cough specimens are preferred.7 If the patient is not able to expectorate adequately, expectoration can be induced by having the patient inhale nebulized water or saline solution. Sputum induction increases the detection of lung cancer.8 When prompt preparation of sputum is not possible, the patient can expectorate into a 70% ethanol solution, which prefixes the specimen. A simple method of sputum preparation is known as the “pick and smear” technique, whereby fresh sputum is

• Fig. 2.1  Normal Ciliated Bronchial Cells (Bronchial Brushing).  These

columnar cells have oval nuclei and finely stippled chromatin. Numerous cilia project from the apical surface (Papanicolaou stain).

A

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B • Fig. 2.2  Anthracotic Pigment and a Mimic (Black Particles).  (A) Pulmonary macrophages have abun-

dant foamy cytoplasm that often contains black carbon particles (anthracotic pigment), as seen here (Papanicolaou stain). (B) Abundant extracellular black metallic particles from an endobronchial ultrasoundguided procedure should not be construed as anthracotic pigment (hematoxylin and eosin stain).

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examined for tissue fragments, blood, or both. Smears are prepared from areas that contain these elements and immediately fixed in 95% ethanol. A modification of this is the Saccomanno method, which calls for sputum to be collected in 50% ethanol and 2% carbowax9; it must be performed in a biologic safety hood because of the risks of infection from aerosolization. The specimen is then homogenized in a blender and concentrated by centrifugation. Improved sensitivity has also been demonstrated by the use of dithiothreitol, N-acetyl-L-Cysteine, or CytoRich Red for mucolysis and homogenization.10 Smears are made from the concentrated cellular material. Sputum can also be processed using thin-layer methods or embedded in paraffin for cell block sections.11 The adequacy of a sputum sample is established by finding numerous pulmonary macrophages.7 Specimens consisting merely of squamous cells, bacteria, and Candida organisms are unsatisfactory because they represent only oral contents. Even ciliated cells, which also line the sinonasal passages, do not guarantee that a sample is from the lower respiratory tract. The presence of numerous macrophages indicates that a satisfactory, deep cough specimen of the lower respiratory tract has been obtained. In an adequate sample they should not be difficult to find: if they are absent or few in number, the sample should be reported as unsatisfactory. The sensitivity of sputum cytology for the diagnosis of malignancy increases with the number of specimens examined, from 42% with a single specimen to 91% with five specimens.12 The specificity of sputum examination is high, ranging from 96% to 99%, and the positive and negative predictive values are 100% and 15%, respectively.13 Thus negative sputum results do not guarantee the absence of a malignancy, especially in a patient suspected of having lung cancer. The sensitivity of sputum cytology depends also on the location of the malignant tumor: 46% to 77% for central lung cancers but only 31% to 47% for peripheral cancers.14,15 Sensitivity is independent of tumor stage and histologic type. Accuracy in tumor classification is 75% to 80%16 and is tumor-type dependent.17 

Bronchial Specimens A pivotal improvement in sampling the lower respiratory tract occurred with the development of the flexible bronchoscope in the late 1960s. Today, any part of the respiratory mucosa can be sampled with this device. Complications of bronchoscopy are rare (0.5% and 0.8% for major and minor complications, respectively)18 and include laryngospasm, bronchospasm, disturbances of cardiac conduction, seizures, hypoxia, and sepsis. The incidence of major complications is higher for transbronchial biopsy (6.8%).18

Bronchial aspirations and washings Bronchial secretions can be aspirated directly from the lower respiratory tract through the bronchoscope, but an alternative (and more common) method is to “wash” the mucosa

by instilling 3 to 10 mL of saline solution and suctioning the washings. The fluid is spun in a centrifuge, and the concentrate is used to make smears, thin-layer preparations, or cell blocks; the latter are particularly useful when special stains are needed. 

Bronchial brushings Fiberoptic bronchoscopy allows direct visualization and sampling of the tracheobronchial tree. A brush is applied to the surface of an endobronchial lesion, and the entrapped cells are either smeared onto a glass slide or rinsed in a collection medium for thin-layer or cell block preparation or both. If smears are made, immediate fixation (by immersion into 95% ethanol or by spray fixation) of the smears is essential to preserve morphologic detail.7 The diagnostic accuracy of bronchial washing/brushing cytology is comparable to that of bronchial biopsy.19 Brushings with cell block preparation sometimes detect malignancy more reliably than bronchial biopsy.20 Accuracy improves when clinical history is provided with the specimen.21 The diagnostic yield also improves when several different sampling methods are used in concert.22,23 

Bronchoalveolar lavage The choice between bronchoalveolar lavage (BAL) and bronchial washing depends on the location of the airway one desires to sample. With BAL, the bronchoscope is wedged into position as far as it will go to sample the distal airways, which are flushed with sterile saline solution. BAL is particularly useful for the diagnosis of opportunistic infections in immunocompromised patients. The specimen can be examined cytologically and a portion also submitted for microbiologic studies. The distinction between oral contamination and a real bacterial infection can be difficult, but an abundance of normal squamous cells usually indicates contamination by oral flora, whereas neutrophils imply a real infection.24 In immunocompromised patients, the diagnostic yield for infectious pathogens is 39%, the sensitivity 82%, and the specificity 53%.25 In patients with acquired immunodeficiency syndrome, BAL has a sensitivity for documenting infection comparable to that for transbronchial biopsy (86%); when used in combination with biopsy, sensitivity increases to 98%.26 Historically, the most common pulmonary pathogens detected by BAL in human immunodeficiency virus (HIV)–seropositive individuals were Pneumocystis carinii (78%) and bacteria (19%); the remainder were Mycobacterium tuberculosis, atypical mycobacteria, Histoplasma, and Cryptococcus.27 The frequency and distribution of infections has changed since the widespread use of highly active antiretroviral therapy to treat HIV.28 Among HIV-seropositive individuals with nonspecific cytologic results, 27% prove to have pathogens, usually bacterial or fungal, by either culture or biopsy,29 which emphasizes the importance of a multimodal approach to diagnosis in this setting. BAL is also used for the diagnosis of malignancy, with a sensitivity that ranges from 29% to 69%.30,31 The sensitivity

CHAPTER 2  Respiratory Tract and Mediastinum

of BAL for detecting malignancy is higher for multifocal or diffuse tumors,32 and lower in peripheral tumors that cannot be seen bronchoscopically.30 Liquid-based cytology preparations yield a slightly higher sensitivity.33 Falsepositive results are occasionally encountered due to atypical type II pneumocytes in the setting of pneumonia, diffuse alveolar damage,34 bone marrow transplantation,35 and chemotherapy.36 

Transbronchial FNA (“Wang Needle”) Transbronchial FNA is especially useful for pulmonary lesions hidden beneath the bronchial surface and for suspicious mediastinal lymph nodes.37-39 Transbronchial FNA eliminates the need for an addition surgical procedure in 20% of patients, at one-third the cost of mediastinoscopy.40 The lesion is aspirated with a retractable (Wang) needle passed through a flexible catheter that is sent down the bronchoscope. At least a moderate number of lymphocytes must be present to ensure the adequacy of lymph node sampling and avoid a false-negative result.41 Ciliated respiratory epithelial cells are common contaminants because the respiratory mucosa needs to be breached to reach the target. For this reason, ciliated cells should not be taken as evidence of adequate sampling of a mediastinal lymph node. Complications from transbronchial aspiration are rare and include endobronchial bleeding, which is usually controlled by suctioning. Contraindications are coagulopathy, respiratory failure, and uncontrollable coughing.42 Transbronchial FNA augments the diagnostic accuracy of bronchial washings, brushings, and endoscopic biopsy for the detection of primary pulmonary neoplasms.43,44 The sensitivity of transbronchial FNA by itself is 56% but increases to 72% when combined with bronchial brushing, washing, and biopsy. Specificity is 74%, and the positive and negative predictive values are 100% and 53% to 70%, respectively. Transbronchial FNA is accurate in distinguishing small cell from non-small cell lung cancer.45 For mediastinal staging of bronchogenic carcinoma, the negative predictive value of transbronchial FNA increases from 36% to 78% when negative specimens without sufficient lymphocytes are regarded as unsatisfactory for evaluation.41 The most common cause of false-negative results is sampling error.44 

Endobronchial Ultrasound-Guided (EBUS) FNA The accuracy of mediastinal staging by FNA improves with the use of ultrasound guidance.37,38 EBUS FNA is an enhanced procedure for sampling mediastinal and paratracheal lymph nodes and peribronchial lung or mediastinal lesions that is increasingly used in the United States.46 Its primary indication is nodal staging of non-small cell lung cancer,47 but it is also indicated for sarcoidosis and metastases from extrapulmonary primaries.48 This minimally invasive procedure is a safer alternative to cervical

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mediastinoscopy in selected patients. Using a bronchoscope equipped with an ultrasound probe tip, the operator performs an FNA with real-time ultrasound imaging of a lymph node or central lung mass. Sensitivity and specificity are 88% and 100%, respectively.48,49 To optimize sensitivity, a cytologist can assess the sample on site for adequacy. In the absence of lesional cells, adequacy is defined as the presence of lymphocytes (if a lymph node is being sampled) or pigmented macrophages (in the case of a lung mass). Some laboratories have developed and adopted quantitative adequacy criteria for mediastinal lymph node sampling (presence of anthracotic pigment, germinal center fragments, a minimum number of lymphocytes),50-52 but no single set of criteria has yet been universally adopted. As with transbronchial (Wang needle) FNA, ciliated respiratory epithelial cells are common contaminants and are not evidence of adequate sampling. Microscopic metallic dust from the needle (Fig. 2.2B) mimics anthracotic pigment. It can be recognized as a contaminant because of its haphazard distribution. The advantage of EBUS is that it increases accessibility to lower station lymph nodes: transbronchial (Wang needle) FNA can sample lymph node stations 2 to 4 and 7, and transesophageal ultrasound–guided FNA can reach stations 2 to 4 and 7 to 9, whereas EBUS can sample stations 2 to 4, 7, and 10 to 12.47 Thus esophageal FNA is often combined with EBUS for full accessibility. Although the EBUS FNA procedure itself is more expensive than transbronchial (Wang) FNA, it saves downstream costs because of its greater sensitivity and the reduced need for more surgical staging, and it has largely supplanted Wang biopsy in many practices.53 EBUS FNA may result in biopsy site changes, including intranodal or perinodal fibrosis and displacement of airway cartilage into lymph nodes. These artifacts, seen on subsequent lymph node excisions, should not be misconstrued as tumor-related changes.54 

Transesophageal FNA Mediastinal lymph node sampling can also be done endoscopically by passing the needle through the esophagus.55-57 The addition of ultrasound guidance improves the accuracy of mediastinal lymph node sampling.58 Like bronchoscopic FNA, endoscopic FNA realizes significant cost savings57 and reduces the number of unnecessary thoracotomies.56 The advantages of endoscopic transesophageal FNA include improved image quality and the ability to sample stations 8 and 9, which are not accessible via EBUS FNA,47 but the primary lesion usually cannot be sampled via this route. Endoscopic transesophageal FNA, when used in combination with transbronchial FNA, improves the diagnostic yield for mediastinal staging greatly, with a sensitivity of 93% and negative predictive value of 97%.59 As with transbronchial FNA, a moderate number of lymphocytes must be present to ensure the adequacy of a lymph node specimen and avoid a false-negative result. 

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Percutaneous FNA

ACCURACY OF PERCUTANEOUS FNA

The ease, rapidity of diagnosis, and minimal morbidity of percutaneous FNA make it an attractive alternative to surgical biopsy in the evaluation of the patient with a peripheral pulmonary mass. FNA is of greatest benefit to patients for whom it spares a more invasive surgical procedure. Surgical intervention, in fact, can be avoided in up to 50% of patients with clinically suspected lung cancer.60 There are some contraindications, however.

• • • • • •

RELATIVE CONTRAINDICATIONS TO PERCUTANEOUS FNA • • • • • • • • •

 hronic obstructive pulmonary disease C Emphysema Uncontrollable coughing Uncooperative patient Bleeding diathesis (e.g., anticoagulant therapy) Severe pulmonary hypertension Arteriovenous malformation Cardiac disease Suspected echinococcal cyst61   

The most common complication of percutaneous FNA is a pneumothorax. A radiographically detectable pneumothorax occurs in 19% of patients62; only 4%, however, require intercostal drainage tubes. The risk of a pneumothorax increases with larger needle size and smaller target lesions and decreases if the needle path does not traverse aerated lung.62 Pulmonary hemorrhage occurs in 6% of patients, and transient hemoptysis occurs in 2%.62 Other complications are rare and include hemopericardium, hemothorax, air embolism, tumor seeding, and death.62,63 Percutaneous FNA is usually performed by a radiologist using computed tomography or ultrasound guidance, with the latter best reserved for lesions that abut the pleura or chest wall. The needle gauge ranges from 18 to 25, and many different types of needle devices are available. Although many radiologists prefer 22-gauge Chiba needles, these require repuncture if more than one pass is needed. Another choice is the coaxial needle, with a large-bore outer needle serving as the guide for a small-bore inner needle. Once the outer needle is positioned, more than one aspiration can be performed using the inner needle. It can be helpful if a cytotechnologist, cytopathologist, or both attend the FNA procedure to assist with specimen handling and assess its adequacy on-site. After smears are prepared, the needle is rinsed in a balanced electrolyte solution, Saccomanno’s fixative, 50% ethanol, or commercial preservative solution. The cellular suspension can be processed as a cytospin, thin-layer preparation, or cell block, and it can be apportioned for flow cytometric analysis if needed. Formalin-fixed cell blocks are ideal for histochemical and immunohistochemical stains. A decision regarding the need, if any, for special studies can be made by the cytotechnologist or cytopathologist after examination of the smears. Percutaneous FNA is a reliable and accurate way to diagnose many pulmonary neoplasms.

 ensitivity: 89% S Specificity: 96% Positive predictive value: 98% Negative predictive value: 70% False-positive rate: 0.85% False-negative rate: 8%   

In a study of more than 13,000 FNA specimens from 436 institutions, the diagnostic sensitivity was 89% for the procedure itself and 99% for the pathologist’s interpretation.64 This difference indicates that most false-negative results are due to sampling error. The reliability of a negative FNA result is a matter of controversy, given that negative predictive values range from 50% to 91%.63,65 About 15% of false-positive diagnoses and 5% of false-negative diagnoses have a significant, permanent, or grave influence on patient outcome.64 For this reason, most investigators recommend a repeat aspiration or tissue biopsy when a specific benign diagnosis that accounts for the lesion cannot be made with certainty. The performance of small, cutting (“core”) biopsy is similar to that of FNA,63,66 but it is performed instead of FNA in some centers. With regard to the management of patients with primary lung cancer, it is important to discriminate small cell from non-small cell carcinoma, and adenocarcinoma from squamous cell carcinoma. The distinction between small cell and non-small cell carcinoma is possible in more than 95% of cases67 and between adenocarcinoma and squamous cell carcinoma in 88% of cases diagnosed by FNA.68 A variety of benign cells occasionally contaminate a percutaneous FNA. Such cells need to be recognized as contaminants and not misconstrued as lesional. In particular, mesothelial cells from the pleura are common, and in some cases they can be numerous (Fig. 2.3). They resemble the cells of a well-differentiated adenocarcinoma (see “Adenocarcinoma”) but are identified as benign mesothelial cells by their relative flatness, cohesion, and the characteristic slit-like “windows” that separate the mesothelial cells from each other. CONTAMINANTS OF PERCUTANEOUS FNA • • • • •

 esothelial cells M Cutaneous squamous cells Skeletal muscle Fibroconnective tissue Hepatocytes (trans-diaphragmatic needle path)   

If the specimen consists only of one (or more) of these contaminants, it should be interpreted as insufficient for evaluation (nondiagnostic) rather than negative because there is no evidence that the lesion itself has been sampled. 

CHAPTER 2  Respiratory Tract and Mediastinum

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• Fig. 2.3  Mesothelial Cells (Fine-Needle Aspiration).  Benign mesothelial cells are occasionally seen in

percutaneous fine-needle aspirates. They are arranged in flat, cohesive sheets. The cells have a round or oval nucleus, a small nucleolus, and a moderate amount of cytoplasm. A slit-like space between the cells (“windows”) can be appreciated (Papanicolaou stain).

A

B • Fig. 2.4  Reactive Bronchial Cells.  (A) Reactive bronchial cells can show marked nuclear size variation. Note that cilia—evidence of their benign nature—are retained (bronchial brushing, Papanicolaou stain). (B) In chronic lung diseases, as in this case of a patient with asthma, clusters of reactive bronchial cells can assume a spherical shape (“Creola body”) and resemble the cells of an adenocarcinoma. Normal nuclear features and cilia indicate their benign nature (bronchial washing, Papanicolaou stain).

Benign Cellular Changes Reactive Squamous Cell Changes Benign squamous cells from the oral cavity often contaminate sputum and bronchial cytology specimens. Inflammatory conditions of the mouth caused by trauma, candidiasis, or pemphigus can exfoliate mildly atypical squamous cells with hyperkeratinization and nuclear degeneration, usually in small numbers. Such minimal changes should not be misinterpreted as squamous cell carcinoma. More marked (but still benign) squamous cell atypias occur adjacent to cavitary fungal infections and stomas, and with almost any injury to the lung (e.g., infarction, radiation, chemotherapy, sepsis, and diffuse alveolar damage) and might result

in a false-positive interpretation of squamous cell carcinoma.35,36 Another, uncommon source of false-positive results is malignant cells from head and neck cancers that contaminate sputum and bronchial specimens.69 

Reactive Bronchial Cell Changes Benign, reactive bronchial cell changes occur in response to noxious stimuli such as radiation, chemotherapy, and severe inflammation. Under such conditions, ciliated columnar cells can increase their nuclear area many times over, with multinucleation, coarsely textured chromatin and large nucleoli (Fig. 2.4A). Large clusters of bronchial cells known as Creola bodies (named after the first patient in whom they were recognized)

64 CHA P T ER 2    Respiratory Tract and Mediastinum

• Fig. 2.5  Benign Treatment Effect (Bronchoalveolar Lavage).  Bronchial cells exposed to cytotoxic chemotherapy or radiation undergo marked cellular changes that mimic adenocarcinoma and/or squamous cell carcinoma. Nucleomegaly, smudgy hyperchromasia, vacuolization (cytoplasmic and nuclear), and a low nuclear-to-cytoplasmic ratio are characteristic (hematoxylin and eosin stain).

are commonly seen in chronic airway diseases like asthma (Fig. 2.4B). Bronchial cells may also undergo squamous metaplasia under conditions of chronic injury (e.g., aspiration, smoking), demonstrating an immature squamous phenotype with mild nucleomegaly and nuclear hyperchromasia. Bronchial cells with markedly reactive changes due to thoracic irradiation or cytotoxic chemotherapy mimic carcinoma (Fig. 2.5). Malignancy can be excluded if the atypical cells have cilia or demonstrate a spectrum of changes (from benign to markedly atypical) rather than the two distinct cell populations typical of a malignant sample obtained bronchoscopically. Note that in sputum and FNA specimens, a helpful dual cell population (malignant cells and bronchial cells) is usually not apparent. 

Bronchial Reserve Cell Hyperplasia As the surface epithelium of the respiratory tract is shed during lung injury, reserve cells proliferate and are seen in bronchial washings and brushings (Fig. 2.6). CYTOMORPHOLOGY OF RESERVE CELL HYPERPLASIA • • • • • •

 ightly packed cells T Very small cells Smudged, dark chromatin Nuclear molding Scant cytoplasm No mitoses or necrosis

• Fig. 2.6  Reserve Cell Hyperplasia (Bronchial Brushing).  These small

cells have dark nuclei and show nuclear molding. They are distinguished from small cell carcinoma by their extremely small size and the lack of necrosis. Compare these cells with the adjacent bronchial columnar cells (Papanicolaou stain).

Reserve cell hyperplasia (RCH) is a mimic of small cell carcinoma but is usually easily distinguished from it. Although they can be abundant and may appear as a “second population,” the cells of RCH show greater cohesiveness than small cell carcinoma, are typically smaller (approximately the size of a red blood cell), and show no mitoses or necrosis. 

Repair

  

Repair represents re-epithelialization of an ulcer created by trauma, radiation, burns, pulmonary infarction, or infection. Typical (and atypical) repair of the respiratory tract is very similar to that seen in the cervix and gastrointestinal tract.

CHAPTER 2  Respiratory Tract and Mediastinum

When floridly hyperplastic, as in diffuse alveolar damage, the cells of type II pneumocyte hyperplasia resemble those of adenocarcinoma (Fig. 2.7).

CYTOMORPHOLOGY OF REPAIR • • • • •

 lat, cohesive sheets F Abundant cytoplasm Enlarged, often hypochromatic nuclei Enlarged nucleoli Mitoses

CYTOMORPHOLOGY OF TYPE II PNEUMOCYTE HYPERPLASIA   

Reparative epithelium is most commonly seen in tracheobronchial brushings and washings. The differential diagnosis of repair includes malignancy: the non-small cell lung cancers, metastases, and other, less common tumors. Malignant cells are usually less cohesive and less orderly than reparative epithelium, and they are usually more numerous. Correlation with clinical history can be helpful; a conservative approach is recommended if the findings are not conclusive and there is a history of mucosal trauma or other lung injury. 

Type II Pneumocyte Hyperplasia Because type II pneumocytes function as alveolar reserve cells, they proliferate after lung injury. CAUSES OF TYPE II PNEUMOCYTE HYPERPLASIA • • • • • • •

65

 neumonia P Sepsis (diffuse alveolar damage) Pulmonary embolus with infarction Chemotherapeutic and immunotherapeutic drugs Radiation therapy Inhalant damage (e.g., oxygen toxicity) Interstitial lung disease

• • • • •

Isolated cells and three-dimensional clusters Large nuclei Coarse chromatin Prominent nucleoli Scant to abundant, sometimes vacuolated cytoplasm   

The only clue to avoiding an incorrect diagnosis of malignancy in a patient with type II pneumocyte hyperplasia may be the clinical history of respiratory distress and diffuse infiltrates in the context of an inciting stimulus such as upper respiratory infection, recent radiation, or recent administration of therapeutic drugs. Thus, in an acutely ill patient with diffuse pulmonary infiltrates, markedly atypical cells should be interpreted cautiously, particularly in the setting of an acute inflammatory background.35 Sequential respiratory specimens can be helpful because hyperplastic pneumocytes are not present in BAL specimens more than 1 month after the onset of acute lung injury.34 

Noncellular Elements and Specimen Contaminants

  

Noncellular and extraneous elements in respiratory material can be inhaled, produced by the host, formed as a host response to foreign material, or introduced as laboratory contaminants.

• Fig. 2.7  Type II Pneumocyte Hyperplasia (Bronchoalveolar Lavage).  In patients with lung injury, type II

pneumocytes are markedly enlarged and mimic adenocarcinoma, as seen here. (A hemosiderin-laden macrophage is also present.) This patient had diffuse infiltrates and marked respiratory distress due to diffuse alveolar damage. In such clinical settings, an unequivocal diagnosis of malignancy should be avoided, inasmuch as most patients with lung cancer are not usually seriously Ill at presentation (Papanicolaou stain).

66 CHA P T ER 2    Respiratory Tract and Mediastinum

Curschmann’s spirals are coiled strands of mucus that stain purple with the Papanicolaou stain (Fig. 2.8). In the past they have been associated with chronic respiratory diseases, but they are, in fact, a nonspecific finding not worth mentioning in the report. Ferruginous bodies are mineral fibers encrusted with ferroproteins. Usually dumbbell-shaped, ranging from 5 to 200 μm in length, they stain golden-yellow to black with Papanicolaou stains (Fig. 2.9). Some but not all ferruginous bodies contain a core of asbestos. So-called asbestos bodies

• Fig. 2.8  Curschmann’s Spiral (Sputum).  These coils of inspissated

mucus are commonly seen in respiratory specimens and are a nonspecific finding (Papanicolaou stain).

•

are distinguished from other ferruginous bodies by their clubbed ends and thin, straight, lucent core. Asbestos fibers are not visible by light microscopy but are usually much more numerous than asbestos bodies. Patients with known asbestos exposure usually have high numbers of ferruginous bodies in BAL fluid.70 Charcot–Leyden crystals are rhomboid-shaped, orangeophilic structures derived from degenerating eosinophils in patients with severe allergic disorders like asthma or other eosinophilic conditions (Fig. 2.10). Psammoma bodies are concentrically laminated calcifications seen in malignant tumors that have papillary architecture, such as primary pulmonary adenocarcinoma, mesothelioma, or metastatic thyroid or ovarian cancer. They are also seen in benign conditions such as pulmonary tuberculosis and alveolar microlithiasis. Corpora amylacea are spherical structures with circumferential and radiating lines. They measure between 30 and 200 μm and are indistinguishable from those seen in the prostate. They have no known significance but are more commonly seen in older individuals (Fig. 2.11). Amorphous protein in respiratory specimens may be a clue to the diagnosis of amyloidosis or alveolar proteinosis. Specimen contaminants include metallic particles (see Fig. 2.2B), vegetable matter (Fig. 2.12), pollen, and the pigmented fungus Alternaria (Fig. 2.13A–B). 

Fig 2.9  Ferruginous Bodies (Bronchoalveolar Lavage).  Patients with asbestos exposure and a high burden of asbestos fibers in lung tissue demonstrate ferruginous bodies in bronchoalveolar lavage fluid: golden-brown, beaded, rod-like structures with bulbous ends. Inflammatory cells such as macrophages may be associated with ferruginous bodies (Papanicolaou stain).

CHAPTER 2  Respiratory Tract and Mediastinum

67

• Fig. 2.10  Charcot–Leyden Crystals (Bronchial Washing).  These needle-shaped crystals from a patient with asthma are a byproduct of eosinophil degranulation (Papanicolaou stain).

• Fig. 2.11  Corpora Amylacea (Bronchoalveolar Lavage).  These large

acellular bodies are somewhat variable in appearance. They may be spherical, as seen here, or angulated. They have fine radial striations and may have concentric laminations. Occasionally, there may be a central pigmented core. They are produced in the lung and other organs and have no known significance. Pulmonary corpora amylacea are not calcific, which distinguishes them from psammoma bodies and the laminated spheres of pulmonary alveolar microlithiasis (Papanicolaou stain).

Infections Cytology plays an important role in diagnosing infectious diseases, particularly those in immunocompromised patients, and is being used more frequently than ever because of improved sampling methods. It is important to know that conventional inflammatory responses can be much reduced, absent, or greatly altered in patients with immune deficiencies.

•

Fig. 2.12  Vegetable Cells (Sputum).  Some vegetable cells have elongated shapes and large nuclei, resembling the cells of keratinized squamous cell carcinoma. Their rectangular shape, uniform size, and refractile cellulose wall, however, help identify them as vegetable cells (Papanicolaou stain).

Viral Infections Herpes Simplex Herpes simplex virus (HSV) pharyngitis, laryngotracheitis, and pneumonia most commonly affect immunocompromised patients and neonates, and HSV-1 is the most common serotype to involve the respiratory tract. Ulcerative/ necrotizing infections can involve the pharynx, larynx, tracheobronchial tree, or pulmonary parenchyma, and cytopathic changes are identical to those seen in other sites: multinucleation, nuclear molding, chromatin margination, and large nuclear (Cowdry A) inclusions (Table 2.1). The

68 CHA P T ER 2    Respiratory Tract and Mediastinum

A

B • Fig. 2.13  Alternaria (Bronchoalveolar Lavage).  This pigmented fungus is rarely pathogenic. It can con-

taminate virtually any cytologic specimen, including cervicovaginal smears, cerebrospinal fluids, and urines. (A) The slender septate stalks (conidiophores) are occasionally branched (not shown). (B) The conidia are snowshoe shaped and have both transverse and longitudinal septations (Papanicolaou stain).

TABLE 2.1    Pulmonary Viral Infections

Virus

Nuclear features

Inclusions

Other changes

Herpes simplex and herpes zoster

Multinucleation; molding; peripheral margination of chromatin

Eosinophilic, intranuclear (Cowdry type A)

—

Cytomegalovirus

Enlargement

Large intranuclear, baso­ philic, with halo; small cytoplasmic, basophilic

Cytoplasmic enlargement

Measles virus

Multinucleation

Eosinophilic, intranuclear; multiple eosinophilic intracytoplasmic

Giant cells

Respiratory syncytial virus

Multinucleation

Cytoplasmic, basophilic, with halo

Giant cells; necrosis

Adenovirus

Smudged appearance as a result of large inclusion that fills entire nucleus

Large intranuclear, basophilic

Ciliocytophthoria

cytopathic changes of HSV are identical to those of herpes zoster. If the cytomorphologic changes are equivocal, the diagnosis can be confirmed by viral culture, immunohistochemistry, or in situ hybridization.71 

Cytomegalovirus Cytomegalovirus (CMV) is one of the most common of opportunistic infections. Patients with CMV pneumonia often present with fever, dyspnea, cough, and diffuse nodular or reticular interstitial infiltrates. Viral cytopathic changes (cytomegaly and large basophilic nuclear and small

basophilic cytoplasmic inclusions) are found in bronchial cells, pneumocytes, macrophages, endothelial cells, and fibroblasts (Table 2.1). The diagnosis can be confirmed by viral culture, immunohistochemistry, in situ hybridization, or the polymerase chain reaction.71-73 

Measles Virus and Respiratory Syncytial Virus Measles is a highly contagious, usually self-limited disease caused by the rubeola virus. The incidence has been curtailed due to the widespread use of a vaccine. Measles pneumonia occurs as an opportunistic complication, however,

CHAPTER 2  Respiratory Tract and Mediastinum

in children immunocompromised due to premature birth, cystic fibrosis, malignancy, or an immunologic disorder. Infection causes a pneumonia characterized by enormous multinucleated epithelial cells (pneumocytes) with cytoplasmic and nuclear inclusions (Table 2.1).74 Similar findings are seen with infection by the respiratory syncytial virus (RSV). The diagnosis is usually confirmed by detecting RSV antigen in BAL specimens. 

Adenovirus Adenovirus infection usually produces only a minor febrile illness, but adenovirus pneumonia can be severe and fatal, particularly in the immunocompromised. The virus causes two types of nuclear inclusions. One is the smudge cell, in which a large basophilic inclusion usually fills the entire nucleus and obscures chromatin detail. The other is eosinophilic inclusions that resembles the Cowdry A inclusion of HSV infection. A curious morphologic decapitation of ciliated columnar cells, called ciliocytophthoria, can be prominent.75 The detached cell apex, represented by only the terminal bar and cilia, without its nucleus, resembles a floating tuft of hair or eyelash (Table 2.1). 

Bacterial Pneumonias Bacterial pneumonias are caused by a large number of bacteria, but most are characterized by a neutrophilic exudate. Common organisms include Streptococcus pneumoniae (pneumococcus), other Streptococci, Staphylococcus aureus, Haemophilus influenzae, Klebsiella pneumonia, Pseudomonas sp., Legionella sp., Nocardia sp., Actinomyces sp., and some anaerobic bacteria. Many but not all bacteria can be seen with routine stains as well as with the Gram stain. Cytologic examination is not usually used for the diagnosis of a bacterial pneumonia, which is typically established by correlating clinical findings with microbiologic studies. Bacterial pneumonias often have a characteristic lobar or lobular distribution, but some pneumonias appear as

A

69

round and circumscribed masses on imaging studies and thus mimic the appearance of a malignancy. In such cases, a cytologic specimen might be obtained because the working clinical diagnosis is a suspected malignancy. Several bacteria deserve special mention. Actinomyces species are a common inhabitant of the tonsillar area and thus a common contaminant of sputum and bronchial specimens (but not FNAs). Infection by Actinomyces, however, is uncommon. Pulmonary infection occurs by aspiration of oral contents or by direct extension from subdiaphragmatic abscesses. Actinomycosis is usually a chronic infection that may result in sinus tracts. The bacteria aggregate into grossly visible sulfur granules (so-called because they look yellow on gross examination) and evoke a brisk neutrophilic response. When they appear in cytologic specimens as just an oral contaminant, Actinomyces bacteria are large blue “cotton balls” often associated with squamous cells, with no neutrophilic infiltrate, similar to what is occasionally encountered in Pap specimens (see Fig.1.23). A true thoracopulmonary actinomycosis should be considered if the bacteria are associated with abundant neutrophils. Nocardia are aerobic, filamentous bacteria that inhabit the soil and are acquired by inhalation. Nocardia asteroides accounts for 80% of Nocardial infections. Nocardia infection is most common in the setting of immunosuppression, and most patients have a subacute presentation. Cavitary nodules are seen in one-third of patients. The organisms are found among abundant neutrophils. They are thin, filamentous, and beaded, with such extensive, predominantly right-angle branching that they resemble Chinese characters. They are Gram-positive (Fig. 2.14A) and stain with silver stains but are only weakly acid-fast and thus require modified acid-fast stains like the Fite-Faraco for visualization. (Fig. 2.14B). The diagnosis is established by culture of a biopsy or BAL fluid. Legionella pneumonia is caused by the aerobic Gramnegative bacteria Legionella sp., of which the most common is Legionella pneumophila. The organisms are seen well

B • Fig. 2.14  Nocardia (Bronchoalveolar Lavage).  (A) The filamentous, beaded bacterial forms are visualized with Gram stains. (B) They are positive with modified acid-fast stains.

70 CHA P T ER 2    Respiratory Tract and Mediastinum

with silver stains like the Steiner, Warthin-Starry, or Dieterle stains. A specific identification of L. pneumophila can be made in BAL samples using immunohistochemical or immunofluorescent methods. 

tuberculosis, there may be an abundance of acid-fast organisms but few well-formed granulomas. If Romanowsky-type stains are used in such cases, the abundant acid-fast organisms can be identified as negative images. 

Tuberculosis

Pulmonary Fungal Infections

Infection by M. tuberculosis commonly results in granulomatous inflammation (Fig. 2.15). Cytologic specimens contain aggregates of epithelioid histiocytes, lymphocytes, and Langhans’ giant cells. Necrosis may or may not be evident. Granulomas by themselves, however, are a nonspecific finding and can be seen in other conditions like fungal infections and sarcoidosis. A definitive diagnosis of tuberculosis rests on identifying the organisms with the help of histochemical stains (Ziehl-Neelsen or auramine-­rhodamine),76 immunohistochemical stains,77 or by microbiologic culture. Cell block preparations are particularly useful for special stains, but rarely are more than one or just a few organisms identified (by contrast, infection by Mycobacterium avium intracellulare, as seen in immunocompromised patients, often yields innumerable acid fast organisms). A sensitive and specific assay called the Mycobacterium tuberculosis direct test is also available for the detection of M. tubercu­ losis and can be applied to respiratory specimens like sputum.76 The assay amplifies M. tuberculosis ribosomal RNA by the polymerase chain reaction and has been approved by the Food and Drug Administration (FDA) for use in both Mycobacterium smear-positive and smear-negative respiratory samples. Other highly sensitive polymerase chain reaction (PCR)–based assays and matrix-assisted laser desorption ionization-time of flight mass spectrometry for the distinction between tuberculous and nontuberculous mycobacteria are used increasingly because nontuberculous mycobacterial infections have become more frequent in developed countries.76,78 In countries where M. tuberculosis is prevalent, the yield of acid-fast bacteria among all clinically suspicious lung masses can be very high.79,80 In immunocompromised patients with

Pulmonary fungal infections are readily diagnosed by cytology, particularly in transthoracic FNA, and should be suspected whenever there is granulomatous inflammation, necrosis, or both. Cell block material can be used for silver or periodic acid–Schiff stains. Many fungi have a characteristic microscopic appearance that enables a rapid, specific diagnosis (Table 2.2).

A

Cryptococcosis Cryptococcus neoformans is an inhabitant of the soil and is found in bird droppings. It can act as both a primary and an opportunistic pathogen and may involve numerous body sites. Pulmonary invasion by this fungus is heralded clinically by a productive cough, fever, and weight loss. 

Histoplasmosis Histoplasma capsulatum, another inhabitant of soil enriched by bird and bat droppings, is contracted by the inhalation of spores and more commonly affects the immunocompromised. In the United States, it occurs most commonly in the Mississippi and Ohio river valleys, but it can occur anywhere. The disease can mimic tuberculosis clinically, in that peripheral nodular lesions and mediastinal lymphadenopathy are relatively common. The organism, often present within the cytoplasm of macrophages, is so small that it may be overlooked if silver stains are not used. 

Blastomycosis Blastomyces dermatitidis inhabits wooded terrain. Although the lung is the primary target of infection, there may be distant spread to other organs, such as skin, bone, and the urinary tract. 

B • Fig. 2.15  Granulomas (Fine-Needle Aspiration).  (A) Granulomas are tight aggregates of epithelioid histio-

cytes. They have a syncytial appearance because individual cell borders are indistinct (Papanicolaou stain). (B) Note the curved, elongated, boomerang-like shapes of some of the nuclei (Romanowsky stain).

CHAPTER 2  Respiratory Tract and Mediastinum

71

TABLE 2.2    Pulmonary Fungal Infections

Immunocompetent response Appearance

Disease

Organism

Demographics

Morphology

Size

Cryptococcosis

Cryptococcus neoformans

Worldwide

Yeast; variably sized; narrow-based budding; mucin capsule (immuno­ competent); refractile center

4–15 μm Granulomatous (diameter)

Histoplasmosis

Histoplasma capsulatum

Americas, espe­ cially Ohio and Mississippi river valleys

Small budding yeast; 1–5 μm Granulomatous intracellular (diameter)

Blastomycosis

Blastomyces dermatitidis

North America

Broad-based budding yeast; thick cell wall

8–20 μm Neutrophilic/ (diameter) granulomatous

Coccidioido­ mycosis

Coccidioides immitis

North American deserts

Spherules; endospores

15–60 μm; Granulomatous 1–2 μm (diameter)

Paracoccidioid­ omycosis

Paracoccidioides Central and braziliensis South America

Sporotrichosis

Sporothrix schenckii

Worldwide

Intracellular ovoid yeast with slight halo

2–4 μm Neutrophilic/ (diameter) granulomatous

Aspergillosis

Aspergillus fumigatus; A. flavus

Worldwide

Hyphae septate; 45-degree angle branching; occasional fruiting bodies in cavities

Hyphae Tissue/vascular 10–30 μm invasion; coloni­ (width) zation; fungus balls

Phycomycosis Mucor; Absidia; Worldwide (zygomycosis) Rhizopus; Cun­ ninghamella

Hyphae variably sized, ribbonlike nonseptate; 90-degree angle branching

Hyphae Tissue/vascular 10–30 μm invasion (width)

Candidiasis

Budding yeast form­ 2–10 μm ing pseudohyphae (width) (“sausage links”)

Candida albicans; Worldwide C. tropicalis; C. glabrata

Yeast with multiple 4–40 μm Neutrophilic/ budding (“mariner’s (diameter) granulomatous wheel”)

Coccidioidomycosis Coccidioides immitis infection is very common in endemic areas of the Southwest and Western United States, giving rise to positive skin test results in more than 80% of individuals in these areas. It produces a respiratory infection that usually resolves spontaneously but persists as a pulmonary mass in about 2% of patients. Multiorgan dissemination is more common in the immunocompromised patient. Because BAL or bronchial washings detect less than 50% of culture-positive cases,81 cytologic diagnosis is best documented by transthoracic FNA. The organisms appear as mature (sporulating) or immature spherules, often with a fractured (broken ping-pong ball) appearance (Fig. 2.16), and as free endospores (Table 2.2) in a background of granular eosinophilic debris with little inflammation. Hyphae

Respiratory space colonization; tissue invasion

are seen in 5% of cases. FNA cytology is diagnostically far superior to the culture of aspirated materials.82 

Paracoccidioidomycosis Paracoccidioidomycosis, also known as South American blastomycosis, is caused by the dimorphic fungus Paracoc­ cidioides brasiliensis (Fig. 2.17) and is the most common systemic mycosis in Latin America.83 It frequently involves the lungs and mucocutaneous areas of healthy individuals and clinically simulates tuberculosis. There may be squamous metaplasia with atypia of the bronchial epithelium overlying granulomas, which may lead to an erroneous diagnosis of squamous cell carcinoma in cytologic material.84 There is a high sensitivity (50%–90%) for the detection of the organism in cell block preparations of sputum.85 

72 CHA P T ER 2    Respiratory Tract and Mediastinum

A

B • Fig. 2.16  Coccidioides Immitis.  (A) A fractured spherule releases endospores, resulting in the appear-

ance of a broken ping-pong ball (Papanicolaou stain). (B) A similar fractured spherule is seen in sections from the cell block (hematoxylin and eosin stain).

• Fig. 2.17  Paracoccidioidomycosis.  A silver stain highlights the ship’s-

wheel appearance of yeast forms budding off from the central parent yeast. The broad-based budding resembles that of Blastomyces species; hence, its alternative term, South American blastomycosis.

Sporotrichosis Pulmonary infection caused by Sporothrix schenckii is uncommon and occurs mainly in immunocompromised patients, including diabetics and alcoholics. The clinical symptoms are nonspecific. Although often self-limited, these infections can become chronic, with mass lesions or cavitary nodule formation. The yeasts resemble Cryptococcus, Histoplasma, and Candida, and therefore culture or fluorescent antibody staining is necessary for definitive diagnosis.86 

Invasive Fungi This subgroup of fungi characteristically invades pulmonary tissue, especially blood vessels, of immunocompromised patients. They are readily diagnosed by transthoracic FNA. Aspergillosis

Aspergillus species can cause a variety of pulmonary disorders. Bronchopulmonary aspergillosis is characterized by the

• Fig. 2.18  Aspergillus Niger Fruiting Body.  This sample was obtained by fine-needle aspiration of a lung mass (cell block, periodic acid–Schiff stain).

expectoration of mucus plugs containing fungal organisms, numerous eosinophils, and Charcot–Leyden crystals. When invasive, there may be hemorrhagic necrosis caused by mycelial invasion of vessels. In cavitary lesions, the organisms sporulate, producing fruiting heads (Fig. 2.18), and are associated with polarizable calcium oxalate crystals. There may be an associated squamous cell atypia that is indistinguishable from carcinoma, making clinical correlation imperative.36  Zygomycosis

Zygomycosis is caused by several mycelia-forming fungi, including Mucor (Fig. 2.19), Absidia, Cunninghamella, and Rhizopus. They are angioinvasive and often cause infarctions in the debilitated patient.  Candidiasis

Candida pneumonia is a common opportunistic infection. An elevated level of Candida antigen in BAL fluid suggests true infection rather than colonization.87 

CHAPTER 2  Respiratory Tract and Mediastinum

73

Pneumocystis Jirovecii

• Fig. 2.19  Mucor Spp. (Fine-Needle Aspiration).  Broad, ribbon-like,

aseptate hyphae with right-angle branching are characteristic of this fungus (Papanicolaou stain).

The taxonomy of this organism has been debated, but microbiologists favor classifying it as a fungus, based in part on molecular evidence.88,89 The pneumonia caused by Pneumo­ cystis jirovecii is particularly common in immunocompromised individuals, but has decreased in frequency since the advent of novel therapies.28 The clinical presentation includes dry cough, fever, and dyspnea. Pulmonary infiltrates are usually bilateral. The organisms are well demonstrated in BAL material, which has a sensitivity that compares favorably with transbronchial biopsy.90 They can also be detected in bronchial washings and induced sputum.91 With Papanicolaou stains, the organisms themselves are not visible, but masses of organisms enmeshed in proteinaceous material result in green, foamy alveolar casts that are more circumscribed than debris or lysed red blood cells (Fig. 2.20A). The cysts are visualized with silver stains. They are cup-shaped,

A

B

C

D • Fig. 2.20  Pneumocystis Jirovecii (Bronchoalveolar Lavage).  (A) With the Papanicolaou stain, the pneu-

mocystis organisms are not seen, but foamy proteinaceous spheres characteristic of this infection are identified. (B) The cysts, which have a cup-shaped configuration and a central dark zone, are seen with the methenamine silver stain. (C) The Giemsa stain outlines the cysts as negative images and stains the intracystic bodies or trophozoites. Each cyst, as seen here, contains eight intracystic bodies. (D) The direct immunofluorescence test is highly sensitive, revealing green fluorescent-stained organisms and their extracellular products.

74 CHA P T ER 2    Respiratory Tract and Mediastinum

measure 5 to 7 μm in diameter, and often have a central dark zone (Fig. 2.20B). No budding occurs, which helps distinguish them from Histoplasma. The Giemsa stain highlights the cysts as negative images, but the eight 1.5μm intracystic bodies or trophozoites are stained as discrete blue dots either within the cysts or as free organisms (Fig. 2.20C). In some cases, the foamy alveolar casts are absent, and the organisms may be present only in vacuolated macrophages.92 Direct immunofluorescence has higher sensitivity (up to 92%) than Giemsa, toluidine blue, and silver stains and remains the gold standard for detection of P. jirovecii in sputum samples.93,94 Application of this method to induced sputum (Fig. 2.20D) is the preferred initial diagnostic step because it is noninvasive and highly accurate. Real-time PCR tests have comparable or better sensitivity than direct immunofluorescence and are commercially available, but have not yet been adopted widely.93,94 

Parasitic Infections Strongyloidiasis Pulmonary strongyloidiasis is caused by the nematode Strongyloides stercoralis. It can affect immunocompetent persons but is more common in the immunosuppressed patient and presents as a pneumonitis with hemoptysis. Infection of the lungs is caused by the hematogenous migration of the infective larva (filariform larva) from the gut or skin. Histologically, there is a hemorrhagic pneumonia. This organism is identified in sputum or bronchial material by its large size and is differentiated from other hookworms by its notched tail and short buccal cavity (Fig. 2.21). 

Dirofilariasis Dog heartworm (Dirofilaria immitis) infection of the human lung has been documented by FNA.95 This microfilarial disease is transmitted from dogs to humans via mosquitos, resulting in entrapment within small pulmonary vessels and subsequent pulmonary infarction. Diagnosis is made by aspiration of the discrete peripheral nodule, which shows necrotic material, fragments of infarcted pulmonary tissue, chronic inflammation, a granulomatous response, and rarely the worm itself. 

Echinococcosis (Hydatid Disease) The clinical and cytologic features of Echinococcosis are described in Chapter 13 (see Fig. 13.4). Sputum may contain scoleces if pulmonary hydatid cysts rupture. Because of the risk of anaphylactic shock, aspiration of a clinically suspected hydatid cyst may be hazardous.61,96 

Nonneoplastic, Noninfectious Pulmonary Diseases Sarcoidosis This common disease of unknown cause is characterized by noncaseating granulomas in many organs, most commonly the lung. CYTOMORPHOLOGY OF SARCOIDOSIS • A  ggregates of epithelioid histiocytes • Multinucleated giant cells • Lymphocytes

• Fig. 2.21  Strongyloides (Sputum).  These large roundworms are distinguished by their short buccal cavities (arrow) (Papanicolaou stain).

  

CHAPTER 2  Respiratory Tract and Mediastinum

Cytologic specimens show noncaseating granulomas comprised of epithelioid histiocytes, with scattered lymphocytes and multinucleated, giant histiocytes (see Fig. 2.15).97 The epithelioid histiocytes have round, oval, curved (boomerang-shaped), or spindle-shaped nuclei, with translucent, vacuolated cytoplasm and are haphazardly arranged in a pseudosyncytial pattern. 

Granulomatosis with Polyangiitis Granulomatosis with Polyangiitis, formerly known as Wege­ ner granulomatosis, is a necrotizing vasculitis that may present clinically as patchy radiologic opacities or as a discrete lung mass, with or without involvement of other organs like the nasal passages and kidneys. The histologic diagnosis of granulomatosis with polyangiitis rests on the identification of necrosis, granulomatous inflammation, and vasculitis. CYTOMORPHOLOGY OF GRANULOMATOSIS WITH POLYANGIITIS • • • •

 eutrophils N Giant cells Necrotic collagen (“pathergic necrosis”) Epithelioid histiocytes   

The cytomorphologic findings are nonspecific but include chunks of necrotic tissue (Fig. 2.22), giant cells, granulomas, and neutrophils. Eosinophils may also be present in significant numbers and do not necessarily signify an eosinophilic vasculitis (i.e., eosinophilic granulomatosis with polyangiitis, previously known as Churg-Strauss

75

syndrome). The differential diagnosis includes a necrotizing infection (e.g., tuberculous, fungal), other vasculitides, lymphomatoid granulomatosis, and other uncommon pulmonary diseases. If suspected on the basis of its characteristic cytomorphology, the diagnosis can be substantiated with serologic studies. The serum immunofluorescent antineutrophil cytoplasmic antibody (ANCA) test greatly aids in establishing the diagnosis of granulomatosis with polyangiitis. Although the classic (cytoplasmic) pattern (c-ANCA) is considered more specific, neither the c-ANCA nor the perinuclear (p-ANCA) pattern is entirely sensitive or specific for the diagnosis of granulomatosis with polyangiitis, which is best made through multidisciplinary assessment of pathologic, clinical, and radiologic features.98 

Pulmonary Amyloidosis Pulmonary amyloidosis can be limited to the lung or represent a localized manifestation of systemic disease. Although it affects a wide age range, most patients are in their 50s and 60s. Pulmonary amyloidosis manifests itself as nodular parenchymal amyloidosis, tracheobronchial amyloidosis (in which the deposits are mostly submucosal and result in dyspnea, wheezing, and recurrent pneumonia), or diffuse parenchymal amyloidosis (with diffuse or multifocal deposits). Nodular parenchymal amyloidosis is the most common form, characteristically presenting as one or multiple wellcircumscribed mass lesions with or without internal calcifications that may mimic malignancy or granulomatous disease. Approximately 50% of these lesions are associated with a lymphoplasmacytic infiltrate showing clonal immunoglobulin gene rearrangement, and they may occasionally

• Fig. 2.22  Granulomatosis with Polyangiitis (Fine-Needle Aspiration).  A granular background consisting of necrotic collagen without acute inflammation is characteristic (Papanicolaou stain).

76 CHA P T ER 2    Respiratory Tract and Mediastinum

be associated with systemic plasma cell neoplasms (e.g., multiple myeloma)99; nevertheless, most patients have only localized disease and do well clinically after resection. FNA yields irregular, waxy, amorphous, hypocellular material with a scalloped, occasionally cracked appearance (Fig. 2.23A and B). These deposits appear blue-green with Papanicolaou stains (Fig. 2.23A) and show apple-green dichroism under polarized light after staining with the Congo red stain (Fig. 2.23B inset). In the nodular parenchymal type there may be multinucleated giant cells, and calcification and ossification are common. 

Pulmonary Alveolar Proteinosis Pulmonary alveolar proteinosis (PAP) is a rare disease chara­cterized by an accumulation of a lipid-rich material within alveoli due to impaired surfactant clearance. Most cases are the result of impaired macrophage function due to autoimmune production of neutralizing autoantibodies to ­ granulocyte-macrophage-colony-stimulating factor (GM-CSF).100 Less-common causes include genetic defects (resulting in alterations of the GM-CSF receptor, surfactant proteins, or thyroid transcription factor–1), immunocompromise (like HIV infection or lung transplantation), and environmental exposures that result in systemic or local inhibition of macrophage activity or maturation.98,100 The onset is insidious, and one-third of patients are symptom free despite impressive radiologic abnormalities. There can be a nonproductive cough, dyspnea, and expectoration of gelatinous material. Chest computed tomography (CT) characteristically shows a combination of ground glass opacities and interlobular septal thickening termed the “crazy paving” pattern, although this finding is not entirely specific for PAP.100 Molecular testing or enzymelinked immunosorbent assays of serum and BAL fluid can help determine the cause of PAP by identifying mutations or autoantibodies, respectively,101 but gross and cytologic examination of BAL specimens are the mainstay of initial diagnosis. The characteristic findings include an opaque,

A

milky gross appearance; large, acellular, eosinophilic, proteinaceous blobs (Fig. 2.24A and B); and pulmonary macrophages filled with periodic acid–Schiff–positive material. The differential diagnosis includes pulmonary edema, P. ­jirovecii pneumonia, acute silicosis, and alveolar mucinosis. The diagnosis is made by correlating clinical and imaging findings with laboratory results and the characteristic cytologic findings. Patients are both diagnosed and treated with whole lung lavage, which typically results in symptomatic improvement.100 Treatment with inhaled or subcutaneous GM-CSF replacement in patients with autoimmune PAP is also effective, with an improved rate of relapse compared to whole lung lavage (22% vs 30% to 66%).100,102 

Common Inflammatory Processes, Including Organizing Pneumonia Some pneumonias respond slowly to antibiotic treatment and undergo fibroblastic organization. The histologic pattern of organizing pneumonia can be seen also with drug reactions, connective tissue disease, adjacent malignancy, and other clinical scenarios. When a clinical cause is not apparent, organizing pneumonia is termed “cryptogenic organizing pneumonia” (formerly known as bronchiol­ itis obliterans organizing pneumonia). Organizing pneumonias may result in chronic mass lesions that mimic a neoplasm radiographically and therefore are occasionally sampled by FNA. CYTOMORPHOLOGY OF ORGANIZING PNEUMONIA • T  issue fragments comprised of fibroblasts and collagen • Masson bodies • Numerous pulmonary macrophages, some with hemosiderin • Lymphocytes • Pneumocytes   

B • Fig. 2.23  Nodular Pulmonary Amyloid (Fine-Needle Aspiration).  (A) Amyloid has a glassy, amorphous appearance with embedded stromal cells. (B) The cell block shows dense, eosinophilic, paucicellular amyloid. (Inset) A Congo red stain examined with polarized light demonstrates green birefringence.

CHAPTER 2  Respiratory Tract and Mediastinum

A

77

B • Fig. 2.24  Pulmonary Alveolar Proteinosis (Bronchoalveolar Lavage).  (A) Hematoxylin and eosin (H&E)–

stained cell block sections show numerous acellular eosinophilic aggregates. (B) Globule, tear drop, and carrot-shaped aggregates are pathognomonic of pulmonary alveolar proteinosis (H&E stain).

Cytologic preparations contain tissue fragments, often crushed, comprised of spindle-shaped fibroblasts embedded in collagen (Fig. 2.25A–D). Because the fibrous proliferation often fills alveoli, some fragments have a well-­circumscribed, saccular appearance that corresponds histologically to Masson bodies (Fig. 2.25B). Pulmonary macrophages, lymphocytes, and pneumocytes are often numerous; when numerous, they may provide the first indication that the lesion is inflammatory in nature rather than neoplastic. Pneumocytes may demonstrate florid reactive atypia. Because the findings are relatively nonspecific, however, at best one can only suggest the diagnosis of organizing pneumonia. The possibility of sampling error cannot be entirely excluded, and it is prudent to mention this in the report, because organizing pneumonia may be seen adjacent to malignancies. There is considerable overlap in the clinical, histologic, and cytologic features of resolving bacterial pneumonia, cryptogenic organizing pneumonia, diffuse alveolar damage, and transplant rejection. All show fibrous tissue, pneumocytes, macrophages, and lymphocytes in various proportions.103-107 Reactive pneumocytes are especially prominent in organizing pneumonia and diffuse alveolar damage (see Fig. 2.7). In the setting of lung transplantation, BAL is a routine monitoring procedure, especially for detecting infections, the most common of which are Candida, CMV, and HSV.108 Neutrophils are normally seen within the first 3 months of transplantation107 and increased numbers of macrophages for years after the transplant. 

Benign Neoplasms of the Lung Pulmonary Hamartoma Despite their time-honored name, pulmonary hamartomas are, in fact, neoplasms. Recurrent clonal rearrangements

have been identified involving the HMGA2 and HMGA1 genes on chromosome 6p21.109,110 Most pulmonary hamartomas are discovered incidentally as a solitary discrete, round mass in the lung periphery on thoracic imaging studies. Less commonly, they are central and endobronchial; multiple hamartomas are rare. CYTOMORPHOLOGY OF PULMONARY HAMARTOMA • • • •

 enign glandular cells B Immature fibromyxoid matrix and bland spindle cells Mature cartilage with chondrocytes in lacunae Adipocytes   

FNA specimens show a mixture of mesenchymal (fibromyxoid and cartilaginous material) and benign epithelial elements (Fig. 2.26A–D). In some hands, FNA has a sensitivity of 78% and a specificity of 100% in the diagnosis of a hamartoma.111 A nationwide self-assessment testing program, however, has revealed a general lack of familiarity with this lesion, with a troubling false-positive rate (22%); the most common misdiagnoses are carcinoid tumor, adenocarcinoma, and small cell carcinoma.112 Hamartoma should be considered for any well-circumscribed neoplasm that contains epithelial cells with low-grade nuclear features. 

Langerhans Cell Histiocytosis Pulmonary Langerhans cell histiocytosis (LCH) is a clinically distinct process from systemic LCH and typically presents in adult smokers as multiple, bilateral, predominantly upper lobe nodules with a cystic component.113 Patients may be symptom free or present with respiratory symptoms. Although some patients progress to pulmonary fibrosis, smoking cessation often results in resolution of the nodules and symptoms.

78 CHA P T ER 2    Respiratory Tract and Mediastinum

A

B

C

D • Fig. 2.25  Organizing Pneumonia (Fine-Needle Aspiration).  (A) Large, crushed tissue fragments com-

prised of fibroblasts and collagen are characteristic (Romanowsky stain). (B) Fibroblasts and collagen fill alveolar spaces, resulting in nodular masses (Masson bodies) (Romanowsky stain). (C) High magnification reveals a mix of fibroblasts, pneumocytes, macrophages, and collagen (Papanicolaou stain). (D) Cell block preparations show fragments of collagenous tissue with loosely arranged fibroblasts admixed with chronic inflammatory cells (hematoxylin and eosin stain).

CYTOMORPHOLOGY OF LANGERHANS CELL HISTIOCYTOSIS • V  ariable cellularity • Histiocytoid cells with convoluted nuclei • Background: mixed inflammatory cells, including eosinophils • Pigmented macrophages

suggest that pulmonary LCH is a reactive lesion, at least a subset demonstrate the BRAF V600E mutation in multiple nodules in the same individual, suggesting that some pulmonary LCHs are clonal neoplasms.115 

Sclerosing Pneumocytoma   

Pulmonary LCH, like systemic LCH, is characterized by a proliferation of Langerhans cells that are immunoreactive for S100, CD1a, and Langerin and demonstrate abundant cytoplasm and convoluted nuclei (see Fig. 18.14B).114 The lesional cells are intermixed with pigmented macrophages and numerous other inflammatory cells, including significant numbers of eosinophils, which often provide the first clue to the diagnosis. Although the association with smoking and the regression of lesions on smoking cessation

Sclerosing pneumocytoma is a rare benign neoplasm of uncertain lineage thought to derive from primitive respiratory precursor cells. It typically presents in middleaged women as a peripheral, well-circumscribed nodule with a mean size of 2.3 cm (range 0.25–5.2 cm) and low 18F-­fluorodeoxyglucose (FDG) avidity.116,117 Histologically, it is characterized by a proliferation of two cell types: cuboidal surface cells and round stromal cells, both of which are considered to be neoplastic.118 The stromal cells have pale cytoplasm and are immunoreactive for thyroid transcription factor 1 (TTF-1) and epithelial membrane antigen (EMA), yet predominantly negative for keratins (rare CK7-positive

CHAPTER 2  Respiratory Tract and Mediastinum

A

B

C

D

79

• Fig. 2.26  Pulmonary Hamartoma (Fine-Needle Aspiration).  (A) Loose myxoid material is often present

(smear, Romanowsky stain). (B) The transition between fibromyxoid material and cartilage is seen. Benign epithelial cells are also present (cell block, hematoxylin and eosin [H&E] stain). (C) Mature cartilage may be a prominent component (cell block, H&E stain). (D) The epithelial component can be monomorphic and cuboidal, as seen here, resembling benign mesothelial cells (smear, Romanowsky stain).

and CAM5.2-positive cells have been reported) and neuroendocrine markers. The cuboidal cells demonstrate the immunophenotype of II pneumocytes (positive for keratin and TTF-1).119 On histologic sections, the tumors may have hemorrhagic, sclerotic, and papillary components, and the lesional cells are surrounded by epithelial “surface” cells with immunohistochemical characteristics of type II pneumocytes. CYTOMORPHOLOGY OF SCLEROSING PNEUMOCYTOMA • • • •

 onomorphic ovoid cells M Smooth nuclear contours and fine chromatin pattern Second population of pneumocytes Papillary microarchitecture   

Both components (surface and stromal) are seen on cytologic specimens, typically demonstrating no mitotic activity, with the monomorphic stromal cells predominating; occasional poorly formed papillae and a hemorrhagic background

may also be seen120 (Fig. 2.27). Because the lesional cells are positive for TTF-1 and yield cellular specimens, sclerosing pneumocytoma resembles lung adenocarcinoma and should be considered whenever a round, slow-growing pulmonary nodule demonstrates marked monomorphism. 

Papillomas and Related Lesions In the lung, papillomas are rare, benign endobronchial tumors that account for less than 1% of all lung tumors and approximately 7% of benign lung tumors.121 The lesions are often incidental, but patients may present with cough, hemoptysis, or postobstructive pneumonia. Three histologic variants are recognized: squamous, glandular, and mixed squamous and glandular papillomas. Although rates vary between studies, a significant number (more than 50%) of pure squamous papillomas have been reported to contain HPV 6 or 11 genetic material, suggesting a potential etiologic role for human papillomavirus.122 Evolution into invasive squamous cell carcinoma or adenocarcinoma has

80 CHA P T ER 2    Respiratory Tract and Mediastinum

A

B • Fig. 2.27  Sclerosing Pneumocytoma (Fine-Needle Aspiration).  (A) Papillary formations have a lining of ­ neumocyte-like cells around a cellular core of round stromal cells (Papanicolaou stain). (B) The stromal p cells are monomorphic, with a round nucleus and one or more small nucleoli (Papanicolaou stain). (Figures courtesy Dr. Natasha Rekhtman, Department of Pathology, Memorial Sloan-Kettering Cancer Center, New York, NY.)

also been described as a rare event and is most common in the squamous and mixed subtypes.122 Histologically, papillomas are characterized by fibrovascular cores lined by either mature squamous epithelium, ciliated and mucinous glandular epithelium, or a mix of the two. A relationship to an airway is typically apparent. Mass-forming proliferations termed “ciliated mucopapillary tumor” and “bronchial adenoma” demonstrate similar histology but grow along alveolar septa without relationship to an airway and have varying proportions of ciliated and mucous cells or cuboidal cells. With endobronchial papilloma, these may represent a spectrum of benign lesions originating in the epithelium of proximal or distal airways and may harbor v-raf murine sarcoma viral oncogene homolog B (BRAF) V600E, Kirsten rat sarcoma viral oncogene homolog (KRAS), or epidermal growth factor receptor (EGFR) mutations.123 Nuclei in this spectrum of lesions are small, with smooth borders, and mitoses are few or absent. Cytologic samples can be very cellular and may show varying degrees of reactive atypia, a pitfall for the misdiagnosis of squamous cell carcinoma, adenocarcinoma, or adenosquamous carcinoma.124 Cytologic specimens suggestive of papilloma should be therefore reported descriptively, and definitive diagnosis best reserved to an excisional specimen to rule out low-grade papillary carcinoma or malignant transformation of papilloma, which has been reported in up to 10% of cases.122 

unpredictably: most patients have an excellent prognosis, but some tumors are locally aggressive.125 The neoplastic nature of this lesion was confirmed by the cloning of translocations involving the anaplastic lymphoma kinase (ALK) gene (chromosome 2p23) and the two tropomyosin genes, TPM3 and TPM4.126 ALK fusion partner variants include the clathrin heavy chain gene127 and ATIC gene.128 IMT can be effectively treated with crizotinib, an ALK-inhibitor that is also effective in EML4-ALK-positive non-small cell lung cancer.129

Inflammatory Myofibroblastic Tumor

Endobronchial Granular Cell Tumor

Inflammatory myofibroblastic tumor (IMT), which acco­ unts for the majority of lesions previously termed “inflammatory pseudotumor,” is a neoplasm of cytologically bland spindle cells that occurs in the lungs and at other sites. It is most common under the age of 40. IMT is usually a discrete solitary peripheral nodule. These tumors behave

Endobronchial granular cell tumors account for a small proportion of all granular cell tumors, which are thought to originate from Schwann cells. They are usually covered by bronchial epithelium and are composed of clusters of tumor cells with small nuclei and abundant, granular, eosinophilic cytoplasm and are surrounded by a thickened basement membrane.

CYTOMORPHOLOGY OF INFLAMMATORY MYOFIBROBLASTIC TUMOR • • • •

 pindle cells S Storiform pattern Polymorphous inflammatory cells Minimal if any necrosis   

Cytologic preparations show bland spindle cells arranged as fascicles or in a storiform pattern.130 There is little pleomorphism and few mitoses. Admixed with the spindle cells is an impressive infiltrate of inflammatory cells, including lymphocytes, plasma cells, histiocytes, and Touton type giant cells (defined as having a peripheral ring of nuclei). The differential diagnosis includes organizing pneumonia and sarcomatoid carcinoma. 

CHAPTER 2  Respiratory Tract and Mediastinum

CYTOMORPHOLOGY OF ENDOBRONCHIAL GRANULAR CELL TUMOR • S  mall clusters of macrophage-like cells • Abundant granular cytoplasm • Small, uniform, round to oval nuclei   

On cytologic preparations, the cells are easily overlooked because of their similarity to macrophages (see Fig. 17.39).131,132 

Precursor Lesions of the Respiratory Epithelium Squamous cell carcinoma of the lung is preceded by a precursor lesion akin to that of cervical cancer, with a progression from benign squamous metaplasia through dysplasia to invasive cancer. Unlike cervical cancer, however, lung cancer is only very rarely associated with human papillomavirus infection,133 and there is no successful method to screen for and treat precursor pulmonary lesions. Aberrant expression of specific cancer-associated proteins like p53 and the EGFR tyrosine kinase in dysplastic respiratory epithelium precedes the development of invasive carcinoma.134,135 Although the classification criteria of dysplasia as mild, moderate, severe, or carcinoma in situ are subjective,118 they predict the risk of developing bronchogenic carcinoma.136,137 Patients with severe dysplasia or carcinoma in situ are more likely to have persistent disease or progress to invasive carcinoma.137 The presumed precursor of pulmonary adenocarcinoma is atypical adenomatous hyperplasia (AAH).118 AAH and nonmucinous adenocarcinoma of lepidic type have similar morphologic features and probably represent a continuum of pulmonary alveolar intraepithelial neoplasia. AAH, unlike adenocarcinoma, is usually an incidental finding because AAHs are small (usually less than 5 mm in diameter) and lack interstitial inflammation and fibrosis. In AAH, the alveoli are lined by cuboidal cells with features of club cells and type II pneumocytes; ciliated cells and mucus cells are not seen. Intranuclear inclusions and binucleation are common. Because AAH lesions are small and the cytologic atypia mild, cytologic samples may contain only a few mildly atypical cells, but this pneumocyte atypia has significant overlap with neoplastic lesions. An outright interpretation of AAH is restricted to larger tissue samples and is not used in cytologic samples. 

Lung Cancer Carcinoma of the lung is the most common cancer in the world today. Its geographic distribution parallels exposure to tobacco smoking, and, as a result, lung cancer is more common in developed countries. It is the most common fatal malignancy in both men and women in the United States, where it accounts for 29% of all cancer deaths in men and 25% in women.138 Although 1-year survival has

81

increased over the past 30 to 40 years, 5-year survival in patients with advanced disease remains dismal and has improved only marginally despite advances in treatment.139 The causes of lung cancer are many. In men, 85% of lung cancers are attributed to cigarette smoking.118 Tobacco smoke contains numerous carcinogens, tumor initiators, and radioactive compounds, but the biologic mechanism by which tobacco substances initiate oncogenic genetic alterations in lung cancer is not known. The mutational landscape pertaining to lung cancer–related genes is characterized by G–C to T–A transversions induced by cigarette smoke.140 Exposure to asbestos has a synergistic effect with smoking: asbestos workers who smoke increase their risk of developing pulmonary cancer by at least 50-fold. The development of lung cancer has also been linked to exposure to other substances, including radon, crystalline silica, nickel compounds, and organic compounds like benzene and vinyl chloride. Universal screening for lung cancer with chest radiographs and sputum cytology is not cost effective. The most comprehensive trial in the United States was undertaken by three medical centers under the auspices of the National Cancer Institute. Approximately 30,000 men were screened, and the overall mortality rate of the screened group was no lower than that of the control group.141 Nevertheless, highrisk individuals, such as workers in the asbestos or uranium industries, persons over the age of 65 with a 20-year history of smoking, and those whose radiographs show a persistent abnormality142 do benefit from periodic screening. More recently, the National Lung Screening Trial evaluated low-dose CT as a modality for screening high-risk individuals and found that mortality rates could be reduced by 20%. Nodules greater than 4 mm were detected in 39% of enrolled subjects; 72% of the nodules were sampled, but only 1% were malignant.143 These findings underscore the importance of safe and effective sampling techniques and the need for shared decision-making between patients and health care providers, with a focus on risk factors, potential harms, and potential benefits. Additional studies have resulted in the current American Cancer Society guidelines, which suggest judicious low-dose CT scan screening of adults ages 55 to 74 with a 30 pack-year smoking history or greater, performed at centers with expertise in lung imaging and treatment.144 Patients with lung cancer often present with shortness of breath, cough, chest pain, hoarseness, hemoptysis, or pneumonia. Some tumors, particularly adenocarcinomas, are detected incidentally in symptom-free individuals. Three histologic types account for 95% of all pulmonary tumors: squamous cell carcinoma, adenocarcinoma, and small cell carcinoma. Because it is usually metastatic at the time of detection, small cell carcinoma is typically treated with systemic chemotherapy. The non-small cell carcinomas (squamous cell carcinoma, adenocarcinoma, and large cell carcinoma) are more likely to be localized at the time of diagnosis, which allows for curative resection by lobectomy or pneumonectomy. With the advent of targeted cancer

82 CHA P T ER 2    Respiratory Tract and Mediastinum

therapies, accurate histologic classification of non-small cell carcinomas has become essential for patient management.

Molecular Testing of Lung Cancers With the development of advanced gene sequencing technologies, it has become evident that lung cancers are genetically heterogeneous and comprise multiple molecularly defined entities with distinct clinical and histologic correlates. The identification of driver genes that render tumors susceptible to “targeted therapies” has resulted in a paradigm shift in the management of lung malignancies, which increasingly relies on treatment with tumor- and gene-specific drugs. Consequently, accurate pathologic and molecular classification is needed. Because many molecular techniques incorporate amplification methods, large amounts of tissue are not necessary, and molecular classification can be performed successfully on different cytologic specimens and preparation types.145,146 A minimum number of tumor cells and a minimum proportion of tumor to normal nuclei, however, is required for testing. Thus assessment of the adequacy of a cytologic sample for molecular testing is a growing part of the cytologist’s job. Many of the new therapies target genetic aberrations in receptor tyrosine kinase pathways. Abnormally activated receptor tyrosine kinases are linked to cascades of downstream effector pathways (Fig. 2.28) that result in transcriptional activation of genes involved in tumor growth,

RTK (EGFR, HER2, VEGFR, IGF-1R, MET)

EML4-ALK PI3K

RAS RAF (BRAF) MEK

AKT

ERK

Nucleus

Growth, invasion, angiogenesis

• Fig. 2.28  Receptor Tyrosine Kinase Signaling in Cancer.  This sim-

plified diagram illustrates the pathways by which receptor tyrosine kinases (RTKs) stimulate growth, invasion, and angiogenesis (see text for details). BRAF, v-raf murine sarcoma viral oncogene homolog B; EGFR, epidermal growth factor receptor; EML4-ALK, echinoderm microtubule–associated protein-like 4-anaplastic lymphoma kinase; ERK, extracellular-signal-regulated kinase; HER2, protein kinase erbb2; IGF-1R, insulin-like growth factor 1 receptor; PI3K, phosphatidylinositol 3-kinase; RAS, Rat sarcoma viral oncogene homolog; VEGFR, vascular endothelial growth factor receptor.

invasion, and angiogenesis.147 Oncoproteins implicated in lung adenocarcinoma pathogenesis include the KRAS, EGFR, ALK, ROS1, hepatocyte growth factor receptor (encoded by the proto-oncogene MET ), rearranged during transfection (RET), BRAF, and protein kinase erbB-2 (HER2).148 Squamous cell carcinomas of the lung generally lack mutations in adenocarcinoma-related genes and are characterized by alterations of genes encoding components within the PI3K-AKT-mTOR pathway, most frequently in the PIK3CA and phosphatase and tensin homolog (PTEN ) genes.149 Although several tyrosine kinase inhibitors have shown significant antitumor activity in patients with advanced lung adenocarcinoma and have become standard of care, targeted therapy for squamous cell carcinoma has not yet shown similar efficacy. A minimum panel of molecular and cytogenetic tests for EGFR, ALK, and ROS1 alterations is therefore currently recommended only in nonsquamous non-small cell lung cancers,150 with subsequent expanded testing as clinical need dictates. Several solid tumors have been shown to repress endogenous antitumor immune-mediated responses via the PD-1/PD-L1 pathway,151,152 spurring new focus on drugs that target the interactions between tumor and the associated inflammatory microenvironment. Evaluation of tumor PD-L1 expression is therefore part of the standard biomarker evaluation of lung tumors. 

EGFR Constitutive activation of the EGFR tyrosine kinase drives tumor growth, invasion, and angiogenesis through activation of PI3K/AKT and RAS/RAF/MEK signaling.153 Activating mutations of EGFR that affect the EGFR tyrosine kinase domain are present in approximately 15% and 30% of lung adenocarcinomas among white and Asian populations, respectively.140,154 Tyrosine kinase inhibitors demonstrate significant antitumor activity in previously treated and untreated patients with EGFR-mutant non-small cell lung cancer and are associated with a favorable toxicity profile.155,156 Routine testing for EGFR mutation is therefore recommended in patients with advanced non-small cell lung cancer. Although mutation-specific antibodies that detect the most common L858R and exon 19 deletions are commercially available, they do not detect some less common but potentially sensitizing EGFR mutations and are therefore not recommended as a primary or screening tool to determine EGFR status.150 Unfortunately, most patients ultimately develop resistance to tyrosine kinase inhibitor therapy. Multiple mechanisms of resistance have been described, most frequently via acquisition of an additional T790M amino acid substitution in exon 20, within the tyrosine kinase domain of EGFR, followed by amplification of the MET gene, histologic “transformation” to small cell carcinoma and other less-common secondary genetic alterations.157 Osimertinib, an EGFR inhibitor that bypasses the T790M mutation, has positive effects on progression-free survival and overall response rates in patients with T790Mpositive advanced non-small cell carcinoma, including those

CHAPTER 2  Respiratory Tract and Mediastinum

with brain metastases158 and is approved by the FDA for use in patients with metastatic T790M-positive disease. 

MET MET is a proto-oncogene that encodes another receptor tyrosine kinase, hepatocyte growth factor receptor (see Fig. 2.28). MET amplification or mutation is seen as a mechanism of resistance to EGFR inhibitors in 5% to 20% of EGFRmutated, EGFR-resistant non-small cell lung ­cancers.159-161 Additionally, MET activation via exon 14 skipping mutations is seen in 4% of untreated lung adenocarcinomas148 and 2% of lung squamous cell carcinomas.149 Therapeutic antibodies with anti-MET activity have indeed shown some promise in patients with MET protein overexpression or MET exon 14 skipping mutation.162,163 

ERBB-2 (HER2) HER2, which encodes a protein member of the EGFR family, is amplified or overexpressed in up to 23% of non-small cell lung cancers, but, unlike patients with HER2-amplified breast cancers, these patients do not benefit from antiHER2 single-agent therapy.164-166 Oncogenic mutations affecting the HER2 kinase domain, however, have been reported in approximately 3% of lung adenocarcinomas and result in constitutive downstream signaling via the PI3K/ AKT and RAS/MAP/MEK pathways.167 Although some patients with HER2 activating mutations demonstrate clinical response to HER2-directed therapy, the genotypic and clinical characteristics associated therewith are not yet well defined.168 

ALK ALK is a receptor tyrosine kinase that becomes constitutively active on fusion to various partners via translocation, resulting in the activation of the pro-growth PI3K/AKT and RAS/RAF/MEK pathways (see Fig. 2.28). EML4-ALK translocation, the most common, is present in 5% to 7% of lung adenocarcinomas and is associated with a unique clinical profile: the majority of patients are non-smokers, relatively young (median age 52) males.169,170 Solid tumor histology and signet-ring cell features are enriched among patients with these translocations. Immunohistochemical expression of ALK (Fig. 2.29A) is highly sensitive and specific for ALK translocation and is an equivalent alternative to fluorescence in situ hybridization (Fig. 2.29B).150,171 ALK translocations are mutually exclusive of EGFR and KRAS mutations and render tumors susceptible to tyrosine kinase inhibitors such as crizotinib172; ALK is therefore included in the minimum recommended molecular testing panel for patients with non-small cell lung carcinoma.150 

ROS1 ROS1 encodes a receptor tyrosine kinase of the insulin family that, like ALK, undergoes oncogenic rearrangement in a subset of non-small cell carcinomas. One percent to 2% of lung adenocarcinomas harbor fusions between ROS1

83

and CD74, SLC34A2, or other less common partners and are associated with clinical and histologic characteristics similar to those of ALK-rearranged tumors: young neversmokers with solid, cribriform, and signet-ring cell histology.173 ROS1-rearranged tumors can be successfully treated with tyrosine kinase inhibitors, including crizotinib,174 and ROS1 is also included in the minimum recommended molecular testing panel for patients with advanced nonsmall cell carcinoma.150 ROS1 immunohistochemistry (Fig. 2.29C) is highly sensitive and specific for ROS1 rearrangement but may show nonspecific positivity in reactive pneumocytes and should be confirmed by a molecular or cytogenetic method.150,175 

RET Fusions of the RET proto-oncogene, which have been most extensively characterized in thyroid carcinomas, are seen in less than 1% of lung adenocarcinomas.148 Like ALK and ROS1 alterations, rearrangements of RET-encoded tyrosine kinase are more frequently seen in never-­smokers and patients with solid and signet-ring cell histology and render patients susceptible to tyrosine kinase inhibitors.176,177 Because RET alterations are rare and mutually exclusive of EGFR mutation and ALK or ROS1 rearrangements, evaluation of RET is usually performed as part of an expanded panel after evaluation of the more common ­adenocarcinoma-associated mutations. 

BRAF BRAF is a form of RAF, a serine-threonine kinase that activates MEK (see Fig. 2.28) Oncogenic mutations in the BRAF-encoded kinase domain are present in 7% of lung adenocarcinomas, 1% of which represent V600E mutations.148,178 A combination of BRAF inhibitors has been approved for the treatment of patients with metastatic nonsmall cell carcinoma harboring BRAF V600E mutations.178 MEK inhibitors that target other BRAF non-V600 mutations are still under investigation. 

PIK3CA Alterations of the PIK3CA gene that result in increased signaling via the PI3K/AKT/MTOR pathway are not mutually exclusive of EGFR and KRAS mutations and may play a role in resistance to receptor tyrosine kinase inhibitor therapy.157 Amplification of PIK3CA occurs in up to 17% of non-small cell lung cancers,179 and activating mutations of regions encoding its kinase or helix domain are seen in 2% to 13% of non-small cell lung cancers,179-181 including 4% of adenocarcinomas and 16% of squamous cell lung carcinomas.148,149 Loss-of-function mutations of PTEN, which encodes a negative regulator of PI3K, are also more common in squamous cell carcinomas than adenocarcinomas, occurring in 8% of tumors. The PI3K/AKT/mTOR pathway therefore continues to be the most promising target of therapy in squamous cell carcinoma, although phase II clinical trials of PI3K and mTOR inhibitors have been

84 CHA P T ER 2    Respiratory Tract and Mediastinum

ALK

A

p25.2 p24.3 p24.1 p23.2 p22.3 p22.1

ALK29.3 EML4 42.3

p16.3 p16.1 p14 p13.2 p12

q12.1 q12.3 q14.1 q14.3 q21.2 q22.1 q22.2 q23.2 q24.1 q24.3 q31.2 q32.1 q32.3 q33.2 q34

B

q36.1 q36.3 q37.2

ROS1

C

PD-L1

D

• Fig. 2.29  Immunohistochemical and Cytogenetic Testing of Lung Malignancies.  (A) Immunoreactivity for ALK (as seen here) is sensitive and specific

for translocations involving the ALK gene. (B) EML4-ALK intrachromosomal translocation is recognized by dual color split-apart fluorescence in situ hybridization. The arrows (left portion of diagram) point to the breakpoints within chromosome 2 that result in the intrachromosomal translocation. On the right, red and green probes flanking the ALK locus on chromosome 2p23.2 reveal red-green doublets at the wild type ALK loci (arrowhead) but are split apart when ALK is rearranged and fused to EML4 on the same chromosome at 2p21 (arrow). The rearranged probes appear far apart because the chromatin of interphase nuclei is dispersed relative to that of chromosomal DNA (Figure courtesy Dr. Lucian Chirieac, Department of Pathology, Brigham and Women’s Hospital, Boston, Mass.). C, ROS1 immunohistochemistry is recommended as a screening test for tumors with ROS1 translocations, but positive immunoreactivity (as seen here) should be confirmed by molecular or cytogenetic methods. D, In specimens containing at least 100 tumor cells, partial or complete membranous staining for PD-L1 is predictive of response to immune checkpoint inhibitors.

CHAPTER 2  Respiratory Tract and Mediastinum

disappointing in selected and unselected patients with nonsmall cell lung carcinoma, respectively.181,182 

demonstrate high concordance of PD-L1 expression with surgical specimens.189,190 

KRAS KRAS-mutated cancers are more common in smokers, connote a worse prognosis, and comprise 30% and 10% to 15% of lung adenocarcinomas in Western and Eastern populations, respectively.140,154 KRAS mutations are mutually exclusive of EGFR, ALK, and ROS1 genetic alterations and typically lead to resistance to tyrosine kinase inhibitor therapy.183 Therefore testing of targetable mutations is typically prioritized, and KRAS is suggested as either part of an expanded secondary molecular testing panel or as a single gene test to exclude patients from additional molecular testing.150 Clinical studies of KRAS-directed therapy have not shown efficacy to date, and treatment of KRAS-mutant lung carcinomas continues under predominantly preclinical investigation.184,185 

PD-L1 Repression of endogenous antitumor immune-mediated responses via disruption of the PD-1/PD-L1 pathway has been described in a number of hematologic and solid malignancies.151 Drugs that inhibit the PD-1/PD-L1 interaction improve overall survival in patients with both adenocarcinoma and squamous cell carcinoma of the lung, particularly in those whose tumors demonstrate membranous PD-L1 protein overexpression in more than 50% of lesional cells (Fig. 2.29D).186,187 As a result of these findings, small molecule inhibitors of the PD-1/PD-L1 pathway (e.g., the anti-PD-1 antibodies Nivolumab and Pembrolizumab) have been approved by the FDA for treatment of lung cancer in first-line and second-line settings.188 To date, tumor PD-L1 status in clinical trials has been determined using biopsy specimens, but cytology specimens that meet the minimum adequacy criterion of 100 tumor cells

A

85

CYTOMORPHOLOGY OF WELLDIFFERENTIATED SQUAMOUS CELL CARCINOMA • A  bundant dyshesive cells • Polymorphic cell shapes: polygonal, rounded, elongated (fiber-like), tadpole-shaped • Dense cytoplasmic orangeophilia (Papanicolaou stain) • Pyknotic nuclei • Frequent anucleate cells   

Squamous Cell Carcinoma Squamous cell carcinoma of the lung accounts for approximately one-third of all primary pulmonary malignancies. Two-thirds of these tumors occur in a central location, and the remainder arise in smaller bronchi. Postobstructive pneumonia and cavitation of the tumor are common. Of all the histologic subtypes, squamous cell carcinoma is the one most commonly associated with hemoptysis. Apical squamous cell carcinomas in the superior sulcus are called Pancoast tumors, characterized by posterior rib destruction and neural invasion, resulting in severe pain and Horner’s syndrome (enophthalmos, ptosis, miosis, and ipsilateral decreased sweating). Specific histologic features (papillary, clear cell, small cell, basaloid) may be present but are no longer considered separate subtypes because they do not have any prognostic significance.118 The cytologic features vary depending on the degree of squamous differentiation. In a well-differentiated squamous cell carcinoma, the malignant cells are noncohesive and come in a variety of shapes (polygonal, spindle, tadpole, Fig. 2.30A and B), with abundant smooth, dense cytoplasm filled with keratin. The cytoplasm stains green, yellow, or

B • Fig. 2.30  Well-Differentiated Squamous Cell Carcinoma (Fine-Needle Aspiration).  (A) Well-differentiated

squamous cell carcinomas are composed of cells with dense, orangeophilic cytoplasm and hyperchromatic, often pyknotic nuclei, some with angular contours (Papanicolaou stain). (B) Bizarre, elongated, spindle-shaped cells are common. Often there is abundant granular debris, seen here, and inflammation (Papanicolaou stain).

86 CHA P T ER 2    Respiratory Tract and Mediastinum

orange with the Papanicolaou stain and robin’s egg blue with Romanowsky stains. Nuclei are usually small, hyperchromatic, and smudgy, and nucleoli are often inconspicuous. CYTOMORPHOLOGY OF MODERATELY AND POORLY DIFFERENTIATED SQUAMOUS CELL CARCINOMAS • • • • •

 arge, cohesive clusters of spindle cells L Rare to absent keratinization Large nuclei Coarse chromatin texture (“Idaho potato”) ± Prominent nucleoli   

In moderately and poorly differentiated squamous cell carcinomas, keratinization is less apparent or absent altogether. The best clue to a potential squamous origin is the presence of spindled cells within dense clusters. These clusters can have the appearance of a “school of fish,” with nuclei streaming in parallel along their long axes, and demonstrating the typical nuclear features of malignancy. The cytoplasm may retain the characteristic dense, smooth appearance of most squamous cell carcinomas, but in some poorly differentiated squamous cell carcinomas, it is granular and scant. Nuclei are larger and nucleoli are prominent. Chromatin, rather than smudgy, is very coarsely textured and resembles the mottled and pitted surface of an Idaho potato (Fig. 2.31). The basaloid variant of squamous cell carcinoma shows prominent palisading of nuclei around the perimeter of cell groups. DIFFERENTIAL DIAGNOSIS OF SQUAMOUS CELL CARCINOMA • • • • • • • • • •

 quamous metaplasia S Degenerative changes Reactive squamous atypia (e.g., Aspergilloma) Vegetable cells Contamination from an upper airway cancer Adenocarcinoma Small cell carcinoma NUT carcinoma SMARCA4-deficient thoracic sarcoma Metastatic squamous cell carcinoma

when the atypical squamous cells are few in number, poorly visualized, degenerated, or associated with a stoma, fungal infection, or any manner of severe lung injury. Vegetable food particles occasionally resemble keratinized squamous cells, but their cellulose wall and the regularity of their shape usually permit correct identification (see Fig. 2.12). Occasionally, malignant cells from a squamous cell carcinoma of the upper airway may contaminate a specimen of the lower respiratory tract.69 Because the distinction between squamous cell carcinoma and adenocarcinoma determines eligibility for molecular testing and the choice of targeted therapies, it is important to refine a malignant diagnosis as much as possible, even with limited samples.191 Most squamous cell carcinomas and adenocarcinomas of the lung are easily distinguished from each other by cytomorphology, based on the presence

•

Fig. 2.31  Poorly Differentiated Squamous Cell Carcinoma (FineNeedle Aspiration).  Poorly differentiated squamous carcinoma cells are often arranged in thick groups of pulled out, spindle-shaped cells, rather than as isolated cells. The nuclei are enlarged, and chromatin is coarsely granular, in contrast to the dense, frequently pyknotic nuclei of well-differentiated (keratinizing) squamous cell carcinomas.

  

Squamous metaplastic cells (from chronic irritation to the bronchial epithelium) are recognizably benign because of their small, round, normochromatic nuclei. In sputum samples, degenerative changes impart a pyknotic, condensed appearance to the nuclei of benign squamous cells that mimics the pyknotic, smudgy nuclei of squamous cell carcinoma. Benign degenerated nuclei, however, are small and do not vary in size and shape as much as those of squamous cell carcinoma. Reactive squamous cell atypias occur adjacent to cavitary fungal infections (Fig. 2.32), stomas, and almost any injury to the lung (e.g., infarction, radiation, chemotherapy, sepsis, and diffuse alveolar damage).35,36 To avoid a false-positive result, caution is advised

•

Fig. 2.32  Atypical Squamous Metaplasia (Fine-Needle Aspiration).  Cavitary fungal infections, like this one by an Aspergillus species (inset), are among the causes of reactive squamous atypia, a mimic of squamous cell carcinoma (hematoxylin and eosin stain).

CHAPTER 2  Respiratory Tract and Mediastinum

of obvious keratinization (in squamous cancers) and abundant mucin and/or glandular structures (in adenocarcinomas). With poorly differentiated cancers, however, the distinction is more difficult. In general, nuclear chromatin is more finely textured in adenocarcinomas and coarser in squamous cell carcinomas. The cytoplasm is thinner, more foamy (vacuolated), and transparent in adenocarcinomas and denser in squamous cell carcinomas. Of note, focal intracellular mucin can be present in squamous cell carcinomas and cannot be relied on for this distinction. If the morphology is not clear-cut in small biopsy or cytology specimens, the International Association for the Study of Lung Cancer and World Health Organization (WHO) guidelines recommend immunohistochemistry for TTF-1 and p63 (or p40) as the first-line stains in the distinction between squamous cell carcinoma and adenocarcinoma (Table 2.3)191,192 Cytologically indeterminate non-small cell carcinomas immunoreactive for TTF-1 alone are reported as “non-small cell carcinoma, favor adenocarcinoma,” and those immunoreactive for p63 or p40 only are reported as “non-small cell carcinoma, favor squamous cell carcinoma.” Tumors positive for both TTF-1 and either p63 or p40 are reported as “non-small cell carcinoma, favor adenocarcinoma,” unless they are positive in different popula­ tions of tumor cells, in which case the tumor is best reported as “non-small cell carcinoma, not otherwise specified,” with a comment that it might represent an adenosquamous carcinoma. If all markers are negative, the tumor is reported as “non-small cell carcinoma, not otherwise specified.” The report should clarify whether the interpretation was made based on cytomorphology alone or whether special stains were required. Tumors that are classified as “non-small cell carcinoma, favor adenocarcinoma” or “non-small cell carcinoma, not otherwise specified” using these guidelines most frequently harbor adenocarcinoma-associated genetic

87

alterations193 and may be triaged for molecular testing accordingly. It is acceptable to substitute p40 for p63; p40 is much more specific for squamous cell carcinoma than p63, which is at least focally positive in 30% of pulmonary adenocarcinomas.194 The basaloid and “small cell” variants of squamous cell carcinoma are comprised of cells with a higher nucleocytoplasmic ratio than the usual squamous cell carcinoma. They are distinguished from small cell carcinoma because the cells of squamous cell carcinoma with these features have coarse or pale chromatin, more prominent nucleoli, and distinct cell borders. The possibility of a NUT carcinoma should be considered for any poorly differentiated malignancy that lacks glandular differentiation. Cytologic preparations show monomorphic, noncohesive cells with vesicular chromatin, variably prominent nucleoli, and moderate-to-scant cytoplasm (Fig. 2.33). Positive nuclear immunoreactivity is virtually diagnostic of NUT carcinoma because, outside of the testis, it is 100% specific and 87% sensitive for NUT carcinoma.195 SMARCA4-deficient thoracic sarcoma (SMARCA4DTS), a poorly differentiated thoracic malignancy lacking epithelial markers that may be related to small cell carcinoma of the ovary, hypercalcemic type, resembles a poorly differentiated carcinoma. SMARCA4-DTS predominantly affects men with a wide age range (28–90 years old) and typically presents with a large mediastinal mass, but it may arise in the pleura or lung parenchyma proper.196,197 Tumors characteristically demonstrate loss of nuclear SMARCA4 staining, which correlates well with an underlying SMARCA4 mutation or copy number variation, and are immunoreactive for SOX2.196,197 TP53 mutations are frequent, and canonical adenocarcinoma driver mutations are lacking.196 SMARCA4-DTS should be a diagnosis of

TABLE 165 2.3    TTF-1 and P63 in the Diagnosis of Squamous Cell Carcinoma and Adenocarcinoma of the Lung

Scenario

TTF1

p63

Diagnosis

Frequency

Next steps?

1

+ to +++

–

NSCLC, favor ACA

∼90% of ACA

2

+++

+ to +++ (in same cells)

NSCLC, favor ACA

0% of SQC

3

–

+++

NSCLC, favor SQC*

>95% of SQC 0% of ACA

4

+++

+++ (in different cells)

NSCLC, NOS, possible adenosquamous carcinoma

Rare (∼1% of NSCLC)

Formal classification deferred to resection

5

–

–

NSCLC, NOSa

∼10% of ACA 0% of SQC

Mucin stain, Napsin

6

–

+

NSCLC, NOSa

1% ACA 1% SQC

Mucin stain, CK5/6

ACA, Adenocarcinoma; NOS, not otherwise specified; NSCLC, non-small cell lung cancer; SQC, squamous cell carcinoma ; TTF1, thyroid transcription factor 1. aExclude metastatic disease.

88 CHA P T ER 2    Respiratory Tract and Mediastinum

exclusion after rigorous immunohistochemical evaluation, as it shows clinical, morphologic, and immunophenotypic overlap with poorly differentiated thoracic carcinomas, including 10% of lung adenocarcinomas,198,199 Metastatic squamous cell carcinomas are morphologically indistinguishable from primary squamous cell carcinomas of the lung. The distinction usually relies on clinical impression. In selected cases, molecular studies can be helpful and may reveal a transversion-predominant smoking signature, ultraviolet radiation–associated signatures, or other signatures that can be helpful within clinical context. 

on clinically evident metastases. Adenocarcinomas rarely show cavitation. To improve on the prior classification system for lung cancers and link therapeutically relevant molecular alterations with radiologic, histologic, and cytologic findings, a consortium consisting of the International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society reclassified adenocarcinomas of the lung, and the changes are reflected in the most recent WHO classification of thoracic tumors.118 A major change was the elimination of the term bronchioloal­ veolar carcinoma and substitution of the term adenocarci­ noma in situ to reflect the natural history, treatment, and molecular underpinnings of lepidic-predominant tumors. The new classification divides adenocarcinoma into four groups based on surgical excision specimens: preinvasive lesions (including AAH and adenocarcinoma in situ), mini­ mally invasive adenocarcinoma, invasive adenocarcinoma, and variants of invasive adenocarcinoma. Adenocarcinoma in situ (formerly bronchioloalveolar carcinoma) is defined

Adenocarcinoma Adenocarcinoma is the most common histologic subtype of lung cancer. Most adenocarcinomas occur in the periphery of the lung, and they may be associated with a desmoplastic reaction and pleural puckering. Of all the histologic types, adenocarcinomas are most likely to be discovered incidentally in a symptom-free individual. Some are detected based

A

B NUT

C

D •

Fig. 2.33  NUT Carcinoma of the Mediastinum (Fine-Needle Aspiration).  (A) Tumor cells have a dispersed pattern on smears, with uniform, rounded nuclei, distinct nucleoli, and minimal cytoplasm (Papanicolaou stain). (B) NUT carcinoma mimics other tumors, including small cell carcinoma, lymphoma, and germ cell tumors due to its dyscohesive nature and scant cytoplasm. (C) Hematoxylin and eosin stains may show pale or clear cytoplasm. (D) Diffuse nuclear NUT protein expression is diagnostic and correlates with NUT gene rearrangement and mis-expression. Here the characteristic “speckled” pattern is seen.

CHAPTER 2  Respiratory Tract and Mediastinum

by its growth along alveolar septa (lepidic growth) without destruction of the underlying alveolar architecture. There are two adenocarcinoma in situ subtypes: non-­mucinous (the great majority) and mucinous (a rarity). Invasive adenocarcinomas are further described based on their predominant architectural pattern: lepidic predomi­ nant, acinar predominant, papillary predominant, micropap­ illary predominant, and solid predominant. It is important to recognize the solid and micropapillary patterns because they have a poorer prognosis than the others, even in stage I tumors.200 Under “variants of adenocarcinoma,” the new classification lists the previously included fetal and colloid types and adds two new variants: invasive mucinous (formerly mucinous bronchioloalveolar carcinoma) and enteric. Three prior variants—mucinous cystadenocarcinoma, clear cell, and signet ring—are now regarded as nondistinct entities. The rare fetal adenocarcinoma resembles the epithelial component of the pulmonary blastoma (see “Sarcomatoid Carcinoma” below).201 Mucinous adenocarcinoma is now recognized as a clinically and genetically distinct variant of adenocarcinoma, harboring KRAS mutations in 76% of cases191,202-205 and lacking EGFR mutations.202-205 Mucinous adenocarcinomas and enteric adenocarcinomas are easily confused with metastatic adenocarcinoma of gastrointestinal origin, because a significant number of these tumors lack TTF-1 expression and may express CK20 and CDX-2.206-208 The new classification also provides guidelines for nomenclature in cytology and small biopsy specimens, with the understanding that specific adenocarcinoma patterns and variants usually cannot be precisely identified and reported on limited samples. The principal recommendation of the new classification is that the diagnosis “nonsmall cell carcinoma” be minimized and that a reasonable attempt be made to establish (or “favor”) either adenocarcinoma or squamous cell carcinoma. The rationale behind this expectation is that adenocarcinomas should be tested for therapeutically-­relevant targetable mutations. If the distinction is not possible by cytomorphology, limited, “first line” immunohistochemistry for just two markers (TTF-1 and p63; p40 may be substituted for p63) is recommended (Table 2.3). Cytologically indeterminate cases that are positive for TTF-1 and negative for p63 and p40 should be reported as “non-small cell carcinoma, favor adenocarcinoma.” Cytologically indeterminate cases positive for p63 or p40 and negative for TTF-1 should be classified as “nonsmall cell carcinoma, favor squamous cell carcinoma.” Cases positive for both TTF-1 and p63 or p40 should be classified as “non-small cell carcinoma, favor adenocarcinoma.” It is imperative that special stains be performed sparingly, so as to preserve tissue for molecular studies, if indicated. It is acceptable to substitute p40 for p63; p40 is much more specific for squamous cell carcinoma than p63, which is at least focally positive in 30% of pulmonary adenocarcinomas.194 Other stains (CK5/6, CK7, Napsin, mucicarmine) are less sensitive and specific in this differential and should not be considered first line tools.

89

CYTOMORPHOLOGY OF ADENOCARCINOMA • H  oneycomb-like sheets, three-dimensional clusters, acini, papillae • Eccentrically placed, round or irregular nucleus • Finely textured chromatin • Large nucleolus • Mucin vacuoles • Translucent, foamy cytoplasm   

There is great heterogeneity in the cytomorphology of lung adenocarcinomas, depending on the predominant histologic pattern. The malignant cells can be arranged in flat sheets with or without acinar formations, densely packed solid masses, true papillae with fibrovascular cores, or small, tight micropapillary balls (Figs. 2.34–2.38). These patterns are generally better appreciated on cell block preparations. Scattered isolated tumor cells are also frequently present. Cytoplasm is usually abundant, with well-defined borders. It can be thin and translucent or foamy. Nuclei usually have round, smooth contours, but in some tumors the nuclear membranes are highly irregular. Chromatin is finely textured in most adenocarcinomas, but some poorly differentiated adenocarcinomas have coarsely textured chromatin. Nucleoli are prominent in many adenocarcinomas. Psammoma bodies can be seen in the micropapillary-­predominant pattern. Cytologic preparations from a mucinous adenocarcinoma (invasive or in situ) show honeycomb-like sheets of uniform cells with pale, optically clear nuclei and an inconspicuous nucleolus, and often pose a morphologic pitfall, because the lesional cells are often low grade, mitotically inert, and sparsely distributed in abundant mucin. Occasionally, the cells of mucinous adenocarcinomas may be dispersed and resemble muciphages. Grooves and nuclear pseudoinclusions are often present but are not entirely specific: they are also seen in nonmucinous adenocarcinoma in situ and nonmucinous invasive adenocarcinomas. The distinction between an adenocarcinoma in situ, a minimally invasive adenocarcinoma, and an invasive carcinoma cannot be made on a cytologic specimen. The cytologic interpretation “positive for malignant cells, adenocarcinoma” implies all three possibilities.118 DIFFERENTIAL DIAGNOSIS OF ADENOCARCINOMA • • • • • • • • • •

 eactive bronchial cells R Creola bodies Reactive pneumocytes Atypical adenomatous hyperplasia Mesothelial cells (FNA specimens) Vegetable cells Hamartoma Squamous cell carcinoma Small cell carcinoma Epithelioid hemangioendothelioma and epithelioid angiosarcoma • Metastatic adenocarcinoma   

90 CHA P T ER 2    Respiratory Tract and Mediastinum

•

Fig. 2.34  Adenocarcinoma (Acinar Predominant).  The cells of this tumor are relatively uniform in size and shape, with small or medium-sized nucleoli. The finely textured chromatin is characteristic of adenocarcinomas as compared with squamous cancers. There are two prominent acinar spaces. Note the indistinct cell borders, imparting a syncytial-like appearance to this sheet of malignant cells. Contrast with the mesothelial cells in Fig. 2.3, where slit-like spaces separate adjacent cells (smear, Papanicolaou stain).

A

B •

Fig. 2.35  Adenocarcinoma (Lepidic Predominant).  (A) Nonmucinous. These cells are hyperchromatic and have a high nuclear-to-cytoplasmic ratio, with scant microvacuolated cytoplasm. There are no clues to suggest a lepidic predominant pattern (bronchoalveolar lavage, Papanicolaou stain). (B) Mucinous. The flat honeycomb-like sheet of these uniform tumor cells is characteristic, as are the intranuclear pseudoinclusions (arrows). Nuclear grooves are better seen with the Papanicolaou stain (not shown). Overall, the nuclear changes are reminiscent of papillary carcinoma of the thyroid (smear, Romanowsky stain).

Benign bronchial cell atypia and hyperplasia (anisonucleosis due to inflammation or injury, Creola bodies) can be recognized as a harmless mimic of adenocarcinoma if the atypical/hyperplastic cells have cilia (see Fig. 2.4) or demonstrate a continuous spectrum of changes (from benign to markedly atypical). In a bronchial specimen, where

malignant cells are often intermixed with benign bronchial cells, it is usually straightforward to identify two distinct cell populations, one malignant, the other benign. The cells of a florid type II pneumocyte hyperplasia resemble those of adenocarcinoma (see Fig. 2.7). Attention to the clinical history (e.g., respiratory distress and diffuse

CHAPTER 2  Respiratory Tract and Mediastinum

A

91

B • Fig. 2.36  Adenocarcinoma (Solid Predominant).  (A) These malignant cells are arranged in large, densely

packed, solid clusters (Romanowsky stain). (B) High magnification of this tumor reveals marked nuclear enlargement, with irregular nuclear contours and prominent nucleoli. Note the microvacuolated cytoplasm, a characteristic feature of adenocarcinomas as compared with squamous cancers (Romanowsky stain).

• Fig. 2.37  Adenocarcinoma (Papillary Predominant).  These crowded malignant cells line a fibrovascular stalk (cell block, hematoxylin and eosin stain).

infiltrates) can provide a clue that the cells are reactive. In an acutely ill patient with diffuse pulmonary infiltrates, markedly atypical cells should be interpreted with caution. Sequential respiratory specimens can help, because hyperplastic pneumocytes are not present in BAL specimens more than a month after the onset of acute lung injury.34 AAH resembles reactive type II pneumocyte hyperplasia in cytologic samples. Unlike adenocarcinoma, however, AAH is usually an incidental finding because AAHs are small (usually 30% of tumors cells (Fig. 4.28). Amplification or overexpression of HER2 is also seen in up to one-third of gastric and gastroesophageal junction (GEJ) cancers, and patients with HER2-positive gastric and GEJ cancers treated with trastuzumab have a significant increase in overall survival.57,117 Thus HER2 testing of effusions positive for metastatic gastric or GEJ cancer might also help guide therapy.118,119  Molecular Testing of Effusions with Metastatic Lung Cancer

Advanced-stage lung cancers are treated with therapies targeted toward a specific genetic abnormality of the tumor; such therapies, if available, are more effective and less toxic than conventional chemotherapy. Activating mutations in the epidermal growth factor receptor (EGFR), for example, are seen in 10% to 26% of non-small cell lung cancers, and patients with an activating mutation respond to tyrosine kinase inhibitors (TKIs) like erlotinib and ­gefitinib.120-122 Randomized trials have shown significantly improved progression-free survival in patients with activating EGFR mutations treated with erlotinib as compared with standard chemotherapy, and patients treated with erlotinib have fewer severe adverse effects.123 Similarly, patients with activating mutations in the ALK and ROS1 genes (less common than the activating EGFR mutations) respond to targeted TKI therapy with crizotinib. Accurate molecular classification of the tumor is essential to determine treatment. Lung cancer patients with positive pleural fluid cytology at the time of presentation are ideal candidates for such treatment because curative surgical resection is not an option.124 Current guidelines recommend testing all advanced-stage lung adenocarcinomas for EGFR,

ALK, and ROS1.124 Immunohistochemistry is equivalent to FISH for ALK testing. For ROS1, immunohistochemistry is recommended as a screening test, but positive results need to be confirmed by a molecular or cytogenetic test.124 Immunohistochemistry is not recommended for EGFR mutation detection.124 The above immunohistochemical, molecular, and cytogenetic tests can be performed successfully on small biopsy and cytology specimens,124,125 but the molecular tests often require that the sample have a minimum proportion of tumor to normal nuclei. Thus assessing the adequacy of the cytologic sample and triaging it for molecular testing is a growing part of the cytologist’s job. Advanced-stage non-small cell lung cancers and other malignancies (melanomas, adenocarcinomas of the gastrointestinal tract) can also be assessed for potential response to immune checkpoint inhibitors like pembrolizumab (see Chapter 2). This can be done by immunohistochemistry for programmed cell death ligand 1, with a growing body of data supporting the use of cytologic preparations of effusions for this evaluation.126,127 

Squamous Cell Carcinoma Squamous cell carcinomas rarely metastasize to the pleura, pericardium, or peritoneum. Those that do are most commonly carcinomas of the lung, larynx, and female genital tract.128 The primary tumor is known in virtually all cases before an effusion develops. CYTOMORPHOLOGY OF SQUAMOUS CELL CARCINOMA • L  arge clusters or isolated cells • Keratinized or nonkeratinized • Dense cytoplasm   

160 CHA P T ER 4    Pleural, Pericardial, and Peritoneal Fluids

A A

B •

Fig. 4.29  Squamous Cell Carcinoma of the Cervix (Pericardial Fluid).  Nonkeratinizing squamous cell cancers shed large spheres of malignant cells (A, Papanicolaou-stained cytocentrifuge Preparation. B, Hematoxylin and eosin–stained cell block preparation).

The cells of squamous cell carcinoma are arranged either in large clusters (Fig. 4.29A and B, 4.30A) or as isolated cells (Fig. 4.30B). The cytologic appearance differs depending on the degree of keratinization of the tumor. The cytoplasm is usually dense and occasionally orangeophilic. Cells with a tadpole or spindle shape are uncommon. Nuclei are enlarged, hyperchromatic, and coarsely granular; nucleoli are usually not prominent. Nuclear pyknosis and karyorrhexis are seen in some cases. Anucleated squames may be present. Cytoplasmic vacuolization is sometimes seen and should not be interpreted as indicative of glandular differentiation.128 A panel of immunostains that includes p40, MOC-31, and the mesothelial markers calretinin and WT-1 can be useful to distinguish squamous cell carcinoma from reactive mesothelial cells and mesothelioma (see Table 4.3). 

Small Cell Carcinoma Small cell carcinoma of the lung, despite its predilection for widespread metastases, causes a pleural effusion in less than 3% of patients.129 The cells are dispersed as isolated cells and arranged in clusters and chains. They are small, with a diameter approximately two to three times that of small lymphocytes. Cytoplasm is scant. The nuclei are dark and have a finely granular chromatin texture; nucleoli are inconspicuous. Karyorrhexis

B • Fig. 4.30  Squamous Cell Carcinoma of the Lung (Pleural Fluid).  (A)

The malignant cells have coarsely textured chromatin and plate-like cytoplasm (Papanicolaou stain). (B) Some of the malignant cells are isolated rather than clustered. Compare the large malignant cell in the center with the two mesothelial cells on either side (Papanicolaou stain).

is common. When arranged in groups, the cells are crescentshaped and angulated as they wrap themselves around one another (Fig. 4.31A and B). CYTOMORPHOLOGY OF SMALL CELL CARCINOMA • S  mall cells (isolated and often in chains and clusters) • Nuclear molding • Scant cytoplasm   

The differential diagnosis includes lymphoma. The cells of small cell carcinoma are distinguished by their tendency to form clusters. Although the nuclei of lymphoma cells can be irregular in shape, they are rarely sharply angular like the cells of small cell carcinoma. Most small cell lung cancers, unlike lymphomas, are immunoreactive for TTF-1, but a negative result does not exclude the diagnosis. The differential diagnosis also includes other small cell malignancies like small cell carcinoma of the prostate102 and Merkel cell

CHAPTER 4  Pleural, Pericardial, and Peritoneal Fluids

A

161

B • Fig. 4.31  Small Cell Carcinoma of the Lung (Pleural Fluid).  (A) Some of the small malignant cells are isolated, and others are molded together in clusters. The mesothelial cell on the right provides a size comparison (Papanicolaou stain). (B) Similar features are seen with air-dried, Romanowsky-stained preparations. In this image, a macrophage in the lower left provides a size comparison.

carcinoma. In most cases, the clinical history of a specific malignancy points in the correct direction. Immunohistochemistry can be helpful if needed: small cell carcinomas are immunoreactive for CK7 and negative for CK20; the reverse is true for Merkel cell carcinoma. 

Melanoma Most malignant melanomas arise in the skin, but extracutaneous tumors like ocular melanomas do occur. In 5% of cases, patients present with metastatic disease without a known primary or with only a remote history of a pigmented cutaneous lesion. To compound the problem, the diagnosis of melanoma can be subtle in effusions. The malignant cells can resemble mesothelial cells: they are often isolated round cells with prominent nucleoli (Fig. 4.32A). In some cases, cells show a fine brown cytoplasmic pigmentation, intranuclear pseudoinclusions, or both. Cell clusters are uncommon (Fig. 4.32B), and cell blocks rarely show pericellular lacunae. The distinction from reactive mesothelial cells is difficult in cases that show little nuclear pleomorphism and hyperchromasia and no cytoplasmic pigmentation.130 Immunocytochemistry is helpful: melanomas are positive for S-100, HMB-45, and SOX10 and usually negative for keratin proteins. Although not entirely specific, melanomas show cytoplasmic staining with WT1.131 

A

Non-Hodgkin Lymphoma A malignant effusion complicates the course of disease in many patients with non-Hodgkin lymphoma. In fact, 10% to 15% of malignant effusions are caused by lymphoma; the proportion is significantly higher in the pediatric population.29,132 Most represent secondary involvement of serosal surfaces; very few are primary effusion lymphomas. Certain subtypes have a greater propensity for involving serosal surfaces (e.g., lymphoblastic lymphoma). Cytologic preparations are highly cellular and composed of dispersed lymphoid cells. Lacunar spaces are not seen in

B •

Fig. 4.32  Melanoma (Pleural Fluid).  (A) Nonpigmented melanoma cells are usually isolated, not clustered, and resemble mesothelial cells (Papanicolaou stain). (B) Less often, they aggregate into large clusters (Papanicolaou stain).

cell block sections with lymphomas. In many cases, mesothelial cells are conspicuously absent. The cells of DLBL are easiest to recognize because they are larger than histiocytes and their nucleoli are usually prominent (see Fig. 4.33).

162 CHA P T ER 4    Pleural, Pericardial, and Peritoneal Fluids

• Fig. 4.33  Diffuse Large B-Cell Lymphoma (Peritoneal Fluid).  The lymphoma cells have predominantly round nuclei with prominent nucleoli. Note the karyopyknosis and karyorrhexis, characteristic of most lymphomas (Papanicolaou stain).

•

Fig. 4.34  Lymphoblastic Lymphoma.  Because lymphoblastic lymphoma cells are only slightly larger than normal lymphocytes, they are easily misdiagnosed. They are recognized by their more finely dispersed chromatin texture, better appreciated on air-dried preparations (Romanowsky stain).

Nuclei are round or highly irregular, and the chromatin is coarsely textured. Cytoplasm is often abundant, pale, and vacuolated. The cells of small cell lymphomas are only slightly larger than normal lymphocytes. Those of follicular lymphomas have irregular and cleaved nuclei and scant cytoplasm. Lymphoblastic lymphomas are also composed of relatively small cells, with round or irregularly shaped

nuclei, finely dispersed chromatin, and scant or moderate amounts of cytoplasm (Fig. 4.34). Small lymphocytic lymphoma rarely involves serosal cavities. Burkitt and Burkittlike lymphomas are high-grade neoplasms composed of lymphoid cells of intermediate size with round nuclei, prominent nucleoli, and coarsely textured chromatin. Mitoses are numerous.

CHAPTER 4  Pleural, Pericardial, and Peritoneal Fluids

A

kappa

B

163

lambda

• Fig. 4.35  Non-Hodgkin Lymphoma.  In equivocal cases, immunostains for immunoglobulin light chains,

performed on cytocentrifuge preparations, can be helpful. Here the malignant lymphoid cells are immunoreactive for κ light chains (A) and negative for λ (B).

Karyorrhexis is a conspicuous feature of many lymphomas (see Fig. 4.33). It is seen in both treated and untreated patients and is an uncommon finding in benign effusions or malignant effusions due to other tumors. Precise classification of the lymphoma based on its morphology is rarely necessary with effusions because most patients have biopsy-proven disease before the effusion develops. Nevertheless, some degree of subclassification is usually possible based on the size of the cells, the degree of nuclear membrane irregularity (cleaved or noncleaved), and whether the cells resemble lymphoblasts or Burkitt cells. Such features can be appreciated especially well on air-dried Romanowsky-stained preparations (Fig. 4.34). DIFFERENTIAL DIAGNOSIS OF SECONDARY INVOLVEMENT BY LYMPHOMA • • • • • • •

 rimary effusion lymphoma P Benign lymphocytic effusion Posttransplant lymphoproliferative disorder Carcinoma Mesothelioma Melanoma Small round blue-cell tumors   

Secondary involvement by a large-cell non-Hodgkin lymphoma like DLBL or ALCL is morphologically indistinguishable from a PEL. The absence of a documented primary or of any mass lesion, lymphadenopathy, or organomegaly should raise the possibility of PEL, which can be confirmed by the presence of HHV-8. The differential diagnosis of secondary involvement by a lymphoma composed predominantly of small cells (e.g., lymphoblastic lymphoma) includes a benign lymphocytic effusion. The diagnosis of tuberculosis should be considered if there are characteristic clinical findings and the fluid is composed predominantly of small mature lymphocytes. The cells of small lymphocytic lymphoma are virtually impossible to distinguish from small, mature lymphocytes with

Papanicolaou-stained preparations, even with the help of computer-assisted morphometry.133 Given that serosal involvement by small lymphocytic lymphoma (or its leukemic counterpart CLL) is a highly rare event (some authors who have reviewed large series of malignant lymphomas could find no autopsy-documented cases),134 it is wise to document immunoglobulin light chain restriction before rendering a diagnosis of malignancy. The κ and λ light chain expression can be examined on cytocentrifuge preparations using immunocytochemistry8 or by flow cytometry.18 With immunocytochemistry, clonal excess is determined by visual inspection of the proportion of B cells that express κ or λ light chains (Fig. 4.35A and B). This method is a useful adjunct for the cytologic evaluation of lymphocyte-rich effusions that are cytologically equivocal for malignancy.19 In the case of the less common T-cell lymphoma, a T-cell phenotype with aberrant markers can be demonstrated by immunocytochemistry or flow cytometry, confirming a malignant diagnosis.8 In a transplant recipient, the possibility of a PTLD must be considered whenever an effusion contains a prominent lymphoid population. PTLDs are associated with EBV, best demonstrated by EBER in-situ hybridization, and are negative for HHV-8. Lymphomas are rarely confused with other malignancies because other tumors tend to form cell clusters in effusions. In some preparations, a crowding of the lymphoma cells mimics cluster formation, but such artifactual clustering is usually recognizable for what it is. Conversely, some carcinomas, mesotheliomas, and melanomas shed in a noncohesive pattern and can resemble the cells of a large-cell lymphoma (see Figs. 4.10C and 4.21) If there is any doubt concerning the lymphoid nature of a malignant effusion, a panel of immunomarkers (eg, CD45, keratin proteins, calretinin, WT1, TTF-1, S-100, HMB-45) can be helpful. Similarly, small round blue-cell tumors (e.g., neuroblastoma, Ewing sarcoma) can be ruled out with the appropriate immunohistochemical panel. 

164 CHA P T ER 4    Pleural, Pericardial, and Peritoneal Fluids

A •

Fig. 4.36  Hodgkin Lymphoma (Pleural Fluid).  The classic Reed– Sternberg cell is a large, multinucleated cell with very prominent (“owl’s eye”) nucleoli (Papanicolaou stain).

Hodgkin Lymphoma Patients with Hodgkin lymphoma can develop benign and malignant effusions. The more common benign effusions are likely due to thoracic duct obstruction or impaired lymphatic drainage due to tumor that is nearby but not directly involving the serosal surface. Malignant effusions are relatively uncommon and are almost never the initial manifestation of the disease. The cytologic hallmark is the Reed–Sternberg cell, a large multinucleated cell with huge (inclusion-like) nucleoli (Fig. 4.36). Mononuclear variants are often present, together with a mixed population of inflammatory cells that includes lymphocytes, plasma cells, eosinophils, neutrophils, and histiocytes. In a patient with a history of Hodgkin lymphoma, a fluid composed of a mixed population of inflammatory cells but without Reed–Sternberg cells is considered suggestive of malignancy. 

Multiple Myeloma Multiple myeloma rarely involves the serosal cavities. The pleura is involved more commonly than the peritoneum or pericardium. Pleural involvement is sometimes a direct extension of the tumor from an erosive rib lesion. The degree of plasmacytoid differentiation varies from one tumor to another. The cells are large and isolated. Nuclei are round and have coarsely textured chromatin with prominent nucleoli. Cytoplasm is usually abundant, nuclei are eccentrically positioned within the cell, and there is a perinuclear clear zone (Fig. 4.37A). The cells of poorly differentiated myelomas have less cytoplasm and vary in size and shape.135 Immunocytochemistry reveals monotypic expression of either κ or λ light chains. Cell block sections are particularly well suited because the immunoglobulin is cytoplasmic in these tumors. Curiously, although the cells are negative for keratin proteins, they often express epithelial membrane antigen.135 They are also positive for the plasma cell marker CD138 (Fig. 4.37B). 

B

CD138

•

Fig. 4.37  Multiple Myeloma (Pleural Fluid).  (A) The fluid is composed mostly of malignant plasma cells with abundant cytoplasm and eccentrically placed nuclei (Romanowsky stain). (B) The malignant cells are immunoreactive for CD138.

Acute and Chronic Leukemias Acute lymphoblastic and myeloblastic leukemias occasionally involve the serosal surfaces. Blasts are round, two or three times the diameter of lymphocytes, and dispersed as isolated cells. The nuclei are round or irregularly shaped. The chromatin is pale and finely dispersed, and nucleoli are usually prominent. Lymphoblasts cannot be distinguished from myeloblasts on Papanicolaou-stained preparations, but this is rarely if ever important, because the type of leukemia has usually been determined long before the patient develops an effusion. Auer rods, pink rod-like structures in the cytoplasm, are diagnostic of myeloid differentiation and can be appreciated on air-dried Romanowsky-stained preparations. The cells of CLL are indistinguishable from small, mature lymphocytes. Thus immunophenotyping is necessary to establish this diagnosis. In patients with circulating blasts, the possibility that the fluid was contaminated by peripheral blood during a traumatic tap is likely if the specimen contains red blood cells. 

Myeloproliferative Neoplasms Myeloproliferative neoplasms like primary myelofibrosis can lead to extramedullary hematopoiesis (EMH). The

CHAPTER 4  Pleural, Pericardial, and Peritoneal Fluids

A

165

B CD61

C • Fig. 4.38  Extramedullary Hematopoiesis of Pleura (Pleural Fluid).  (A) This patient had a recent diag-

nosis of a squamous cell carcinoma of the lung and developed a pleural effusion. The sample contained scattered megakaryocytes, some with hyperlobulated and complex nuclei, typical of patients with myeloproliferative neoplasms. A history of primary myelofibrosis was uncovered in the electronic medical record (Papanicolaou stain). (B) A hyperlobulated nucleus with thread-like connections between lobes is apparent (hematoxylin and eosin stain). (C) Megakaryocytes are immunoreactive for CD61. (The fluid was negative for metastatic squamous cell carcinoma.)

most common sites of EMH in patients with primary myelofibrosis are the spleen and liver, but the pleura and other organs can also be involved.95,136 Megakaryocytes are the most conspicuous component of EMH and readily seen on alcohol-fixed, Papanicolaou-stained preparations and cell block sections. Megakaryocytes are rare in pleural and peritoneal effusions and thus usually an unexpected and often puzzling finding137 (Fig. 4.38). Because of their large size and unusual morphology, they mimic malignant cells. Their identification can be aided with immunocytochemistry for the megakaryocyte marker CD61. In patients with a myeloproliferative neoplasm, the megakaryocytes of EMH may be enlarged, hyperlobulated and complex, or hypolobulated. 

Sarcomas Virtually any sarcoma can metastasize to the serosal surfaces, although sarcomas do so much less frequently than other tumors. When they do, it is usually late in the course of the disease. Sarcomas in effusions range widely in morphology and include spindle cell sarcomas such as leiomyosarcoma, synovial sarcoma, and malignant nerve sheath tumors; epithelioid sarcomas such as epithelioid angiosarcoma; and round-cell sarcomas such as Ewing sarcoma, neuroblastoma, and embryonal rhabdomysarcoma.54 Not surprisingly, the cytomorphology of sarcomas in effusions is highly variable, depending on the sarcoma involved. The cells can be cohesive (see Fig. 4.18 and Fig. 4.39A)

166 CHA P T ER 4    Pleural, Pericardial, and Peritoneal Fluids

A

B • Fig. 4.39  (A) Epithelioid Angiosarcoma (Peritoneal Fluid). The patient had a history of squamous cell

carcinoma of the cervix. The new diagnosis of an intra-abdominal angiosarcoma was made on this fluid and confirmed by positive staining for the vascular markers ERG and CD31. (B) Pleomorphic rhabdomyosarcoma (peritoneal fluid). The malignant cells are noncohesive. Binucleation, eccentrically placed nuclei, and dense cytoplasm are typical of rhabdomyosarcoma. The cells were negative for keratin but positive for desmin (Papanicolaou stain).

B

A •

Fig. 4.40  Adult Granulosa Cell Tumor (Ascites).  (A) The malignant cells are uniform and bland and resemble benign mesothelial cells. The arrangement of cells in large clusters is suspicious, as is the prominence of nuclear grooves and focal follicle formation (Papanicolaou stain). (B) Comparison with the original histologic sections was helpful in establishing the diagnosis (hematoxylin and eosin stain).

or noncohesive (Fig. 4.39B), small or large. Round-cell sarcomas such as embryonal rhabdomyosarcoma exfoliate predominantly as dispersed cells, mimicking the pattern of a non-Hodgkin lymphoma. Immunocytochemistry is helpful in resolving equivocal cases. 

Germ Cell Tumors Like sarcomas, germ cell tumors rarely cause malignant effusions. When they do, it is usually late in the course of widely disseminated disease. The seminoma of the testis and its counterpart the dysgerminoma of the ovary are composed of noncohesive, uniform cells that resemble enlarged mesothelial cells. They have less cytoplasm than mesothelial cells, however, and more prominent nucleoli (see Fig. 5.15). Dysgerminoma/seminoma can

be distinguished from embryonal carcinoma because the former has a characteristic membranous staining pattern for CD117. Non-seminomatous germ cell tumors, which include embryonal carcinoma, endodermal sinus (yolk sac) tumor, choriocarcinoma, and malignant teratoma, are composed of large pleomorphic cells with pale, finely granular chromatin and prominent, often multiple, nucleoli. A cytologic diagnosis of malignancy in this group of tumors is straightforward, but correct subtyping and distinction from a poorly differentiated epithelial malignancy requires correlation with serologic markers and immunocytochemical studies. Embryonal carcinoma, yolk sac tumor, and choriocarcinoma are keratin-positive but can be distinguished from epithelial malignancies because of their staining for

CHAPTER 4  Pleural, Pericardial, and Peritoneal Fluids

PLAP and SALL4. Additionally, embryonal carcinoma, like dysgerminoma, is immunoreactive for Oct-3/4 and NANOG. Immunohistochemistry is rarely necessary, however, because these tumors rarely manifest initially as a malignant effusion. Rarely, sex cord-stromal tumors involve serosal surfaces and cause an effusion, sometimes many years after treatment of the primary tumor (Fig. 4.40). Immunohistochemistry for inhibin, a marker of many sex cord-stromal tumors, along with comparison of the effusion specimen with sections from the original tumor, can be indispensable for correct interpretation.

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93. Chen Y, Chen JQ, Katz RL. Epithelioid hemangioendothelioma: a study of 14 cytopathology cases. J Am Soc Cytopathol. 2015;4:148–159. 94. Doyle LA, Fletcher CD, Hornick JL. Nuclear expression of CAMTA1 distinguishes epithelioid hemangioendothelioma from histologic mimics. Am J Surg Pathol. 2016;40(1):94– 102. 95. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of the Haematopoietic and Lymphoid Tissues. Revised 4th ed. Lyon: IARC; 2017. 96. Brimo F, Michel RP, Khetani K, Auger M. Primary effusion lymphoma: a series of 4 cases and review of the literature with emphasis on cytomorphologic and immunocytochemical differential diagnosis. Cancer. 2007;111(4):224–233. 97. Wakely Jr PE, Menezes G, Nuovo GJ. Primary effusion lymphoma: cytopathologic diagnosis using in situ molecular genetic analysis for human herpesvirus 8. Mod Pathol. 2002;15(9):944– 950. 98. Ansari MQ, Dawson DB, Nador R, et  al. Primary body cavity-based AIDS-related lymphomas. Am J Clin Pathol. 1996;105:221–229. 99. Alexanian S, Said J, Lones M, Pullarkat ST. KSHV/HHV8negative effusion-based lymphoma, a distinct entity associated with fluid overload states. Am J Surg Pathol. 2013;37(2):241– 249. 100. Wu W, Youm W, Rezk SA, Zhao X. Human herpesvirus 8-unrelated primary effusion lymphoma-like lymphoma: report of a rare case and review of 54 cases in the literature. Am J Clin Pathol. 2013;140(2):258–273. 101. Ohori NP, Whisnant RE, Nalesnik MA, Swerdlow SH. Primary pleural effusion posttransplant lymphoproliferative disorder: distinction from secondary involvement and effusion lymphoma. Diagnostic Cytopathology. 2001;25(1):50–53. 102. Renshaw AA, Nappi D, Cibas ES. Cytology of metastatic adenocarcinoma of the prostate in pleural effusions. Diagn Cytopathol. 1996;15(2):103–107. 103. Parwani AV, Chan TY, Ali SZ. Significance of psammoma bodies in serous cavity fluid: a cytopathologic analysis. Cancer. 2004;102(2):87–91. 104. Onur I, Siddiqui MT, Wakely PE, Ali SZ. Pseudomyxoma peritonei: cytomorphologic findings and clinicopathologic correlates. J Am Soc Cytopathol. 2016;5:43–49. 105. Esteban JM, Yokota S, Husain S, Battifora H. Immunocytochemical profile of benign and carcinomatous effusions. Am J Clin Pathol. 1990;94:698–705. 106. Nasser H, Kuntzman TJ. Pleural effusion in women with a known adenocarcinoma: the role of immunostains in uncovering another hidden primary tumor. Acta Cytol. 2011;55(5):438– 444. 107. Gurel B, Ali TZ, Montgomery EA, et al. NKX3.1 as a marker of prostatic origin in metastatic tumors. Am J Surg Pathol. 2010;34(8):1097–1105. 108. Jia L, Jiang Y, Michael CW. Performance of different prostate specific antibodies in the cytological diagnosis of metastatic prostate adenocarcinoma. Diagn Cytopathol. 2017;45(11):998– 1004. 109. Laury AR, Perets R, Piao H, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 2011;35(6):816–826. 110. Laury AR, Hornick JL, Perets R, et  al. PAX8 reliably distinguishes ovarian serous tumors from malignant mesothelioma. Am J Surg Pathol. 2010;34(5):627–635.

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111. Tong GX, Devaraj K, Hamele-Bena D, et al. PAX8: a marker for carcinoma of Mullerian origin in serous effusions. Diagn Cytopathol. 2011;39(8):567–574. 112. Mito JK, Conner JR, Hornick JL, Cibas ES, Qian X. SOX10/ keratin dual-color immunohistochemistry: an effective firstline test for the workup of epithelioid malignant neoplasms in FNA and small biopsy specimens. Cancer Cytopathol. 2018;126(3):179–189. 113. Nelson ER, Sharma R, Argani P, Cimino-Mathews A. Utility of SOX10 labeling in metastatic breast carcinomas. Hum Pathol. 2017;67:205–210. 114. Hammond ME, Hayes DF, Dowsett M, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134(7):e48–72. 115. Wolff AC, Hammond ME, Schwartz JN, et al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med. 2007;131(1):18–43. 116. Kinsella MD, Birdsong GG, Siddiqui MT, Cohen C, Hanley KZ. Immunohistochemical detection of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 in formalin-fixed breast carcinoma cell block preparations: correlation of results to corresponding tissue block (needle core and excision) samples. Diagn Cytopathol. 2013;41(3):192–198. 117. Fox SB, Kumarasinghe MP, Armes JE, et al. Gastric HER2 Testing Study (GaTHER): an evaluation of gastric/gastroesophageal junction cancer testing accuracy in Australia. Am J Surg Pathol. 2012;36(4):577–582. 118. Wong DD, de Boer WB, Platten MA, Jo VY, Cibas ES, Kumarasinghe MP. HER2 testing in malignant effusions of metastatic gastric carcinoma: is it feasible? Diagn Cytopathol. 2015;43(1):80–85. 119. Bartley AN, Washington MK, Colasacco C, et al. HER2 testing and clinical decision making in gastroesophageal adenocarcinoma: guideline from the College of American Pathologists, American Society for Clinical Pathology, and the American Society of Clinical Oncology. J Clin Oncol. 2017;35(4):446–464. 120. Pao W, Miller V, Zakowski M, et al. EGF receptor gene mutations are common in lung cancers from “never smokers” and are associated with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad Sci U S A. 2004;101(36):13306–13311. 121. Lynch TJ, Bell DW, Sordella R, et al. Activating mutations in the epidermal growth factor receptor underlying responsiveness of non-small-cell lung cancer to gefitinib. N Engl J Med. 2004;350(21):2129–2139. 122. Paez JG, Janne PA, Lee JC, et al. EGFR mutations in lung cancer: correlation with clinical response to gefitinib therapy. Science. 2004;304(5676):1497–1500. 123. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung

cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol. 2012;13(3):239–246. 124. Lindeman NI, Cagle PT, Aisner DL, et  al. Updated molecular testing guideline for the selection of lung cancer patients for treatment with targeted tyrosine kinase inhibitors: guideline from the College of American Pathologists, the International Association for the Study of Lung Cancer, and the Association for Molecular Pathology. Arch Pathol Lab Med. 2018;142(3):321–346. 125. Smouse JH, Cibas ES, Janne PA, Joshi VA, Zou KH, Lindeman NI. EGFR mutations are detected comparably in cytologic and surgical pathology specimens of nonsmall cell lung cancer. Cancer. 2009;117(1):67–72. 126. Russell-Goldman E, Kravets S, Dahlberg SE, Sholl LM, Vivero M. Cytologic-histologic correlation of programmed deathligand 1 immunohistochemistry in lung carcinomas. Cancer Cytopathol. 2018;126:253–263. 127. Mansour MSI, Seidal T, Mager U, Baigi A, Dobra K, Dejmek A. Determination of PD-L1 expression in effusions from mesothelioma by immuno-cytochemical staining. Cancer Cytopathol. 2017;125(12):908–917. 128. Smith-Purslow MJ, Kini SR, Naylor B. Cells of squamous cell carcinoma in pleural, peritoneal, and pericardial fluids: origin and morphology. Acta Cytol. 1989;33:245–253. 129. Chhieng DC, Ko EC, Yee HT, Shultz JJ, Dorvault CC, Eltoum IA. Malignant pleural effusions due to small-cell lung carcinoma: a cytologic and immunocytochemical study. Diagn Cytopathol. 2001;25(6):356–360. 130. Walts AE. Malignant melanoma in effusions: a source of falsenegative cytodiagnoses. Diagn Cytopathol. 1986;2:150–153. 131. Conner JR, Cibas ES, Hornick JL, Qian X. Wilms tumor 1/ cytokeratin dual-color immunostaining reveals distinctive staining patterns in metastatic melanoma, metastatic carcinoma, and mesothelial cells in pleural fluids: an effective first-line test for the workup of malignant effusions. Cancer Cytopathol. 2014;122(8):586–595. 132. Das DK. Serous effusions in malignant lymphomas: a review. Diagn Cytopathol. 2006;34(5):335–347. 133. Walts AE, Marchevsky AM. Computerized interactive morphometry: an expert system for the diagnosis of lymphoid-rich effusions. Am J Clin Pathol. 1989;92:765–772. 134. Billingham ME, Rawlinson DG, Berry PF, Kempson RL. The cytodiagnosis of malignant lymphomas and Hodgkin’s disease in cerebrospinal, pleural, and ascitic fluids. Acta Cytol. 1975;19:547–556. 135. Sasser RL, Yam YT, Li CY. Myeloma with involvement of the serous cavities: cytologic and immunochemical diagnosis and literature review. Acta Cytol. 1990;34:479–485. 136. Silverman JF. Extramedullary hematopoietic ascitic fluid cytology in myelofibrosis. Am J Clin Pathol. 1985;84(1):125–128. 137. Kumar NB, Naylor B. Megakaryocytes in pleural and peritoneal fluids: prevalence, significance, morphology, and cytohistological correlation. J Clin Pathol. 1980;33(12):1153–1159.

5

Peritoneal Washings EDMUND S. CIBAS

P

eritoneal washing cytology (PWC) was introduced in the 1950s as a way to identify microscopic spread of cancer not visible by gross inspection of the peritoneal surface.1 In some cancer patients, positive PWC may be the only evidence of metastatic disease to the peritoneum. Because positive results correlate with poorer prognosis,2,3 cytologic findings are included in the staging system for ovarian and fallopian tube cancers.4 The yield, however, is low: positive washings by themselves change the surgical stage of only 3% to 5% of women with gynecologic cancers.5,6 PWC is also used to exclude an occult cancer in patients who undergo laparoscopy or laparotomy for presumed benign gynecologic conditions (e.g., endometriosis, leiomyomata) and in women with BRCA1 and BRCA2 mutations undergoing risk-reducing salpingo-oopherectomy.7,8 PWC can be used to monitor a patient’s response to treatment for advanced ovarian cancer and other malignancies (the “­second-look” procedure), but this is usually limited to patients in research protocols.9-13 In some instances, PWC is used to detect peritoneal spread of nongynecologic malignancies such as pancreatic and gastric cancer. INDICATIONS FOR PERITONEAL WASHINGS • S  taging gynecologic malignancies • Ruling out occult cancer • Assessing response to treatment (the “second-look” procedure) • Staging nongynecologic malignancies   

Specimen Collection, Preparation, and Reporting Terminology On entering the peritoneal cavity, the surgeon evacuates any preexisting peritoneal fluid and submits it separately for cytologic examination. Washings are obtained by instilling 50–200 mL of sterile saline or other physiological solution into several different areas, usually the pelvis, the right

and left paracolic gutters, and the undersurface of the diaphragm. The fluid is aspirated and submitted for cytologic evaluation. Segregating washings from different abdominal cavity sites and sending them separately for cytologic evaluation does not have any advantage over combining them into a single specimen.14 The specimen should be delivered to the laboratory unfixed and refrigerated at 4°C until slides can be prepared. If a significant delay before cytopreparation is anticipated, an equal volume of 50% ethanol can be added. To prepare slides, the specimen is thoroughly mixed, and an aliquot (often 50 mL) is spun in a centrifuge to a cell sediment/ pellet. From this sediment one can prepare smears, cytocentrifuge preparations, or thin-layer preparations (e.g., ThinPrep, SurePath), depending on the resources and preferences of the laboratory.15 The remaining (or separately centrifuged) cell sediment can also be fixed in 10% formalin, embedded in paraffin, and processed as a histologic specimen (“cell block”). Cell block sections are very useful, especially for morphologic comparison to the patient’s resected neoplasm.15 Results of PWC are commonly reported as “negative for malignant cells,” “atypical,” “suspicious for malignancy,” or “positive for malignant cells.”16 “Atypical” (connoting a low degree of suspicion for malignancy) and “suspicious for malignancy” (connoting a high degree of suspicion) interpretations should be avoided whenever possible because they are not helpful to a physician faced with making a treatment decision.16,17 In most cases, only an unequivocally positive diagnosis is used for staging purposes—anything less is treated as a negative result. Side-by-side comparison with the corresponding resection specimen often helps resolve an equivocal case. For involvement of peritoneal washings by a known borderline tumor, “neoplastic cells present” is preferred over “positive for malignant cells” because borderline tumors, strictly speaking, are not malignancies. Criteria for specimen adequacy have not been established, but it seems reasonable to require some benign mesothelial cells before considering a peritoneal washing 171

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specimen adequate and negative for malignant cells.18 A specimen with malignant cells is always adequate. Because peritoneal washings are obtained as part of a cancer staging procedure, there is usually a concurrent histologic specimen. For example, when washings are obtained as part of ovarian cancer staging, an oophorectomy is obtained by the laboratory at the same time. Representative slides from the oophorectomy specimen can be helpful in a side-by-side comparison of a diagnostically difficult washing specimen. 

Accuracy Not all patients with metastases to the peritoneum have positive PWC results. In fact, 23% to 52% of patients with biopsy-proven peritoneal involvement have negative results.19-23 When peritoneal washings are examined as part of a second-look procedure, the false-negative rate is even higher, ranging from 31% to 86% of patients with biopsyproven metastases.9,11,20,22,24-26 The higher false-negative rate of second-look procedures may be due to the poor distribution of fluid when the abdominal cavity is altered by adhesions. False-positive diagnoses are uncommon but well documented,2,3,20,27-30 occurring in less than 5% of cases.2,23,31 Causes include reactive mesothelial proliferation with psammoma bodies23,27,28 and endometriosis.22,29 In particular, eosinophilic metaplastic atypia in endometriosis can be a source of atypical (but benign) cells that might be misconstrued as malignant.32 In one instance, ectopic pancreatic tissue was misinterpreted as adenocarcinoma in a patient with a mucinous ovarian cancer.33 

The Normal Peritoneal Washing Peritoneal washings differ morphologically from peritoneal fluid (ascites) in several easily recognizable ways. First, the washing procedure mechanically strips the peritoneal surface of entire sheets of mesothelial cells, whereas sheets of mesothelial cells are not seen in a benign peritoneal effusion. (One exception: peritoneal and pleural effusions suctioned during abdominal or thoracic surgery contain sheets of peritoneal cells that are pulled in by the suctioning process.) Second, skeletal muscle and adipose tissue fragments are common in peritoneal washings, especially in cell block preparations. CYTOMORPHOLOGY OF BENIGN PERITONEAL WASHINGS • • • • •

 esothelial cells in sheets M Collagen balls Histiocytes Skeletal muscle Adipose tissue   

Mesothelial cells in peritoneal washings are arranged predominantly in flat sheets, often large and occasionally folded (Fig. 5.1). In cell block sections the sheets are transected and appear as long, thin ribbons (Fig. 5.2). The cells are evenly spaced, with a moderate amount of cytoplasm. Nuclear membranes are thin, and the chromatin is pale and evenly dispersed; small nucleoli are often present. Although usually round or oval (Fig. 5.3A), the nuclei are sometimes scalloped, wrinkled, or grooved,34 possibly due to fixation

• Fig. 5.1  Mesothelial Cells. Normal mesothelial cells in peritoneal washings are often arranged in cohesive, sometimes folded, sheets. Isolated mesothelial cells are present in the background (Papanicolaou stain).

CHAPTER 5  Peritoneal Washings

artifact. In extreme cases the nuclei have a flower-like appearance (“daisy cells,” Fig. 5.3B).35 Isolated mesothelial cells, similar to those seen in ascites, are also encountered. Spherical masses of collagen surrounded by benign, flattened mesothelial cells, known as collagen balls, are seen in up to 50% of peritoneal washings.34,36 Aqua in color with the Papanicolaou stain, they have round or bosselated contours (Fig. 5.4). They are usually few in number, but occasionally they can be abundant. They have no known significance (and therefore do not deserve mention on a cytology report). It has been suggested that they result from a pinching off of mesothelial-lined stromal projections known as micropapillomatosis on the surface of the ovaries.36,37 Numerous histiocytes, scattered either as isolated cells or in variably-sized aggregates, are often present (Fig. 5.5).

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Because they look different from mesothelial cells and are often haphazardly aggregated, they might be misinterpreted as metastatic cancer cells. Attention to the typical nuclear and cytoplasmic features of histiocytes (oval, folded, and kidney-shaped nuclei; pale chromatin; granular and microvacuolated cytoplasm) is helpful in correctly identifying them. Skeletal muscle and adipose tissue fragments are sometimes seen: they fall into the peritoneal cavity when the abdominal incision is made and are suctioned along with the fluid. Detached ciliary tufts, presumably of fallopian tube origin, are a relatively common incidental finding, especially when the washings are obtained during the secretory (luteal) phase of the menstrual cycle.38 A typical interpretation of a benign peritoneal washing might read as follows: “Negative for malignant cells. Mesothelial cells and histiocytes.” 

Benign Conditions Endosalpingiosis and Similar Benign Proliferations

• Fig. 5.2  Mesothelial Cells (Cell Block). In cell block sections, a sheet

of mesothelial cells is transected and looks like a string of pearls. When it curls around on itself, it has a pseudoglandular appearance (hematoxylin & eosin stain).

A

A group of benign conditions involving the peritoneal and ovarian surfaces results in a proliferation of fallopian tubal-type epithelial cells or mesothelial cells, often with psammoma bodies. These conditions mimic peritoneal involvement by a serous borderline tumor and serous carcinoma; familiarity with them is thus important. Endosalpingiosis is a proliferation of benign glands and cysts lined by ciliated, fallopian tube–like epithelium. Common locations include the ovarian cortex, uterine serosa, peritoneal surface, and omentum. Psammoma bodies are often seen. Some authors reserve the term endosalpingiosis for proliferations that include tubal-type stroma, as well as glands, and use the term Müllerian inclusion cyst for cases

B • Fig. 5.3  Mesothelial Cells. (A) Benign mesothelial cells usually have round or oval nuclei, but in some

cases there are irregularities in nuclear contour. Note the spaces (“windows”) that separate adjacent cells, a common finding (Papanicolaou stain). (B) Benign mesothelial cells sometimes have grooved or even lobulated nuclei, in extreme cases resembling the petals of a flower (“daisy cells”). This finding has no diagnostic significance (ThinPrep, Papanicolaou stain). (Figure 5.3B courtesy Charles D. Sturgis, MD, Cleveland Clinic, Cleveland, OH.)

174 CHA P T ER 5    Peritoneal Washings

A

B •

Fig. 5.4  Collagen Ball. These spheres of collagen are surrounded by flattened mesothelial cells. (A) Papanicolaou stain; (B) hematoxylin & eosin–stained cell block section.

that lack tubal-type stroma.37,39 Both endosalpingiosis and Müllerian inclusion cysts, however, are usually incidental microscopic findings in a patient undergoing surgery for something else. Histologically, they raise the possibility of metastatic disease but are identified as benign proliferations because the cells are uniform and bland and lack mitotic activity. Serous adenofibromas of the ovarian surface are benign ovarian tumors that, like endosalpingiosis, are composed of benign tubal-type glands, cysts, or both, except that they are often a visible mass rather than a microscopic finding, and they have a broad, fibrous, stromal component. The epithelial portion of a serous adenofibroma, such as endosalpingiosis, often contains psammoma bodies and can be confused with peritoneal involvement by a serous borderline tumor or even adenocarcinoma. Prior surgery, pelvic inflammatory disease, a ruptured cyst, and other conditions cause florid mesothelial hyperplasia, sometimes accompanied by psammoma body formation.16

A

CYTOMORPHOLOGY OF ENDOSALPINGIOSIS AND SIMILAR BENIGN PROLIFERATIONS • C  uboidal cells (± cilia) with minimal to mild atypia • Psammoma bodies   

The conditions described above all have a similar appearance in peritoneal washings. Clusters of cuboidal mesothelial-like cells (Fig. 5.6), some arranged around a psammoma body (Figs. 5.7 and 5.8) or nondescript calcification, are present and can be abundant.28 Cilia may be present, but often they are absent or impossible to identify. Nuclei are round or oval, with a pale chromatin pattern and small nucleoli. Nuclear atypia is mild, and mitoses are very uncommon. These benign conditions are a potential cause of a falsepositive interpretation of involvement by a serous carcinoma

B • Fig. 5.5  Histiocytes. Histiocytes are either isolated or in groups, as

seen here. They have indistinct cell borders (no “windows”), granular or vacuolated cytoplasm, and occasionally folded nuclei. A short strip of mesothelial cells is also present on the cell block section. (A) Papanicolaou stain; (B) hematoxylin & eosin–stained cell block.

CHAPTER 5  Peritoneal Washings

or borderline tumor.27 To avoid a false-positive interpretation, a diagnosis of malignancy should not be based on the presence of psammoma bodies alone.22,27 Correlation with the concurrent histologic material is helpful in most cases. Certainly, if the patient does not have a carcinoma or borderline tumor, one should be cautious rendering such a diagnosis on the basis of the peritoneal washings alone. Some patients with endosalpingiosis or benign mesothelial proliferations, however, can also have a serous borderline tumor confined to the ovary.17 Comparison of the peritoneal washings, particularly cell block preparations, with the histologic sections from the tumor often resolves equivocal cases, but the final interpretation is not always straightforward, and the pathologist is sometimes required to make the best judgment possible. There are few helpful morphologic clues, particularly because many serous borderline tumors have only mild cytologic atypia, equivalent to what can be seen with florid mesothelial hyperplasia and

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endosalpingiosis. One possible clue: in cell block sections, some papillary fragments of serous borderline tumors have broad stromal cores, a feature not seen in cell blocks containing only mesothelial hyperplasia or endosalpingiosis. 

Endometriosis Endometriosis, the presence of ectopic benign endometrial glands and stroma in the omentum, peritoneum, ovary, and elsewhere, affects 6% to 10% of women of reproductive age, causing pelvic pain and infertility. The pathogenesis is controversial, but endometriotic lesions are considered benign, nonneoplastic lesions, although there is growing evidence that many harbor somatic cancer-associated driver mutations.39 CYTOMORPHOLOGY OF ENDOMETRIOSIS • • • •

 emosiderin-laden macrophages H Endometrial glandular cells Endometrial stromal cells Tissue fragments containing endometrial glands and stroma (cell blocks)   

•

Fig. 5.6  Reactive Mesothelial Cells (Ovarian Torsion). The mesothelial cells are enlarged, with irregular nuclei and prominent nucleoli. Exploratory laparotomy revealed torsion of the right ovary with intraperitoneal adhesions.

A

The purpose of peritoneal washings in this setting is to exclude an occult malignancy, not to diagnose endometriosis. A diagnosis of endometriosis, in fact, is rarely made by examining peritoneal washings alone. Hemosiderinladen macrophages are seen in one-third of women with endometriosis.40 In a young woman, hemosiderin-laden macrophages in peritoneal washings are most likely from endometriosis,40 but by themselves they are a nonspecific finding seen in any condition associated with intraperitoneal bleeding. Some cases of endometriosis show a population of cells morphologically similar to but distinct (in subtle ways)

B • Fig. 5.7  Endosalpingiosis. (A) The psammoma body has concentric lamination and is surrounded by

cuboidal cells with small nucleoli and vacuolated cytoplasm. Cilia are not identified (Papanicolaou stain). (B) The surface of the resected ovary shows benign ciliated glandular cells associated with psammoma bodies. There was no evidence of tumor (hematoxylin & eosin stain).

176 CHA P T ER 5    Peritoneal Washings

A

B • Fig. 5.8  Serous Adenofibroma of the Ovary.  (A) A psammoma body is surrounded by evenly spaced

mesothelial cells with minimal nuclear atypia (Papanicolaou stain). (B) The resected ovary shows a serous adenofibroma involving the ovarian surface, with an occasional psammoma body (arrow; hematoxylin & eosin stain).

from mesothelial cells and macrophages; these likely represent endometrial glandular or stromal cells or both. The endometrial-like glandular and stromal cells may, in fact, resemble the clusters of exfoliated endometrial cells seen in menstrual-phase cervical-vaginal preparations (Fig. 5.9A), but a definitive distinction between mesothelial cells and endometrial cells is difficult.40 On rare occasions, a diagnosis can be made when tissue fragments with endometrialtype glands and stroma are identified in cell block sections41 (Fig. 5.9B–C); immunohistochemistry for CD10 can be useful to confirm the presence of endometrial stromal cells. Occasionally, the endometrial glandular cells are enlarged and atypical and might suggest a malignancy. Correlation with concurrent histologic material is very helpful in preventing a misdiagnosis.29,32 Care must be taken in interpreting immunostains in this context. In particular, PAX8 can be positive in endometriosis (Fig. 5.9D), benign mesothelial cells, and some peritoneal mesotheliomas, and by itself is not diagnostic of a Müllerian malignancy.42,43 

Malignant Tumors Ovarian and Fallopian Tube Cancers Ovarian cancer is the 11th most common cancer in women in the United States and the fifth most common cause of cancer-related death (behind lung, breast, colorectal, and pancreatic cancers).44 The peak incidence is in the fifth and sixth decades of life. Most women (70% to 75%) with ovarian cancer present with stage III or IV disease (tumor spread beyond the pelvis). Fallopian tube cancers resemble ovarian cancers in many ways: they have similar risk factors, share most of the same histologic tumor types, and have a similar biologic behavior. For these reasons, the staging system for fallopian tube cancers (and primary peritoneal cancers) is the same as for

ovarian cancers. PWC is an important staging component for these tumors: positive PWC or malignant ascites modifies stage I tumors to stage IC3.4,45 Primary treatment for the common epithelial cancers is surgical staging and surgical debulking.46 Most women receive postoperative chemotherapy, either systemic or intraperitoneal, depending on the stage of their disease; observation is an option for some patients with early stage, grade 1 tumors, for whom survival is greater than 90% with surgery alone.46 Platinum drugs (cisplatin, carboplatin) and taxanes (paclitaxel, docetaxel) are the most efficacious agents and are often used in combination. The most common type of ovarian and tubal cancer—and the type that most often produces positive cytologic findings—is the high-grade serous carcinoma.

CYTOMORPHOLOGY OF HIGH-GRADE SEROUS CARCINOMA • • • • • • •

 arge or small clusters and isolated cells L Large cells Marked variation in nuclear size Nuclear hyperchromasia Prominent nucleoli Mitoses Scant or abundant vacuolated cytoplasm   

High-grade serous carcinoma is usually easy to recognize in peritoneal washings. The malignant cells are often numerous, isolated, and arranged in clusters (Fig. 5.10). Nuclei are enlarged and vary markedly in size. There is nuclear hyperchromasia, with coarsely textured chromatin and very prominent nucleoli (Fig. 5.11). Mitoses are common. Cytoplasm may be scant but is often abundantly vacuolated. Psammoma bodies may be present. Morphologically

CHAPTER 5  Peritoneal Washings

A

B PAX8

C

D •

Fig. 5.9  Endometriosis. (A) A cluster of cells has scalloped borders and contains nuclear fragments. Individually, the cells resemble mesothelial cells, but the nuclear debris suggests that they are degenerating endometrial cells. Such clusters are often recognized as endometrial cells only in retrospect, after reviewing cell block sections or correlating with laparoscopic or histologic evidence (Papanicolaou stain). (B) The cell block from the same case as (A) shows endometrial glands surrounding a core of degenerated endometrial stromal cells (hematoxylin & eosin stain). (C) Occasionally, the cell block of peritoneal washings from a patient with endometriosis shows diagnostic tissue fragments composed of intact endometrial-type glands and stroma (hematoxylin & eosin stain). (D) Endometriosis, like many Müllerian malignancies, is positive for PAX8.

A

B • Fig. 5.10  High-Grade

Serous Carcinoma of the Ovary. (A) The malignant cells are often arranged in large, irregularly shaped three-dimensional clusters of crowded cells (Papanicolaou stain). (B) Higher magnification reveals crowded large cells with round, dark nuclei, large nucleoli, and pale vacuolated cytoplasm. Mitoses are often present (Papanicolaou stain).

177

178 CHA P T ER 5    Peritoneal Washings

• Fig. 5.11  High-Grade Serous Carcinoma of the Peritoneum. These tumors are morphologically indistinguishable from high-grade serous carcinomas of the ovary (Papanicolaou stain).

identical tumors occur as primary neoplasms of the peritoneum (Fig. 5.11). Low-grade serous carcinomas are uncommon, accounting for 5% of all serous carcinomas.47 By definition, they have low-grade nuclei like those seen with serous borderline tumors, and many contain a component of serous borderline tumor. They cannot be distinguished from serous borderline tumors cytologically; the distinction is based on identifying stromal invasion in the primary tumor histologically. Peritoneal biopsy in patients with a serous borderline tumor may reveal involvement of peritoneal surfaces, classified as either noninvasive or invasive implants; this distinction is important for prognosis and management. Peritoneal washing cytologic study cannot discriminate between noninvasive and invasive implants.48 Although the distinction cannot be made on the basis of peritoneal washings, some differences between high-grade serous carcinoma and serous borderline tumor/low-grade serous carcinoma are apparent cytologically.34,49 CYTOMORPHOLOGY OF SEROUS BORDERLINE TUMOR AND LOW-GRADE SEROUS CARCINOMA • • • • •

 arge or small clusters and isolated cells L Minimal-mild nuclear atypia Cytoplasmic vacuoles Psammoma bodies Fibrovascular cores (cell block)   

Positive peritoneal washings from patients with serous borderline tumors and low-grade serous carcinomas usually show large (Fig. 5.12A) or small (Fig. 5.12B), cohesive, three-dimensional, often branching, clusters. Isolated cells are less conspicuous than with high-grade serous carcinoma. Pleomorphism is slight or moderate. Cytoplasmic vacuoles can be seen, and the nuclear-to-cytoplasmic ratio is high. Nucleoli are inconspicuous, and mitoses are rare. As with

high-grade serous carcinomas, psammoma bodies may be present. Cell blocks sometimes show thin or broad fibrovascular cores lined by neoplastic epithelium (Fig. 5.13). The differential diagnosis for serous borderline tumor and low-grade serous carcinoma includes endosalpingiosis and similar benign proliferations.48 A malignant diagnosis should never be made solely on the basis of psammoma bodies alone. The evaluation of peritoneal washings in patients with a serous borderline tumor depends on a careful comparison with the resected primary tumor. Cell block sections are especially useful in this regard. Fibrovascular cores lined by atypical epithelium are characteristic of serous borderline tumors and low-grade serous carcinomas and are very rarely seen in patients with endosalpingiosis or mesothelial hyperplasia (see Fig. 5.13). Positive washing results in a woman with a serous borderline tumor do not have the same clinical significance as in a woman with a carcinoma. The overall survival rate is very good with surgery alone; chemotherapy is considered for patients with invasive peritoneal implants. In fact, “positive” washing results in women with borderline ovarian tumors raise a question regarding terminology. Although the staging system for ovarian cancers is also used for ovarian borderline tumors, borderline tumors are not, strictly speaking, carcinomas. It is therefore preferable to report positive washing results in a woman with a borderline tumor as “Neoplastic cells present. Consistent with involvement by the patient’s known serous borderline tumor.” The interpretation “positive for malignant cells” should be avoided because it might imply that the cytologist is converting the borderline diagnosis (made after examination of the ovary) to a carcinoma diagnosis. Less common are the other malignant epithelial tumors: mucinous, endometrioid, clear cell, and Brenner tumors. There is significant overlap in the morphologic features of these tumors when they appear in peritoneal washings, such that distinction among them is not reliable (nor is it necessary) based on PWC. It is sufficient to report results as “Positive for malignant cells. Consistent with carcinoma.” Alternatively, if one holds reporting of the washings until the concurrent ovarian tumor resection has been examined and subtyped (e.g., as a clear carcinoma), one can report the washings as “Positive for malignant cells. Consistent with clear cell carcinoma.” A mucinous tumor in the ovary, particularly one associated with pseudomyxoma peritonei, is often a metastasis (the appendix is a likely primary site) rather than a primary ovarian neoplasm (Fig. 5.14). Primary versus metastatic mucinous tumors of the ovary are difficult to distinguish by conventional histologic examination. Germ cell (Figs. 5.15 and 5.16) and sex cord–stromal tumors of the ovaries can also involve peritoneal surfaces.50 

Endometrial Cancer Unlike ovarian cancers, most endometrial cancers are detected at an early stage and are therefore potentially

CHAPTER 5  Peritoneal Washings

A

179

B • Fig. 5.12  Serous Borderline Tumor. (A) Some neoplastic cells exfoliate as large branching micropapillary clusters with smoothly contoured or slightly scalloped edges. The centers are occupied by microcalcifications (Papanicolaou stain). (B) A smaller cluster of cuboidal cells surrounding a cracked psammoma body from the same case demonstrates the minimal atypia characteristic of the serous borderline tumor. Biopsy specimens showed implants of identical cells on the omentum and peritoneum. Compare with Fig. 5.7: the cytologic distinction between endosalpingiosis and a borderline tumor is often very difficult (Papanicolaou stain).

•

Fig. 5.13  Serous Borderline Tumor. Cell block sections are helpful because they may show papillary structures lined by atypical cells with stratification and tufting, features that are characteristic of these tumors (hematoxylin & eosin stain).

curable. In 1988 the International Federation of Gynecology and Obstetrics (FIGO) adopted a surgical staging system in which peritoneal washings played an important role: positive results signified stage IIIA disease. In 2009, however, FIGO revised the surgical staging of endometrial cancer and eliminated PWC from the staging system, presumably because of controversy over the prognostic significance of PWC in women with endometrial cancer.51-53 Nevertheless, FIGO and the American Joint Committee on Cancer continue to recommend the collection of peritoneal washings (and recording of results) in women with endometrial cancer because it may provide useful information for treatment decisions.54 Positive PWC is seen in 11% of patients with endometrial cancer (range 5% to 21%).55 The risk is lower in

patients with low-risk features (stage I, grade 1 or 2, 25%), and nodules that were previously aspirated and yielded an unsatisfactory sample.1

CHAPTER 10 Thyroid

289

A

B •

Fig. 10.1  (A) Transverse sonogram of a thyroid nodule. The tip of a needle (thin arrows) is in the middle of a nodule (large arrow). (Courtesy Dr. Carol Benson, Brigham and Women’s Hospital, Boston, Massachusetts, USA.) (B) Ultrasound gel. Ultrasound gel has a purple, mesh-like or spiderweb-like appearance. If not wiped off the skin before aspiration, it can cover large areas of the slide surface and interfere with cellular evaluation (Papanicolaou stain).

The aspiration technique is essentially the same whether palpation or ultrasound is used for guidance. To reduce the risk of bleeding, a very fine (25- or 27-gauge) needle is ideal for most thyroid nodules.10 Depending on the circumstances and operator preference, the aspiration can be performed with or without suction applied by a syringe. Local anesthesia by subcutaneous lidocaine injection is commonly used but is optional.10 The skin is wiped clean with an alcohol swab. If ultrasound guidance is used, the needle should not pass through gel, which obscures cytomorphology (Fig. 10.1B).10 The dwell time of each pass should be 2 to 5 seconds, with rapid back-and-forth oscillations of the needle (three per second) within the nodule.10 If suction is used, it should be released before the needle is withdrawn from the nodule. The number of passes needed to ensure adequacy

varies. If the specimen is evaluated on site for adequacy, one or two passes may be sufficient. But on-site evaluation is time-consuming,11 and many aspirations that are performed in outpatient settings are too far removed from a laboratory for such evaluation. Most practitioners recommend between 2 and 6 passes for each nodule aspirated.10-12 Even minor complications such as a transient hematoma are uncommon; site infections are almost never seen. Post-FNA infarction of the nodule occurs to some degree in up to 10% of cases.13 Other histologic alterations in the remaining gland include pseudoinvasion, vascular proliferation, and cytologic atypia, but these are usually easily recognized by virtue of their proximity to the linear biopsy tract.14 Serious complications such as needle track seeding are virtually nonexistent.

290 CHA P T ER 1 0   Thyroid

TABLE 10.1    The 2017 Bethesda System for Reporting Thyroid Cytopathology

Diagnostic category

Risk of malignancy if NIFTP = CA (%)

Risk of malignancy if NIFTP ≠ CA (%)

Nondiagnostic or Unsatisfactory

5–10

5–10

Repeat FNA with ultrasound guidance

Benign

f

None

Liver/spleen/blood

Indolent, incurable

Mantle cell

60/ m>f

Rare

Many

Aggressive, incurable

Follicular

59/ f>m

Rare

Variable

Indolent, rarely curable

MALT

61/ m=f

Often

Usually

Indolent, curable

MALT, Mucosa-associated lymphoid tissue. aExcluding bone marrow.

CHAPTER 12  Lymph Nodes

up to 95% of cases. This translocation can be detected by FISH on cytologic material in over 80% of cases,109 and FISH appears to be more sensitive than flow cytometry in diagnosing FL.110 By contrast, antibody staining for BCL2, which is extremely helpful in distinguishing FL from follicular hyperplasia in tissue sections, is ineffective with smears because the spatial relationship of follicle center versus paracortical staining cannot be evaluated, unless a two-color BCL6/BCL2 double stain is used.12 CYTOMORPHOLOGY OF FOLLICULAR LYMPHOMA • P  redominantly small irregular/cleaved lymphocytes • Large cleaved/noncleaved lymphocytes (more in highgrade follicular lymphoma) • Few tingible-body macrophages • Lymphoid cell aggregates (one-third or more of cases)   

Although some claim that a follicular pattern can be appreciated in well-made smears, our experience leads us to conclude that this is uncommon. Aspirates may be composed almost exclusively of small centrocytes, but they are more apt to show a mixture of centrocytes and some centroblasts (large noncleaved lymphocytes).94 Cell nuclei are commonly contorted into notches and grooves. In some aspirates the clefts are so exaggerated as to appear to bisect or trisect the nucleus, whereas in others the nuclear folds are more subtle (Fig. 12.16). Follicular aggregates occur in smears of FL in about one-third of cases,94 but the dendritic cells are sometimes difficult to discern.57,94 Several variants of FL have been described: FL with monocytoid B cells,

397

FL with rosettes, floral pattern of FL, and FL with cerebriform nuclei. These variants have no clinical significance and generally are not recognizable by FNA. A signet-ring–cell variant of FL, however, is cytologically distinct because the cells have a single, large, optically clear cytoplasmic vacuole filled with immunoglobulin that can create confusion with a signet-ring-cell carcinoma (Fig. 12.16C).111 FLs must be assigned a histologic grade; to avoid sampling error, one should select the largest or most rapidly enlarging site of disease for sampling (perhaps with correlation of metabolic imaging, such as positron emission tomography).12 For practical therapeutic purposes, FLs are divided into low-grade (a combination of WHO grades 1 and 2) and high-grade (WHO grade 3) tumors based on the quantity of large nucleolated cells (centroblasts) in tissue sections. Unfortunately, there is no standardized method for grading the FL sample obtained by FNA.112,113 If grading is essential (e.g., if an additional sample is impractical to obtain), most sources advise visual estimation of the percentage of small and large lymphocytes.114 Low-grade FL is favored if the percentage of large cells is less than 20%,115 and high-grade follicular lymphoma if the percentage is greater than 40% to 50%115,116; grading is indeterminate if the percentage is between 20% and 40%.115 The distinction between grade 3A or 3B FL and diffuse large B-cell lymphoma is not possible by FNA but may not have great clinical relevance.26  Marginal Zone Lymphoma

Marginal zone lymphoma is an indolent, low-grade B-cell lymphoma that is divided into nodal and extranodal types. The extranodal type is synonymous with mucosa-associated lymphoid tissue (MALT) lymphoma and is more common

TABLE 12.6    Differential Immunophenotype and Genetics of Small B-Cell Lymphomas

Small lymphocytic

Mantle cell

Follicular

Marginal zone

CD5

+ (dim)

+/−

−

−

CD10

−

−/+

+/−

−

CD20

+ (dim)

+

+

+

CD23

+

−/+

−/+

−

BCL6

−

−

+

−

Cyclin D1/SOX11

−/+a

+

−

−

CD43

+

+

−

+/−

LEF1

+

−

−

−

CD200

+

−

−

−

TdT

−

−

−

−

Genetics

Trisomy 12 (30%), others

t(11;14)

t(14;18)

Various

+/−, Majority of cases are positive, small percentage negative; −/+, Majority of cases are negative, small percentage positive. aProliferation centers in small lymphocytic lymphoma may express cyclin D1 but not SOX11.

398 CHA P T ER 1 2    Lymph Nodes

A

B

C •

Fig. 12.16  Follicular Lymphoma.  (A) Note the nuclear irregularities and occasional clefted nucleus. Lymphoglandular bodies are numerous (Romanowsky stain). (B) FISH reveals a (14;18) translocation in the abnormal cell on the right. A green probe for IgH (immunoglobulin heavy chain) at 14q32 and a red probe for BCL2 at 18q21 come together to give a yellow signal when there is a translocation. Note that the green and red signals are far apart in the normal cell. (Dual color interphase fluorescence in situ hybridization. Courtesy Paola dal Cin, PhD, Brigham and Women’s Hospital, Boston, MA, USA). (C) Follicular lymphoma with signet-ring cells. Some cells have a single, large, optically clear cytoplasmic vacuole filled with immunoglobulin that can create confusion with a signet-ring-cell carcinoma (Romanowsky stain).

CHAPTER 12  Lymph Nodes

than the nodal type, accounting for 7% to 8% of all B-cell lymphomas. Although MALT lymphomas occur in a variety of sites, FNA cytologists typically only encounter them in specimens from the salivary gland, lung, lacrimal gland, thyroid, breast, and skin. There is a strong association with autoimmune disorders such as Hashimoto thyroiditis and Sjögren syndrome. A variety of genetic alterations are seen, but the incidence of these varies by site, and none are sufficiently common in FNA sites to be useful.10 Patients with MALT lymphoma often have stage I or II disease and thus are potentially curable by surgery or regional radiotherapy. Histologically, a mixed background including reactive follicular hyperplasia is the rule. The neoplastic cells are small lymphocytes, some of which have more abundant cytoplasm, leading to a monocytoid appearance. Neoplastic cells with the appearance of plasma cells are present in many cases. In glandular organs, nests of neoplastic cells invade the epithelium, resulting in characteristic lymphoepithelial lesions.

CYTOMORPHOLOGY OF MARGINAL ZONE LYMPHOMA • • • •

 olymorphous population P Small lymphocytes Round or slightly irregular nuclei “Monocytoid” cells (moderate amount of pale cytoplasm) • Plasma cells • Follicular dendritic cells, tingible-body macrophages, follicular aggregates, immunoblasts (reactive elements)   

•

399

The heterogeneous cell population makes it difficult to recognize the lesion as a lymphoma.10,117 Unfortunately, the diagnostically useful lymphoepithelial lesions are difficult (or impossible) to appreciate in smears. A mixed population of small lymphocytes with pale staining cytoplasm (monocytoid cells) and plasma cells, together with reactive elements in a site where lymphoid tissue is normally sparse, should raise the suspicion of MALT lymphoma118 (Fig.12.17). Flow cytometric immunophenotyping (for clonality) is critical to establish the diagnosis in most cases, but a minority of cases may have no detectable surface light chain expression.11 With regard to subtyping, the neoplastic cells are usually negative for CD5 and CD10, making the diagnosis of marginal zone lymphoma one of immunophenotypic exclusion.  Small Lymphocytic Lymphoma

Small lymphocytic lymphoma (SLL) comprises about 7% of non-Hodgkin lymphoma.26 It is an indolent and typically incurable neoplasm of older adults. Disease is widespread (nodally and extranodally) at the time of diagnosis, usually including peripheral blood and bone marrow involvement (the latter perversely referred to by a different name: chronic lymphocytic leukemia (CLL)).10 Tissue sections show effacement of nodal architecture by the neoplastic cells, with pseudofollicular proliferation centers that contain prolymphocytes and paraimmunoblasts. The CD5+, CD23+, CD10− immunophenotype is highly characteristic of SLL/CLL, but it is important to know that CD5 expression (and CD20 and light chain expression) is usually dim/ weak.10 Caution is advised before diagnosing SLL if there

Fig. 12.17  Marginal Zone Lymphoma.  These small- and intermediate-size lymphocytes have irregular nuclear contours, and occasional monocytoid cells are present (arrow). Lymphoglandular bodies are numerous (Romanowsky stain).

400 CHA P T ER 1 2    Lymph Nodes

A

B • Fig. 12.18  Small Lymphocytic Lymphoma.  (A) The monomorphous neoplastic small lymphocytes have

scant cytoplasm; some bare nuclei are present. The mostly round nuclei have very dense (“clotted”) chromatin. Lymphoglandular bodies are present in the background (Romanowsky stain). (B) “Soccer-ball–like” chromatin is more evident with alcohol-fixed smears. A few (large) paraimmunoblasts are also present (Papanicolaou stain).

is no lymphadenopathy greater than 1.5 cm and no proliferation centers are found because such cases may represent a “nodal equivalent” of monoclonal B-cell lymphocytosis, rather than a full-fledged lymphoma.26 CYTOMORPHOLOGY OF SMALL LYMPHOCYTIC LYMPHOMA • • • • • • •

 onomorphous small lymphocytes M Clumped (soccer-ball–like) chromatin Smooth or minimally irregular nuclear contour Inconspicuous or absent nucleoli Scant cytoplasm Prolymphocytes and paraimmunoblasts Rare or absent tingible-body macrophages and follicular aggregates   

Nuclei are round and have coarse, clumped chromatin—so-called clotted or soccer-ball–like chromatin. Nuclear borders are smooth and usually round. So-called smudge cells (crushed cells) may be common on smears. Prolymphocytes are larger and have a distinct nucleolus and a modest amount of pale cytoplasm, whereas paraimmunoblasts are larger still, with a more prominent nucleolus, and more basophilic cytoplasm (Fig. 12.18). These two types of transformed lymphocytes are not easily distinguished from each other in smears and need not be. Morphologic transformation to diffuse large-B cell lymphoma occurs in up to 20% of patients with SLL/CLL and is an important indicator of prognosis. In fact, clinical concern for transformation is the most common indication for lymph node FNA in patients with SLL. Transformation is assessed by determining the proportion of large cells, identifying necrosis, or a Ki-67 index of >30%.119 Development of HL is also observed in heavily treated

patients and may be diagnosable by FNA,120 provided one takes care to identify immunophenotypically and morphologically typical RS cells in the appropriate background. RS-like cells can occur in SLL without HL, but the appropriate background is lacking.  Mantle Cell Lymphoma

Mantle cell lymphoma (MCL) is usually a biologically aggressive B-cell lymphoma comprising 3% to 10% of nonHodgkin lymphoma.26 It occurs in adults over 50 years of age, with a male predominance. MCL is commonly extranodal, and most patients have disseminated disease (including variably subtle peripheral blood involvement) at initial diagnosis. MCL is typically resistant to standard lymphoma therapeutic regimens and has a poor prognosis. A t(11;14) (q13;q32) translocation is present in the vast majority of patients, resulting in overexpression of the protein cyclin D1, which drives cell proliferation. CYTOMORPHOLOGY OF MANTLE CELL LYMPHOMA • • • • • • • • •

 onomorphous small to intermediate size cells M Fine nuclear chromatin Irregular nuclear contours Inconspicuous/absent nucleoli Scant cytoplasm No centroblasts or immunoblasts “Pink” histiocytes Lymphoid cell aggregates (one-third of cases) Blastoid variant   

Aspirates of MCL are comprised of uniform, small- to medium-sized121 cells that have a very high nuclear-to-cytoplasmic ratio and somewhat resemble centrocytes,122,123

CHAPTER 12  Lymph Nodes

401

A

normal pattern

abnormal pattern

B •

Fig. 12.19  Mantle Cell Lymphoma.  (A) The smear shows an isomorphic population of small cells with round-to-oval nuclei and scant cytoplasm. Note the neutrophil at upper right for size comparison (Romanowsky stain). (B) FISH reveals a t(11;14) translocation in the abnormal cells on the right. A green probe for IgH (immunoglobulin heavy chain) at 14q32 and a red probe for the cyclin D1 gene come together to give a yellow signal when there is a translocation. Note that the green and red signals are far apart in the normal cells. (Dual color interphase fluorescence in-situ hybridization. Courtesy Paola dal Cin, PhD, Brigham and Women’s Hospital, Boston, MA.)

although the nuclei are more often subtly notched rather than deeply clefted (Fig.12.19A). Nucleoli are generally small/absent. Lymphoid cell aggregates occur in about one-third of cases.94,121 They are similar to dendritic-lymphocytic aggregates, except that dendritic cells are inconspicuous.57,94 Unlike other small cell lymphomas, there are virtually no transformed lymphocytes, and monotony

is the rule. Occasional histiocytes with moderately abundant eosinophilic cytoplasm (“pink histiocytes”) are noted. In typical cases, the cytologic interpretation can be confirmed by demonstrating the characteristic CD5+, CD23−, CD10−, CD200− immunoprofile by flow cytometry,10 but approximately 10% to 20% of cases deviate from this immunophenotype.12,121,124 Therefore demonstration of

402 CHA P T ER 1 2    Lymph Nodes

immunoreactivity for cyclin D1 or SOX11 on cytocentrifuge or cell block preparations, or the t(11;14) translocation by FISH (Fig. 12.19B) is helpful for primary diagnosis/ subclassification.125-127 Ki67 staining is useful for prognostication.26 Rarely, the blastoid variant of MCL is observed; this variant has a more aggressive course,26 potentially causing confusion with lymphoblastic lymphoma (because of immature-appearing chromatin)128 or DLBCL (because of nuclear pleomorphism and nucleoli.121 Differential Diagnosis: Small Cell Lymphomas

The differential diagnosis of the small cell lymphomas includes reactive hyperplasia, HL, and each of the four small cell lymphomas themselves and their in situ counterparts. DIFFERENTIAL DIAGNOSIS OF SMALL CELL LYMPHOMAS • • • • • • •

 eactive hyperplasia R Hodgkin lymphoma Follicular lymphoma Marginal zone/MALT lymphoma Small lymphocytic lymphoma Mantle cell lymphoma In situ follicular or mantle cell neoplasia

marginal zone lymphoma and MCL and may be seen in FL (particularly in the head and neck region), but it is rarely the presenting site of SLL. Morphologically, SLL and MCL are more monomorphous than FL and marginal zone lymphoma. In particular, SLL is composed of uniform small round cells, with relatively infrequent larger cells. MCL is also composed of monomorphous small cells, with more finely textured chromatin and more irregular nuclear membranes than SLL. Monocytoid B cells are a clue to the diagnosis of marginal zone lymphoma, and cleaved nuclei are characteristic of FL. An infiltrate that includes plasma cells and plasmacytoid cells is characteristic of marginal zone lymphoma. Although morphologic and clinical features are helpful, the immunophenotype, particularly the expression of CD5, CD10, CD23, and CD200, is paramount in subclassifying the small cell lymphomas. ALGORITHM FOR DISCRIMINATING AMONG SMALL CELL LYMPHOMAS BASED ON FLOW CYTOMETRY IMMUNOPHENOTYPE

  

First and foremost, the four small cell lymphomas must be distinguished from a reactive hyperplasia. There are helpful morphologic clues, but they are subtle, and, in practice, immunophenotyping is commonly relied on to make this distinction (Table 12.6). If a smear is comprised of a monomorphous population of small lymphoid cells, lymphoma is likely, but a reactive lymph node cannot be excluded without immunophenotyping, because a minimally reactive lymph node can also show little lymphocyte polymorphism.129 Although small round lymphocytes predominate in reactive hyperplasia, in most cases there is an expanded range of lymphoid cells, including plasmacytoid lymphocytes, plasma cells, and immunoblasts. Dendritic-lymphocytic aggregates and tingible-body macrophages, although common in reactive hyperplasia, are rare or absent in the small cell lymphomas, with the exception of FL. Marginal zone lymphoma, in particular, is an exception to the rule of morphologic homogeneity in the small cell lymphomas. Marginal zone lymphoma resembles a reactive hyperplasia because of its associated nonneoplastic polymorphous infiltrate of small lymphocytes, centrocytes, and monocytoid B cells.27 Ultimately, the distinction between lymphoma and reactive hyperplasia rests on immunophenotyping, usually by flow cytometry. Immunophenotyping also helps in the distinction between non-Hodgkin lymphoma and a Hodgkin lymphoma with very few RS cells: the small B cells of HL show polyclonal expression of immunoglobulin light chains. The small cell lymphomas are distinguished from one another by a combination of clinical, morphologic, immunophenotypic, and genetic features. Clinical features are summarized in Table 12.5. An extranodal location is common in

• C  D5+ (small lymphocytic lymphoma and mantle cell lymphoma) • CD23+, CD200+ small lymphocytic lymphoma • CD23−, CD200− mantle cell lymphoma • Light chain, CD20, and CD5 dim favor small lymphocytic lymphoma • CD5− (follicular lymphoma and marginal zone lymphoma) • CD10+ follicular lymphoma • CD10− unresolved   

The CD5+, CD23+, CD10−, CD200+ immunophenotype is relatively specific for small lymphocytic lymphoma, and the CD5+, CD23−, CD10−, CD200−10 immunophenotype is relatively specific for MCL, but exceptions occur.121,124 With SLLs, fluorescence for light chains, CD20, and CD5 is characteristically weak (commonly referred to as dim). By contrast, increased cyclin D1 (BCL1) expression by immunocytochemistry is relatively specific for MCL, but caution is advised because cells from proliferation centers in SLL may express cyclin D1.55 SOX11 expression (by immunohistochemistry) is even more sensitive and specific for MCL,130,131 whereas LEF-1 is specific for SLL.131,132 The CD5−, CD10+ immunophenotype is characteristic of FL (CD23 can be positive or negative), but many cases of FL are negative for CD10 (at least by flow cytometry, which seems to have imperfect sensitivity for detecting CD10). The CD5−, CD10− profile is therefore nonspecific because it does not distinguish between FL and marginal zone lymphoma; immunohistochemistry on cell block sections for CD10 and BCL6 is recommended in such cases. Aberrant immunophenotypes occur, so the immunophenotype must be correlated with morphologic, clinical, and, where appropriate, molecular genetic studies, if available. Molecular genetic studies are most helpful (see Table 12.6) when the immunophenotype is aberrant or ambiguous.

CHAPTER 12  Lymph Nodes

About 30% of SLLs show trisomy 12, and other aberrations may also be seen (e.g., del 13q14.3, del 11q22-23, or del 17p13, the latter also being a significant prognostic feature). The t(11;14)(q13;q32) translocation is a hallmark of MCL and is detected in most cases. FL is characterized by a t(14:18)(q32;q21) translocation in up to 95% of cases. These characteristic abnormalities can be identified by FISH on cytologic preparations.50,126,127 Caution is advised when using sensitive methods such as flow cytometry, FISH, and cell block immunohistochemistry for the diagnosis of follicular or mantle cell lymphoma on FNA, because of the recently characterized entities “in situ follicular neoplasia” and “in situ mantle cell neoplasia,” which, although not considered malignant processes, may be associated with a lymphoma-like immunophenotype on flow cytometry.32,55 Careful attention to clinical context (in particular, extent of disease) and to the percentage of immunophenotypically aberrant cells in the FNA specimen should help to minimize the possibility for error, with judicious use of excisional biopsy if doubt remains. 

Lymphomas of Large Cells A broad and heterogeneous group of neoplasms that arise from B cells, T cells, or NK (natural killer) cells, this category is nonetheless dominated by a single common entity: diffuse large B-cell lymphoma. Unlike the previous group of small B-cell lymphomas, which are tumors of adults, many of the large cell lymphomas occur in children, as well as adults. In children, the most common B-cell lymphomas are diffuse large B-cell lymphoma, lymphoblastic lymphoma, and Burkitt lymphoma.133 Note that the designation “large cells” for the entities described in this section is used here as a convenience; indeed, large in this context refers to nuclear size relative to histiocyte nuclei, and therefore large lymphocytes often are actually smaller than the epithelial cells that cytologists are more familiar with. Moreover, although some of the neoplasms discussed below are indeed comprised predominantly of large lymphoid cells, others contain a mixture of small and large neoplastic cells, or large neoplastic cells with reactive (nonlesional) small cells. Some are actually comprised of intermediate-sized cells (e.g., Burkitt lymphoma). Lymphoblastic lymphoma cells are often smaller cells, but this entity is considered here under the broader heading of T-cell lymphomas. Also, note that not all posttransplant lymphoproliferative disorders are even lymphomatous, but they are discussed here because most of them are neoplastic, and those that are neoplastic usually contain at least some, if not a preponderance of, large cells.  Diffuse Large B-cell Lymphoma

DLBCL is an aggressive but potentially curable subtype of non-Hodgkin lymphoma that comprises 35% of adult lymphomas. It is also one of the three major lymphomas in children. About 40% of patients present with extranodal disease. The histologic hallmark of DLBCL is the presence of confluent areas of large malignant B cells. Several morphologic subtypes were recognized in the past (e.g., centroblastic,

403

immunoblastic, and anaplastic large B-cell), but subclassification into these categories is not clinically relevant134 except as noted below. Gene expression profiling, however, has identified different genetic subtypes of DLBCL—germinal center B-cell–like and activated B-cell–like.135 These have clinical significance in that they may predict survival after chemotherapy.136 Although gene expression profiling can be performed on FNA,54 a more practical algorithm for identifying these subtypes is the use of immunohistochemistry (“Hans algorithm”137) for CD10 and, if necessary, BCL6 and IRF4/ MUM110,138 (with positivity for these markers defined by expression in at least 30% of cells). It is also useful to perform immunohistochemistry for MYC, because MYC immunopositivity (usually defined as MYC expression in at least 40% of cells) is a poor prognostic factor131 and because immunohistochemical expression of MYC suggests a MYC translocation. A MYC translocation that is paired with a BCL2 or BCL6 translocation defines a distinct entity, high-grade B-cell lymphoma. If MYC and BCL2 immunopositivity is present without the corresponding double translocations, the lymphoma remains in the DLBCL category and is termed a “double-expressor.”55 CYTOMORPHOLOGY OF DIFFUSE LARGE B-CELL LYMPHOMA • P  redominantly large cells (nucleus 2.5–5 times that of a small lymphocyte or greater than that of a histiocyte) • Distinct to large nucleoli • Lymphoglandular bodies • Variable number of tingible-body macrophages • Paucity or absence of dendritic-lymphocytic aggregates   

The cellularity is variable. Aspirates of mediastinal DLBCL and extranodal sites can be very sparsely cellular due to the extensive sclerosis or sparse distribution of neoplastic cells common to these tumors. Most FNAs of DLBCL are at least moderately cellular, however, and smears are usually easily recognizable as abnormal because of the abundance of large atypical cells (Fig. 12.20). They can have a round or highly irregular nucleus with multiple nucleoli and scant cytoplasm (centroblastic type); a round or irregular nucleus with a single very prominent nucleolus and more abundant cytoplasm (immunoblastic type); or a bizarre, pleomorphic nucleus with frequent multinucleation that resembles the nucleus of RS cells (anaplastic type). A population of small, nonneoplastic T cells is always present but usually represents a minority of the cell sample. When T cells or histiocytes are few in number (or preparation artifact is present), it may be difficult to judge the size of the neoplastic cells. Attention to the presence of nucleoli helps to exclude one of the lymphomas of small cells in such cases. Characterization of DLBCL by flow cytometry can be challenging (requiring careful correlation of cytomorphology with flow results) because the large, atypical cells may appear outside the usual “lymphoid gate” on flow cytometry scattergrams.11 Less commonly, the large cells may not survive processing because of fragility.139 Additionally, DLBCL often lacks detectable surface light chain expression,11 hampering

404 CHA P T ER 1 2    Lymph Nodes

A

B •

Fig. 12.20  Diffuse Large B-Cell Lymphoma (DLBCL).  (A) A uniform population of lymphocytes with large, almost vesicular nuclei, obvious nucleoli, and a moderate amount of cytoplasm is admixed with neutrophils and small lymphocytes (Papanicolaou stain). (B) By definition, the nucleus of DLBCL cells (black arrow) is at least the size of a histiocyte nucleus (black arrowhead on foam-cell nucleus); the background T-cell nuclei (red arrow) are much smaller (Romanowsky stain).

confirmation of clonality. This apparent stumbling block, however, can in fact be a clue to the correct diagnosis, because lack of surface light chain on B cells outside the marrow is an abnormal finding, usually (but not invariably140) associated with neoplasia.141 Nevertheless, detemination of clonality by PCR may be required if doubt remains and if the neoplastic nature of the cells is not evident morphologically. With respect to other markers, DLBCL cells usually show bright CD20 staining by flow cytometry or immunohistochemistry, but rituximab-treated patients (i.e., recurrences) may show reduced CD20 staining; in these circumstances PAX5/BSAP staining by immunohistochemistry may be helpful to confirm the B-cell phenotype of the cells. 

Variants of Diffuse Large B-Cell Lymphoma

Several clinically distinct variants of DLBCL are worth noting. Primary mediastinal (thymic) large B-cell lymphoma predominates in young adult females and may cause superior vena cava obstruction. FNA has been used successfully in diagnosing this variant,46,142 but the sclerosis may limit FNA sampling to a few inconspicuous cells.10 Expression of CD30, IRF4/MUM1, TRAF, REL,143 MAL, PD-L1,26 and CD200144 is characteristic. A second variant, T-cell/histiocyte-rich large B-cell lymphoma,145 is rarely (if ever) specifically diagnosed by FNA because of the small proportion of neoplastic large B cells in a background of nonneoplastic cells, but careful screening

CHAPTER 12  Lymph Nodes

for large atypical cells should at least prompt further tissue sampling. Two other DLBCL variants are primary effusion large B-cell lymphoma (see Chapter 4) and intravascular large B-cell lymphoma (see Chapter 15).  High-Grade B-Cell Lymphoma

High-grade B-cell lymphoma (HGBL) consists of mature B-cell lymphomas with blastoid or Burkitt-like morphology, or, more commonly, lymphomas with DLBCL-like morphology and a MYC translocation combined with either a BCL2 or BCL6 translocation (previously termed “double hit

lymphoma.”)55,146-148 The latter subtype of HGBL deserves mention because of its relatively high incidence in older patients with advanced/aggressive disease and its poor prognosis. Clinically, a subset of these patients may have a history of a low-grade FL, with sudden progression, but many present de novo. Double hit lymphoma usually has a “germinal center” immunophenotype131 and shows immunohistochemical expression of BCL2, a moderately high Ki67 index (usually greater than 80%), and sometimes reduced CD20 expression by flow cytometry.149 The cytologic features are not specific (Fig. 12.21), but, in an older patient, a highly aggressive or proliferative BCL2-positive B-cell lymphoma should prompt special attention to MYC immunohistochemistry,150,151 with confirmatory FISH or cytogenetic studies to detect a MYC translocation and either a IGH-BCL2 or IGH-BCL6 translocation (Table 12.7). Alternatively, all lymphomas with DLBCL morphology can be tested by FISH for a MYC, BCL2, and BCL6 translocation to maximize detection of HGBL.55  Burkitt Lymphoma

• Fig. 12.21  High-Grade B-Cell Lymphoma.  A monomorphous popu-

lation of intermediate-sized cells closely mimics Burkitt lymphoma; note the tingible-body macrophage at upper right. FISH evaluation showed both MYC and BCL2 translocations (not pictured) (Romanowsky stain).

Burkitt lymphoma (BL) is a highly aggressive but potentially curable B-cell malignancy with a characteristic morphology and a cytogenetic translocation that constitutively activates the MYC oncogene. The diagnosis of BL has great clinical importance because it necessitates a chemotherapeutic regimen that is more aggressive than that used for DLBCL. BL occurs in three clinical settings: (1) an endemic form found in Africa and Asia and rare in the United States and Europe; (2) a sporadic form, which occurs in the United States and other countries; and (3) an immunodeficiencyassociated form. The endemic and sporadic forms are more common in children, and the immunodeficiency-associated

TABLE 12.7    Differential Features of DLBCL and Mimics

DLBCL

High grade B-cell lymphoma

Burkitt lymphoma

Lymphoblastic lymphoma

CD20

+

+a

+

−/+

CD10

+/−

+

+

+

BCL6

+/−

+

+

NR

BCL2

+/−

+/−

−/+

NR

IRF4/MUM1

−/+

−/+

−

NR

sIg

+/−

+/−

+

−

−/+

+/−

+

NR

TdT

−

−

−

+

Ki67

40-100%

30-100%

100%

NR

cytogenetics

variable

MYC translocation and BCL2 or BCL6 translocation

MYC translocation

variable

nuclear

MYC150,151

405

+/−, Majority of cases are positive, minority negative; −/+, majority of cases are negative, minority positive; DLBCL, diffuse large B-cell lymphoma; NR, not relevant for dfferential diagnosis. aCD20 may be weak in high-grade B-cell by flow cytometry.

406 CHA P T ER 1 2    Lymph Nodes

form is more common in adults. Virtually all the endemic cases show latent infection of the tumor cells by EBV, whereas only a subset of the sporadic and immunodeficiency-associated cases are EBV-associated. The malignant cells involve lymph nodes or extranodal tissues (the gastrointestinal tract, liver, and bone marrow are common sites in the nonendemic form). Cerebrospinal fluid is involved in 20% to 30% of cases at presentation. CYTOMORPHOLOGY OF BURKITT LYMPHOMA • • • •

 niform intermediate-sized cells U Round nuclei, coarse chromatin Two to five small nucleoli per nucleus Scant blue cytoplasm with small vacuoles (Romanowsky stain) • Apoptosis and numerous mitoses • Numerous tingible-body macrophages   

Smears are monotonously hypercellular. Tingible-body macrophages are randomly dispersed throughout the smear, mimicking the “starry sky” pattern of tissue sections. Because individual cell necrosis (apoptosis) is common, a “dirty” background is typical. The cells are intermediate in size, with a round nucleus, coarse chromatin, multiple (but small) nucleoli, and scant cytoplasm (Fig. 12.22A). With Romanowsky-type stains, the cytoplasm is deep blue with small lipid vacuoles,152 but similar small vacuoles are common in DLBCL as well. BL cells express the B-cell–specific surface antigens CD19 and CD20, the immunoglobulin heavy chain, and monotypic surface immunoglobulin light chains. BL also expresses CD10 and BCL6 but not BCL211 or terminal

A

deoxynucleotidyl transferase (TdT). Nearly 100% of the cells are positive for Ki67. BL is almost invariably associated with a chromosomal translocation involving the MYC protooncogene on chromosome 8 with either an IgG heavy-chain or κ or λ lightchain gene. The most reliable method for identifying the specific translocation in BL is karotyping by conventional cytogenetics, but the presence of any of these three translocations can also be suggested by nuclear immunoreactivity for MYC150,151 or documented by FISH.153,154 In the latter assay, probes to the flanking regions of the MYC locus show a split signal, indicating a rearrangement (Fig. 12.22B). A MYC rearrangement is highly sensitive but not specific for BL because it is also seen in high-grade B-cell lymphoma, occasionally in large B-cell lymphoma, and some late-stage multiple myelomas. For this reason, the diagnosis of BL is based on an integration of clinical, morphologic, immunophenotypic, and genetic findings (Table 12.7). Plasmablastic Lymphoma

Plasmablastic lymphoma is a rare tumor, occurring in HIVpositive or otherwise immunodeficient patients, typically in the oral cavity or other mucosal sites, but also in lymph nodes. This tumor is unique among hematolymphoid neoplasms because the constituent cells resemble B immunoblasts (i.e., large, noncohesive cells with large central nucleoli, Fig. 12.23) but have an immunophenotype similar to plasma cells (i.e., CD138+, CD38+, IRF4/MUM1+, CD45−, CD20−, and PAX5/BSAP−, but, unlike myeloma, usually CD56−) and often express EBVencoded RNA by in situ hybridization.155 Caution should be used before overinterpreting CD138-positivity in a large cell neoplasm, however, because this antigen is widely distributed in nonhematolymphoid neoplasms, including carcinomas. 

B •

Fig. 12.22  Burkitt Lymphoma.  (A) The intermediate-sized neoplastic lymphocytes have round nuclei, multiple indistinct nucleoli, blue cytoplasm, and small cytoplasmic vacuoles. Apoptotic bodies (arrows) are present (Romanowsky stain). (B) Detection of MYC translocation by FISH. Red and green fluorescent probes to regions flanking MYC are split apart. This occurs in all three of the common MYC translocations. (Dual-color interphase fluorescence in situ hybridization. Courtesy Paola dal Cin, PhD, Brigham and Women’s Hospital, Boston, MA.)

CHAPTER 12  Lymph Nodes

T-Cell Lymphomas T-cell lymphomas comprise about 10% of all non-Hodgkin lymphomas in Western countries. The WHO classification26 lists numerous subtypes (Table 12.8) that are broadly divided into precursor T-cell lymphoblastic lymphoma and mature T-cell lymphomas. The mature T-cell and NK-cell neoplasms comprise a diverse group of neoplasms defined primarily by their clinical features. In contrast to B-cell lymphomas, many T-cell lymphomas are not associated with specific immunophenotypic profiles. Also unlike B-cell lymphomas, where light-chain restriction is a marker of malignancy, there are no convenient antigenic markers of monoclonality. The diagnosis, nevertheless, is suggested by an aberrant T-cell immunophenotype,

407

such as loss of one or more of the pan T-cell markers CD2, CD3, CD5, CD7; loss of both CD4 and CD8; or coexpression of both CD4 and CD8.44 Care must be taken, however, not to base the diagnosis of T-cell lymphoma solely on the presence of a double-positive (CD4+, CD8+) T-cell population, which can be seen in nodular lymphocyte-predominant HL,66 in the lymphocytes accompanying a thymoma, and, uncommonly, in a reactive lymph node.2 Conversely, a double negative (CD4−, CD8−) population can be observed in the lymphocytes accompanying a thymoma156 and in the autoimmune lymphoproliferative syndrome.2 Indeed, molecular genetic studies, most commonly PCR for rearrangement of the T-cell receptor genes, are advisable to confirm the clonality of a T-cell proliferation92 with any aberrant phenotype.11 Peripheral T-cell lymphoma unspecified, anaplastic large cell lymphoma, angioimmunoblastic lymphoma, lymph node involvement by mycosis fungoides, and adult T-cell leukemia/lymphoma are the most common mature T-cell lymphomas enocountered by the cytologist.  Peripheral T-cell lymphoma, NOS. Peripheral T-cell lymphoma of unspecified type (PTCL, NOS) is much more common in Asia than Europe or North America. In the United States, patients are typically elderly and systemically ill, with fever, night sweats, and bulky lymphadenopathy. CYTOMORPHOLOGY OF PERIPHERAL T-CELL LYMPHOMA, UNSPECIFIED

• Fig. 12.23  Plasmablastic Lymphoma.  Large malignant cells are dispersed in a noncohesive pattern. The eccentric position of the nucleus is the only “plasmacytic” feature. Because this image is virtually impossible to distinguish from a plasmablastic myeloma or melanoma, correlation with clinical history and ancillary testing are necessary for definitive diagnosis (Romanowsky stain).

TABLE   World Health Organization Classification of 12.8  T- and NK-cell Neoplasms26 relevant to FNAa Precursor T-cell lymphoblastic leukemia/lymphoma Mature T-cell and NK-cell neoplasms Aggressive NK-cell leukemia Extranodal NK / T-cell lymphoma, nasal type Mycosis fungoides Sézary syndrome Angioimmunoblastic T-cell lymphoma Follicular T-cell lymphoma/nodal PTCL with TFH phenotype Peripheral T-cell lymphoma (PTCL), unspecified Adult T-cell leukemia/lymphoma (human T-cell leukemia virus I positive) Anaplastic large cell lymphoma, ALK-positive Anaplastic large cell lymphoma, ALK-negative Breast implant-associated anaplastic large cell lymphoma Subcutaneous panniculitis-like T-cell lymphoma Hepatosplenic T-cell lymphoma FNA, Fine-needle aspiration; TFH, T-follicular helper. aMore common entities are in bold.

• M  onomorphous small or large lymphocytes, or a mixture of small and large lymphocytes • Irregular nuclei • Histiocytes, plasma cells, and eosinophils • Reed-Sternberg–like cells   

PTCL, NOS cases show marked variety in cell composition. Most aspirates (70%) have a polymorphous cell population with histiocytes, eosinophils, plasma cells, and intermediate and large lymphocytes (Fig. 12.24), whereas others resemble a pure large cell lymphoma; a smaller number resemble SLL.35,44,157 Nuclei of both small and large lymphocytes frequently display nuclear indentations, grooves, and knob-like projections.  Anaplastic Large Cell Lymphoma. Anaplastic large cell lymphoma (ALCL) is a subtype of non-Hodgkin lymphoma that was recognized after the discovery of the CD30 antigen. In the past, many of these were misdiagnosed as carcinomas and other nonlymphoid malignancies or as undifferentiated malignant neoplasms. ALCL accounts for about 3% of adult non-Hodgkin lymphoma and 10% to 20% of childhood lymphomas.26 It is usually a T-cell neoplasm and often expresses one or more T-cell antigens, but most cases lack CD3,26 and some have a “null cell” phenotype. Their T-cell lineage is revealed by demonstrating clonal T-cell receptor rearrangement. There is a broad morphologic spectrum, but in the common type the cells are large and pleomorphic. Histologically, a bizarre cell with a horseshoe-shaped nucleus (the

408 CHA P T ER 1 2    Lymph Nodes

•

Fig. 12.24  Peripheral T-Cell Lymphoma, Unspecified.  A picture of lymphocyte heterogeneity is produced by this mixture of small, intermediate, and large cells. In other fields of the smear the large cells were more numerous, but taken in isolation this image is easily misinterpreted as representing reactive hyperplasia (Romanowsky stain).

so-called hallmark cell) is characteristic. Reflecting the fact that lymph nodes are often only partially involved (with a sinusoidal growth pattern in histologic sections), the FNA sample can contain a mixed lymphoid background that obscures the hallmark cells. In addition to this classic form, there is a lymphohistiocytic variant, characterized by a large number of histiocytes that may mask the malignant cells, and a small cell variant, which resembles a PTCL. Some cases of ALCL are associated with a t(2;5)(p23;q35) or similar ALK translocation, which fuses the ALK gene with the NPM (nucleophosmin) gene,43 but many of these tumors in adults and some children lack this translocation. Immunostaining for the ALK protein (detectable in 60% to 85% of cases) is helpful both in diagnosis and prognosis; patients with ALK positivity have a better prognosis than a subset of ALK-negative patients.158 These biologic and clinical differences have led the WHO to divide ALCL into two separate entities: ALCL, ALK-positive and ALCL, ALK-negative (the latter distinguished, with difficulty, from PTCL by morphology, strong/diffuse staining for CD30, and positivity for EMA and cytotoxic markers).26 CYTOMORPHOLOGY OF ANAPLASTIC LARGE CELL LYMPHOMA • Intermediate and large cells • Irregular nuclei—horseshoe, “donut,” and embryoid shapes • Reed-Sternberg–like cells with smaller nucleoli • Histiocytes, neutrophils • No tingible-body macrophages or dendritic-lymphocytic aggregates • Few or absent lymphoglandular bodies   

Aspirates show moderate to high cellularity; necrosis and inflammation can be prominent. The hallmark cell has a horseshoe-shaped nucleus, but a spectrum of abnormal large cells varying in number, shape, and lobation of nuclei, particularly ring-shaped (“donut”) nuclei, are seen159-161 (Fig. 12.25). Necrosis can be a prominent feature.162 The tumor cells are positive for CD30 in a membranous pattern and in the Golgi region, but CD30 is nonspecific. Many cases are positive for ALK and EMA, but negative results do not exclude the diagnosis. EBV RNA sequences are absent. A break-apart FISH probe permits identification of the t(2;5) translocation in FNAs,43,163 but immunohistochemistry is generally sufficient for confirming ALK positivity, if present. Flow cytometry results have been described,164 but flow cytometry is rarely useful. Confirmation of a T-cell receptor gene rearrangement may be necessary in more difficult cases. Angioimmunoblastic T-Cell Lymphoma (AITL). AITL is a typically an advanced stage lymphoma of T cells with a T follicular helper immunophenotype (e.g., expressing PD1, CD10, BCL6, CXCL13, and others). Morphologically, these lymphomas display a polymorphous cytomorphology and, as a pitfall, usually include atypical EBV-positive B cells and even cells simulating RS cells.26  Lymph Node Involvement by Mycosis Fungoides. Mycosis fungoides is a primary cutaneous T-cell lymphoma, but in advanced stages lymph nodes and viscera may be involved. Such nodes may exhibit small or large lymphocytes with cerebriform nuclei, but the cytologic features alone are insufficient for accurate diagnosis of lymph node involvement.165 FNA is, however, a useful tool for confirming such involvement, provided that flow cytometry and a molecular study for clonality are performed on the sample.49 

CHAPTER 12  Lymph Nodes

409

• Fig. 12.25  Anaplastic Large Cell Lymphoma.  Very large cells with marked variation in nuclear shape, including “donut” forms, are seen (Romanowsky stain).

A

B •

Fig. 12.26  Adult T-Cell Leukemia/Lymphoma.  (A) The most distinctive cell is the floret cell, a large lymphocyte with multiple radiating nuclear lobes (Romanowsky stain), but (B) a wide range of neoplastic forms are typically seen, including large nucleolated cells (liquid-based preparation, Papanicolaou stain).

Adult T-Cell Leukemia/Lymphoma. Adult T-cell leukemia/lymphoma is a disease of adult patients in endemic regions (including the Caribbean islands in the Western Hemisphere) caused by latent infection by the retrovirus HTLV-I. Adult T-cell leukemia/lymphoma usually presents with lymph node involvement and, frequently, skin lesions.26 The FNA cytologist may be called on to make the initial diagnosis if the characteristic circulating cells are absent or not specifically appreciated on routine peripheral

blood testing. Large cells with multilobulated “floret-like” nuclei, prominent nucleoli, and deeply basophilic cytoplasm with occasional vacuoles are characteristic (Fig. 12.26), but in practice the cytomorphologic spectrum is wide. Although most aspirates appear malignant, specific diagnosis rests on appreciation of the characteristic clinical features (ethnic background, hypercalcemia, and atypical lymphocytosis in the peripheral blood) coupled with flow cytometry (showing the CD2+, CD3+, CD4+, CD5+, CD25+, CD7−,

410 CHA P T ER 1 2    Lymph Nodes

• Fig. 12.27  Lymphoblastic Lymphoma.  A pure population of blasts is present with finely dispersed chromatin and nuclear molding. A mitotic figure is seen. Compare the size of the blasts with the neutrophil at the lower right (Romanowsky stain).

CD8− immunophenotype) and follow-up serologic testing for HTLV-I.166  Precursor T- and B-Cell Lymphoblastic Lymphoma. Lymphoblastic lymphoma is an aggressive lymphoma that comprises almost one-half of childhood non-Hodgkin lymphoma, is more common in males, and is predominantly (90%) of T-cell lineage. Lymphadenopathy almost always occurs above the diaphragm. An anterior mediastinal mass is commonplace (up to 80% of patients) and can induce clinical symptoms mimicking bronchial asthma (due to tracheal compression) or the superior vena cava syndrome. Because these patients often have a palpable neck mass and a pleural effusion and are critically ill (many with some degree of respiratory compromise), the expediency of FNA or thoracentesis makes cytology the procedure of choice to establish the diagnosis (see Fig. 4.34). Combined with immunophenotypic studies, FNA has near-perfect accuracy.167-170 CYTOMORPHOLOGY OF LYMPHOBLASTIC LYMPHOMA •

Lymphoblasts: • Round or convoluted nucleus • Finely granular chromatin • Inconspicuous nucleolus • Scant or moderate amount of cytoplasm   

Samples are nearly always very cellular and contain monotonous lymphoblasts to the near exclusion of any other cell type. Blasts are 1.5× to 2× the size of a small lymphocyte,170 and the nucleus may be round but is more often indented/ irregular. Nucleoli are either absent or inconspicuous (Fig.

12.27). Cytoplasm is usually scant and may contain tiny vacuoles. Mitotic figures are variable, and their detection depends on smear quality. Tingible-body macrophages are variably present. In most cases, blasts are positive for TdT (a nuclear enzyme) and CD10 and negative for light chains. Immunophenotyping, coupled with cell morphology and clinical features, is nearly always diagnostic.2  Posttransplant Lymphoproliferative Disorders

Posttransplant lymphoproliferative disorders (PTLDs) are a heterogeneous group of lesions that include both nonneoplastic lymphoid proliferations, as well as a variety of lymphomas. They occur in about 1% to 2% of solid organ (e.g., kidney, liver, lung) and bone marrow transplant recipients.26 Up to 80% of PLTDs are associated with EBV infection and represent EBV-induced monoclonal or (less often) polyclonal B-cell proliferations; a small percentage are T-cell proliferations. PTLDs usually occur within the first year of transplantation, but they can also occur many years later. PTLDs tend to involve lymph nodes and the gastrointestinal tract, but other extranodal sites can also be involved, including the allograft itself. A significant proportion of PTLDs resolve after reduction of immune suppression. Some fail to regress, however, and are treated with cytotoxic chemotherapy. Histologically, PTLDs are divided into 4 major categories26: (1) Nondestructive PTLDs are polyclonal, nonneoplastic lymphoid proliferations that preserve the underlying lymph node architecture and may show plasma cell hyperplasia, an infectious mononucleosis-like picture, or florid follicular hyperplasia. (2) Polymorphic PTLD is a destructive, neoplastic proliferation that shows the full range of B-cell maturation, including immunoblasts, plasma cells,

CHAPTER 12  Lymph Nodes

411

EBER

A

B •

Fig. 12.28  Posttransplant Lymphoproliferative Disorder, Polymorphic Type.  (A) The smear shows a polymorphic population of small lymphocytes, plasmacytoid lymphocytes, plasma cells, and centrocytes (Romanowsky stain). (B) In situ hybridization for Epstein-Barr virus (EBV)–encoded RNA (EBER), performed on cell block sections, shows a positive reaction in many of the lesional cells.

centrocytes, and small and medium-sized lymphocytes. Immunophenotyping by flow cytometry may appear polytypic, but molecular tests reveal clonal rearrangements of the immunoglobulin heavy chains and monoclonal episomal EBV. (3) Monomorphic PTLD is also a destructive, neoplastic proliferation, but it usually exhibits large, cytologically malignant lymphoid cells with prominent nucleoli or sheets of plasma cells, indicating non-Hodgkin lymphoma or plasmacytoma. Most monomorphic PTLDs are B-cell neoplasms like DLBCL, but some arise from T-cells. (4) Classic Hodgkin lymphoma PTLD. The cytologic features mirror the varied histology of PTLD.171 Nondestructive PTLDs and polymorphic PTLD have overlapping features cytologically (a mixture of immunoblasts, plasma cells, centrocytes, and small and mediumsized lymphocytes) (Fig. 12.28A) but can be distinguished by molecular studies for clonality. The monomorphic PTLDs are cytologically malignant and are classified according to the WHO classification system26 of nontransplantrelated lymphomas, but the term PTLD should be included in the diagnosis. The PTLD moniker is important because it prompts the oncologist to include reduction of immunosuppression in the therapeutic armamentarium. Light chain restriction is sometimes absent by flow cytometry or immunocytochemistry in polymorphic and monomorphic PTLDs; molecular testing is often needed to demonstrate clonality. Thus, the absence of light chain restriction does not exclude the diagnosis of PTLD. In situ hybridization for EBV-encoded RNA (EBER) can be very helpful: a positive result for EBER in a majority of the lesional cells provides strong support for the diagnosis of PTLD171 (Fig. 12.28B). At least 20% of PTLDs are EBV negative, however. In patients who received a bone marrow transplant for lymphoma, the differential diagnosis includes recurrent lymphoma. A positive result for EBER in a majority of the lesional cells helps establish the diagnosis of PTLD in this setting. 

DIFFERENTIAL DIAGNOSIS OF LARGE CELL LYMPHOMAS • • • • • • • • • • • • •

 onhematopoietic tumors N Reactive hyperplasia Hodgkin lymphoma Diffuse large B-cell lymphoma Burkitt lymphoma High-grade B-cell lymphoma Blastic mantle cell lymphoma Peripheral T-cell lymphoma, not otherwise specified Anaplastic large cell lymphoma Precurser T- and B-cell lymphoblastic lymphoma Thymoma Posttransplant lymphoproliferative disorder Myeloid sarcoma (synonymous with granulocytic sarcoma and chloroma) • Histiocytic and dendritic cell neoplasms   

Differential Diagnosis: Large Cell Lymphomas

The group of large cell lymphomas can mimic and be mimicked by nonhematopoietic neoplasms. Poorly differentiated large cell carcinomas, epithelioid sarcomas, melanoma, and germ cell tumors all can resemble DLBCL. With rare exceptions (nasopharyngeal carcinoma and seminoma), the nonlymphoid tumors lack lymphoglandular bodies. Conversely, ALCL is very carcinoma-like. ANAPLASTIC LARGE CELL LYMPHOMA IS CARCINOMA-LIKE BECAUSE OF: • • • • • •

 paucity of lymphoglandular bodies162 A Cell clustering Marked pleomorphism Spindle cells172 Immunoreactivity for EMA Scant smaller lymphocytes   

412 CHA P T ER 1 2    Lymph Nodes

Immunostains are particularly useful in the distinction between lymphoma and a nonhematopoietic neoplasm, particularly keratins (for carcinoma), S-100 and SOX10 (for melanoma), OCT3/4 (for seminoma and embryonal carcinoma), and CD45, CD3, and CD20 (for lymphoma). Of note, some ALCL are negative for CD45, but these will often stain for at least some T-cell markers, clusterin, or the ALK protein. Nonhematopoietic neoplasms are also in the differential diagnosis of lymphoblastic lymphoma and BL. The cells of these lymphomas are intermediate in size rather than truly large.170 For this reason, and because they are common in children, the differential diagnosis includes “small roundcell malignancies” such as neuroblastoma, rhabdomyosarcoma, and Ewing sarcoma. Morphologic features are helpful: the nonhematopoietic neoplasms lack lymphoglandular bodies, and the cells form aggregates in smears. The cells of neuroblastoma form rosettes, have fibrillary neuropil, and show nuclear molding. The cells of rhabdomyosarcoma have a greater degree of anisonucleosis than lymphoblastic lymphoma or BL; more abundant, tapered cytoplasm; and they are frequently binucleated or multinucleated.173 Immunostains for neural, muscle, and lymphoid markers help to confirm the morphologic impression. Occasionally, one of the large cell lymphomas resembles reactive lymphoid hyperplasia. This is particularly true of a subtype of DLBCL, the T-cell/histiocyte–rich DLCBL.92 In this tumor, the neoplastic large cells are outnumbered by reactive T cells. To identify the monoclonal population by flow cytometry, it is necessary to gate on the large cells using forward scatter. Even so, flow may give a false-negative result. Some cases of PTCL, those composed of a mixture of small and large cells, also mimic reactive hyperplasia. In most cases, however, there is a sufficient number of atypical small and large cells, with nuclear membrane irregularities (indentations, grooves, knobs), to prompt consideration of neoplasia.35,157 Immunophenotyping often demonstrates aberrant patterns of expression of T-cell antigens, supporting the neoplastic nature of the lesion. Mononuclear and classic (binucleate) RS cells are frequently seen in ALCL. The sheer number of these pleomorphic cells107 and the paucity of inclusion-like macronucleoli help to exclude HL. In borderline cases, immunostaining for CD15 and BSAP/PAX5 (positive in RS cells and negative in the hallmark cells of anaplastic large cell lymphoma) and, if necessary, T-cell receptor rearrangements (detectable by molecular testing in ALCL) can be helpful. Extensive sclerosis in some cases of DLBCL, particularly those in the mediastinum, may yield sparsely cellular smears. The few lymphoid cells present are often crushed, and the remaining fragments of fibrous tissue might be mistaken for a spindle-cell neoplasm142 or granulomatous inflammation. Knowledge of this pitfall (and thus carefully searching for large, atypical lymphoid forms, especially in young adult female patients) is usually rewarding. The large cell type of PTCL is morphologically identical to DLBCL; the distinction depends on immunophenotyping. The distinction between DLBCL, particularly T-cell/ histiocyte-rich DLBCL, and HL can also be problematic.

Again, immunomarkers are useful: in general, RS cells (except in the nodular lymphocyte predominant HL) are positive for CD15 and CD30, and most non-Hodgkin lymphomas are negative.26 ALCL express a T-cell or null cell phenotype and may or may not express the ALK protein, and thus the differential diagnosis includes both ALK-positive and ALK-negative entities. Morphologically similar B-cell tumors exist, and some of these may even express CD30, but because the anaplastic B-cell neoplasms have clinical characteristics and behavior similar to other DLBCL, they are categorized as DLBCL, not as ALCL. An additional entity in the immunophenotypic differential diagnosis is the very rare ALK-positive large B-cell lymphoma, which is usually negative for CD30, CD20, and PAX5.26 T-cell lymphoblastic lymphoma is almost always positive for TdT, a highly specific and sensitive marker of lymphoblasts that is not present in other lymphomas or reactive lymphocytes in a lymph node. If blastoid morphology is present in a B-cell lymphoma without TdT, a diagnosis of high-grade B-cell lymphoma or blastic MCL should be considered (the latter showing at least focal staining for cyclin D1, SOX11, or both).130 In the mediastinum, thymoma is an important clinical and partial immunophenotypic mimic that includes TdTpositive lymphocytes with an “immature” (CD4+, CD8+) immunophenotype, but careful inspection of the flow cytometry data174 typically reveals an immunophenotypically heterogeneous population of T cells with variable expression of CD4, CD8, and surface CD32,170,174 and typically mature cytomorphology. The epithelial component of a thymoma is often inconspicuous on FNA smears, requiring immunocytochemistry (for keratin) for confident identification.156,170,175 The diagnosis of BL is based on a combination of clinical, morphologic, immunophenotypic, and cytogenetic features. The endemic, sporadic, and HIV-associated forms have characteristic clinical presentations: extranodal sites are involved in the endemic (jaw/orbit) and sporadic (abdomen) forms, as well as the immunodeficiency-associated forms. BLs express B-cell antigens, as well as CD10 and BCL6, and are negative for BCL2 and TdT (the latter two antibodies aiding in exclusion of DLBCL and lymphoblastic lymphoma, respectively). The morphologic features (cellular uniformity, intermediate cell size, round nuclei, clumped chromatin, small nucleoli, high mitotic rate, apoptosis, tingible-body macrophages) are somewhat characteristic. If any morphologic features of BL are present, however, or the Ki67 index is high, immunohistochemistry for nuclear MYC150,151 with confirmatory FISH for a MYC translocation may be helpful, albeit not completely specific, because these studies will detect both BL and high-grade B-cell lymphoma, with the latter diagnosis supported by FISH for an IGH-BCL2 translocation, and, if necessary, an IGH-BCL6 translocation.147,148 PTLDs occur only in solid organ or bone marrow transplant recipients. In such patients, this diagnosis should always be considered. In situ hybridization for EBER is very helpful: a positive result for EBER in many of the lesional cells provides strong support for the diagnosis of PTLD, although a negative result does not exclude this diagnosis, because at least 20% are EBV-negative.

CHAPTER 12  Lymph Nodes

413

A

C

B •

Fig. 12.29  Myeloid Sarcoma.  (A) At low and medium magnification, blasts mimic lymphoma cells. Recognition of these cells as myeloid is often precluded when a tumor lacks cytoplasmic granules (Romanowsky stain). (B) Myeloid blasts are also difficult to identify as such with the Pap stain, even if they show characteristic (but nonspecific) folded nuclear membranes and dispersed chromatin (liquid-based preparation, Papanicolaou stain). (C) Blasts are more easily recognized with Romanowsky stains if the characteristic chromatin pattern, nucleoli, cytoplasmic granules (upper left), and accompanying granulocytic/eosinophilic forms are all present (Romanowsky stain).

Myeloid sarcomas, tumors of immature myeloid cells that occur in bones or extramedullary locations, may mimic non-Hodgkin lymphoma (Fig. 12.29),176,177 even to the point of exhibiting lymphoglandular bodies.178 These tumors may be identifiable on routine “lymphoma” flow cytometry because blasts typically form a population with dim CD45.10 Further immunophenotyping for myeloid antigens (CD13, CD33, CD34, CD117) or histochemistry for myeloid enzymes (myeloperoxidase, chloroacetate esterase) is, however, essential for final diagnosis. In particular, positivity for CD123 (now often included in flow cytometry leukemia panels) may suggest the separate entity blastic plasmacytic dendritic cell neoplasm, particularly if peripheral cytoplasmic microvacuoles or pseudopodia are seen.26 Once a diagnosis of myeloid sarcoma is established, full genetic analysis, by conventional cytogenetics, FISH, and molecular assays, is essential for clinical management,

because the genetic profile stratifies the acute myeloid leukemias and myeloid sarcomas into prognostically and therapeutically relevant subgroups.26,179 A repeat FNA can easily be performed to obtain material for such genetic studies if none was available from the initial specimen. Finally, the differential diagnosis includes the rare histiocytic and dendritic cell neoplasms (Table 12.9), which together represent less than 1% of tumors in lymph nodes.26 They are diagnosed on the basis of their phenotypic resemblance to their presumed cell of origin. Histiocytic sarcoma can be morphologically indistinguishable from a DLBCL or an ALCL. Unlike the latter two tumors, however, it expresses the histiocyte markers CD68 and CD163 and may be positive for a BRAF V600E mutation.180 Because myeloid lesions, like myeloid sarcomas, are also positive for CD68, the absence of myeloid markers (e.g., myeloperoxidase) and demonstration of a more mature histologic appearance and

414 CHA P T ER 1 2    Lymph Nodes

TABLE   Histiocytic and Dendritic Cell Neoplasms and 12.9  their Identifying Markers

Tumor

Identifying markers

Histiocytic sarcoma

CD68, CD163, BRAF V600E (subset)

Langerhans cell histiocytosis

S-100, CD1a, langerin, BRAF V600E (subset)

Langerhans cell sarcoma

S-100, CD1a, langerin

Interdigitating dendritic cell sarcoma

S-100

Follicular dendritic cell sarcoma

CD21, CD23, CD35

immunophenotype (e.g., CD34 negativity) must be established before the diagnosis of histiocytic sarcoma is made. The family of dendritic cells, whose function is antigen presentation to other cells of the immune system, includes the Langerhans cells (in the skin and mucosa), interdigitating dendritic cells (in the lymph nodes), and follicular dendritic cells (in lymph node follicles). Both Langerhans cells and interdigitating dendritic cells are positive for S-100 protein, but in other ways they are phenotypically distinct. Langerhans cell histiocytosis can affect children or adults and be unifocal or multifocal. Common sites are bones, skin, and lungs. The key to diagnosis is recognizing the Langerhans cell, which has a grooved, indented, or lobulated vesicular nucleus and abundant cytoplasm (imparting an epithelioid appearance), in a background of eosinophils, histiocytes, neutrophils, and lymphocytes (see Fig. 18.14).80 Immunohistochemical positivity for CD1a and langerin confirm the diagnosis. A majority of cases show BRAF V600E (or, less commonly, MAP2K1) mutation181; the former is potentially detectable by immunohistochemistry with BRAF V600E mutationspecific antibody. Tumors of Langerhans cells with overtly malignant cytologic features are Langerhans cell sarcomas.26 Interdigitating dendritic cell sarcoma is an extremely rare tumor that occurs predominantly in adults.182 The cells can have an epithelioid or spindle-cell appearance. Like the Langerhans cells, interdigitating dendritic cells are positive for S-100 protein, but they lack CD1a, CD21, CD35, B- and T-cell markers, and keratin. Because follicular dendritic cell sarcomas usually have a spindle-cell appearance, they are discussed under “Sarcomas” below. 

Nonlymphoid Neoplasms Carcinomas, melanomas, germ cell tumors, and sarcomas can all metastasize to lymph nodes; carcinomas are the most frequent. Because there is often a clinical history of a malignancy, and because the cytomorphology of these tumors is so alien to that of the normal lymph node milieu, metastases to lymph nodes are readily recognized as malignant on FNA. Moreover, the metastatic tumor cells are usually present in abundance relative to the background lymphocytes (if any); rare atypical cells in a sea of lymphocytes (e.g., cells

found only by screening) are likely to be benign cells (such as dendritic cells, histiocytes, or large lymphocytes) rather than metastatic tumor cells.

Carcinomas Small cell carcinoma of the lung (Fig. 12.30) commonly metastasizes to lymph nodes; its principle mimic is metastatic Merkel cell carcinoma (Fig. 12.31), easily distinguished by its immunoreactivity for CK20. Large cell carcinomas of the aerodigestive tract, lung, thyroid, breast, kidney, pancreas, and colorectal region also metastasize to the lymph nodes, and in some instances the primary tumor is occult. In such instances, a useful rule of thumb is that primaries below the diaphragm tend to metastasize to the left supraclavicular lymph node,183 rather than the right, because of the thoracic duct anatomy. Thus the primary site for a right supraclavicular lymph node metastasis should be sought above the diaphragm. More generally, smears may contain clues that point to a likely primary site. CYTOMORPHOLOGY OF LARGE CELL CARCINOMAS • • • • •

 ells predominantly in clusters C No lymphoglandular bodies Abundant necrosis—favor colorectal carcinoma Signet-ring cells—favor gastric and breast carcinoma Abundant clear cytoplasm—favor renal cell carcinoma and ovarian carcinoma • Intranuclear inclusions—favor papillary thyroid carcinoma and melanoma   

Some aspirates from metastatic squamous cell carcinomas with cystic degeneration contain only necrotic parakeratotic cells and amorphous debris and thus imitate a branchial cleft cyst or epidermal inclusion cyst. In the absence of convincingly malignant cells, smudgy nuclear hyperchromasia and pyknosis should prompt the diagnosis “suspicious for squamous cell carcinoma.” Human papillomavirus (HPV)-associated head and neck squamous cell carcinomas deserve special mention. They are often diagnosed first by lymph node FNA, because onethird of these tumors manifest as an enlarged cervical lymph node in the context of an occult primary in the oropharynx. They tend to occur in middle-aged adult male nonsmokers, and cystic degeneration is common. Thus a cystic lesion in the neck is an imaging clue that the mass might be an HPVassociated squamous cell carcinoma. CYTOMORPHOLOGY OF HPV-ASSOCIATED SQUAMOUS CELL CARCINOMAS • • • • • •

“ Basaloid pattern” (most common) Immature, nonkeratinizing cells Cystic debris, necrosis, mitoses Variant patterns Keratinizing Nasopharyngeal carcinoma-like   

CHAPTER 12  Lymph Nodes

415

• Fig. 12.30  Small Cell Carcinoma.  When cell clusters are sparse and there is smearing of cell nuclei in a background of necrotic debris, the smears mimic lymphoma. Fragments of cytoplasm from necrotic cell “ghosts” masquerade as lymphoglandular bodies (Papanicolaou stain).

•

Fig. 12.31  Merkel Cell Carcinoma.  Meager cytoplasm and round nuclei with finely granular chromatin instead of nucleoli characterize these malignant cells. Smear background shows fragments of cells in various stages of necrosis that can be confused for large lymphoglandular bodies (Papanicolaou stain).

Cytomorphologically, they show a number of different patterns. The most common is the “basaloid” pattern: sheets of medium-sized, immature cells with indistinct cell borders, a round to oval nucleus, an inconspicuous nucleolus, and scant, nonkeratinized cytoplasm. (Fig. 12.32A, B). This pattern recapitulates the morphology of nonneoplastic tonsillar crypt epithelium. Cystic debris is common, and there can be necrosis and abundant mitoses. Abundant tumorinfiltrating lymphocytes may obscure the neoplastic cells.

Variant morphologies occur: some HPV-associated squamous cell carcinomas are keratinized, and others resemble a nasopharyngeal carcinoma. The differential diagnosis includes a poorly differentiated adenocarcinoma, a basaloid neoplasm of the salivary gland, and a small-cell neuroendocrine carcinoma. All metastatic squamous carcinomas in a level II or III cervical lymph node and any squamous carcinoma concurrent with an oropharyngeal primary should be tested for HPV, because HPV-associated squamous cell carcinomas of the head and neck have a better prognosis than those unassociated with HPV, impacting the choice of therapy.184 Optimal HPV testing for FNA specimens has not been established and needs to be standardized in individual laboratories.184,185 Testing options include p16 immunohistochemistry, HPV DNA or RNA in situ hybridization, HPV PCR testing, and liquid-phase assays designed for HPV detection in Pap tests. If immunohistochemistry for the surrogate marker p16 is used (Fig. 12.32C), the threshold for a positive result may differ in cell block material from the criteria for a tissue sample (at least 70% of tumor cells with nuclear and cytoplasmic staining). A positive p16 result for a nonkeratinizing tumor or one associated with a clinically apparent oropharyngeal primary can be confidently classified as an HPV-associated squamous cell carcinoma.184,185 If the tumor is keratinizing and no clinically apparent oropharyngeal primary is evident, a more specific follow-up test for HPV, such as RNA in situ hybridization (Fig. 12.32D) or HPV PCR, is warranted.184,185 Nasopharyngeal carcinoma commonly manifests itself, like lymphoma, as an enlarged cervical lymph node.

416 CHA P T ER 1 2    Lymph Nodes

A

B HPV 16/18 RNA

p16

C

D •

Fig. 12.32  Human Papillomavirus (HPV)–Associated Squamous Cell Carcinoma.  (A) The neoplastic cells form large crowded sheets of immature cells (Romanowsky stain). (B) Nuclei are round to oval, and cytoplasm is scant. Cell borders are indistinct and intercellular bridges are not apparent (Papanicolaou stain). (C) An immunostain for p16 shows positive nuclear and cytoplasmic staining. (D) In situ hybridization for HPV 16 and 18 RNA is positive.

CYTOMORPHOLOGY OF NASOPHARYNGEAL CARCINOMA • • • • • •

 lusters of undifferentiated large cells C Large nuclei with pale chromatin ± Prominent nucleolus Moderate amount of cytoplasm Lymphocytes, often commingled with epithelial cells Lymphoglandular bodies

CD45-negative. Virtually all nasopharyngeal carcinomas are positive for EBV DNA or RNA (Fig. 12.33 inset). Because some HPV-associated squamous cell carcinomas mimic nasopharyngeal carcinoma morphologically, both HPV and EBV testing should be performed if there is no known primary. 

Malignant Melanoma   

An overwhelming population of lymphocytes can sometimes obscure the clusters of malignant epithelial cells of metastatic nasopharyngeal carcinoma, which then resemble dendritic-lymphocytic aggregates186 (Fig. 12.33). The malignant cells are not keratinized; thus the cytoplasm is thin and transaparent on Papanicolaou-stained preparations. In lymph node samples with few metastatic nasopharyngeal carcinoma cells, the rare large malignant cells with their prominent nucleoli can be mistaken for the RS cells of HL. Immunostains can be very helpful. The cells of nasopharyngeal carcinoma are cytokeratin-positive and

Malignant melanoma is aptly named the great masquerader. Lymph nodes are among the most commonly aspirated metastatic sites.187 CYTOMORPHOLOGY OF MELANOMA • • • • • • •

 ispersed single cells and loose clusters D Epithelioid, spindle, or pleomorphic shapes Nuclei eccentrically placed, commonly binucleated Nuclear inclusions Single small to large nucleoli Cytoplasm: melanin pigment variable, vacuoles variable No lymphoglandular bodies   

CHAPTER 12  Lymph Nodes

417

• Fig. 12.33  Nasopharyngeal Carcinoma.  These neoplastic cells have a large round-to-oval nucleus, a

large nucleolus, and abundant cytoplasm with indisctinct cell borders; small lymphocytes are in the background (Papanicolaou stain). The malignant cells show nuclear staining for Epstein Barr virus–encoded ribonucleic acids by in situ hybridization (inset).

Melanin pigment is seen in less than 50% of aspirate smears.187 The differential diagnosis of amelanotic malignant melanoma includes DLBCL because both show a single cell pattern of monomorphic large cells. The eccentric nuclear placement (plasmacytoid nuclei), lack of lymphoglandular bodies, and frequent binucleation are clues to the diagnosis of malignant melanoma (Fig. 12.34), as is the presence of abundant tiny vacuoles in the cytoplasm (best visualized with air-dried smears). Three immunostains, SOX10, CD45, and keratin, are highly efficient at distinguishing melanoma, lymphoma, and carcinoma.12 

Seminoma/Germinoma Testicular and mediastinal seminomas/germinomas commonly metastasize to deep lymph nodes of the abdomen and chest, respectively. CYTOMORPHOLOGY OF SEMINOMA/ GERMINOMA • D  ispersed large cells • Macronucleolus • Voluminous cytoplasm, peripherally placed vacuoles with large blister-like quality • Small round lymphocytes and lymphoglandular bodies • Granulomas • Tigroid background   

Much of the literature regarding seminoma has focused on the smear background, in which strands of cytoplasm

•

Fig. 12.34  Malignant Melanoma.  Large nonpigmented cells are distributed in a noncohesive pattern. Tiny cytoplasmic bubbles (best seen with Romanowsky stains) and binucleated cells with wide spacing between the mirror-image nuclei are clues to the diagnosis (Romanowsky stain).

and proteinaceous fluid are arranged in a reticular or linear network termed the tigroid pattern (Fig. 12.35). Although helpful in the proper clinical context, it is not entirely specific for seminoma; it is also seen with clear cell carcinomas. Lymphocytes are common in seminoma, and thus lymphoglandular bodies may be present, so that confusion with a lymphoma is possible. Foci of cell clustering and the marked vacuolization of germ cells is helpful in this regard. FNA smears of other malignant germ cell tumors (e.g., yolk sac tumor and embryonal carcinoma) mimic those of nonsmall

418 CHA P T ER 1 2    Lymph Nodes

cell carcinoma. The tigroid pattern is not evident, nor is a background population of lymphocytes present. Sometimes one finds hyaline globules in aspirates of yolk sac tumor, where they appear as spherical glassy structures. Immunohistochemical positivity for OCT3/4 is relatively specific for seminoma and embryonal carcinoma.188 

SARCOMAS THAT MORE COMMONLY INVOLVE LYMPH NODES INCLUDE • • • • • •

 ynovial sarcoma S Epithelioid sarcoma Angiosarcoma Rhabdomyosarcoma Kaposi sarcoma Follicular dendritic cell sarcoma   

• Fig. 12.35  Seminoma.  Partially vacuolated detached strips of cyto-

plasm create a striking “tigroid” background. Large germ cells are mixed with small lymphocytes (Romanowsky stain).

Sarcomas Most sarcomas tend not to metastasize to lymph nodes: less than 3% of patients with sarcoma develop lymph node metastases.189 A subset of sarcomas breaks rank with the “antilymph node” imperative, however.

One of the more common sarcomas to metastasize to lymph nodes, synovial sarcoma, usually appears as large, loosely cohesive syncytia of small cells with monomorphic, ovoid nuclei with finely granular chromatin, smooth nuclear outlines, and scant tapering cytoplasm. Lymph node involvement by Kaposi sarcoma occurs in an endemic form and sporadically in immunodeficient states such as kidney transplantation and AIDS. Smears are variably cellular with abundant red cells, and the cytomorphology is that of a nondescript spindle cell neoplasm.190 Hyaline globules, a typical histologic feature, are usually difficult to find in smears. Definitive diagnosis nearly always requires immunocytochemistry: the spindle cells are positive for CD31, CD34, and (more specifically) HHV8. Follicular dendritic cell sarcoma is a rare tumor of young to middle-aged adults that arises in lymph nodes, as well as in extranodal sites. It is a slow-growing tumor with metastatic potential. Smears show loose, flat aggregates and single cells with oval and spindle shapes.191,192 Intermediate-sized nuclei have smooth borders and small inapparent nucleoli (Fig. 12.36). Background lymphocytes are usually present.

• Fig. 12.36  Follicular Dendritic Cell Sarcoma.  Delicate cytoplasmic processes extend from these clustered cells with smooth, ovoid nuclei (Romanowsky stain).

CHAPTER 12  Lymph Nodes

The neoplastic cells are positive for one or more of the follicular dendritic cell markers CD21, CD23, and CD35, and are negative for CD45.

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Liver BARBARA S. DUCATMAN AND KURT D. BERNACKI

F

ine-needle aspiration (FNA) and small core needle biopsy are mainstays in the evaluation of liver masses. They are usually performed percutaneously with guidance by computed tomography (CT), ultrasonography, or magnetic resonance imaging (MRI), and its principal value is in the diagnosis of malignancies. FNA can also be performed with the aid of endoscopic ultrasonography (EUS).1,2 The large core needle biopsy is generally reserved for diffuse liver diseases such as hepatitis and cirrhosis, for which large-scale architectural details are important; however, modern EUS needles can retrieve tissue for histologic study, as well as material for cytologic assessment.3,4 The sensitivity of FNA ranges from 71% to 94% and specificity from 87% to 100%, with an accuracy of 90% to 94%.1,2,5-26 False-positive diagnoses are very uncommon; hepatic dysplasia, bile duct hamartoma, and focal nodular hyperplasia have been misdiagnosed as malignant.11,27,28 Cell block preparations facilitate the subtyping of tumors.29 Obtaining brushings during endoscopic retrograde cholangiopancreatography is preferred for diagnosing tumors at the hilum of the liver, particularly cholangiocarcinoma.30,31 When brushings are negative or inconclusive, EUS-guided FNA is used.20-22,30,32-34 A sample can also be obtained using a small forceps biopsy, thereby combining histologic with cytologic study of tissue squashed between two slides (smash technique).35 FNA has also been used in the routine monitoring of liver transplants for acute cellular rejection.36-42 Slides are air-dried and stained with a Romanowsky stain. Lymphocytes are examined for evidence of activation (enlargement, “blast” forms), and hepatocytes for signs of injury (swelling, vacuolization, necrosis) and cholestasis.41 The technique is not useful for diagnosing causes of chronic rejection.38 Complications of liver FNA are rare and include hemorrhage,22,43,44 pain,22 bile peritonitis, infection,45 and anaphylactic shock (after aspiration of an echinococcal cyst). FNA is associated with a very low risk (0.1%–0.6%) of tumor seeding44,46-51 and recurrence of hepatocellular carcinoma after liver transplantation.52 Death is very uncommon (0.6%).22,53-56

Normal Liver Benign liver cells are often seen in aspirates of liver masses and even, occasionally, in aspirates of adjacent organs such as the pancreas, right kidney, right adrenal gland, and the right lower lobe of the lung, when the liver is penetrated en route to the target organ. The components of a normal liver aspirate commonly include hepatocytes, bile duct epithelium, Kupffer cells, and sheets of mesothelial cells.

CYTOMORPHOLOGY OF HEPATOCYTES (Fig. 13.1A and B) • L  arge polygonal cells • Isolated cells, thin ribbons (trabeculae), or large tissue fragments • Centrally placed, round to oval, variably sized nuclei • Binucleation common • Prominent nucleolus • Intranuclear pseudoinclusions • Abundant granular cytoplasm • Pigment: • Lipofuscin (common; a normal pigment related to cellular aging): golden with the Papanicolaou stain and green-brown with a Romanowsky-type stain • Hemosiderin (uncommon; when present in large quantities it suggests a disorder of iron metabolism): dark brown with the Papanicolaou stain and blue with a Romanowsky-type stain. • Bile (seen in cholestasis): dark green with both Papanicolaou and Romanowsky stains (Fig. 13.2)   

CYTOMORPHOLOGY OF BILE DUCT EPITHELIUM • C  ohesive flat sheets • Cuboidal cells smaller than hepatocytes • Evenly spaced nuclei (“honeycomb appearance”)   

425

426 CHA P T ER 1 3   Liver

A

B • Fig. 13.1  Normal Hepatocytes.  (A) Hepatocytes have abundant granular cytoplasm, a round and regular nucleus (or two), and a prominent nucleolus. They are arranged as a ribbon of two cells’ width. Lipofuscin, the normal “wear and tear” pigment, is present (arrow) (Papanicolaou stain). (B) Lipofuscin appears greenish with the Romanowsky stains. A pseudoinclusion is present (Romanowsky stain).

CYTOMORPHOLOGY OF KUPFFER CELLS • R  esemble macrophages • Vacuolated cytoplasm and may contain pigment, most commonly hemosiderin   

DIFFERENTIAL DIAGNOSIS OF NORMAL LIVER CELLS • • • • •

 epatic adenoma H Focal nodular hyperplasia Regenerative nodule in cirrhosis Nodular regenerative hyperplasia Steatosis   

The first four conditions listed in the differential diagnosis have characteristic clinical or imaging findings and should be considered when an FNA of the liver is composed entirely of normal liver cells (predominantly hepatocytes) and the patient has a focal lesion. The possibility of sampling error must also be considered. In steatosis (fatty metamorphosis), many hepatocytes have large cytoplasmic vacuoles filled with lipid (Fig. 13.3). This alteration of the liver is seen with toxic-metabolic injuries such as those caused by alcohol consumption, diabetes, obesity, drugs (e.g., methotrexate), total parenteral nutrition, post-jejunoileal bypass, and hepatitis C. Although usually a diffuse liver abnormality, some cases of steatosis show areas of low attenuation on CT that suggest an infiltrative tumor. Steatosis can be a component of focal nodular hyperplasia and hepatic adenoma, thus correlation with imaging and clinical findings is essential. 

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427

• Fig. 13.3  Steatosis.  This patient had a long-standing history of alcohol abuse, and diffuse nodularity was noted on computed tomography. Fine-needle aspiration demonstrated abundant fatty changes (Papanicolaou stain).

•

Fig. 13.2  Bile.  Bile pigment is not usually seen unless there is bile stasis. With the Romanowsky stains, bile pigment is a dense, dark greenish-black pigment in the cytoplasm of hepatocytes (arrow). This case is from a patient with cirrhosis (Romanowsky stain).

Infections Hepatic Abscess Hepatic abscesses can be bacterial, fungal, or amebic, the latter principally due to Entamoeba histolytica. Bacterial abscesses result from ascending cholangitis and sepsis. The most common organisms are streptococci, staphylococci, and enteric bacteria. Fungal abscesses are most common in immunocompromised patients: Candida species are the most common pathogens. CYTOMORPHOLOGY OF BACTERIAL AND FUNGAL ABSCESSES • A  bundant polymorphonuclear leukocytes and necrotic debris • May see bacteria and fungi with routine stains, but special stains (i.e., silver or Gram) and culture are helpful   

Amebic abscesses are rare in the United States but common in other countries, particularly developing nations. They are caused by the protozoan E. histolytica and are usually a sequela of colonic infection. CYTOMORPHOLOGY OF AMEBIC ABSCESS • • • •

“ Anchovy paste” (macroscopic appearance) Necrotic debris Little if any acute inflammation Amebic trophozoites resemble histiocytes: • Round nucleus • Peripheral chromatin margination • Abundant ovoid cytoplasm containing ingested red blood cells   

Microbiologic culture helps to confirm a presumptive diagnosis of infection. For this reason, an on-site evaluation is crucial to ensure that aspirated material is sent for culture. A careful search for malignant cells is essential whenever an FNA shows abundant necrotic debris, acute inflammation, or both. Charcot-Leyden crystals have been reported in amebic abscess.57 

Echinococcal Cyst (Hydatid Cyst) The larval form of Echinococcus granulosus, a dog tapeworm, causes infection in a variety of organs in humans, chiefly the liver. In one series of hepatic cysts greater than or equal to 4 cm in diameter, 10% were echinococcal cysts.58 The disease is endemic in the countries bordering the Mediterranean and Baltic seas, in South America, and in Australia and New Zealand. It is also seen in North America. Infection can be asymptomatic. Imaging studies reveal a solitary cyst, often with a fluid level. An outer, acellular, laminated membrane lines the cyst. The internal, germinal layer gives rise to daughter cysts, each of which contains scolices with numerous hooklets. Hooklets resist degeneration but scolices can be lost in longstanding cysts with degeneration. CYTOMORPHOLOGY OF ECHINOCOCCAL CYST • F  ragments of the laminated membrane (Fig. 13.4A) • Hooklets (Fig. 13.4A inset, Fig. 13.4B) • Scolices (Fig. 13.4B)   

Although anaphylactic shock has been reported as an occasional complication of FNA,53 its incidence has not been established, and successful aspiration without serious complications has been observed.40,59-61 Other parasitic diseases that present as a liver mass include schistosomiasis, clonorchiasis, and visceral larva migrans.62 

428 CHA P T ER 1 3   Liver

Benign Lesions Solitary Cysts Solitary, nonparasitic cysts of the liver include the simple unilocular cyst, which is lined by cuboidal or columnar epithelium that resembles biliary epithelium. The aspirated fluid is typically hypocellular and nondiagnostic. The ciliated foregut cyst is also solitary and unilocular and is lined by respiratory-type epithelium. CYTOMORPHOLOGY OF CILIATED FOREGUT CYST

A

• C  iliated columnar cells • Mucus cells   

DIFFERENTIAL DIAGNOSIS OF CILIATED FOREGUT CYST • B  ile duct cystadenoma • Bile duct cystadenocarcinoma   

B • Fig. 13.4  Echinococcal Cyst.  (A) Fragments of the laminated mem-

brane of the cyst appear as parallel, acellular striations and are diagnostic (hematoxylin and eosin–stained cell block). Inset: Some cysts contain mostly acellular debris without scolices. A diligent search uncovers the pale dagger-like hooklets, which do not stain with the Papanicolaou stain (Papanicolaou stain). (B) Rarely, an intact scolex and numerous hooklets are identified (Papanicolaou stain). (Case B courtesy Dr. Edward B. Stelow, University of Virginia School of Medicine, Charlottesville, VA.)

Other Infections Infection by the hepatotropic viruses (hepatitis A, B, and C) is generally not evaluated by FNA because the diagnostic changes of acute, subacute, and chronic hepatitis caused by these viruses are primarily architectural and not cytologic. Immunocompromised patients can develop hepatitis caused by cytomegalovirus and herpes viruses, but these also result in diffuse liver disease and are rarely diagnosed by FNA. Granulomas are seen in miliary tuberculosis, sarcoidosis, primary biliary cirrhosis, Hodgkin lymphoma, and drug reactions. Cytologic features are described elsewhere (see Figs. 2.15 and 12.4). The differential diagnosis of granulomatous inflammation includes a hepatic angiomyolipoma (AML) because the myoid cells of an AML have a syncytium-like appearance similar to that of epithelioid histiocytes. The presence of adipocytes and extramedullary hematopoiesis are a clue to the diagnosis of AML. 

Bile duct cystadenomas and cystadenocarcinomas are multilocular tumors lined by mucinous epithelium and filled with watery or viscous fluid. The cystadenoma is lined by a single layer of benign mucin-producing cells, whereas the cystadenocarcinoma shows nuclear pleomorphism and stromal infiltration; the latter cannot be evaluated on cytologic preparations. 

Cirrhosis Cirrhosis, whether caused by alcoholic hepatitis, viral hepatitis, or other diseases, results in a disruption of normal liver architecture, with bands of fibrosis separating nodules of regenerating hepatocytes. Some regenerative nodules are larger than others and, on imaging studies, raise the specter of malignancy, primarily that of hepatocellular carcinoma, because patients with cirrhosis are at increased risk for developing hepatocellular carcinoma. Focal lesions in the setting of cirrhosis are often biopsied by FNA, although accuracy may be higher with needle biopsy or a combination of the two.6 CYTOMORPHOLOGY OF CIRRHOSIS • N  ormal-appearing hepatocytes, sometimes with steatosis (see Fig. 13.3) • Focal atypia in some cases • Marked variation in nuclear size • Prominent nucleolus • Binucleation   

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DIFFERENTIAL DIAGNOSIS OF CIRRHOSIS • • • • •

 epatocellular carcinoma H Hepatic adenoma Focal nodular hyperplasia Normal liver Nodular regenerative hyperplasia   

The distinction between a cirrhotic nodule and a welldifferentiated hepatocellular carcinoma can be problematic. When hepatocyte atypia is due to cirrhosis, there is a wide morphologic spectrum ranging from normal hepatocytes to markedly atypical ones, whereas a hepatocellular carcinoma is generally more monomorphic. Other features of hepatocellular carcinoma, rare or absent in cirrhosis, include an increased nuclear-to-cytoplasmic ratio, a thickened trabecular arrangement of hepatocytes surrounded by endothelial cells, acinar architecture, and atypical naked nuclei. Larger tissue fragments comprised of normal hepatocytes are characteristic of cirrhosis, whereas trabeculae of variable thickness are seen in hepatocellular carcinoma.63 The hepatocyte dysplasia of cirrhosis features loose hepatocyte clusters with minimal or no endothelial wrapping, cellular enlargement with preservation of a normal nuclear-to-cytoplasmic ratio, and cytoplasmic basophilia.64 A combination of features permits the diagnosis of most cases of hepatocellular carcinoma.28,65,66 Specimens composed of hepatocytes without atypia are cytologically indistinguishable from the remainder of the conditions listed in the differential diagnosis; the diagnosis requires clinical-pathologic correlation. Nodular regenerative hyperplasia is a poorly understood condition in which small nodules of regenerating liver are scattered diffusely throughout the liver. Unlike cirrhosis, these nodules are not separated by bands of fibrosis, but patients often have portal hypertension and ascites. 

Focal Nodular Hyperplasia Focal nodular hyperplasia (FNH) is a benign liver lesion that possibly results from focal vasculopathy. It usually presents as a solitary mass or, less commonly, several masses. Most patients are women in their third or fourth decade. The nodules usually contain a central scar, which can be appreciated on imaging studies. Histologic examination reveals nodules of hepatocytes that are separated by radiating fibrous septae that contain bile ductules. CYTOMORPHOLOGY OF FOCAL NODULAR HYPERPLASIA • • • •

 epatocytes without significant atypia (Fig. 13.5A) H Normal trabeculae (two cells thick) Steatosis (some cases) Bile ductular cells (Fig. 13.5B)   

A

B •

Fig. 13.5  Focal Nodular Hyperplasia.  (A) Findings include benign hepatocytes, indistinguishable from those of normal liver (Romanowsky stain). (B) In some cases, there may be numerous benign ductal cells. This constellation of findings is nonspecific, however (Papanicolaou stain).

DIFFERENTIAL DIAGNOSIS OF FOCAL NODULAR HYPERPLASIA • H  epatic adenoma • Regenerating nodule in cirrhosis • Normal liver   

These conditions are cytologically indistinguishable, and radiologic and clinical correlation is necessary. FNHs express the usual markers of hepatocellular differentiation, including HepPar 1, TTF-1 (cytoplasmic staining), ARG-1 (arginase-1), CAM5.2, and canalicular staining for polyclonal carcinoembryonic antigen (CEA). A core biopsy can help by suggesting or confirming the diagnosis of FNH if bile ductules are present within the lesion: bile ductules rule out an adenoma. Thus, if FNH is suspected by imaging and the rapid on-site assessment shows only normal hepatocytes, ductal cells, or both, the cytologist can recommend a concurrent core biopsy.

430 CHA P T ER 1 3   Liver

The distinction between FNH and hepatic adenoma is a difficult but important one because hepatic adenomas, unlike FNH, have an increased risk of life-threatening hemorrhage and are linked to hepatocellular carcinoma. Molecular studies have confirmed the distinction between FNH and hepatic adenoma and have identified different subtypes of hepatic adenoma (see Hepatic Adenoma).67,68 Data from molecular studies have led to the application of selected immunohistochemical markers in the distinction between FNH and hepatic adenoma in needle biopsies.69 For example, glutamine synthetase has a characteristic map-like staining pattern in FNH. They have not been widely applied to FNAs, however, and interpretation of these markers, especially in limited samples, can be problematic and is best deferred to specialists with experience in liver biopsies. 

A

Hepatic Adenoma Hepatic adenomas or hepatocellular adenomas (HCA) are rare benign neoplasms usually encountered in women under the age of 30, especially those with a history of longterm use of oral contraceptives. Patients often present with abdominal pain.70 These lesions may rupture through the liver capsule, resulting in intraperitoneal hemorrhage, and they have been linked to hepatocellular carcinoma: malignant transformation to hepatocellular carcinoma is rare but well-documented.68 Histologically, they lack portal triads and are composed exclusively of hepatocytes. So-called “naked arterioles”—arterioles surrounded by scant connective tissue without bile ducts—are characteristic. CYTOMORPHOLOGY OF HEPATIC ADENOMA (Fig. 13.6A and B) • • • •

 ormal-appearing hepatocytes N Steatosis (some cases) Normal trabeculae (two cells thick) “Naked arterioles” (in cell blocks and core biopsies)   

DIFFERENTIAL DIAGNOSIS OF HEPATIC ADENOMA • • • •

 ormal liver N Focal nodular hyperplasia Regenerating nodule in cirrhosis Hepatocellular carcinoma   

HCAs express the usual markers of hepatocellular differentiation, including HepPar 1, ARG-1 (arginase-1), CAM5.2, and canalicular staining for polyclonal CEA. Cytoplasmic expression of TTF-1 has been described in normal hepatocytes, HCA, and hepatocellular carcinoma; however, this phenomenon is clone dependent.71,72 With regard to the distinction between FNH and HCA, the presence of bile duct epithelium in the lesion helps to exclude

B •

Fig. 13.6  Hepatic Adenoma.  Anisonucleosis and binucleation are no different from that seen with normal hepatocytes. (A, smear, Romanowsky stain; B, cell block, hematoxylin and eosin stain).

HCA,73 but in practice clinical and radiologic correlation is necessary. Molecular studies have confirmed the distinction between FNH and HCA and have identified several different subtypes of HCA: HNF1α-inactivated (H-HCA), β-catenin-activated (b-HCA), inflammatory (IHCA), and unclassified (UHCA).67,68 Immunohistochemically, H-HCA is characterized by loss of liver fatty-acid binding protein expression. b-HCA shows nuclear and cytoplasmic staining for β-catenin and diffuse staining for glutamine synthetase. IHCA expresses serum amyloid A and C-reactive protein. Of note, a subset of IHCAs have a β-catenin mutation (b-IHCA) and express β-catenin, glutamine synthetase, and serum amyloid A.69,74 Subtyping of HCA is important for recognizing tumors with an increased risk of malignant transformation (b-HCA, IHCA, and b-IHCA) and those with an association with an inflammatory syndrome (IHCA), which may be treated surgically. With core biopsy material, an immunohistochemical panel of β-catenin, glutamine synthetase, and serum amyloid A can help in identifying these clinically important subtypes.74 The cells of most hepatocellular carcinomas have a

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higher nuclear-to-cytoplasmic ratio and more architectural derangement than is seen in adenomas. 

Bile Duct Hamartoma and Adenoma The bile duct hamartoma (von Meyenberg complex) is characterized by multiple small nodules dispersed throughout the liver and composed of haphazardly arranged bile ductules and fibrous stroma. The bile duct adenoma is usually a solitary subcapsular nodule less than 1 cm in diameter.  CYTOMORPHOLOGY OF BILE DUCT HAMARTOMA AND ADENOMA • H  ypocellular specimens • Benign ductal cells in tubules and cohesive sheets • Benign hepatocytes   

Hemangioma The hemangioma is the most common benign tumor of the liver. Histologically, dilated vascular spaces are lined by benign endothelial cells. Because most hemangiomas are recognized radiologically with sufficient accuracy, an FNA is rarely necessary. Occasional hemangiomas have unusual imaging findings, however, and an FNA is performed to exclude a more significant lesion. If well sampled, cytologic findings are characteristic. Some cases, however, show only blood or benign hepatocytes.75 If a spindle cell lesion of the liver is encountered, it is most likely a hemangioma. The next most likely lesions are metastatic gastrointestinal stromal tumor (GIST), metastatic leiomyosarcoma, and granulomatous hepatitis.76 Epithelioid hemangioendothelioma is rare in the liver, with larger, epithelioid cells, greater cytologic atypia, and more conspicuous loss of cohesion.77  CYTOMORPHOLOGY OF HEMANGIOMA • B  lood only (some cases) • Hepatocytes only (some cases) • Three-dimensional arcades of bland elongated spindle cells • Compact, dense coils of spindle cells (Fig. 13.7A) • Rare isolated spindle-shaped cells • Vascular channels lined by endothelial cells (cell block sections) (Fig. 13.7B).   

Angiomyolipoma AML is the most common benign tumor of the kidney. It is seen less often in other locations but does occur in the liver, mediastinum, heart, spermatic cord, vaginal wall, fallopian tube, oral cavity, pharynx, nasal cavity, skin, and parotid gland. Of the extrarenal sites, the liver is the most common; to date, more than 100 hepatic AMLs have been reported worldwide.78

431

Hepatic AML shares many clinical features with its renal counterpart. The average age at diagnosis is about 50 years. Although 60% of patients are symptomatic, and many tumors, particularly small ones, are diagnosed incidentally by imaging studies. Hepatic AMLs can rupture spontaneously, just like their renal counterparts, but this is uncommon. Some patients with a hepatic AML have tuberous sclerosis, as do some patients with a renal AML. AMLs of the liver range in size from 0.3 to 36 cm in diameter (mean 9 cm) and are well circumscribed. Hemorrhage, necrosis, and calcification are rare. As with AMLs of the kidney, many are not biopsied because their high fat content permits an accurate diagnosis by CT or MRI studies. Only the radiologically atypical AMLs (usually due to low fat content) undergo biopsy. A study of 49 sporadic hepatic AMLs found no loss of heterozygosity or microsatellite instability in any of the cases.79 The proportions of the components vary considerably from patient to patient. Some tumors are composed predominantly of adipose tissue, whereas others have virtually no fat and are almost exclusively myoid. The myoid cells may cause diagnostic difficulty because they can be epithelioid and markedly pleomorphic with bizarre giant cells, and they may show mitotic activity. In contrast to renal AMLs, extramedullary hematopoiesis is a frequent feature, seen in 40% of cases. CYTOMORPHOLOGY OF HEPATIC ANGIOMYOLIPOMA • • • •

 lusters of epithelioid or spindle cells (myoid cells; Fig. 13.8) C Fat cells Blood vessels Extramedullary hematopoiesis (see Fig. 13.8)   

Hepatic AMLs can be diagnosed accurately by FNA.80 The correct diagnosis rests on identifying the triad of fat, vessels, and smooth muscle, but the myoid component is the only specific and diagnostic component of AML. The myoid cells have indistinct cell membranes and abundant granular cytoplasm. Sheets of myoid cells therefore have a syncytium-like appearance that resembles that of a granuloma. Adipose tissue is not consistently present.81 The positivity of the myoid cells for HMB-45 and Melan A is a very helpful diagnostic feature (see Fig. 15.5D).82,83 Almost onehalf of AMLs of the liver have extramedullary hematopoiesis, a striking finding that should make one consider the diagnosis of an AML. DIFFERENTIAL DIAGNOSIS OF HEPATIC ANGIOMYOLIPOMA • • • • • •

 epatocellular carcinoma H Other carcinomas Granulomatous inflammation Myelolipoma of the liver Nodular hematopoiesis Sarcoma   

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A

B

• Fig. 13.7  Hemangioma.  (A) Smears show occasional tangles of spindle-shaped cells that are difficult to classify (Papanicolaou stain). (B) The diagnosis is easier with cell block sections, where the vascular architecture is more readily apparent (hematoxylin and eosin stain).

have a definitive diagnosis may be considered for resection. Nevertheless, most patients are spared surgical resection, and their tumors are monitored by periodic imaging studies. 

Malignant Tumors Hepatocellular Carcinoma

• Fig. 13.8  Angiomyolipoma of the Liver.  The myoid cells have indis-

tinct cell borders and, grouped together, resemble the epithelioid histiocytes of a granuloma. Note the prominent component of extramedullary hematopoiesis, including a megakaryocyte on the right (Papanicolaou stain).

The differential diagnosis includes hepatocellular carcinoma. The cells of hepatocellular carcinoma have distinct cytoplasmic borders, unlike the myoid cells of AML, which have a fibrillar cytoplasm with indistinct cell membranes. Because hematopoietic elements are seen in many hepatic AMLs, the differential diagnosis also includes myelolipoma and nodular hematopoiesis of the liver, but cytologic samples from these lesions lack the myoid cells of AML. Because the myoid cells vary widely in their appearance, they can mimic a variety of spindle- and epithelioid cell neoplasms of the liver. Their indistinct cell membranes and arrangement in nodular aggregates may cause them to be mistaken for the epithelioid histiocytes of granulomas, especially when hematopoietic elements are interspersed. Ultimately, the diagnosis rests on showing that the large neoplastic cells are positive for HMB-45, MelanA (Mart-1), or both. Because a small percentage of hepatic AMLs rupture, a patient who is symptomatic, has a large tumor, or does not

Hepatocellular carcinoma (HCC) accounts for 90% of all primary cancers of the liver. It is less common in the United States and Western Europe than in Africa and Asia. In the United States, most cases are seen in the setting of cirrhosis. Most patients are over the age of 50, but children and younger adults can be affected. An α-fetoprotein (AFP) serum level greater than 4000 ng/mL is highly suggestive of HCC, but elevated titers are not present in all cases. The tumor can present as a solitary nodule, as multiple nodules, or as diffuse liver enlargement. Histologically, HCCs are divided into classical and “special types.”68 The classical HCC has three architectural patterns that often occur in combination and can be appreciated cytologically, especially with cell block preparations: trabecular, pseudoglandular (acinar), and compact. In addition, classical HCC has a myriad of cytological variants, including pleomorphic cells, clear cells, spindle cells, and fatty change. Bile is prominent in a minority of tumors, and a variety of cytoplasmic inclusions are occasionally encountered (Mallory hyaline bodies, globular hyaline bodies, pale bodies, and ground glass). The special types of HCC include fibrolamellar carcinoma, scirrhous HCC, undifferentiated carcinoma, lymphoepithelioma-like carcinoma, and sarcomatoid HCC. There is a wide range of differentiation, from well-differentiated tumors that resemble normal liver, to poorly differentiated ones with marked nuclear pleomorphism and tumor giant cells (Fig. 13.9A and B). Because the cytomorphology and differential diagnosis are distinctly different at the two ends of the spectrum of HCC (i.e., well-to-moderately vs poorly differentiated), they are presented separately. HCCs express the usual markers

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433

A • Fig. 13.10  Hepatocellular Carcinoma.  A markedly cellular ­preparation with abundant naked nuclei is characteristic of many hepatocellular carcinomas (Romanowsky stain).

CYTOMORPHOLOGY OF WELL- AND MODERATELY DIFFERENTIATED HEPATOCELLULAR CARCINOMA

B •

Fig. 13.9  The Spectrum of Hepatocellular Carcinoma.  (A) Welldifferentiated tumors are clearly of hepatocyte derivation but can be difficult to distinguish from benign hepatocytes (Papanicolaou stain). (B) Poorly differentiated tumors show little if any hepatocellular differentiation; they resemble poorly differentiated cholangiocarcinomas and metastatic carcinomas (Papanicolaou stain).

of hepatocellular differentiation, including HepPar 1, TTF-1 (cytoplasmic staining, clone dependent), ARG-1 (arginase-1), CAM5.2, and canalicular staining for polyclonal CEA. To standardize the imaging diagnosis of HCC, the American College of Radiology developed the Liver Imaging Reporting and Data System (LI-RADS).84 Since 2011 it has been adopted by many practices throughout the world. LI-RADS is not intended for the general population but rather for individuals at increased risk for HCC. It categorizes nodules examined by CT or MRI as definitively benign (LR-1), probably benign (LR-2), intermediate probability of being HCC (LR-3), probably HCC (LR-4), and definitively HCC (LR-5). LR-5 means that the radiologist is certain the lesion is HCC. Many HCCs will not meet LR-5 criteria and will be categorized LR-4 or less. If imaging suggests that the mass might be a malignancy other than HCC (e.g., cholangiocarcinoma), it is categorized LR-M (probable malignancy, not specific for HCC).

• H  ighly cellular smears • Isolated cells and large naked nuclei • Thickened cell cords (trabeculae) wrapped by endothelial cells • Pseudoglandular structures (acini) • Transgressing vessels • Increased nuclear-to-cytoplasmic ratio • Large, round nucleus with prominent nucleolus • Intranuclear pseudoinclusions • Intracellular bile   

The cells of well- and moderately differentiated HCCs are often recognizably hepatocytic in origin: they are polygonal, with moderate to abundant amounts of granular cytoplasm, and their nuclei are round, centrally placed, with a prominent nucleolus and an occasional pseudoinclusion. The cells of HCCs are often dispersed as numerous isolated cells (see Fig. 13.9A) or naked nuclei (Fig. 13.10). Thickened trabeculae of neoplastic hepatocytes are a prominent feature of some HCCs. In contrast to normal liver, cirrhotic nodules, FNH, and hepatic adenoma, where hepatocytes are predictably arranged in thin ribbons no more than 2 or 3 cells across, the trabeculae in HCC can be markedly thickened (Fig. 13.11A and B). Thick trabeculae, often outlined by flattened endothelial cells, are virtually pathognomonic of HCC.63,85 Some HCCs have a predominantly acinar pattern: instead of thickened trabeculae wrapped by endothelial cells, the cords of neoplastic cells form acini. The acinar pattern is more easily recognized on cell block preparations (Fig. 13.12A and B). An increased nuclear-to-cytoplasmic ratio is characteristic of HCC (see Figs. 13.11A and 13.12A), although it may not be prominent in some well-differentiated HCCs (see Fig. 13.9A).65,86-91 Cellular monomorphism, nuclear crowding, macronucleoli, multinucleated cells, mitoses, and capillaries

434 CHA P T ER 1 3   Liver

A

A

B

B

•

Fig. 13.11  Hepatocellular Carcinoma (Trabecular Type).  (A) Thick (>2 cells across) trabeculae are a characteristic feature of the classic hepatocellular carcinoma with a prominent trabecular pattern. Note the increased nuclear-to-cytoplasmic ratio of the neoplastic hepatocytes and the flat endothelial cells with spindle-shaped nuclei that wrap the trabeculae (Papanicolaou stain). (B) Thickened trabeculae are cut across in cell block sections. Note the prominence of the wrapping endothelial cells, a helpful diagnostic feature (hematoxylin and eosin stain).

traversing neoplastic tissue fragments (Fig. 13.13) are also characteristic features of HCC; some features are better seen on cell block sections than on smears.9,90,92 Irregularly arranged, overlapping cells in sheets are also typical of HCC, in contrast with the evenly spaced arrangement of benign lesions.93 DIFFERENTIAL DIAGNOSIS OF WELLAND MODERATELY DIFFERENTIATED HEPATOCELLULAR CARCINOMA • R  egenerating nodule in cirrhosis • Hepatic adenoma • Focal nodular hyperplasia   

The distinction between benign conditions and a wellor moderately differentiated HCC can be challenging.

•

Fig. 13.12  Hepatocellular Carcinoma (Acinar Type).  (A) The cords of neoplastic cells are arranged in acini. Note the increased nuclearto-cytoplasmic ratio, a key feature in the diagnosis of hepatocellular carcinoma (Papanicolaou stain). (B) Acini are easily appreciated in cell block preparations (hematoxylin and eosin stain).

Abundant bare hepatocyte nuclei are rarely seen in benign conditions and should prompt a search for other characteristic features, like intact cells with an increased nuclearto-cytoplasmic ratio and thickened trabeculae lined by endothelial cells. Although endothelial cells can be seen within large fragments of benign hepatocytes, thickened trabeculae are not apparent.94 Histochemistry and immunohistochemistry can be helpful.95 A reticulin stain outlines the normal, thin ribbons of hepatocytes in benign lesions, versus thickened trabeculae in HCC (Fig. 13.14A and B).90,96-98 CD34 highlights the endothelial cells that wrap the thickened cords of HCC.88,99 Immunohistochemistry for glypican-3, an oncofetal protein important in cell growth and differentiation, is also useful: benign hepatocytes are negative, and most HCCs are positive (Fig. 13.15).100-104 The definite distinction between a benign nodule and HCC is not always possible, and the interpretation “suspicious for hepatocellular carcinoma” is appropriate in some cases.

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CYTOMORPHOLOGY OF POORLY DIFFERENTIATED HEPATOCELLULAR CARCINOMA • • • • • • •

 ighly cellular smears H Isolated cells or clusters Moderate to marked pleomorphism Atypical mitoses Spindled-shaped cells Tumor giant cells Bile   

•

Fig. 13.13  Hepatocellular Carcinoma.  Prominent transgressing vessels are characteristic of hepatocellular carcinoma (Papanicolaou stain).

The most notable features of hepatocytic differentiation— polygonal cell shape, a centrally placed nucleus, cytoplasmic bile (Fig. 13.16), and trabecular architecture—are either lost or barely apparent in poorly differentiated HCC. Atypia and pleomorphism can be marked (Fig. 13.17A and B). glypican

• Fig. 13.15  Hepatocellular Carcinoma.  Glypican-3 is a useful marker A

that distinguishes benign from malignant hepatocytes: benign hepatocytes are virtually always negative, whereas most hepatocellular carcinomas show cytoplasmic and membranous staining (immunostain for glypican-3).

B •

Fig. 13.14  Reticulin for Distinguishing Benign From Malignant Hepatocellular Proliferations.  (A) Benign Liver. The reticulin stain sharply outlines the normal, two-cell-thick hepatic cords. (B) Hepatocellular Carcinoma. The reticulin strands are attenuated and outline large, chunky masses of neoplastic cells (A and B, reticulin stain).

• Fig. 13.16  Hepatocellular Carcinoma.  Bile stains dark green-brown

and is a useful marker of hepatocyte differentiation (Papanicolaou stain).

436 CHA P T ER 1 3   Liver

B

A

• Fig. 13.17  Hepatocellular Carcinoma, Poorly Differentiated.  A, Bizarre, atypical forms including spindle cells with intracytoplasmic globules are noted (smear, Romanowsky stain). (B) Pronounced nuclear atypia and numerous mitoses are seen (cell block, hematoxylin and eosin stain).

DIFFERENTIAL DIAGNOSIS OF POORLY DIFFERENTIATED HEPATOCELLULAR CARCINOMA • C  holangiocarcinoma • Metastatic carcinoma   

Poorly differentiated HCCs, if well sampled, are usually obviously malignant lesions. The difficulty is in distinguishing them from cholangiocarcinoma and metastatic carcinoma. To complicate matters, the existence of a rare combined HCC and cholangiocarcinoma presents a special diagnostic challenge.105-107 The clear cell (Fig. 13.18A and  B) and fatty change (Fig. 13.19) variants of HCCs mimic metastatic tumors, most notably those of renal, adrenal, or germ cell origin.108,109 Hyaline globules are seen in HCC, but also in other tumors such as renal cell carcinoma.110 Special stains are helpful.111,112 Intracytoplasmic mucin is extremely uncommon in HCC but present in most adenocarcinomas, including cholangiocarcinoma. The markers of hepatocellular differentiation—canalicular staining for polyclonal CEA, HepPar 1, TTF-1 (cytoplasmic staining, clone dependent), ARG-1 (arginase-1), and glypican-3— are very useful in distinguishing HCC from cholangiocarcinoma and metastatic adenocarcinoma (Fig. 13.20A–D). Polyclonal CEA show a distinctive linear staining pattern outlining bile canaliculi (Fig. 13.20A); this is not seen with adenocarcinomas, which instead show diffuse cytoplasmic staining.113-119 HepPar1 has high sensitivity and specificity for HCC (Fig. 13.20B), but there may not be uniform staining,114,120-122 and aberrant HepPar1 staining is sometimes seen in tumors other than HCC.121-123 Glypican-3 is another very useful marker for HCC124-128 and may be a more sensitive marker of hepatocellular

A

B • Fig. 13.18  Hepatocellular

Carcinoma, Clear Cell Variant.  (A) As with many hepatocellular carcinomas, abundant bare nuclei are present. The cells have been disrupted, and the abundant spilled cytoplasmic glycogen gives a mottled appearance to the background that is reminiscent of the “tigroid” background seen with another glycogen-rich tumor, the seminoma (see Fig. 2.48) (Romanowsky stain). (B) Intact clear cells are better seen in the cell block sections (hematoxylin and eosin stain).

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•

Fig. 13.19  Hepatocellular Carcinoma, Fatty Change Variant.  The malignant cells are distended by sharply outlined, large fat vacuoles (Romanowsky stain).

differentiation than HepPar1 (see Fig. 13.15).129,130 Clonedependent TTF-1 expression in normal hepatocytes and HCCs is in a granular cytoplasmic (rather than nuclear) staining pattern and is a useful marker of hepatocytic differentiation (Fig. 13.20C).72,131,132 The cytoplasmic staining of TTF-1 appears to be mitochondrial, similar to that of HepPar1.133,134 ARG-1 (Arginase-1) is the newest marker of hepatocellular differentiation and effectively distinguishes between HCC and metastatic carcinomas (Fig. 13.20D)135138; however, a small subset (10%) of well-differentiated HCCs can be ARG-1 negative.139 It is important to note that polyclonal CEA, HepPar 1, cytoplasmic TTF-1, and ARG-1 are merely markers of hepatocytic differentiation; they do not distinguish benign from malignant hepatocytes. CD34 is useful for highlighting the endothelial cells that wrap the thickened cords of HCC.88,99 Numerous spider-shaped, vimentin-positive Kupffer cells are noted in HCCs and not in metastatic carcinomas.140 Organ-specific

HepPar1

pCEA

A

B ARG-1

TTF-1

C

437

D • Fig. 13.20  Immunomarkers of Hepatocytic Differentiation.  The markers illustrated here help distinguish hepatocellular carcinoma (HCC) from cholangiocarcinoma and metastatic carcinoma. (All images are of HCCs.) (A) Immunoreactivity with polyclonal antibodies to carcinoembryonic antigen leaves the malignant cells unstained but outlines the bile canaliculi as short, discontinuous linear threads and small lumina. (B) Most HCCs are immunoreactive for HepPar1. (C) Most HCCs show cytoplasmic immunoreactivity for TTF1. (D) Most HCCs show cytoplasmic and sometimes nuclear reactivity for ARG-1.

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immunohistochemical markers (e.g., PAX8, prostate specific antigen) can be helpful in selected circumstances as dictated by clinical and imaging findings. The fibrolamellar variant of HCC deserves special mention because of its unusual cytomorphology and histomorphology and clinical presentation. It is seen predominantly in young patients (average age, mid-20s) who do not have cirrhosis, and their prognosis is favorable. Histologically, the malignant cells are large and polygonal, with abundant eosinophilic cytoplasm. Dense bands of fibrosis surround tumor cells. This tumor has a recurrent somatic intrachromosomal deletion on chromosome 19, leading to a DNAJB1-PRKACA fusion transcript. The DNAJB1PRKACA fusion can be detected by the polymerase chain reaction and by fluorescence in situ hybridization (FISH) and is a very sensitive and specific finding in support of the diagnosis of fibrolamellar carcinoma.141

CYTOMORPHOLOGY OF FIBROLAMELLAR HEPATOCELLULAR CARCINOMA • • • • • •

 arge cells with abundant cytoplasm L Large nucleus Very prominent nucleolus Mostly isolated cells or loose clusters Hyaline intracytoplasmic globules (some cells) Bands of fibrosis separating neoplastic cells   

The cells of fibrolamellar HCC are much larger than normal hepatocytes and larger than the cells of a welldifferentiated HCC (Fig. 13.21A and B).142 Notably, the nuclear-to-cytoplasmic ratio is lower in fibrolamellar HCC than in ordinary HCC.142 In contrast to typical HCC, fibrolamellar HCC and pediatric HCCs are more likely to stain positively with CK7 than adult HCCs.143-145 

A

B

• Fig. 13.21  Fibrolamellar Hepatocellular Carcinoma.  (A) Large, atypical hepatocytes are accompanied

by fibrous tissue. (B) The malignant cells have abundant granular cytoplasm and intracytoplasmic hyaline globules (arrows) (Papanicolaou stain).

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439

Cholangiocarcinoma Cholangiocarcinoma is the second most common primary hepatic malignancy after HCC, representing 5% to 15% of primary liver cancers.68 Risk factors include primary sclerosing cholangitis, hepatolithiasis (more common in the Far East), parasitic infestation of the liver by Clonorchis sinensis, nonbiliary cirrhosis, and deposition of Thorotrast. Cholangiocarcinomas can arise from the intrahepatic and extrahepatic bile ducts; a tumor arising from the right or left hepatic ducts near their junction is called a hilar cholangiocarcinoma (or Klatskin tumor). Obstructive jaundice is the most common presentation in patients with extrahepatic tumors. Significant elevation of Ca19-9 and CEA can be seen. The diagnosis is made in 60% to 70% of patients using bile cytology, biliary brush cytology, or FNA.146,147 Increasingly, EUS FNA is used to diagnose this tumor,148-155 and transperitoneal biopsy has been associated with an increased risk of peritoneal metastases.156

A

CYTOMORPHOLOGY OF CHOLANGIOCARCINOMA • Isolated cells, crowded sheets and cell clusters • Glandular differentiation (e.g., mucin vacuoles, acinar structures) • Nuclear enlargement, with variation in size and shape   

Histologically and cytologically, cholangiocarcinomas resemble adenocarcinomas of the pancreas and demonstrate a similar variety of morphologic types and range of differentiation (Fig. 13.22A and B). Smears typically show crowded sheets of cells with marked anisonucleosis, and atypical glands can be seen in cell block sections. DIFFERENTIAL DIAGNOSIS OF CHOLANGIOCARCINOMA • H  epatocellular carcinoma • Metastatic adenocarcinoma   

The cells of most cholangiocarcinomas are cuboidal or columnar rather than polygonal. Neither bile nor endothelial wrapping of thickened trabeculae, so typical of HCC, are seen with cholangiocarcinoma. Cholangiocarcinoma is a gland-forming tumor; mucin production may be abundant.157 Positive staining for mucicarmine, AE1 keratin, and diffuse cytoplasmic staining for polyclonal CEA are characteristic of cholangiocarcinoma and very uncommon in HCC. (The canalicular staining pattern for polyclonal CEA is not seen in cholangiocarcinoma.) Immunoreactivity for CK7, CK17, and CK19 is typical of cholangiocarcinomas and less so for HCC, whereas the reverse is true for CK8/18.158 Hepatitis B viral antigens are uncommon in cholangiocarcinoma.

B • Fig. 13.22  Cholangiocarcinoma.  (A) The tumor cells are in crowded

sheets. Haphazard cellular arrangement and glandular differentiation are apparent (Papanicolaou stain). (B) Anisonucleosis and nuclear membrane irregularity are present (Romanowsky stain).

The distinction between cholangiocarcinoma and metastatic adenocarcinoma is challenging. Because cholangiocarcinomas and pancreatic cancers are morphologically and immunophenotypically similar, the distinction between these entities is based on anatomic location rather than pathologic characteristics. Of note, overexpression of p53 and loss of SMAD4 by immunohistochemistry are seen in both cholangiocarcinomas and pancreatic adenocarcinomas.159 The presence of proliferating ductules, in particular more than 10 ductular clusters, has been suggested as a cytologic feature to distinguish cholangiocarcinoma from metastatic adenocarcinoma.160 In general, the diagnosis of cholangiocarcinoma is made only after ruling out other primary sites clinically. 

Hepatoblastoma Hepatoblastoma is a rare malignant tumor of infancy and early childhood: 90% of these tumors present before the age of 5 years. Although rare, it is the most common malignant liver tumor in the pediatric population. Histologically, it shows a wide range of hepatocellular differentiation, from

440 CHA P T ER 1 3   Liver

small undifferentiated cells to well-differentiated cells that resemble fetal hepatocytes. The myriad histopathologic patterns include small cell undifferentiated, embryonal, fetal (with several subdivisions), macrotrabecular, and cholangiocellular.161 The most common patterns are the embryonal (30% of hepatoblastomas) and fetal (80% to 90% of hepatoblastomas); most tumors have two or more epithelial patterns. The small cell undifferentiated component confers a worse prognosis, whereas a tumor comprised exclusively of the fetal pattern with low mitotic activity has an excellent prognosis. Up to 40% of hepatoblastomas have a mesenchymal component, usually monomorphic spindle cells, but some cases have cartilage, osteoid, or both. Hepatoblastomas, especially the embryonal pattern, can show nuclear expression of β-catenin. Loss of nuclear INI-1 expression is seen in the small cell undifferentiated pattern of some hepatoblastomas. At many institutions, patients with hepatoblastoma receive chemotherapy before resection. Whether an FNA is sufficient for the diagnosis of hepatoblastoma is controversial: an international consortium sponsored by the Children’s Oncology Group Liver Tumor Committee recommends against FNA for diagnosis,162 but others have found that FNA accurately diagnoses and reliably subtypes hepatoblastomas.163 Cytologic findings mirror the histopathology, and there is often a mixture of different patterns.163-167 The neoplastic cells of the fetal pattern are usually monomorphic; although smaller than normal adult hepatocytes, they have a low nuclear-to-cytoplasmic ratio, resembling normal fetal hepatocytes or a well-differentiated hepatocellular neoplasm. Nuclei are round and nucleoli are small. Cytoplasmic glycogen can be abundant.168 The embryonal pattern is characterized by clusters, sheets, and rosettes of neoplastic cells with an increased nuclear-to-cytoplasmic ratio, nuclear crowding, prominent nucleoli, and hyperchromasia (Fig. 13.23). The small cell undifferentiated pattern shows noncohesive small round cells with irregular nuclear membranes,

inconspicuous nucleoli, hyperchomasia, and a very high nuclear-to-cytoplasmic ratio. The mesenchymal pattern may consist of fascicles of spindle cells and osteoid.163,167 The distinction from HCC can be challenging. The diagnosis rests entirely on an assessment of the cytomorphologic features in the context of clinical findings; immunohistochemistry is not useful. Serum levels of AFP are elevated in almost all cases of hepatoblastoma (small cell undifferentiated hepatoblastomas are an exception), and therefore a liver tumor in a patient with a normal serum AFP level is most likely not a hepatoblastoma. (Elevated AFP levels are not useful as a discriminant because they can be seen in HCC and other liver diseases.) Age over 5 years and underlying liver disease favor HCC. Childhood HCC usually arises in older individuals compared with hepatoblastoma and is usually of fibrolamellar type. The neoplastic cells of a fibrolamellar HCC are larger than those of hepatoblastoma and more polygonal; they have a prominent nucleolus, and intracytoplasmic hyaline deposits and stromal fragments are characteristic. Although some classical HCCs are composed of patterns similar to those of hepatoblastoma (e.g., fetal, macrotrabecular), in many cases the cells of HCC are large and variable in size, with a large nucleus and prominent nucleolus. 

Angiosarcoma Angiosarcoma is an uncommon malignant tumor of endothelial cells that represents less than 1% of primary hepatic malignancies. Most cases arise in adults, but the tumor can occur in persons of any age. Like HCC, angiosarcoma is associated with cirrhosis: about one-third of adult cases arise in this setting. It is seen with increased frequency in workers who are exposed to polyvinyl chloride and in patients who received thorium dioxide (Thorotrast) as a radiographic contrast agent. Histologically, these are spindle or epithelioid cell neoplasms that range from well-differentiated tumors with well-formed vascular channels to poorly differentiated tumors. Massive bleeding is a potential complication of FNA.169 CYTOMORPHOLOGY OF ANGIOSARCOMA • W  ell-differentiated tumors: elongated cells; isolated cells, tightly cohesive clusters, and syncytia • P  oorly differentiated tumors: large, spindle-shaped or epithelioid cells, pleomorphic nuclei, multinucleated giant cells • “Rhabdoid” forms in some epithelioid angiosarcomas 170 • Abundant finely or coarsely vacuolated cytoplasm, often with intracytoplasmic lumina 171 • Red blood cells in cytoplasm or vacuoles 172 • Bar/bullet-shaped nucleoli and chromatin stranding 172 • Cell blocks useful in demonstrating the characteristic anastomosing vascular pattern   

•

Fig. 13.23  Hepatoblastoma.  The embryonal pattern is illustrated here, exemplified by crowded, overlapping cells with prominent nucleoli. The patient was a 7-month-old baby girl with a large liver mass and a markedly elevated serum AFP level. (Case courtesy Dr. Martha Pitman, Massachusetts General Hospital, Boston, MA.)

Angiosarcoma is a rare neoplasm and difficult to recognize by cytomorphology. The epithelioid variant is especially tricky, inasmuch as it is often misinterpreted, at least initially, as a carcinoma.173 Expression of keratins in epithelioid

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angiosarcoma is well described and further complicates distinction from carcinoma.172 DIFFERENTIAL DIAGNOSIS OF ANGIOSARCOMA • E  pithelioid hemangioendothelioma • Other malignant neoplasms   

Angiosarcoma cells are larger, more atypical and more monomorphous than those of an epithelioid hemangioendothelioma, and they are more likely to be cohesive.174 They may have a large nucleolus and intracytoplasmic vacuoles.174 Positive immunoreactivity for the endothelial markers CD34, CD31, and ERG is helpful for confirming the diagnosis of a vascular neoplasm.171,173,175-179 

Epithelioid Hemangioendothelioma Epithelioid hemangioendothelioma (EHE) arises in the liver and at other sites. Like angiosarcoma, it is a tumor of endothelial cells, but its course is less aggressive. The cytologic findings are suggestive,77,174,180 and immunostains for endothelial markers (CD31, CD34, ERG, Fli-1) are very useful in confirming the diagnosis.181,182 A translocation resulting in a WWTR1-CAMTA1 gene fusion is present in approximately 90% of EHEs from all primary sites, and can be detected by various methods, including FISH and reverse transcription-polymerase chain reaction.182,183  CYTOMORPHOLOGY OF EPITHELIOID HEMANGIOENDOTHELIOMA (see Fig. 4.18) • H  ypocellular (some cases) with a clean background • Isolated, large polymorphous cells with a folded nuclear outline • Fragments of metachromatic stroma77 • Frequent binucleated or multinucleated giant cells • Round or irregular nucleoli and prominent nucleoli • Abundant lacy or dense cytoplasm, sometimes with intracytoplasmic lumina77,184   

Metastatic Tumors The liver is a common site for metastases, and most liver masses sampled by FNA prove to be metastatic malignancies, most often metastatic carcinomas.185 Common primary tumors include those of the colon and rectum, lung, pancreas, stomach, and breast. As one might expect, the cytologic picture varies greatly depending on the tumor. If a patient has a known primary and the FNA demonstrates malignant cells, establishing a morphologic match by comparing the FNA with available histologic or cytologic slides from the original tumor is very helpful, and the cost of immunohistochemistry can be avoided in such cases. If there is no history of a primary outside the liver, cytologic

441

features may suggest a specific primary site or a limited differential diagnosis. Correlating the cytomorphology with clinical findings allows one to select a tailored immunohistochemical panel to further refine the interpretation. In some cases, the cytomorphology and immunoprofile prove relatively nonspecific. In such cases, it can be helpful to suggest the most likely primary sites. CYTOMORPHOLOGY OF COMMON METASTATIC MALIGNANCIES TO THE LIVER • • • • • • • • • •

Colorectal cancers • Tall, dark, and necrotic (Fig. 13.24A and B) • Immunoreactive for CK20, CDX2, and SATB2 Gastric cancer • Intestinal or signet-ring cell morphology Breast cancer • Ductal or lobular, variable differentiation • Signet-ring cells • Immunoreactive for GATA-3, mammaglobin, GCDFP15, estrogen receptor, progesterone receptor, HER2neu Prostate (Fig. 13.25A and B) • Microacini • Prominent nucleoli • Immunoreactive for prostatic acid phosphatase, NKX3.1, and prostate-specific antigen Well-differentiated neuroendocrine tumor (Fig. 13.26) • Eccentrically placed nucleus • “Salt-and-pepper” chromatin pattern • Abundant granular cytoplasm • Isolated cells, loosely cohesive clusters and rosettes • Immunoreactive for synaptophysin, chromogranin, and CD56 • Immunoreactive for islet-1 and PAX8 (if pancreatic endocrine tumor) Small cell carcinoma (Fig. 13.27) • Isolated and loosely cohesive cells • Nuclear molding • Hyperchromatic nucleus with finely granular chromatin and small nucleolus • Round, polygonal, or spindled cells • Paranuclear blue bodies (see Fig. 2.41B) Urothelial carcinoma • Small clusters and isolated cells • Cercariform cells • Nuclear enlargement, hyperchromasia, and membrane irregularity • Coarse chromatin and prominent nucleoli (Fig. 13.28) Squamous cell carcinoma • Small, dark nucleus without discernible texture • Abundant cytoplasm with a hard, glassy appearance (orange with Papanicolaou stain, gray with Romanowsky-type stain) • P  oorly differentiated, nonkeratinized: hard to distinguish from other poorly differentiated malignancies • Immunoreactive for keratins and p63 Malignant melanoma (Fig. 13.29) • Prominent isolated cell pattern • Intranuclear pseudoinclusions, macronucleolus • Abundant cytoplasm, sometimes pigmented • Melanin pigment is finely granular (bile pigment more variable in size) • Immunoreactive for S-100, HMB-45, Mart-1, SOX-10 Sarcomas (Fig. 13.30A and B, Fig. 13.31) • Spindle-shaped, round, or pleomorphic cells   

442 CHA P T ER 1 3   Liver

A

B •

Fig. 13.24  Metastatic Colonic Carcinoma.  (A) Colorectal cancer cells often have a “picket fence” arrangement and are associated with a dirty, necrotic background (cell block, hematoxylin and eosin stain). (B) The columnar, picket-fence arrangement is apparent on smears as well. Nuclei are enlarged and ovoid (smear, Papanicolaou stain).

A

B • Fig. 13.25  Metastatic Prostatic Carcinoma.  (A) Acinar structures are characteristic of prostate cancers

(cell block, hematoxylin and eosin stain). (B) Immunoreactivity for prostate-specific antigen confirms the diagnosis (cell block).

Colorectal cancers have a predilection for metastasizing to the liver, and some patients present with liver metastases and an unknown primary. These tumors have a characteristic cytomorphology: the cells are large and columnar (“tall”), nuclei are hyperchromatic (“dark”), and necrosis with abundant nuclear debris (“dirty necrosis”) is often present; hence, the familiar mnemonic for FNAs from colorectal cancers: “tall, dark, and necrotic.” The impression can be

confirmed by demonstrating the typical immunoprofile of colon cancer: positive for CK20, CDX2, and SATB2, and negative for CK7. Esophageal and gastric adenocarcinomas resemble colorectal cancers. A larger proportion, however, have a prominent component of signet-ring cells. Gastric and esophageal adenocarcinomas have a similar immunoprofile and cannot be distinguished from each other by FNA of a

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A

B •

Fig. 13.26  Metastatic Carcinoid Tumor.  (A) The tumor cells are monomorphic and arranged in loose clusters. This appearance should raise the possibility of a neuroendocrine tumor (Romanowsky stain). (B) The cell block shows cords and trabeculae of cells with a granular chromatin pattern (hematoxylin and eosin stain).

A

B • Fig. 13.27  Metastatic Small Cell Carcinoma.  (A) Note the abundant small blue cells with scant cytoplasm and pronounced molding. Nuclear crush artifact is prominent (Romanowsky stain). (B) The cells are isolated and arranged in loosely cohesive clusters. The nuclear-to-cytoplasmic ratio is very high, nuclear molding is seen, and the chromatin is finely granular without nucleoli (Papanicolaou stain).

• Fig. 13.28  Metastatic Urothelial Carcinoma.  The tumor cells are isolated and in small clusters. A cercariform cell (upper right) is a clue that the primary is a urothelial carcinoma (Papanicolaou stain).

•

Fig. 13.29  Metastatic Melanoma.  Melanin is more finely granular than bile. Melanoma cells are usually isolated rather than clustered. The nucleoli of melanoma cells are usually prominent and solitary (Papanicolaou stain).

443

444 CHA P T ER 1 3   Liver

A

B

• Fig. 13.30  Metastatic Gastrointestinal Stromal Tumor.  (A) There are cohesive groups of spindle-shaped cells. Nuclear atypia is minimal in this case (Papanicolaou stain). (B) The spindle-cell proliferation is also apparent on cell block sections. The tumor cells were immunoreactive for CD117 (c-kit) (not shown) (hematoxylin and eosin stain).

•

Fig. 13.31  Metastatic Leiomyosarcoma.  Fascicles of neoplastic spindle cells have cigar-shaped nuclei and homogenous cytoplasm (Papanicolaou stain). (Case courtesy Dr. Edward G. Bernacki Jr., Beaumont Health System, Royal Oak, MI.)

metastatic lesion. Gastric cancers are heterogeneous in their CK7, CK20, and CDX2 profile, although CDX2 immunoreactivity, when present, is often patchy and weaker than the strong and diffuse staining seen in most colorectal cancers. Both ductal and lobular breast cancers are encountered as metastases to the liver. It is very uncommon for a breast primary tumor to be occult when the patient presents with metastases to the liver. Well-differentiated (low- and intermediate-grade) neuroendocrine tumors (NETs), whether carcinoids (of gastrointestinal or lung origin) or pancreatic NETs, all have a similar cytomorphology, and all are usually positive for the generic neuroendocrine markers synaptophysin, chromogranin, or CD56. Islet 1 and PAX8 are relatively specific for NETs of the pancreas, however, and thus helpful in confirming pancreatic origin if one or both is positive. (Of course, the presence of a pancreatic mass by imaging is also helpful!) The proliferative rate is used to grade NETs

because it provides useful prognostic information and may influence clinical management. In some patients, the only pathologic specimen might be a liver metastasis sampled by FNA, thus grading of liver FNA samples is appropriate. Gastrointestinal and pancreatic NETs are graded by assessing the number of mitoses per 10 high power fields (hpf ): low-grade (G1) tumors have less than 2 mitoses per 10 hpf, intermediate-grade (G2) tumors 2 to 20 per 10 hpf, and high-grade tumors (neuroendocrine carcinoma) greater than 20 per 10 hpf. Counting mitoses is challenging with small specimens like FNAs, and therefore staining for Ki67 (MIB-1) is a useful surrogate: it provides an accurate assessment of the proliferative rate and is readily applicable to smaller specimens. If the Ki67 index is less than 3%, the tumor is low-grade (G1); if 3% to 20% it is of intermediate grade (G2); and if the tumor is poorly differentiated and the index is greater than 20% it is a neuroendocrine carcinoma (G3) 186 (see Fig. 14.16A and B). A subset of welldifferentiated pancreatic NETs have a Ki-67 index greater than 20% and are classified as a well-differentiated NET, G3.187 “Well-differentiated neuroendocrine tumor G3” will likely be adopted for other gastrointestinal sites as well. A grade assigned on the basis of an FNA or core biopsy, however, needs to take into account the possibility that a highergrade focus may not have been sampled. GISTs of the stomach and small intestine have a propensity to metastasize to the liver. Immunoreactivity for c-kit and DOG1 distinguishes GISTs from smooth muscle tumors, which are positive for desmin and smooth muscle actin but negative for c-kit and DOG1. Positive staining for c-kit (CD117) and/or DOG1 is virtually essential for the diagnosis of a GIST (see Fig. 7.18B). DOG1 (“discovered on GIST-1”) is a protein of unknown function that was identified from gene expression data as a highly sensitive and specific marker for GIST. It is more sensitive than c-kit; that is, some GISTs are negative for c-kit but positive for DOG1.188

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Extramedullary hematopoiesis in the liver, seen in patients with myeloproliferative disorders, can be diagnosed when megakaryocytes, erythroid precursors, and myeloid precursors are identified. Lymphomas occur as metastatic or primary tumors of the liver,189 and features are described in Chapter 12. Germ cell tumors may metastasize to liver. Seminomas and dysgerminomas are immunoreactive for placental alkaline phosphatase (PLAP) (cytoplasmic and membrane staining), CD117 (c-kit; membrane staining), and the stem cell-related proteins Oct-3/4 (synonymous with Oct-4), NANOG, and SALL4 (nuclear staining for all three) (see Fig. 16.16B–D). PLAP and SALL4 are expressed in most germ cell tumors, whereas Oct-3/4 and NANOG are only expressed in dysgerminoma/seminoma and embryonal carcinoma. The membranous staining pattern for CD117 is very characteristic of seminoma/dysgerminoma and not seen in embryonal carcinoma or yolk sac tumor. Unlike the embryonal and yolk sac tumors, dysgerminomas generally show no immunoreactivity for keratin proteins, epithelial membrane antigen, AFP, or CEA.

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281. Williamson SR, Grignon DJ, Cheng L, et al. Renal cell carcinoma with chromosome 6p amplification including the TFEB gene: a novel mechanism of tumor pathogenesis? Am J Surg Pathol. 2017;41(3):287–298. 282. Comprehensive molecular characterization of clear cell renal cell carcinoma. Nature. 2013;499(7456):43–49. 283. Calio A, Grignon DJ, Stohr BA, Williamson SR, Eble JN, Cheng L. Renal cell carcinoma with TFE3 translocation and succinate dehydrogenase B mutation. Mod Pathol. 2017;30(3):407–415. 284. Gupta K, Nijhawan R. Renal-cell carcinoma in a three-year-old child (letter). Diagn Cytopathol. 2004;30:369–370. 285. Schinstine M, Filie AC, Torres-Cabala C, Abati A, Linehan WM, Merino M. Fine-needle aspiration of a Xp11.2 translocation/TFE3 fusion renal cell carcinoma metastatic to the lung: report of a case and review of the literature. Diagn Cytopathol. 2006;34:751–756. 286. Barroca H, Correia C, Castedo S. Cytologic and cytogenetic diagnosis of pediatric renal cell carcinoma associated with t(X;17). Acta Cytol. 2008;52(3):384–386. 287. El Naili R, Nicolas M, Gorena A, Policarpio-Nicolas ML. Fineneedle aspiration findings of Xp11 translocation renal cell carcinoma metastatic to a hilar lymph node. Diagn Cytopathol. 2017;45(5):456–462. 288. Murphy WM, Grignon EJ, Perlman EJ. Tumors of the Kidney, Bladder and Related Urinary Structures. Vol. 1. Washington, DC: Armed Forces Registry of Pathology; 2004. 289. Marks-Jones DA, Zynger DL, Parwani AV, Cai G. Fine needle aspiration biopsy of renal mucinous tubular and spindle cell carcinoma: report of two cases. Diagn Cytopathol. 2010;38(1):51–55. 290. Owens CL, Argani P, Ali SZ. Mucinous tubular and spindle cell carcinoma of the kidney: cytopathologic findings. Diagn Cytopathol. 2007;35(9):593–596. 291. Huimiao J, Chepovetsky J, Zhou M, et  al. Mucinous tubular and spindle cell carcinoma of the kidney: Diagnosis by fine needle aspiration and review of the literature. Cytojournal. 2015;12:28. 292. Ortega JA, Solano JG, Perez-Guillermo M. Cytologic aspect of mucinous tubular and spindle-cell renal carcinoma in fineneedle aspirates. Diagn Cytopathol. 2006;34:660–662. 293. Santamaria M, Jauregui I, Urtasun F, Bertol A. Fine needle aspiration biopsy in urothelial carcinoma of the renal pelvis. Acta Cytol. 1995;39:443–448. 294. Friedman HD, Nsoulis IS, Krauss DJ, Vohra S, Powers CN. Transitional cell carcinoma arising in a pyelocaliceal cyst. An unusual cystic renal lesion with cytologic and imaging findings. Virchows Arch. 1999;434:459–462. 295. Powers CN, Elbadawi A. “Cercariform” cells: a clue to the cytodiagnosis of transitional cell origin in metastatic neoplasms? Diagn Cytopathol. 1995;13:15–21. 296. Renshaw AA, Madge R. Cercariform cells for helping distinguish transitional cell carcinoma from non-small cell lung carcinoma in fine needle aspirates. Acta Cytol. 1997;41:999–1007. 297. Truong LD, Caraway N, Ngo T, Laucirica R, Katz R, Ramzy I. Renal lymphoma. The diagnostic and therapuetic roles of fineneedle aspiration. Am J Clin Pathol. 2001;115:18–31. 298. Bynum JP, Duffield A, Ali SZ. Importance of flow cytometry in the cytopathologic evaluation of lymphoid lesions involving the kidney. Acta Cytol. 2016;60(2):131–138. 299. Subhawong AP, Subhawong TK, Vandenbussche CJ, Siddiqui MT, Ali SZ. Lymphoproliferative disorders of the kidney on fine-needle aspiration: cytomorphology and radiographic correlates in 33 cases. Acta Cytol. 2013;57(1):19–25.

517

300. Bracken RB, Chica G, Johnson DE. Secondary renal neoplasms, an autopsy study. South Med J. 1979;72:806–807. 301. Gattuso P, Ramzy I, Truong LD, et al. Utilization of fine-needle aspiration in the diagnosis of metastatic tumors to the kidney. Diagn Cytopathol. 1999;21:35–38. 302. Giashuddin S, Cangiarella J, Elgert P, Levine PH. Metastases to the kidney: eleven cases diagnosed by aspiration biopsy with histologic correlation. Diagn Cytopathol. 2005;32:325–329. 303. Khurana KK, Powers CN. Basaloid squamous carcinoma metastatic to renal-cell carcinoma: fine needle aspiration cytology of tumor-to- tumor metastasis. Diagn Cytopathol. 1997;17:379–382. 304. Tabatabai ZL, Staerkel GA. Distinguishing primary and metastatic conventional renal cell carcinoma from other malignant neoplasls in fine-needle aspiration biopsy specimens. Arch Pathol Lab Med. 2005;129:1017–1021. 305. Zhou C, Urbauer DL, Fellman BM, et al. Metastases to the kidney: a comprehensive analysis of 151 patients from a tertiary referral centre. BJU Int. 2016;117(5):775–782. 306. Simsir A, Chhieng D, Wei XJ, Yee H, Waisman J, Cangiarella J. Utility of CD10 and RCCma in the diangosis of metastatic conventional renal-cell adenocarcinoma by fine-needle aspiration biopsy. Diagn Cytopathol. 2005;33:3–7. 307. Laury AR, Perets R, Piao H, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 2011;35(6):816–826. 308. Liu H, Shi J, Wilkerson ML, Lin F. Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas. Am J Clin Pathol. 2012;138(1):57–64. 309. Grignon DJ, Ro JY, Ayala AG. Primary mucin-secreting adenocarcinoma of the kidney. Arch Pathol Lab Med. 1988;112:847–849. 310. Capella C, Eusebi V, Rosai J. Primary oat cell carcinoma of the kidney. Am J Surg Pathol. 1984;8:855–861. 311. Ferry JA, Harris NL, Papanicolaou N, Young RH. Lymphoma of the kidney. Am J Surg Pathol. 1995;19:134–144. 312. Iyer VK, Agarwala S, Verma K. Fine-needle aspiration cytology of clear-cell sarcinoma of the kidney: study of eight cases. Diagn Cytopathol. 2005;33:83–89. 313. Radhika S, Bakshi A, Rajwanshi A, et  al. Cytopatology of uncommon malignant renal neoplasms in the pediatric age group. Diagn Cytopathol. 2005;32:281–286. 314. Nayak A, Iyer VK, Agarwala S. The cytomorphologic spectrum of Wilms tumour on fine needle aspiration: a single institutional experience of 110 cases. Cytopathology. 2011;22(1):50–59. 315. Shet T, Viswanathan S. The cytological diagnosis of paediatric renal tumours. J Clin Pathol. 2009;62(11):961–969. 316. Goregaonkar R, Shet T, Ramadwar M, Chinoy R. Critical role of fine needle aspiration cytology and immunocytochemistry in preoperative diagnosis of pediatric renal tumors. Acta Cytol. 2007;51(5):721–729. 317. Gupta R, Mathur SR, Singh P, Agarwala S, Gupta SD. Cellular mesoblastic nephroma in an infant: report of the cytologic diagnosis of a rare paediatric renal tumor. Diagn Cytopathol. 2009;37(5):377–380. 318. Portugal R, Barroca H. Clear cell sarcoma, cellular mesoblastic nephroma and metanephric adenoma: cytological features and differential diagnosis with wilms tumour. Cytopathology. 2008;19(2):80–85. 319. Thomson TA, Klijanienko J, Couturier J, et al. Fine-needle aspiration of renal and extrarenal rhabdoid tumors: the experience of the Institut Curie regarding 20 tumors in 13 patients. Cancer Cytopathol. 2011;119(1):49–57.

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320. Krishnamurthy S, Bharadwaj R. Fine needle aspiration cytology of clear cell sarcoma of the kidney. A case report. Acta Cytol. 1998;42:1444–1446. 321. Barroca HM, Costa MJ, Carvalho JL. Cytologic profile of rhabdoid tumor of the kidney: a report of 3 cases. Acta Cytol. 2003;47:1055–1058. 322. Li P, Perle MA, Scholes JV, Yang GC. Wilms’ tumor in adults: aspiration cytology and cytogenetics. Diagn Cytopathol. 2002;26:99–103. 323. Nayak A, Iyer VK, Agarwal S, Agarwala S. Fine needle aspiration cytology of fetal rhabdomyomatous and teratoid Wilms tumor. Acta Cytol. 2010;54(4):563–568. 324. Zhang DF, Li ZH, Gao DP, et  al. The CT and US features of Ewing’s sarcoma/primary neuroectodermal tumor of the kidney: two case reports and review of literature. Onco Targets Ther. 2016;9:1599–1603. 325. Serrano R, Rodriguez-Peralto JL, De Orbe GG, Melero C, de Agustin P. Intrarenal neuroblastoma diagnosed by fine-needle aspiration a report of two cases. Diagn Cytopathol. 2002;27:294–297. 326. Parvin S, Ghosh R, Das RN, et al. Primary renal rhabdomyosarcoma: an unusual bone metastasizing tumor of kidney. Fetal Pediatr Pathol. 2016;35(4):251–259. 327. Khayyata S, Grignon DJ, Aulicino MR, Al-Abbadi MA. Metanephric adenoma vs. Wilms’ tumor: a report of 2 cases with diagnosis by fine needle aspiration and cytologic comparisons. Acta Cytol. 2007;51(3):464–467. 328. Kulkarni MP, Gosavi AV, Anvikar AR, Ramteerthakar NA, Lanjewar DN. Cytodiagnosis of congenital mesoblastic nephroma: a case report. Diagn Cytopathol. 2013;41(3):234–238. 329. Bera G, Das RN, Bisht J, et al. Cytological diagnosis of mesoblastic nephroma: a report of three cases with summary of prior published cases. Diagn Cytopathol. 2016;44(10):823–827. 330. Kumar N, Jain S. Aspiration cytology of mesoblastic nephroma in an adult: diagnostic dilemma. Diagn Cytopathol. 2000;23:124–126. 331. Das DK, Shome DK, Garg A, Bhatt NC, Rath B. Pediatric acute leukemia presenting as bilateral rela enlargement: report of a case with fine needle aspirtion cytologic features suggestive of megakaryocytic differentiation. Acta Cytol. 2000;44:819–823. 332. Kumar R, Gautam U, Srinivasan R, et al. Primary Ewing’s sarcoma/primitive neuroectodermal tumor of the kidney: report of a case diagnosed by fine needle aspiration cytology and confirmed by immunocytochemistry and RT-PCR along with review of literature. Diagn Cytopathol. 2012;40(suppl 2):E156–161. 333. Agarwal S, Agarwal K. Rare pediatric adrenocortical carcinoma with oncocytic change: a cytologic dilemma. Endocr Pathol. 2011;22(1):40–43. 334. Rosa M. Cytomorphology of kidney “thyroidization.” Diagn Cytopathol. 2011;39(7):508–509. 335. Sun W, McGregor DK, Ordonez NG, Ayala AG, Caraway NP. Fine needle aspiration cytology of a low grade myxoid renal epithelial neoplasm: a case report. Acta Cytol. 2005;49:525–529. 336. Silverman JF, Nathan G, olson PR, Prichard J, Cohen JK. Fineneedle aspiration cytology of low-grade fibromyxoid sarcoma of the renal capsule. Diagn Cytopatyol. 2000;23:279–283. 337. Vesoulis Z, Rahmeh T, Nelson R, Clarke R, Lu Y, Dankoff J. Fine needle aspiration biopsy of primary renal synovial sarcoma. A case report. Acta Cytol. 2003;47:668–672. 338. Klijanienko J, Colin P, Couturier J, et  al. Fine-needle aspiration in desmoplastic small round cell tumor: a report of 10 new tumors in 8 patients with clinicopathological and molecular correlations with review of the literature. Cancer Cytopathol. 2014;122(5):386–393.

339. Patel YA, Dhalla S, Olson MT, Lennon AM, Khashab MA, Singh VK. Pancreatic metastasis from a solitary fibrous tumor of the kidney: a rare cause of acute recurrent pancreatitis. Pancreatology. 2013;13(6):631–633. 340. Singh C, Xie L, Schmechel SC, Manivel JC, Pambuccian SE. Epithelioid angiosarcoma of the kidney: a diagnostic dilemma in fine-needle aspiration cytology. Diagn Cytopathol. 2012;40(suppl 2):E131–139. 341. Grapsa D, Sakellariou S, Politi E. Fine-needle aspiration cytology of primary renal angiosarcoma with histopathologic and immunocytochemical correlation: a case report. Diagn Cytopathol. 2014;42(10):872–876. 342. Stoll LM, Li QK. Cytology of fine-needle aspiration of inflammatory myofibroblastic tumor. Diagn Cytopathol. 2011;39(9):663–672. 343. Nambirajan A, Jain D, Malik P, Arava S, Mathur SR. Metastatic alveolar soft part sarcoma of the lung: a morphologic pitfall on cytology and aberrant CD10 expression on histology. Diagn Cytopathol. 2016;44(3):250–254. 344. Deodhare S, Saap M. Adrenal histoplasmosis: diagnosis by fine needle aspiration biopsy. diagn Cytopathol. 1997;17:42–44. 345. Silverman SG, Mueller PR, Pinkney LP, Koenker RM, Seltzer SE. Predictive value of image-guided adrenal biopsy: analysis of results of 101 biopsies. Radiology. 1993;187(3):715–718. 346. Hamrahian AH, Ioachimescu AG, Remer EM, et  al. Clinical utility of noncontrast computed tomography attenuation value (Hounsfield units) to differentiate adrenal adenomas/hyperplasias from nonadenomas: Cleveland Clinic experience. J Clin Endocrinol Metab. 2005;90(2):871–877. 347. DeAgustin P, Lopez-Rios F, Alberti N, et al. Fine needle aspiration biopsy of the adrenal glands: a ten year experience. Diagn Cytopathol. 1999;21:92–97. 348. Dusenbery D, Dekker A. Needle biopsy of the aadrenal gland: retrospective review of 54 cases. Diagn Cytopathol. 1996;14:126–134. 349. Kindelberger D, Cibas ES. Evaluating metastatic carcinoma to the adrenal: utility of fine needle aspiration. Pathology Case Reviews. 2005;10:257–262. 350. Fassina AS, Borsato S, Fedeli U. Fine needle aspiration cytology (FNAC) of adrenal masses. Cytopathol. 2000;11:302–311. 351.  NIH State-of-the-Science statement on management of the clinically inapparent adrenal mass (“incidentaloma”). NIH Consens State Sci Statements. 2002;4–6;19(2):1–23. 352. Quayle FJ, Spitler JA, Pierce RA, Lairmore TC, Moley JF, Brunt LM. Needle biopsy of incidentally discovered adrenal masses is rarely informative and potentially hazardous. Surgery. 2007;142(4):497–502; discussion 502–494. 353. McCorkell SJ, Niles NL. Fine-needle aspiration of catecholamine-producing adrenal masses: a possibly fatal mistake. AJR Am J Roentgenol. 1985;145(1):113–114. 354. Baguet JP, Hammer L, Tremel F, et al. Metastatic pheochromocytoma: risks of diagnostic needle puncture and treatment by arterial embolization. J Hum Hypertens. 2001;15:209–211. 355. Min KW, Song J, Boesenberg M, et al. Adrenal cortical nodule mimicking small round cell malignancy on fine needle aspiration. Acta Cytol. 1988;32:543–546. 356. Suen KC, McNeely TB. Adrenal cortical cells mimicking small cell anaplastic carcinoma in a fine-needle aspirate. Mod Pathol. 1991;4(5):594–595. 357. Saboorian MH, Katz RL, Charnsangavej C. Fine needle aspiration cytology of primary and metastatic lesions of the adrenal gland. A series of 188 biopsies with radiologic correlation. Acta Cytol. 1995;39(5):843–851.

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358. Settakorn J, Sirivanichai C, Rangdaeng S, et al. Fine needle aspiration cytology of adrenal myelolipoma: case report and review of the literature. Diagn Cytopathol. 1999;21:409–412. 359. Kumar R, Dey P. Fine-needle aspiration cytology of nonneoplastic adrenal pathology. Diagn Cytopathol. 2016;44(6): 472–476. 360. Medeiros LJ, Weiss LM. New developments in the pathologic diagnosis of adrenal cortical neoplasms. A review. Am J Clin Pathol. 1992;97:73–83. 361. Weiss LM. Comparative histologic study of 43 metastasizing and nonmetastasizing adrenocortical tumors. Am J Surg Pathol. 1984;8:163–169. 362. Aubert S, Wacrenier A, Leroy X, et al. Weiss system revisited: a clinicopathologic and immunohistochemical study of 49 adrenocortical tumors. Am J Surg Pathol. 2002;26(12):1612–1619. 363. Wu HH, Cramer HM, Kho J, et  al. Fine needle aspiration cytology of benign adrenal cortical nodules. A comparison of cytologic findings with those of primary and metastatic adrenal malignancies. Acta Cytol. 1998;42:1352–1358. 364. Sangoi AR, Fujiwara M, West RB, et al. Immunohistochemical distinction of primary adrenal cortical lesions from metastatic clear cell renal cell carcinoma: a study of 248 cases. Am J Surg Pathol. 2011;35(5):678–686. 365. Chen KT. Extraneous cells of hepatic origin in adrenal fine needle aspiration as a diagnostic pitfall: a case report. Acta Cytol. 2005;49(4):449–451. 366. Mondal SK, Dasgupta S, Mandal PK, Sinha MG. Cytodiagnosis of myxoid adrenocortical carcinoma and role of immunocytochemistry to differentiate it from renal cell carcinoma. J Cytol. 2014;31(2):111–113. 367. Misic M, Vidas Z, Skegro D, Kocman B, Jelic-Puskaric B, Kardum-Skelin I. Fine needle aspiration cytology of adrenocortical carcinoma--case report. Coll Antropol. 2010;34(2):665–669.

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368. Jorda M, De MB, Madji M. Calretinin and inhibin are useful in separating adrenocortical neoplasms from pheochromocytomas. Appl Immunohistochem Mol Morph. 2002;10:67–70. 369. Sangoi AR, McKenney JK. A tissue microarray-based comparative analysis of novel and traditional immunohistochemical markers in the distinction between adrenal cortical lesions and pheochromocytoma. Am J Surg Pathol. 2010;34(3):423–432. 370. Wadih GE, Nance KV, Silverman JF. Fine-needle aspiration cytology of the adrenal gland. Fifty biopsies in 48 patients. Arch Pathol Lab Med. 1992;116(8):841–846. 371. Chu P, Wu E, Weiss LM. Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: a survey of 435 cases. Mod Pathol. 2000;13:962–972. 372. Fetsch PA, Powers CN, Zakowski MF, et al. Anit-alpha-inhibin: marker of choice for the consistent distinction between adrenocortical carcinoma and renal cell carcinoma in fine needle aspiration. Cancer. 1999;87:168–172. 373. Loy TS, Phillips RW, Linder CL. A103 immunostaining in the diagnosis of adrenal cortical tumors: an immunohistochemical study of 316 cases. Arch Pathol Lab Med. 2002;126:170–172. 374. Renshaw AA, Granter SR. Comparison of A103 and inhibin reactivity in adrenal cortical tumors: distinction from hepatocellular carcinoma and renal tumors. Mod Pathol. 1998;11:1160–1164. 375. Rishi M, Howard LN, Bratthauer GL, Tavassoli FA. Use of monoclonal antibody against human inhibin as a marker for sex cord stromal tumors of the ovary. Am J Surg Pathol. 1997;21:583–589. 376. Yang B, Ali SZ, Rosenthal DL. CD10 facilitates the diagnosis of metastatic renal cell carcinoma from primary adrenal cortical neoplasms in adrenal fine needle aspiration. Diagn Cytopathol. 2002;27:149–152. 377. Lam KY, Lo CY. Metastatic tumours of the adrenal glands; a 30-year experience in a teaching hospital. Clin Endocrinol. 2002;56:95–101.

16

Ovary EDMUND S. CIBAS

F

ine-needle aspiration (FNA) is a safe, minimally invasive, and relatively inexpensive procedure for the diagnosis of selected ovarian lesions.

INDICATIONS FOR FINE-NEEDLE ASPIRATION OF THE OVARY • C  onfirm the benign nature of an incidental cyst discovered during: • An infertility workup • Pregnancy • Confirm malignancy in a patient with a suspicious pelvic mass • Confirm recurrence of an ovarian tumor treated conservatively • Drain a tuboovarian abscess   

FNA of the ovary is often used for small, incidental cystic masses that appear benign on sonographic or laparoscopic examination.1-3 Incidental ovarian cysts are often discovered in women with infertility or during pregnancy.4 Aspiration cytology, in combination with a benign ultrasound appearance, is used to reassure the patient that an oophorectomy is not necessary.5 In some laboratories, estradiol levels in the fluid are also measured because these are usually elevated in follicle-derived cysts but not in epithelial lesions.6,7 Thus ultrasonography, cytology, and estradiol levels can form an effective “triple test” for distinguishing benign from malignant ovarian cysts. ULTRASOUND FEATURES OF A BENIGN OVARIAN CYST • • • • • •

 mall S Thin walled No septations No papillarity No solid areas Low echogenicity (sonolucency)   

In part because of this preselection, most ovarian FNAs are benign.8 The diagnosis of endometriosis is sometimes established by this means as an unsuspected cause of infertility.4 520

If imaging features suggest a malignancy, the clinician will often forego aspiration and recommend surgery. Because borderline and malignant ovarian tumors are treated by surgical resection, FNA is perceived as unnecessary in many such circumstances. Aspiration of suspicious ovarian masses is therefore uncommon, although it is recommended to confirm malignancy in patients with inoperable or metastatic disease.3,9,10 Nevertheless, based on their positive experience, some authors advocate cytologic assessment for suspicious, potentially early stage ovarian lesions.11

Obtaining the Specimen The aspiration can be carried out transrectally, transvaginally, percutaneously, or during laparascopic or open surgery.12 The method used depends on the size of the lesion, its location, and the resources available to the aspirator. Percutaneous transabdominal aspiration can be performed by palpation13 or, more commonly, with imaging guidance. Transvaginal and transrectal aspiration can be done by palpation using the Franzen needle guide13 originally developed for FNA of the prostate. Transvaginal aspiration under ultrasound guidance, with the probe placed in the vagina, can be performed in the outpatient setting with only intravenous sedation.4,8 Depending on the trajectory of the needle, the cyst fluid may be contaminated with squamous cells, mesothelial cells, or intestinal epithelium. Therefore knowledge of the route taken is important in evaluating the specimen.8,14-16 Aspirations are also performed when cysts are discovered during laparoscopy and laparotomy. Complications are uncommon. More than two decades ago, a pair of authors argued that aspiration of a malignant ovarian tumor causes seeding of the peritoneal cavity,17 but their evidence consisted of only two cases, both with ambiguous circumstances. Since then, there has been no documentation of tumor seeding; on the contrary, practitioners with considerable experience have reported no such cases.18,19 The risk of peritoneal seeding therefore is very low.19 Severe pelvic infection after transvaginal and transrectal FNA is seen in 1.3% of cases, however.18 Acute abdominal or pelvic pain is a contraindication to FNA because it may delay treatment of a serious condition such as torsion of a cyst.1 

CHAPTER 16 Ovary

Preparing the Specimen and Reporting Results Specimens are usually cyst fluids. Standard methods are used in the preparation of direct smears, cytocentrifuge preparations, thinlayer preparations, or cell block sections. A portion of the fresh fluid can be submitted to the clinical laboratory to measure the level of estradiol or tumorassociated antigens CA-125, carcinoembryonic antigen (CEA), and α-fetoprotein. Elevated levels are typical of some ovarian lesions and can be a useful adjunct to cytologic examination.18,20 Specimens that are virtually acellular or uninterpretable for other reasons are reported as nondiagnostic.21,22 The percentage of FNAs of the ovary that are nondiagnostic ranges widely, from a low of 13% to a high of 72%1,4,14,15,21-23; this variation is likely related to the type of lesion selected for aspiration and the definition of a nondiagnostic specimen. The cytology report should state that malignant cells are either absent (“no malignant cells identified”) or present (“positive for malignant cells”). If the findings are equivocal, the report should state that atypical or suspicious cells are present. Most ovarian FNAs are benign (90%); only a small percentage (about 2%) are interpreted as malignant, with the remainder falling into the atypical or suspicious categories.24 If sufficient benign lesional cells (granulosa cells, epithelial cells) are seen, descriptive terms can further categorize the cyst as a follicular, simple, serous, benign mucinous, endometriotic, or dermoid cyst.3 Benign ovarian FNA results fall into two broad categories: (1) follicular/lutein cysts, and (2) epithelial cysts. This has significant clinical relevance because surgery is unnecessary for follicle-derived cysts, which usually regress over time. Surgery may be indicated, however, for a benign-appearing epithelial cyst, particularly if the sonographic findings are worrisome. The lining of an epithelial cyst can vary from one area to another, and some cysts contain benign, borderline, and frankly malignant foci; sampling is not always representative of this heterogeneity. For this reason, an explanatory note accompanying the diagnosis of a benign epithelial cyst is helpful (e.g., “Clinical correlation is advised to ensure that the sample is representative of the underlying lesion”).25 

Accuracy The reported sensitivity for a diagnosis of malignancy ranges widely from 25% to 93%,14,18,23,24,26-31 although some of the higher estimates are likely inflated because borderline tumors were excluded from analysis.14,18 In fact, FNA of a borderline tumor often yields a false-negative result4,18,24,26,32; much of the lesion is acellular cyst fluid, and neoplastic epithelium may not be sampled. When borderline tumors are included, sensitivity is low, ranging from 26% to 54%.24,26,32,33 Low sensitivity is an argument against the aspiration of clinically suspicious ovarian masses. False-positive results have been reported.14,33 Follicle cysts are a notorious cause of false positives because they can

521

yield a cellular and highly mitotic specimen.9,34,35 Familiarity with the laparoscopic and sonographic findings should be taken into account when making a diagnosis. Knowing that a lesion appears benign clinically can help avoid a falsepositive interpretation. FNA of the ovary therefore has its limitations. Not only is there a high false-negative rate, particularly for borderline tumors, but the method cannot reliably distinguish between borderline tumors and carcinomas.21,29,36 (Because both lesions are treated by surgical resection, the inadequacy of FNA in this instance is not very significant.) Furthermore, benign lesions cannot always be histologically categorized by cytologic evaluation.20,29 Although in some cases FNA can establish a specific diagnosis such as endometriosis,8 at present it is most valuable for confirming a clinical impression that an ovarian cyst is benign, thus sparing the patient unnecessary surgery.1,21,37 

Benign Tumor-Like Lesions of the Ovary Nonneoplastic Cysts Benign ovarian cysts are common; most are discovered incidentally by ultrasonography, laparoscopy, or laparotomy. They fall into two major categories: the most common are the so-called “functional cysts” (i.e., cysts of follicular origin—follicle and corpus luteum cysts). The rest are nonfunctional cysts derived from ovarian surface epithelium or endometriosis. Precise classification by cytologic examination is not always possible, particularly when only cyst contents (fluid and macrophages) are obtained.38

Cystic Follicle and Follicle Cyst These two lesions have a similar histogenesis and are distinguished only on the basis of size. The arbitrary size cutoff is variably given as 2 cm, 2.5 cm, and 3 cm; if the size cutoff is exceeded, a cystic follicle is called a follicle cyst. One of the most common, if not the most common, cystic lesions of the ovary, they arise from an ovarian follicle and are physiological, not neoplastic. Ovarian follicles follow a predictable development from primordial follicle (oocyte surrounded by a flattened layer of granulosa cells), to primary follicle (increased size and mitotic activity of granulosa cells), to secondary follicle (stratification of granulosa cells), to the Graafian follicle (characterized by a distinct zone of fluid, an antrum for the oocyte, and proliferation of surrounding theca cells). Cystic follicles/follicle cysts occur when a Graafian follicle does not rupture but rather persists for a variable period of time. The remainder of this discussion focuses on the follicle cyst, which, because of its size, is more likely to manifest clinically. Follicle cysts can be solitary or multiple and range in size up to 8 cm or more in diameter. They look benign on sonographic and laparoscopic examination: they are unilocular, smooth-surfaced, translucent, and thin-walled. Most regress spontaneously within a few months. Multiple follicle cysts are common in juvenile hypothyroidism, ovarian hyperstimulation syndrome, and polycystic ovary disease.

522 CHA P T ER 1 6   Ovary

• Fig. 16.1  Follicle Cyst.  In this histologic section, the follicle cyst is collapsed, and the cavity is slit-like.

Follicle cysts are lined by one or several layers of granulosa cells. Mitotic activity (arrow) is common (hematoxylin and eosin stain).

Histologically, a follicle cyst is lined by an inner layer of attenuated or stratified granulosa cells and an outer layer of theca cells (Fig. 16.1); both may show luteinization. Fluid is clear or hemorrhagic. It may be sparsely cellular, containing only histiocytes and blood, or markedly cellular (sometimes alarmingly so), with numerous granulosa cells that are isolated and in large clusters. Because follicle cysts appear benign sonographically and laparoscopically, they are managed conservatively to preserve the ovary. FNA is commonly used as a minimally invasive method to confirm the diagnosis. CYTOMORPHOLOGY OF FOLLICLE CYST • • • • • •

• Fig. 16.2  Follicle Cyst.  Granulosa cells are clustered in loose aggre-

 parsely or highly cellular S Isolated cells and clusters Coarsely granular chromatin Mix of viable and pyknotic nuclei Foamy cytoplasm Mitoses

gates (Papanicolaou stain).

  

FNA specimens range from sparsely to highly cellular. The highly cellular cases are termed cellular follicle cysts and account for between 23% and 76% of all follicle cysts.21,39 The granulosa cells can be isolated, but they have a tendency to cluster haphazardly in loose spherical aggregates (Fig. 16.2). They have a round nucleus with coarsely granular chromatin and one or two small nucleoli. Nuclear grooves are not usually seen.34 Some nuclei are dark, pyknotic, and eccentrically placed, whereas others are round and vesicular with a prominent nucleolus. Mitotic figures can be absent or numerous

(Fig. 16.3). Luteinized granulosa cells have foamy cytoplasm that may contain yellow pigment. Theca interna cells have an eccentrically placed ovoid nucleus and a moderate amount of cytoplasm. Theca externa cells are indistinguishable from spindle-shaped ovarian stromal cells.40 DIFFERENTIAL DIAGNOSIS OF FOLLICLE CYST • • •

Epithelial cysts • Simple cyst • Serous cyst • Mucinous cyst • Endometriotic cyst Granulosa cell tumor Carcinoma   

CHAPTER 16 Ovary

523

•

Fig. 16.3  Follicle Cyst.  Granulosa cells have a round nucleus and a moderate amount of granular cytoplasm. Degenerated granulosa cells with pyknotic nuclei are a common finding, as are mitoses (arrow) (Papanicolaou stain).

• Fig. 16.4  Corpus Luteum Cyst.  These cysts are lined by very large

Follicle cysts can be a challenge to distinguish from epithelial cysts. When ciliated or mucinous epithelium is identified, the cyst is of surface epithelial origin and not a follicle cyst. Immunostaining for α-inhibin, EMA, and CK7 can be very helpful: granulosa cells are often immunoreactive for inhibin and negative for EMA and CK7; the reverse is true of epithelial cysts.41 Estradiol levels in cyst fluid are also helpful: elevated concentrations correlate strongly with cysts of follicular origin1,6,7,18: 81% to 90% of follicle cysts have an estradiol content greater than 20 nmol/L as measured by radioimmunoassay, and, in 97% to 99% of nonfollicular cysts, it is less than 20 nmol/L. Also helpful are fluid CEA and CA-125 levels, which are low in follicle cysts; one or more of these are usually elevated in serous, mucinous, and endometriotic cysts.20,37 The differential diagnosis also includes a cystic granulosa cell tumor. Fluid from the latter is usually highly cellular.42 Unlike the cells of a follicle cyst, those of a granulosa cell tumor have nuclei with pale, finely dispersed chromatin. Cellular follicle cysts are a potential pitfall: because of their cellularity and conspicuous mitotic activity, they may raise the possibility of a granulosa cell tumor or carcinoma.9,34,35 Knowledge of the invariably benign sonographic and laparoscopic findings of follicle cysts, including the cellular variant, is reassuring and diagnostically helpful. 

The fluid may be hemorrhagic and composed of numerous, predominantly isolated luteinized granulosa cells with round or pyknotic nuclei. The cytoplasm is abundant and finely vacuolated and contains lipids (Fig. 16.4). Macrophages with hemosiderin or the yellow hematoidin pigment and smaller luteinized theca interna cells are also seen.43 Cysts associated with pregnancy contain hyaline bodies and calcifications.21 Fluid from a hemorrhagic corpus luteum cannot always be distinguished cytologically from that of other benign cysts, including endometriotic cysts. 

Corpus Luteum Cyst Corpus luteum cysts are unilocular and histologically similar to follicle cysts, except that the wall is composed of large luteinized granulosa and theca interna cells. CYTOMORPHOLOGY OF CORPUS LUTEUM CYST • • • • •

Isolated very large cells Abundant foamy cytoplasm Intact or pyknotic nuclei Macrophages Hyaline bodies, calcification (pregnancy)   

cells that have a small nucleus and abundant finely vacuolated cytoplasm (Papanicolaou stain).

Endometriotic Cyst Endometriosis affects women of reproductive age and is often associated with infertility. It can result in a cystic tumor-like mass of the ovary, known as an endometriotic cyst or endometrioma. As many as one-half of ovarian endometriomas are bilateral. For an unequivocal diagnosis, identification of two of the following features is required: endometrial glands, endometrial stroma, and hemosiderin. CYTOMORPHOLOGY OF ENDOMETRIOTIC CYST • H  emosiderin-laden macrophages • Endometrial glandular cells • Endometrial stromal cells   

The aspirated cyst fluid contains hemolyzed old blood the color of chocolate (“chocolate cyst”). Hemosiderinladen macrophages are numerous (Fig. 16.5A). Endometrial epithelial cells are usually arranged in clusters or sheets. The cells are small and have indistinct borders; nuclei are round or oval; nucleoli are inconspicuous; and cytoplasm is scant and sometimes vacuolated. Nuclear molding may be seen. Stromal cells with oval nuclei and scant cytoplasm may also be present. Epithelial and stromal cells are best appreciated as such on cell block preparations (Fig. 16.5B).

524 CHA P T ER 1 6   Ovary

A

B • Fig. 16.5  Endometriotic Cyst.  (A) Cytologic preparations often show only macrophages with abundant

intracytoplasmic hemosiderin (Papanicolaou stain). (B) In cell block sections, fragments containing endometrial glands and stroma are diagnostic (hematoxylin and eosin stain).

The diagnosis of endometriosis is easily made when both cytologically benign epithelial and stromal cells are present. It is impossible to distinguish an endometriotic cyst from a hemorrhagic corpus luteum when epithelial cells are absent because luteinized granulosa and theca cells resemble histiocytes.44 Occasional endometriotic cysts have marked cytologic atypia.45 A suspicion of malignancy may be unavoidable in these atypical endometriotic cysts. 

Simple Ovarian, Paraovarian, and Paratubal Cysts Simple cysts of the ovary or fallopian tube are most common in postmenopausal women and result from an invagination of mesothelium or the surface epithelium. They are usually small and multiple and lined by a single layer of benign columnar, cuboidal, or flattened epithelium. When the epithelium is ciliated, the cyst is called a serous cyst.

• Fig. 16.6  Simple Ovarian Cyst.  This sheet of evenly arranged cuboidal cells resembles a sheet of mesothelial cells (Papanicolaou stain).

CYTOMORPHOLOGY OF SIMPLE CYSTS • S  cant cellularity • Mesothelial-like cells • Sheets and clusters   

The cytologic features of simple ovarian, parovarian, and paratubal cysts are identical. They contain clear fluid that is sparsely cellular. When small cuboidal epithelial cells are seen (rarely), they are usually arranged in tight clusters or sheets. The cells resemble benign mesothelial cells (Fig. 16.6), having a small round or oval nucleus, finely granular chromatin, and an inconspicuous nucleolus. The quantity of cytoplasm varies. If ciliated cells are present, however, the lesion is either a serous cyst or a hydrosalpinx. 

Hydrosalpinx Hydrosalpinx, a complication of salpingitis, presents as a large cystic adnexal mass. The distended fallopian tube is

• Fig. 16.7  Detached Ciliary Tufts.  These decapitated tips of ciliated

cells are diagnostically very useful because they exclude a follicle cyst. They are seen in serous cysts, cystic teratomas, and hydrosalpinx (Papanicolaou stain).

filled with clear fluid and is lined by ciliated epithelium. The fluid is hypocellular and may contain histiocytes, ciliated epithelial cells, or detached ciliary tufts (Fig. 16.7). These findings are identical to those seen with serous ovarian and paraovarian cysts. 

CHAPTER 16 Ovary

Tuboovarian Abscess Tuboovarian abscess is an advanced complication of acute salpingitis, known clinically as pelvic inflammatory disease. It presents as a palpable adnexal mass that can be visualized by ultrasonography or computed tomography. Most cases result from an ascending infection of the lower genital tract by sexually transmitted pathogens like Neisseria gonorrhoeae, Chlamydia trachomatis, and Mycoplasma genitalium. Aspirated material is thick and yellow and composed of numerous polymorphonuclear leukocytes and abundant necrotic debris. When these elements are seen during a rapid interpretation of the aspirated material, additional passes should be performed to obtain samples for microbiologic study to identify the causative organism. Surgery is considered for those patients who do not respond to antibiotic therapy. Laparoscopic or radiographically guided drainage is helpful in some cases. 

If the cyst fluid does not contain ciliated cells or detached ciliary tufts, the findings are nonspecific. Analysis of tumor markers may be helpful because elevated CA-125 levels are characteristic of serous cysts.1,18,20,37 

Benign Mucinous Tumors Mucinous cystadenoma and the uncommon mucinous adenofibroma are cystic neoplasms lined by a single layer of mucinous epithelium of gastric foveolar type or intestinal type. They can be very large and multiloculated. CYTOMORPHOLOGY OF MUCINOUS CYSTADENOMA • • • •

Benign Surface Epithelial–Stromal Tumors Tumors derived from the ovarian surface epithelium are the most common neoplasms of the ovary. They are divided histologically into serous (the most common), mucinous, endometrioid, clear cell, Brenner, seromucinous, and undifferentiated types.46 Most have a benign, borderline, and malignant counterpart. Because FNA is rarely used to diagnose these neoplasms, only the more common types are discussed below.

Benign Serous Tumors Benign serous tumors (e.g., serous cystadenoma and serous adenofibroma) are cystic neoplasms lined by an epithelium resembling the fallopian tube lining or ovarian surface, that is, ciliated or nonciliated cuboidal or columnar cells. They are unilocular or multilocular, and the cysts contain clear fluid. The cyst wall may be entirely smooth and round or nodular. Some benign serous tumors have a stromal component, hence, “adenofibroma.” Most ovarian adenofibromas are serous, but endometrioid, mucinous, and clear cell differentiation is occasionally seen.25

Mucinous cells • Gastric foveolar-type cells • Goblet cells Isolated cells, ribbons, sheets Macrophages Extracellular mucin   

The fluid is either gelatinous or watery. The lining cells are isolated, often retaining their columnar shape, or arranged in clusters and honeycomb-like sheets with sharply defined cell membranes. They resemble either benign gastric foveolar cells (Fig. 16.9) or goblet cells (Fig. 16.10). Mucin-filled macrophages and extracellular mucin are present. Diagnosing a mucinous cystadenoma is straightforward when mucinous epithelium is identified. If needed, chemical analysis of the fluid can be helpful; a high CEA level and low estradiol and CA-125 levels are characteristic of mucinous cysts and help to distinguish them from follicle and serous cysts.1,18,20,37 The differential diagnosis also includes a mucinous carcinoma and a mucinous borderline tumor. If nuclear atypia is present, a borderline tumor or carcinoma should be suspected. Surgical excision of the cyst should

CYTOMORPHOLOGY OF A BENIGN SEROUS TUMOR • • • •

 iliated cells C Cuboidal cells Detached ciliary tufts Psammoma bodies (rare)   

FNA of a benign cystic serous tumor yields clear fluid that is usually sparsely cellular but occasionally highly cellular. The cuboidal cyst lining cells, if present, have a uniformly round or oval nucleus and are arranged in crowded clusters. Ciliated columnar cells (Fig. 16.8), psammoma bodies, and detached ciliary tufts (see Fig. 16.7) are sometimes present.47 If the needle has collected a sample from a solid area of an adenofibroma, stromal cells may be seen.

525

•

Fig. 16.8  Serous Cystadenoma.  The cyst is lined by benign ciliated cells that have a basally placed nucleus, terminal bar, and cilia (Papanicolaou stain).

526 CHA P T ER 1 6   Ovary

be considered even when the cytologic findings are benign because the borderline or malignant areas can be focal and may not be sampled by FNA.18,24,26,32 

Benign Brenner Tumor Benign Brenner tumors account for about 5% of benign ovarian epithelial neoplasms.46 Less than 10% of cases occur bilaterally. Most are solid, but some contain small or large cystic areas that may show mucinous differentiation. Benign Brenner tumors are composed of nests of transitionaltype epithelium (resembling urothelial cells) embedded in a dense fibrous stroma. Aspirates show sheets of transitional-type cells, the nuclei of which resemble coffee beans because they are

• Fig. 16.9  Mucinous Cystadenoma.  Admixed with macrophages are fragments of benign mucinous epithelium resembling gastric foveolar epithelium (Papanicolaou stain).

oval and have a prominent longitudinal groove. Globular hyaline structures that stain bright orange with the Papanicolaou stain are seen48; their composition and significance are not known. Brenner tumors are positive for CK7, p63, and GATA3. The differential diagnosis includes a granulosa cell tumor, the cells of which also have longitudinal grooves. Granulosa cell tumors, unlike Brenner tumors, are negative for keratin proteins. 

Malignant Surface Epithelial-Stromal Tumors FNA is rarely used to diagnose borderline and malignant ovarian tumors, as noted earlier: Most of these tumors already have a suspicious laparoscopic or imaging appearance that merits surgery, and FNA has a significant false-negative rate for these tumors. Ovarian tumors, however, sometimes present as pelvic masses so large that they significantly alter pelvic anatomy. In such cases, an FNA is performed because a soft tissue, lymphoid, or other tumor is suspected rather than ovarian cancer. FNA is also recommended to diagnose ovarian tumors presenting at an advanced stage and to document the recurrence or metastasis of ovarian cancer.49,50 Immunohistochemistry is useful in the diagnosis and classification of ovarian tumors. Virtually all epithelial tumors of the ovary express CK7; therefore the absence of CK7 staining suggests a non-epithelial tumor of the ovary or a metastasis. Most ovarian tumors are negative for CK20 and CDX2, with the exception of the intestinal type of mucinous ovarian tumor, which sometimes expresses both. WT1 is expressed by serous carcinomas of the ovary,

• Fig. 16.10  Mucinous Cystadenoma.  Some mucinous cystadenomas contain intestinal-type goblet cells (Papanicolaou stain).

CHAPTER 16 Ovary

fallopian tube, and peritoneum; serous carcinomas of the endometrium tend to be negative. Mesotheliomas are also positive for WT1, as are other uncommon ovarian tumors, including small cell carcinoma of hypercalcemic type and some sex cord–stromal tumors. Loss of BAP1 expression is seen in most abdominal mesotheliomas and virtually never in serous carcinomas.51 PAX8 is a highly sensitive marker of serous tumors of the ovary, fallopian tube, and peritoneum, but it is also expressed in renal cell carcinomas, thyroid cancers, and pancreatic endocrine neoplasms. PLAP, SALL4, Oct-3/4, and NANOG are sensitive and relatively specific markers of germ cell tumors and are discussed in greater detail below. Inhibin is a sensitive and relatively specific marker of sex cord–stromal tumors but also expressed in adrenocortical neoplasms. Calretinin is a more-sensitive but less-specific marker of sex cord– stromal tumors than inhibin. CA-125, on the other hand, is so promiscuous—it is expressed by tumors of the breast, colon, lung, and pancreas, as well as gynecologic cancers—that it has little value in the cytologic diagnosis of ovarian cancer.

Serous Borderline Tumor and Serous Carcinoma The most common malignant ovarian neoplasm is the serous carcinoma. Often bilateral, serous carcinomas commonly contain cystic and solid foci. Serous carcinomas are divided histologically into low-grade and high-grade types. The high-grade serous carcinoma is by far more common, composed of large pleomorphic cells lining the cystic areas, forming papillary projections, and invading the stroma. Low-grade serous carcinomas account for only 5% of all serous carcinomas and are composed of a more uniform population of cells with minimal pleomorphism. As with serous carcinoma, the serous borderline tumor can be both solid and cystic. The degree of nuclear atypia resembles that of a low-grade serous carcinoma, and therefore it is not possible to distinguish between the two cytologically29,36; the diagnosis of carcinoma is based on identifying stromal invasion. Nevertheless, borderline tumors are generally of lower grade than high-grade serous carcinomas, and therefore some generalizations are possible. CYTOMORPHOLOGY OF SEROUS BORDERLINE TUMOR AND LOW-GRADE SEROUS CARCINOMA • • • • • •

 wisted sheets and spheres T Branching clusters Mild to moderate nuclear atypia Large cytoplasmic vacuoles (some cells) Psammoma bodies Stripped fibrovascular cores   

Aspirates from serous borderline tumors in particular are often sparsely cellular because the needle samples only cyst contents, resulting in a false-negative diagnosis.18,26,32 When sampled adequately, the specimen is composed of atypical cells in spheres, branching clusters, and sheets (Fig. 16.11).

527

CYTOMORPHOLOGY OF HIGH-GRADE SEROUS CARCINOMA • • • • •

 lusters and isolated cells C Large pleomorphic cells Round nuclei Prominent nucleoli Psammoma bodies   

Aspirates from high-grade serous carcinomas are usually very cellular, composed of atypical cells in papillary clusters. The nuclei are large and pleomorphic, and nucleoli are prominent (Fig. 16.12). Many atypical bare nuclei are present.48 The cytoplasm, like that of serous borderline tumors, can have large vacuoles. Psammoma bodies accompanied by a rim of malignant cells are seen in some but not all cases.29 Nuclear atypia distinguishes serous borderline tumors and carcinomas from benign serous cystadenomas. Although aspirates of cellular follicle cysts, like those of a serous carcinoma, can be highly cellular and show mitotic activity, follicle cysts have uniform nuclei and negative sonographic or laparoscopic findings. As mentioned above, the distinction between a borderline tumor and a serous carcinoma is possible only on the surgically excised specimen. The distinction between serous carcinoma and other epithelial malignancies such as mucinous carcinoma and clear cell carcinoma, especially with poorly differentiated tumors, is sometimes difficult.29 Serous borderline tumors and carcinomas are almost always immunoreactive for PAX8, which helps to distinguish them from PAX8-negative neoplasms such as mesothelioma.52,53 The other epithelial tumors of the ovary described below are also often positive for PAX8, however, so this marker is not useful in distinguishing them from serous tumors. 

Mucinous Borderline Tumor and Mucinous Carcinoma Mucinous carcinomas are less common than serous carcinomas, accounting for only 3% to 4% of all primary ovarian carcinomas.46 They are usually large, unilateral complex cysts with solid areas and papillary excrescences. Metastatic intestinal adenocarcinomas to the ovary, particularly from the appendix, are histologically very similar to primary mucinous ovarian cancers and need to be excluded by a combination of clinical features, extensive histologic sampling, and immunophenotyping with cytokeratins CK7 and CK20, PAX8, and estrogen and progesterone receptors. CYTOMORPHOLOGY OF MUCINOUS CARCINOMA • C  olumnar mucinous cells with mild atypia or groups of pleomorphic large cells with prominent nucleoli • Cytoplasmic vacuolization • Extracellular mucin • Macrophages   

528 CHA P T ER 1 6   Ovary

A

C

B •

Fig. 16.11  Serous Borderline Tumor.  (A) The cells are arranged in a crowded sheet. There is mild to moderate atypia. (B) In this tight spherical aggregate, some cells have large cytoplasmic vacuoles. (C) Psammoma bodies are a common finding (Papanicolaou stain).

Aspirates from mucinous carcinomas are cellular, composed of isolated cells and cells in sheets or grouped in irregular clusters. Cells from well-differentiated tumors are columnar, contain mucin, and have mild nuclear atypia. The nuclei of poorly differentiated tumors are pleomorphic

and indistinguishable from high-grade serous carcinomas; some tumors show a range of differentiation (Fig. 16.13). As is the case for serous tumors, cytologic samples cannot accurately distinguish a borderline mucinous tumor from a mucinous carcinoma; histologic examination for

CHAPTER 16 Ovary

the presence of invasion is needed. Aspirates from mucinous borderline tumors are a common cause of false-negative results. Spread of a mucinous ovarian tumor into the peritoneum can lead to pseudomyxoma peritonei, but most ovarian tumors associated with pseudomyxoma peritonei are actually metastases from an occult appendiceal primary. Aspirates from pseudomyxoma peritonei are hypocellular, composed predominantly of abundant mucin.54 Atypical mucinous glands are identified in some but not all cases. 

to 20% with a coexisting adenocarcinoma of the endometrium.46 Endometrioid carcinomas are usually cystic and solid tumors with foci of necrosis and hemorrhage. Morphologically similar to the usual type of adenocarcinoma of the endometrium, they are graded using the same criteria. Squamous differentiation may be seen. CYTOMORPHOLOGY OF ENDOMETRIOID CARCINOMA • • • •

Endometrioid Carcinoma Endometrioid carcinoma is the second-most common form of ovarian epithelial malignancy, comprising 10% to 15% of ovarian carcinomas. It is bilateral in 17% of cases.46 Up to 42% are associated with endometriosis, and 15%

529

 umerous isolated cells N Strips, crowded glands, or both Palisading Elongated columnar shape   

Aspirates yield numerous isolated elongated cells, as well as occasional short strips of cells and intact or “broken” glands (Fig. 16.14A). Cell block sections are helpful in revealing the resemblance of the neoplasm to endometrial glands (Fig. 16.14B). The background is usually bloody and contains hemosiderin-laden macrophages. It may be impossible to distinguish an endometrioid carcinoma from other surface epithelial malignancies, such as a serous or mucinous carcinoma, especially with high-grade tumors.1,14,29,55 

Clear Cell Carcinoma

• Fig. 16.12  High-Grade Serous Carcinoma.  The malignant cells often have large, round, and pleomorphic nuclei, and nucleoli are prominent (Papanicolaou stain).

A

Clear cell carcinoma of the ovary resembles the clear cell carcinomas of the endometrium, cervix, and vagina. Tumor cells have a large, pleomorphic, often eccentrically placed nucleus with a prominent nucleolus. The cytoplasm is abundant and vacuolated or scant and eosinophilic. Within or adjacent to the tumor cell clusters there may be hyaline extracellular material that stains pink-purple with

B • Fig. 16.13  Mucinous Carcinoma.  (A) Some sheets of mucinous cells show only mild atypia (compare with Figs. 16.9, 16.10). (B) Other cells are markedly atypical, with little if any mucinous differentiation (Papanicolaou stain).

530 CHA P T ER 1 6   Ovary

Romanowsky stains (“raspberry bodies”).56,57 The background shows necrosis. Clear cell carcinomas are almost always positive for PAX8 and negative for WT1.46 

Germ Cell Tumors Germ cell tumors are most common in the ovary and testis but can also be seen in the retroperitoneum, mediastinum, and the midline of the central nervous system. Neoplasms identical to those of the ovary occur in the testis and extragonadal sites. Germ cell tumors can occur at any age but are most common during the reproductive years. They comprise 30% of all ovarian tumors. The vast majority of those seen in adults (up to 95%) are benign cystic teratomas (dermoid cysts). In contrast, up to one-third of germ cell tumors in children are malignant. Although some germ cell tumors are “pure” tumors, such as the pure dysgerminoma, many are composed of a mixture of germ cell subtypes.

Teratoma Mature Teratoma Mature teratomas are the most common of the germ cell tumors. Although they are seen at any age, they usually occur during the reproductive years. Most are cystic (mature cystic teratoma or dermoid cyst) and composed of tissue derived from ectoderm, endoderm, and mesoderm, with ectodermal derivatives such as skin and hair the most common. Sebaceous glands are prominent. Struma ovarii is a mature teratoma composed exclusively or predominantly of thyroid tissue. Mature teratomas are rarely aspirated because their sonographic features (particularly the tooth) are diagnostic. FNA yields cellular material containing predominantly anucleated squamous cells (Fig. 16.15A). Ciliated cells, detached ciliary tufts, mucinous cells, and hair are seen in some cases (Fig. 16.15B).47 With transvaginal aspirations,

A

B • Fig. 16.14  Endometrioid Carcinoma.  (A) The cells have elongated nuclei and a narrow columnar shape.

Some are arranged in pseudostratified strips and glands (Papanicolaou stain). (B) Cell block sections reveal endometrial-like glands with atypia (hematoxylin and eosin stain).

CHAPTER 16 Ovary

531

B

A •

Fig. 16.15  Mature Cystic Teratoma (Dermoid Cyst).  (A) Mature nucleated and anucleate squamous cells and occasional glandular cells are present (Papanicolaou stain). (B) This cell block section contained several transected hair shafts, recognizable on the basis of their size, shape, and pigmentation (hematoxylin and eosin stain).

contamination of a benign epithelial cyst by normal vaginal squamous cells mimics this picture.8 A confident diagnosis of a mature teratoma can be made; however, if some other route was taken to obtain the specimen. In rare cases, a mature cystic teratoma may undergo malignant transformation, most commonly to squamous cell carcinoma. 

Immature Teratoma Immature teratomas are rare, malignant, rapidly growing neoplasms, usually solid and unilateral, most commonly in the first two decades of life. These tumors are composed of derivatives from all three germ cell layers and contain a variable amount of immature or embryonal-type tissue. Microscopically, immature or embryonal tissue, usually admixed with benign mature elements, is characteristic of these tumors. Immature neuroectoderm is particularly prominent. 

Carcinoid Tumor Carcinoid tumors of the ovary are usually a component of mature teratomas but may also occur in a pure form. Strumal carcinoid is a carcinoid tumor associated with struma ovarii. It is important to distinguish a primary ovarian tumor from a metastatic carcinoid tumor to the ovary, a distinction that requires clinical correlation and is not possible by cytologic examination. Metastatic carcinoid tumors to the ovary almost always originate from a gastrointestinal primary and are most often bilateral. FNA reveals numerous isolated cells and loose clusters. Occasionally, rosettes are seen. Tumor cells have an eccentrically placed round or oval nucleus, and the chromatin has a granular, “salt-and-pepper” appearance. The cytoplasm is eosinophilic and granular, with distinct outlines. Immunocytochemistry for chromogranin and other neuroendocrine markers is positive. 

Dysgerminoma Dysgerminomas are the ovarian analogue of the testicular seminoma. They are the most common type of malignant germ cell tumor but constitute only 1% to 2% of all malignant ovarian tumors.46 They are often large, solid tumors and are bilateral in 15% to 20% of cases. They are most frequent in women under the age of 30. Because they are highly radiosensitive, accurate diagnosis is crucial so that appropriate therapy may be instituted. Large tumors may show cystic areas associated with necrosis and hemorrhage. Histologic examination reveals sheets, nests, and cords of uniform, large, polyhedral tumor cells with a centrally placed round nucleus; a prominent, sometimes bizarre, eosinophilic nucleolus; and coarsely granular chromatin. The cytoplasm of these cells is abundant, well defined, and eosinophilic or vacuolated. It contains both lipid and glycogen and stains positively with the periodic acid-Schiff reaction. The tumor cells are separated by fibrous septa infiltrated by lymphocytes, some arranged as lymphoid follicles with germinal centers. Granulomas are seen in some cases. A small percentage (3%) contain syncytiotrophoblastic cells that secrete human chorionic gonadotropin.58 Aspirates from dysgerminomas are usually highly cellular, composed predominantly of isolated tumor cells, with some loose syncytium-like clusters. In the background are small lymphocytes and, in some cases, granulomas. A characteristic “tiger-stripe” background, similar to that seen in seminomas, is seen on air-dried preparations (see Fig. 2.48). Necrosis and hemorrhage may be present. The tumor cells have a large, round, centrally placed nucleus with one or more prominent, irregularly shaped nucleoli (Fig. 16.16A). The cytoplasm may be clear or granular. Mitoses are present. Dysgerminomas are immunoreactive for placental alkaline phosphatase (PLAP) (cytoplasmic and membrane staining), CD117 (c-kit; membrane staining), and the stem cell-related proteins Oct-3/4 (synonymous with Oct-4),

532 CHA P T ER 1 6   Ovary

SALL4

A

B Oct-3/4

C

NANOG

D • Fig 16.16  Dysgerminoma.  (A) Tumor cells have a large round nucleus with a prominent nucleolus and clear cytoplasm (hematoxylin and eosin–stained cell block). They show nuclear immunoreactivity for the stem cell proteins SALL4 (B), Oct-3/4 (C), and NANOG (D).

NANOG, and SALL4 (nuclear staining for all three) (Fig. 16.16B–D). PLAP and SALL4 are expressed in most germ cell tumors, whereas Oct-3/4 and NANOG are only expressed in dysgerminoma/seminoma and embryonal carcinoma. The membranous staining pattern for CD117 is very characteristic of dysgerminoma and not seen in embryonal carcinoma or yolk sac tumor. Unlike the embryonal and yolk sac tumors, dysgerminomas generally show no immunoreactivity for keratin proteins, epithelial membrane antigen, α-fetoprotein, or CEA. 

Embryonal Carcinoma and Other Malignant Germ Cell Tumors In contrast with their testicular counterparts, embryonal carcinomas, endodermal sinus tumors, and choriocarcinomas of the ovary are extremely rare. They are aggressive neoplasms that often present at an advanced stage of disease. Histologically, they are primitive large cell neoplasms with marked nuclear anaplasia. Necrosis and hemorrhage are a prominent feature. The tumor cells of embryonal carcinoma have a centrally placed, large, round, or highly irregular nucleus with several

nucleoli. The cytoplasm is indistinct and pale. Bizarrely shaped cells and mitoses are common. Cells of yolk sac tumors resemble those of poorly differentiated adenocarcinomas. They are cohesive, pleomorphic cells with prominent nucleoli.15 Some tumor cells contain intracytoplasmic dense hyaline globules composed of α-fetoprotein; others have vacuolated cytoplasm.59 Mucoid and basement membrane-like material may be present in the background.60 Choriocarcinomas are composed of malignant cytotrophoblast and syncytiotrophoblast. These tumors secrete human chorionic gonadotropin. A cytologic diagnosis of malignancy in this group of tumors is straightforward, but correct subtyping and distinction from a poorly differentiated epithelial malignancy require correlation with serologic markers and immunocytochemical studies. Embryonal carcinoma, yolk sac tumor, and choriocarcinoma are keratin-positive but can be distinguished from epithelial malignancies because of their staining for PLAP and SALL4. Additionally, embryonal carcinoma, like dysgerminoma, is immunoreactive for Oct-3/4 and NANOG. Dysgerminoma can be distinguished from embryonal carcinoma because the former has a characteristic membranous staining pattern for CD117. 

CHAPTER 16 Ovary

Sex Cord–Stromal Tumors Sex cord–stromal tumors include tumors derived from granulosa cells, theca cells, Sertoli cells, Leydig cells, and the fibroblasts of the ovarian stroma. Many of these tumors produce and secrete steroid hormones, which results in clinically apparent estrogenic or, less commonly, androgenic changes. 

Granulosa Cell Tumors Granulosa cell tumors account for about 1.5% of all ovarian tumors. There are two major subcategories: the adult and juvenile types.

Adult Granulosa Cell Tumor The adult granulosa cell tumor (AGCT) accounts for about 1% of all ovarian tumors and more than 95% of all granulosa cell tumors. The average age is 53 years, but the tumor occurs over a wide age range.46 Typically unilateral, the tumor grows slowly but is capable of metastasis. Many AGCTs secrete estrogens; prolonged elevated levels of estrogen may result in endometrial hyperplasia or carcinoma. Rarely, androgens are secreted, with the resultant signs of virilization. Although about 10% of these tumors are accompanied by ascites, the fluid does not usually contain malignant cells.42 Although most commonly solid and cystic, with foci of hemorrhage and necrosis, purely solid or cystic tumors are also seen. The tumor is composed of neoplastic granulosa cells arranged in a variety of architectural patterns. Most characteristic is the microfollicular pattern, which shows Call–Exner bodies: tumor cells arranged around small cavities containing eosinophilic fluid. Granulosa cell tumors are typically strongly immunoreactive for inhibin and calretinin; other immunostains that are often or occasionally positive include estrogen receptor, progesterone receptor, SF-1, CD56, CD99, WT1, broad-spectrum cytokeratins, S-100, and smooth muscle actin. AGCTs are typically negative for CK7 and epithelial membrane antigen. CYTOMORPHOLOGY OF ADULT GRANULOSA CELL TUMOR • • • • • • • •

 ighly cellular H Isolated cells, clusters, sheets, trabeculae Call-Exner bodies Small- to medium-sized cells Round, monomorphic nuclei Nuclear grooves Ill-defined cytoplasm Globular basement membrane-like material (best with Romanowsky-type stains)

533

(“coffee bean nuclei”) are present to some degree in virtually all cases61 but are easily visible in only a minority.50,61-63 Nucleoli are prominent in some cases.61 Cytoplasm is scant, poorly defined, and pale.42 Mitoses are rare. Call-Exner bodies (small rings of granulosa cells surrounding fluid or pyknotic nuclei) are seen in some cases.61,63 In some cases, Romanowsky-type stains reveal globular basement membrane-like material.50 Almost one-half of cases show a prominent, arborizing vascular pattern.61,63 Cell block sections are helpful in highlighting one or more of the different growth patterns of granulosa cell tumors: microfollicular, macrofollicular, trabecular, insular, diffuse, and moiré silk (watered silk) (Fig. 16.17B). DIFFERENTIAL DIAGNOSIS OF ADULT GRANULOSA CELL TUMOR • • • • •

 ellular follicle cyst C Other sex cord–stromal tumors Carcinoid tumor Endometrioid carcinoma Small cell carcinoma, pulmonary type   

The cytomorphology of a cellular follicle cyst (see Figs. 16.1–16.3) mimics that of an AGCT.64 Correlation with sonographic and laparoscopic findings can be key to avoiding an erroneous interpretation: cellular follicle cysts are usually unilocular and clinically entirely benign. Other sex cord–stromal tumors such as the Sertoli-Leydig cell tumor and the sex cord tumor with annular tubules cannot be reliably distinguished from an AGCT by cytology or immunohistochemistry.49,50,62 A carcinoid tumor lacks nuclear grooves and is immunoreactive for chromogranin. Call-Exner bodies mimic the appearance of endometrioid tubules, but endometrioid carcinomas lack nuclear grooves and often show squamous metaplasia. Two rare ovarian tumors are composed partly or completely of undifferentiated small cells: small cell carcinoma, hypercalcemic type; and small cell carcinoma, pulmonary type. The hypercalcemic type is more common in young women and therefore needs to be considered in the differential diagnosis of juvenile granulosa cell tumor (discussed next). The small cell carcinoma, pulmonary type, resembles small cell carcinoma of the lung (Fig. 16.18). It typically occurs in postmenopausal women and needs to be considered in the differential diagnosis of AGCT. 

Juvenile Granulosa Cell Tumor   

FNA samples are highly cellular. Small- to medium-sized cells have a centrally placed, round or oval, monomorphous nucleus (Fig. 16.17A). Nuclear chromatin is pale and finely dispersed. Naked nuclei are common.61 Nuclear grooves

Juvenile granulosa cell tumors (JGCTs) occur predominantly in childhood and adolescence. Like AGCT, JGCTs secrete estrogens and thus are often associated with sexual pseudoprecocity. JGCTs are usually solid and cystic, unilateral, and restricted to the ovary. Patients may present with an acute abdomen after rupture of the tumor capsule. The tumor forms follicles lined by granulosa cells and luteinized theca cells. In contrast to AGCT, almost all nuclei

534 CHA P T ER 1 6   Ovary

A

B • Fig. 16.17  Adult-Type Granulosa Cell Tumor.  (A) The small to medium-sized cells are arranged in loose

clusters. They are difficult to distinguish from normal granulosa cells (compare with Figs 16.2 and 16.3) (Papanicolaou stain). (B) Large fragments of tumor in cell block sections may show one of the characteristic architectural patterns, in this case the watered-silk pattern (hematoxylin and eosin–stained cell block).

A

B • Fig. 16.18  Small Cell Carcinoma of the Ovary, Pulmonary Type.  This tumor resembles the small cell carcinoma of neuroendocrine type of the lung. (A) Wright–Giemsa-stained smear; (B) hematoxylin and eosin–stained cell block section.

CHAPTER 16 Ovary

lack grooves. Despite the presence of pleomorphism and frequent mitoses, only 10% behave aggressively. The cells of JGCT are positive for inhibin, calretinin, SF-1, CD99, CD56, and vimentin. On FNA, tumor cells are seen in loose clusters and single cells.63,65 Nuclei are round, with fine chromatin and small to prominent nucleoli. There may be nuclear protrusions, but nuclear grooves are absent. Some mitoses are seen. There is a moderate amount of granular cytoplasm, and the oil-red-O stain demonstrates intracytoplasmic lipid. Call–Exner bodies are usually not found. JGCTs should be distinguished from the small cell carcinoma, hypercalcemic type, which occurs in the same age range. The clinical association of estrogenic manifestations with JGCT and hypercalcemia with this type of small cell carcinoma are helpful clues. Immunostaining for inhibin (positive in JGCT, negative in small cell carcinoma) can provide confirmation. 

Thecoma Thecomas, benign tumors of theca cells, are more common in postmenopausal women and are usually solid and unilateral tumors. They may be functional, secreting estrogens or androgens, or nonfunctional. Microscopically, the tumor is composed of monomorphous oval- or spindle-shaped cells with cytoplasm that shows variable degrees of luteinization. Unlike aspirates from granulosa cell and Sertoli–Leydig cell tumors, aspirates from thecomas are usually hypocellular.36 They are composed of isolated elongated cells and loose clusters. The nuclei are spindle-shaped, with finely granular chromatin. The cytoplasm is clear and contains numerous lipid vacuoles. 

Fibroma Fibromas are benign, nonfunctional, stromal tumors that comprise 4% of ovarian neoplasms.46 These tumors are accompanied by ascites and pleural effusion (Meigs’ syndrome) in 1% of cases. Histologically, fibromas are composed of bundles of spindle-shaped cells arranged in a whorling or storiform pattern. The cells have a fibroblastic appearance, and collagen is usually abundant. Intracellular edema and intracytoplasmic lipid are sometimes observed. Aspirates are hypocellular and contain bundles of fibroblastic spindle-shaped cells similar to those seen in thecomas. The distinction between a fibroma and a thecoma by FNA cytology is impossible because luteinized cells can be a component of both tumors. 

Uncommon Primary Ovarian Tumors Benign and malignant mesenchymal tumors, bone-producing and cartilage-producing tumors, vascular tumors, and primary lymphomas of the ovary are rare, as are tumors of neural origin and those derived from the mesothelium (e.g., adenomatoid tumors). Leiomyomas may have prominent

535

cystic degeneration: aspirates from such tumors contain benign-appearing foam cells suggestive of a benign cyst. When leiomyomas are not cystic, the aspirates are usually very scant and contain only occasional smooth muscle cells. Aspirates from lymphomas yield numerous isolated cells that are usually easily recognizable as lymphoid in origin. 

Metastatic Tumors The most common tumors that metastasize to the ovaries originate in the colon, stomach, appendix, breast, and elsewhere in the female genital tract. Krukenberg tumors are characterized by mucin-filled signet-ring–shaped cells metastatic to the ovary.36 Most arise in the stomach, but tumors of the colon, appendix, and breast also cause this pattern of spread. Because signet-ring cells are rare in primary ovarian tumors, the possibility of a metastasis should be considered when an ovarian tumor is rich in signet-ring cells. In many cases, distinguishing between a primary ovarian carcinoma and a metastasis is impossible by cytomorphology alone. Because primary ovarian epithelial tumor are generally CK7-positive and CK20-negative, the reverse immunophenotype (CK7-negative, CK20-positive) suggests a metastasis, probably from the appendix or intestine. Measuring the concentration of tumor-associated antigens in cyst fluid is useful: unlike ovarian carcinomas, metastatic colon cancers have a high CEA level combined with a low CA 125 level.20

References 1. De Crespigny L, Robinson HP, Davoren RA, Fortune D. The “simple” ovarian cyst: aspirate or operate? Br J Obstet Gynecol. 1989;96:1035–1039. 2. DePriest PD, Shenson D, Fried A, et  al. A morphology index based on sonographic findings in ovarian cancer. Gyn Oncol. 1993;51:7–11. 3. Yee H, Greenebaum E, Lerner J, Heller D, Timor-Trisch IE. Transvaginal sonographic characterization combined with cytologic evaluation in the diagnosis of ovarian and adnexal cysts. Diagn Cytopathol. 1994;10:107–112. 4. Rubenchik I, Auger M, Casper RF. Fine-needle aspiration cytology of ovarian cysts in in vitro fertilization patients: a study of 125 cases. Diagn Cytopathol. 1996;15(4):341–344. 5. Guariglia L, Conte M, Are P, Rosati P. Ultrasound-guided fineneedle aspiration of ovarian cysts during pregnancy. Eur J Obstet Gynecol Rep Biol. 1999;82:5–9. 6. Allias F, Chanoz J, Blache G, Thivolet-Bejui F, Vancina S. Value of ultrasound-guided fine-needle aspiration in the management of ovarian and paraovarian cysts. Diagn Cytopathol. 2000;22:70– 80. 7. Mulvany N, Ostor A, Teng G. Evaluation of estradiol in aspirated ovarian cystic lesions. Acta Cytol. 1995;39:663–668. 8. Greenebaum E, Mayer JR, Stangel JJ, Hughes P. Aspiration cytology of ovarian cysts in in  vitro fertilization patients. Acta Cytol. 1992;36:11–18. 9. Stanley MW, Horwitz CA, Frable WJ. Cellular follicular cyst of the ovary: fluid cytology mimicking malignancy. Diagn Cytopathol. 1991;7:48–52.

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10. American College of Obstetricians and Gynecologists. ACOG Practice Bulletin. Management of adnexal masses. Obstet Gynecol. 2007;110(1):201–214. 11. Walsh DJ, Sills ES, Shkrobot LV, Gleeson NC, Sheppard MN, Walsh AP. Ovarian serous adenocarcinoma identified during IVF: diagnostic approach, surgical management, and reproductive outcome. World J Surg Oncol. 2009;7:46. 12. Abrams J, Silverberg SG. The role of intraoperative cytology in the evaluation of gynecologic disease. Pathol Ann. 1989;24(part 2):167–187. 13. Ganjei P, Nadji M. Aspiration cytology of ovarian neoplasms: a review. Acta Cytol. 1984;28:329–332. 14. Kjellgren O, Angstrom T, Bergman F, Wiklund DE. Fine-needle aspiration biopsy in diagnosis, and classification of ovarian carcinoma. Cancer. 1971;28:967–976. 15. Mulvany NJ. Aspiration cytology of ovarian cysts and cystic neoplasms: a study of 235 aspirates. Acta Cytol. 1996;40:911–920. 16. Ganjei P. Fine-needle aspiration cytology of the ovary. Clin Lab Med. 1995;15(3):705–726. 17. Trimbos JB, Hacker NF. The case against aspirating ovarian cysts. Cancer. 1993;72:828–831. 18. Geier GR, Strecker JR. Aspiration cytology and E2 content in ovarian tumors. Acta Cytol. 1981;25:400–406. 19. Cole L, Mount S, Nuzzo E, Wong C. Aspiration cytology of ovarian cystic masses: histologic correlation and review of the literature. Acta Cytol. 2011;55(1):19–25. 20. Pinto MM, Bernstein LH, Brogan DA, Parikh F, Lavy G. Measurement of CA125, carcinoembryonic antigen, and alpha- fetoprotein in ovarian cyst fluid: diagnostic adjunct to cytology. Diagn Cytopathol. 1990;6:160–163. 21. Wojcik EM, Selvaggi SM. Fine-needle aspiration cytology of cystic ovarian lesions. Diagn Cytopathol. 1994;11:9–14. 22. Tahir Z, Yusuf NW, Ashraf M, Yusuf AW, Aziz F. Fine needle aspiration of unilocular ovarian cysts--a cytohistological correlation. J Pak Med Assoc. 2004;54(5):266–269. 23. Martinez-Onsurbe P, Ruiz Villaespesa A, Sanz Anquela JM, Valenzuela Ruiz PL. Aspiration cytology of 147 adnexal cysts with histologic correlation. Acta Cytol. 2001;45(6):941–947. 24. Zhou AG, Levinson KL, Rosenthal DL, VandenBussche CJ. Performance of ovarian cyst fluid fine-needle aspiration cytology. Cancer Cytopathol. 2018;126(2):112–121. 25. Volmar KE, Herman CM, Creager AJ. Fine-needle aspiration cytology of endometrioid adenofibroma of the ovary. Diagn Cytopathol. 2004;31(1):38–42. 26. Moran O, Menczer J, Ben-Barauch G, et al. Cytologic examination of ovarian cyst fluid for the distinction between benign and malignant tumors. Obstet Gynecol. 1993;82:444–446. 27. Higgins RV, Matkins JF, Marroum MC. Comparison of fineneedle aspiration cytologic findings of ovarian cysts with ovarian histologic findings. Am J Obstet Gynecol. 1999;180(3 Pt 1):550– 553. 28. Gaetje R, Popp LW. Is differentiation of benign and malignant cystic adnexal masses possible by evaluation of cysts fluids with respect to color, cytology, steroid hormones, and tumor markers? Acta Obstet Gynecol Scand. 1994;73(6):502–507. 29. Ganjei P, Dickinson B, Harrison T, Nassiri M, Lu Y. Aspiration cytology of neoplastic and non-neoplastic ovarian cysts: is it accurate? Int J Gynecol Pathol. 1996;15(2):94–101. 30. Vercellini P, Oldani S, Felicetta I, Bramante T, Rognoni MT, Crosignani PG. The value of cyst puncture in the differential diagnosis of benign ovarian tumours. Hum Reprod. 1995;10(6):1465– 1469.

31. Gupta N, Rajwanshi A, Dhaliwal LK, et al. Fine needle aspiration cytology in ovarian lesions: an institutional experience of 584 cases. Cytopathology. 2012;23(5):300–307. 32. Granados R. Aspiration cytology of ovarian tumors. Curr Opin Obstet Gynecol. 1995;7:43–48. 33. Diernaes E, Rasmussen J, Soerensen T, Hasch E. Ovarian cysts: management by puncture? Lancet. 1987;1:1084. 34. Nuñez C, Diaz JI. Ovarian follicular cysts: a potential source of false positive diagnoses in ovarian cytology. Diagn Cytopathol. 1992;8:532–536. 35. Selvaggi SM. Fine-needle aspiration cytology of ovarian follicle cysts with cellular atypia from reproductive-age patients. Diagn Cytopathol. 1991;7:189–192. 36. Uguz A, Ersoz C, Bolat F, Gokdemir A, Vardar MA. Fine needle aspiration cytology of ovarian lesions. Acta Cytol. 2005;49(2):144–148. 37. Pinto MM, Greenebaum E, Simsir A, Kleinman GM, Portnoy LM, Garfinkel R. CA-125 and carcinoembryonic antigen assay versus cytodiagnostic experience in the classification of benign ovarian cysts. Acta Cytol. 1997;41:1456–1462. 38. Ramzy I, Delaney M, Rose P. Fine needle aspiration of ovarian masses: II. Correlative cytologic and histologic study of nonneoplastic cysts and noncelomic epithelial neoplasms. Acta Cytol. 1979;23:185–193. 39. Kovacic J, Rainer S, Levincnik A, Cizelj T. Cytology of benign ovarian lesions in connection with laparoscopy. In: Zajicek J, ed. Aspiration Biopsy Cytology, Part 2: Cytology of Infradiaphragmatic Organs. Basel: S. Karger; 1979. 40. Tao LC, ed. Transabdominal Fine-Needle Aspiration Biopsy. New York: Igaku-Shoin; 1990. 41. McCluggage WG. Value of inhibin staining in gynecological pathology. Int J Gynecol Pathol. 2001;20(1):79–85. 42. Ehya H, Lang WR. Cytology of granulosa cell tumor of the ovary. Am J Clin Pathol. 1986;85:402–405. 43. Selvaggi SM. Cytology of nonneoplastic cysts of the ovary. Diagn Cytopathol. 1990;6:77–85. 44. Tabbara SO, Nayar R. Fluid cytology in nonneoplastic ovarian cysts [Abstract]. Acta Cytol. 1994;38:833–834. 45. Ballouk F, Ross JS, Wolf BC. Ovarian endometriotic cysts: an analysis of cytologic atypia and DNA ploidy patterns. Am J Clin Pathol. 1994;102:415–419. 46. Kurman RJ, Carcangiu ML, Herrington CS, Young RH, eds. WHO Classification of Tumours of Female Reproductive Organs. Lyon: IARC; 2014. 47. Rivasi F, Gasser B, Morandi P, Philippe E. Ciliated bodies in ovarian cyst aspirates. Acta Cytol. 1993;37:489–493. 48. Ramzy I, Delaney M. Fine needle aspiration of ovarian masses: I. Correlative cytologic and histologic study of celomic epithelial neoplasms. Acta Cytol. 1979;23:97–104. 49. Watson B, Siegel CL, Ylagan LR. Metastatic ovarian Sertoli-cell tumor: FNA findings with immunohistochemistry. Diagn Cytopathol. 2003;29(5):283–286. 50. Ylagan LR, Middleton WD, Dehner LP. Fine-needle aspiration cytology of recurrent granulosa cell tumor: case report with differential diagnosis and immunocytochemistry. Diagn Cytopathol. 2002;27(1):38–41. 51. Andrici J, Jung J, Sheen A, et al. Loss of BAP1 expression is very rare in peritoneal and gynecologic serous adenocarcinomas and can be useful in the differential diagnosis with abdominal mesothelioma. Hum Pathol. 2016;51:9–15. 52. Laury AR, Hornick JL, Perets R, et al. PAX8 reliably distinguishes ovarian serous tumors from malignant mesothelioma. Am J Surg Pathol. 2010;34(5):627–635.

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53. Laury AR, Perets R, Piao H, et al. A comprehensive analysis of PAX8 expression in human epithelial tumors. Am J Surg Pathol. 2011;35(6):816–826. 54. Leiman G, Goldberg R. Pseudomyxoma peritonei associated with ovarian mucinous tumors: cytologic appearance in five cases. Acta Cytol. 1992;36:299–304. 55. Nagai S, Nozawa S, Kurihara S, Mukai M. Cytologic and biologic studies of endometrioid carcinoma of the ovary. Acta Cytol. 1983;27:676–682. 56. Atahan S, Ekinci C, Icli F, Erdogan N. Cytology of clear cell carcinoma of the female genital tract in fine needle aspirates and ascites. Acta Cytol. 2000;44(6):1005–1009. 57. Mettler TN, Manivel JC, Pambuccian SE. “Raspberry bodies,” extracellular hyaline globules and branching hyaline stromal fragments in a fine-needle aspirate of metastatic clear cell carcinoma of the ovary. Diagn Cytopathol. 2014;42(3):230–235. 58. Young RH. New and unusual aspects of ovarian germ cell tumors. Am J Surg Pathol. 1993;17:1210–1224. 59. Nadji M, Sevin BU. Pelvic fine needle aspiration cytology in gynecology. In: Linsk JA, ed. Clinical Aspiration Cytology. 2nd ed. Philadelphia: Lippincott; 1989.

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60. Akhtar M, Ali MA, Sackey K, Jackson D, Bakry M. Fine-needle aspiration biopsy diagnosis of endodermal sinus tumor: histologic and ultrastructural correlations. Diagn Cytopathol. 1990;6:184– 192. 61. Ali S, Gattuso P, Howard A, Mosunjac MB, Siddiqui MT. Adult granulosa cell tumor of the ovary: fine-needle-aspiration cytology of 10 cases and review of literature. Diagn Cytopathol. 2008;36(5):297–302. 62. Nguyen GK, Redburn J. Aspiration biopsy of granulosa-cell tumor of the ovary: cytologic findings and differential diagnosis. Diagn Cytopathol. 1992;8:253–257. 63. Lal A, Bourtsos EP, Nayar R, DeFrias DV. Cytologic features of granulosa cell tumors in fluids and fine needle aspiration specimens. Acta Cytol. 2004;48(3):315–320. 64. Dejmek A. Fine needle aspiration cytology of an ovarian luteinized follicular cyst mimicking a granulosa cell tumor. A case report. Acta Cytol. 2003;47(6):1059–1062. 65. Stamp GW, Krausz T. Fine needle aspiration cytology of a recurrent juvenile granulosa cell tumor. Acta Cytol. 1988;32:533–539.

17

Soft Tissue VICKIE YOUNG JO, XIAOHUA QIAN

F

ew areas in cytology arouse such passionate discourse as fine-needle aspiration (FNA) of soft tissue masses. Critics jump at the opportunity to point out the limitations of the technique but pose few valid arguments along with outdated fallacies and academic concerns that do not necessarily consider the best interests of individual patients. Because needle core biopsy has largely replaced incisional biopsy as the initial diagnostic modality for patients presenting with soft tissue lesions, FNA has been increasingly accepted as a means to assess the adequacy of a needle core biopsy and as an alternative to core biopsy in certain settings. One undeniable difficulty with soft tissue FNA is the lack of experience most cytopathologists have with this subject; there are more than 130 different soft tissue lesions, including more than 30 sarcoma types.1 Because the morphologic heterogeneity is so astounding, it has been suggested that at least five soft tissue lesions ought to be sampled weekly to maintain practitioner experience.2 With sarcomas constituting only 0.8% of all malignancies in the United States,3 this recommendation is unrealistic in most practice settings. (The estimated number of new cases of sarcoma in the United States every year is 13,040, roughly the same as new cases of cervical cancer.3) Although it might be desirable to restrict FNA of primary soft tissue lesions to centers with cytological expertise and the capability for a multidisciplinary approach, most patients are first seen in community practices. A systematic approach to differential diagnosis, specimen triage, recommendations, and referrals can enhance the efforts of the cytopathologist, regardless of their level of experience. For this reason, this chapter focuses on pattern recognition, with sections on adipocytic, myxoid, spindle cell, fibrohistiocytoid, round cell, epithelioid, and pleomorphic neoplasms. Sampling error is a problem FNA shares with core and even incisional biopsies.4-6 Ancillary techniques such as immunohistochemistry and molecular/genetics are often indispensable for definitive classification.7-10 Needle tract seeding, a concern in the era of larger needles, however, is exceedingly rare with FNA.11 To prevent this potential risk, a single needle insertion point is recommended for several passes in cases with a high suspicion for sarcoma.12 538

FNA is a useful screening tool for soft tissue masses. It is the least invasive method for sampling a heterogeneous lesion, does not compromise tissue planes for a subsequent excision, and is cost effective.13,14 Although not widely accepted by most soft tissue pathologists as the sole sampling modality for the primary classification of sarcomas, it plays an important role in triaging patients—the initial differential diagnosis for many soft tissue masses is broad and might include lymphoma, carcinoma, and melanoma. FNA can be indispensable as the initial step toward narrowing the possibilities. FNA is especially valuable for confirming a recurrence or metastasis of a sarcoma.15-17 With recent advances in our understanding of mesenchymal lesions and the availability of an increasing number of ancillary diagnostic tests, FNA is suitable for the classification of a growing number of soft tumor tumors that have well-defined cytomorphologic, immunophenotypic, or molecular/genetic features.18 The location, size, and degree of invasiveness of a lesion and its relationship to surrounding structures are important clues to correct diagnosis. With a few exceptions, benign lesions are generally small, circumscribed, and cutaneous or superficial masses, whereas malignant lesions are more often large (greater than 5 cm), infiltrative, and deep seated (intermuscular or intramuscular or retroperitoneal).2,13-15,19-22 Patients with a benign mesenchymal mass lesion, which is at least 100 times more common than a sarcoma, benefit the most from a definitive benign diagnosis provided by FNA, the fastest, least expensive, and least invasive diagnostic procedure.

ADVANTAGES OF FINE-NEEDLE ASPIRATION FOR SOFT TISSUE LESIONS • T  echnical ease • Less invasive; less morbidity • Allows for immediate assessment of specimen adequacy, allocation of tissue for special studies, and initiation of treatment in emergency scenarios • Provides suitable material for ancillary studies • No contamination of tissue planes • Cost-effective

CHAPTER 17  Soft Tissue

Most (87%–100%) cases are correctly diagnosed as benign or malignant, with an average sensitivity and specificity for malignancy of approximately 95%.2,13,14,16,19,21,23,24 The distinction between lymphoma, carcinoma, and sarcoma has specificity rates for sarcoma of 54% to 98% and a positive predictive value of 91% to 99%.16,23,25 Owing to the rarity of most soft tissue tumors, data on the sensitivity and specificity for a specific entity are just emerging.18,26 The three main reasons for a false diagnosis are sampling error, a technically limited specimen, and misinterpretation.13 The false-positive rate for soft tissue FNA is relatively low (5% or less). False-negative rates vary a little more (2%–15%), but any clinically suspicious lesion should be further evaluated by biopsy.2,6,13,14,16,17,21,23 Rapid on-site evaluation of specimen adequacy (ROSE) at the time of the procedure, with triage of tissue for ancillary studies, decreases the rate of inadequate specimens.2,13-15,19,27 SUMMARY: STATISTICAL PERFORMANCE CHARACTERISTICS OF SOFT TISSUE FINENEEDLE ASPIRATION • • • •

 ensitivity rates: as high as 95% S Specificity for sarcoma: 54% to 98% False-positive rate: ≤5% False-negative rate: 2% to 15%

Specimen Collection and Preparation Cytomorphologic evaluation should be based on both alcohol-fixed, Papanicolaou-stained preparations (for nuclear details) and air-dried, Romanowsky-type stained preparations (for cytoplasmic details and matrix material). Thinlayer preparations offer good nuclear detail, optimize results from aspirates performed without the benefit of rapid evaluation, and can be used for adjunct studies, but cells can appear smaller, rounder, and falsely epithelioid, and useful background information (e.g., vascular patterns, chondromyxoid material) is sometimes lost.2,14,24,28-32 Cell block preparations provide architectural information and are the favored substrate for immunohistochemical studies and molecular testing. Unstained smears and cytospin slides are also suitable for ancillary testing, including fluorescence in situ hybridization (FISH) and molecular studies. A concurrent core biopsy is commonly obtained when imaging characteristics or ROSE suggests a primary soft tissue tumor.  SPECIMEN COLLECTION AND PREPARATION • 2  2- to 25-gauge needles • Separate passes for ancillary studies • Alcohol-fixed, Papanicolaou-stained smears for nuclear detail • Air-dried, Romanowsky-stained smears for cytoplasmic and matrix details • Cell block and core needle sample for immunohistochemical studies • Air-dried, unstained cytospin slides for FISH studies • Limited use of thinlayer preparations for morphologic evaluation

539

Ancillary Studies Ancillary techniques such as immunohistochemistry, cytogenetics, and molecular genetics are indispensable for the classification of soft tissue lesions. Electron microscopy is only rarely used, but it may provide assistance in classifying a poorly differentiated neoplasm or soft tissue tumor that exhibits specific ultrastructural features, such as the Weibel-Palade bodies of vascular tumors.2,33,34 Cell block sections are preferred for immunohistochemical studies, but air-dried or alcohol-fixed direct smears and cytocentrifuge or thinlayer preparations can also be used. Flow cytometry is useful when lymphoma is in the differential diagnosis. Ideally, one makes a dedicated pass for each desired ancillary study.23,34-37 Immunohistochemistry is useful in narrowing a differential diagnosis, detecting characteristic immunophenotypes of specific tumors (including correlates of underlying molecular/genetic changes), and identifying cases that need confirmatory molecular testing.38 An increasing number of reproducible, relatively specific genetic/molecular abnormalities can be identified with conventional chromosomal analysis (karyotyping), FISH, and reverse transcriptase-polymerase chain reaction (RT-PCR) techniques (Table 17.1).10,21,23,39,40 Sequencing-based methods, including high-throughput next-generation assays, are useful in detecting somatic gene mutations, insertions and deletions, and structural variants.41 Conventional chromosomal analysis offers the advantages of a full karyotype, but it requires fresh material and is counterbalanced by its low sensitivity and longer turnaround time as compared with molecular methods such as FISH.9,21,39,42 FISH accurately labels targeted chromosomal regions to detect gene rearrangements and amplifications and is a widely used, efficient diagnostic approach for soft tissue FNA. Virtually all cytologic preparations (cytocentrifuge preparations, thinlayer slides, and smears) are ideal substrates for FISH because they contain intact cells and nuclei free of sectioning artifact and truncation. When the diagnostic chromosomal aberration is known for a suspected soft tissue tumor, FISH is preferred because the number of cells needed is far less than for conventional karyotyping.2,43 Because only a specified chromosomal abnormality is targeted, however, a negative result provides minimal information.9,44 RT-PCR is helpful in identifying specific fusion transcripts; although advantageous for small samples, its performance depends on the assay design. 

Reporting Terminology As with other cytologic specimens, the use of general category headings on the report (e.g., benign, atypical, suspicious, positive, nondiagnostic), along with a descriptive interpretation, is useful for clarity of communication. Although histologic typing is desired whenever possible, a descriptive interpretation with a differential diagnosis is

540 CHA P T ER 1 7    Soft Tissue

TABLE 17.1    Soft Tissue Tumors, Their Associated Chromosomal Changes, and FISH Probes

Tumor

Cytogenetic aberration(s)

FISH probe(s)

Alveolar rhabdomyosarcoma

t(2;13)(q35;q14) t(1;13)(p36;q14), double minutes 2q35-rearrangement

FOXO1 FOXO1 N/A

Alveolar soft part sarcoma

der(X)t(X;17)(p11;q25)

TFE3

Angiofibroma of soft tissue

t(5;8)(p15;q13)

N /A

Angiomatoid fibrous histiocytoma

t(12;16)(q13;p11) t(12;22)(q13;q12) t(2;22)(q34;q12)

FUS EWSR1 EWSR1

Atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma

Ring/giant marker chromosomes from chromosome 12q13-15

HMGA2/MDM2

BCOR-CCNB3 sarcoma

inv(X)(p11.4p11.22)

N/A

CIC-rearranged sarcoma

t(4;19)(q35;q13) t(10;19)(q26.3;q13)

CIC

Clear cell sarcoma

t(12;22)(q13;q12) t(2;22)(q33;q12)

EWSR1 EWSR1

Dermatofibrosarcoma protuberans

r(17;22)(q21;q13)/t(17;22)(q21;q13)

PDGFB

Desmoplastic small round cell tumor

t(11;22)(p13;q12)

EWSR1

Embryonal rhabdomyosarcoma

Trisomies 2q, 8, and 20 Loss of heterozygosity at 11p15

N/A

Epithelioid hemangioendothelioma

t(1;3)(p36.3;q25)

N/A

Epithelioid sarcoma

t/der(22)(q11.2)

N/A

Ewing sarcoma

t(11;22)(q24;q12) t(21;22)(q22;q12) t(2;22)(q33;q12) t(7;22)(p22;q12) t(17;22)(q12;q12) t(20;22)(q13;q12)

EWSR1 EWSR1 EWSR1 EWSR1 EWSR1 EWSR1

Extraskeletal myxoid chondrosarcoma

t(9;22)(q22;q12) t(9;17)(q22;q11) t(9;17)(q22;q11) t(3;9)(q11;q22)

EWSR1 NR4A3 NR4A3 NR4A3

Hibernoma

11q13 rearrangement

N/A

Inflammatory myofibroblastic tumor

2p23 rearrangement

ALK

Infantile fibrosarcoma

t(12;15)(p13;q26)

ETV6

Intimal sarcoma

12q12-15 amplification

MDM2

Lipoblastoma

8q12 rearrangement or polysomy 8

PLAG1

Lipoma, chondroid

t(11;16)(9q13;p12-13)

N/A

Lipoma

12q14.3 rearrangement

HMGA2

Low-grade fibromyxoid sarcoma

t(7;16)(q34;p11) t(1;16)(p11;p11)

FUS FUS

Myoepithelial neoplasms of soft tissue

t(19;22)(q13;q12) t(1;22)(q23;q12) t(6;22)(p21;q12)

EWSR1 EWSR1 EWSR1

Myxoid liposarcoma

t(12;16)(q13;p11) t(12;22)(q13;q12)

DDIT3/FUS DDIT3/EWSR1

Myxoinflammatory fibroblastic sarcoma

der(10) t(1;10)(p22;q24)

N/A continued

CHAPTER 17  Soft Tissue

541

TABLE 17.1    Soft Tissue Tumors, Their Associated Chromosomal Changes, and FISH Probes­—cont’d

Tumor

Cytogenetic aberration(s)

FISH probe(s)

Nodular fasciitis

t(17;22)(p13.1;q12.3)

USP6

Pseudomyogenic hemangioendothelioma

t(7;19)(q22;q13)

N/A

Sclerosing epithelioid fibrosarcoma

t(11;22)(p11;q12) t(7;22(q33;q12)

EWSR1 EWSR1

Solitary fibrous tumor

inv12(q13q13)

N/A

Spindle cell/pleomorphic lipoma

Deletions of 13q and 16q

N/A

Synovial sarcoma

t(X;18)(p11;q11)

SS18

Tenosynovial giant cell tumor

t(1;2)(p13;q37)

N/A

FISH, Fluorescence in-situ hybridization; N/A, not available because either the probe is not commercially available or the gene is unknown. (Table 1 adapted from Dal Cin P, Qian X, Cibas ES. The marriage of cytology and cytogenetics. Cancer Cytopathol 2013;121:279-290, with permission from Wiley.)

appropriate when the findings are not conclusive for a specific entity.2,15,16,21,23,45 The most important prognostic consideration with soft tissue tumors is the histologic grade.46 Grading is ideal when a specific diagnosis can be unequivocally established and the tumor is gradable based on cellularity, pleomorphism, mitoses, and necrosis. For many sarcoma types, grade is definitional: Ewing sarcoma, synovial sarcoma, and angiosarcoma are high-grade malignancies, whereas well-differentiated liposarcoma and dermatofibrosarcoma protuberans are low grade. Some sarcomas are not readily gradable, such as epithelioid sarcoma and clear cell sarcoma. If grading is applied, a two-tiered grading system of low versus high is probably sufficient in most cases.2,13,17,46,47 Low-grade lesions have mild nuclear atypia, minimal or absent necrosis, low cellularity with minimal nuclear overlap, and rare or absent mitoses (generally less than 3 per 10 high-power fields). High-grade lesions have moderate to marked nuclear atypia, intermediate to high cellularity with prominent nuclear overlap, definite necrosis, and frequent mitoses, often with atypical forms.17,47 The presence or absence of mitoses and necrosis is an objective finding worthy of separate mention in the report. A mitotic count based on field unit is rarely possible on cytologic preparations. A statement such as “mitoses are absent” or “mitoses are numerous” suffices to avoid falsely high or low counts.17,46 

REPORTING SOFT TISSUE FINE-NEEDLE ASPIRATION RESULTS • G  eneral category • Histologic subtype, or description with differential diagnosis • Grade • Mitoses (including atypical forms) • Necrosis • Ancillary study results • Notes and recommendations

Adipocytic and Lipogenic Neoplasms Neoplasms with lipogenic differentiation occur over a broad age range, but malignant tumors occur almost exclusively in adults. Lesions with well-developed adipocytic morphology are difficult to classify accurately.48 Some authors discourage the use of FNA for large, deep-seated, intramuscular or retroperitoneal lesions that radiographically appear predominantly fatty.23 The diagnosis of atypical lipomatous tumor/well-differentiated liposarcoma can be missed if the (focal) morphologically diagnostic areas are not sampled; thorough sampling is often required for definitive diagnosis. With the help of ancillary studies (particularly molecular/ genetic), however, the classification of most fatty lesions is possible even with cytology and small biopsies (see Table 17.1).14,21-23,49

Lipoma Benign lipomas, occurring mainly in adults over the age of 30 years, are very common and account for about half of all soft tissue tumors. Most are slowly growing, subcutaneous or intramuscular tumors that occur over a wide anatomic distribution but spare the hands and feet; rarely exceed 10 cm in size; and present as soft, painless lumps. Retroperitoneal lipomas are exceedingly rare and usually need molecular/genetic confirmation. Benign-appearing adipose tissue in a retroperitoneal mass aspirate should evoke the differential diagnosis of atypical lipomatous tumor/welldifferentiated liposarcoma, myelolipoma, angiomyolipoma, and retroperitoneal fibrosis.

CYTOMORPHOLOGY OF LIPOMA • S  mall tissue fragments • Large, univacuolated adipocytes of uniform size • Small, bland nuclei without atypia   

  

542 CHA P T ER 1 7    Soft Tissue

CYTOMORPHOLOGY OF HIBERNOMA • L  arge tissue fragments containing many delicate capillaries • Numerous brown fat cells with multiple small cytoplasmic vacuoles • Variable cytoplasm (granular, microvesicular, or macrovesicular) • Small, bland nuclei

•

Fig. 17.1  Lipoma.  Benign lipomas have occasional capillaries, but they are not as numerous as in hibernomas (Papanicolaou stain).

Smears are composed of small tissue fragments; isolated cells are rarely present. Larger fragments contain thin capillaries, and striated or atrophic muscle fibers can be seen in juxtamuscular or intramuscular tumors. The large, univacuolated adipocytes are of uniform size and shape (Fig. 17.1). Their nuclei are small, round and regular, with evenly distributed chromatin.29,50 Scattered foamy macrophages with ingested lipid (called “lipophages”) are common in tumors with fat necrosis and regressive changes. Occasionally, myxoid stroma and metaplastic bone are seen. DIFFERENTIAL DIAGNOSIS OF LIPOMA • S  ubcutaneous adipose tissue • Pleomorphic lipoma • Atypical lipomatous tumor/well-differentiated liposarcoma

Normal subcutaneous adipose tissue lacks the masslike clinical presentation of a lipoma; thus the distinction between the two is most confidently achieved when the cytopathologist performs the aspiration, especially with ultrasound guidance. Atrophic muscle fibers in some lipoma aspirates mimic the floret cells of a pleomorphic lipoma, but the characteristic stromal cell population is absent. Lipomas lack the variability in adipocyte size and atypical stromal cells of atypical lipomatous tumor/well-differentiated liposarcoma.21,29,50-52 

Hibernoma Hibernoma is a rare benign tumor of brown fat that occurs predominantly in adults aged 20 to 50 years. The most common site is the thigh, followed by the trunk/chest, upper extremity, and head and neck. Although most often subcutaneous, it can be intramuscular and large (greater than 10 cm), mimicking an atypical lipomatous tumor radiologically.53 They rarely occur in the abdominal cavity, mediastinum, and retroperitoneum.

Hibernomas yield moderately cellular aspirates of fatty tissue fragments containing many delicate capillaries and numerous hibernoma (brown fat) cells of variable size (Fig. 17.2A). These round to polygonal cells have finely multivacuolated or granular cytoplasm and small, bland nuclei. Some large cells with two or more larger fat vacuoles indenting the small nucleus may resemble lipoblasts (Fig. 17.2B). Regular fat, myxoid stroma, and bland spindle cells may also be present. DIFFERENTIAL DIAGNOSIS OF HIBERNOMA • A  typical lipomatous tumor/well-differentiated liposarcoma • Myxoid liposarcoma • Spindle cell/pleomorphic lipoma • Chondroid lipoma • Lipoma with fat necrosis • Granular cell tumor • Adult rhabdomyoma • Pilosebaceous units

Although lipoblast-like cells are seen in hibernoma (Fig. 17.2B), the atypical lipoblasts (see Fig. 17.6) characteristic of atypical lipomatous tumor/well-differentiated liposarcoma are not present. Capillary vessels can be numerous in hibernomas, but they are less prominent than in a myxoid liposarcoma. Spindle cell lipomas lack the characteristic microvacuolated hibernoma cells. The myxoid stroma and chondromyxoid matrix of a chondroid lipoma are not prominent in a hibernoma. Lipophages in fat necrosis generally have a larger, plumper nucleus, and their fat vacuoles are more variable in size. Granular cell tumors lack adipocytic differentiation and are positive for S-100, SOX10, and NSE, and rhabdomyomas have larger cells with dense, granular cytoplasm and express desmin.51,53,54 Normal pilosebaceous units can contaminate any FNA that traverses hair-bearing skin. They are recognized by the admixture of finely vacuolated sebaceous cells with duct-like epithelial cells that have nonvacuolated cytoplasm. 

Spindle Cell/Pleomorphic Lipoma Spindle cell and pleomorphic lipomas are benign lipoma variants that represent a morphologic continuum. They are solitary, painless, well-circumscribed, and slowly growing tumors that often arise in the subcutis or dermis of the upper back, shoulder,

CHAPTER 17  Soft Tissue

A

B • Fig. 17.2  Hibernoma.  (A) Delicate capillaries (arrows) can be numerous and are closely associated with

tumor cells that have distinct granular and multivacuolated cytoplasm (Papanicolaou stain). (B) Lipoblastlike cells are present, but the nuclei are small and lack atypia (Papanicolaou stain).

543

544 CHA P T ER 1 7    Soft Tissue

neck, or anterior head and neck regions of middle-aged to older men. They rarely exceed 5 cm in greatest dimension. Spindle cell/pleomorphic lipoma is related to cellular angiofibroma and mammary-type myofibroblastoma; they share overlapping morphology and an association with 13q deletions.55 CYTOMORPHOLOGY OF SPINDLE CELL/ PLEOMORPHIC LIPOMA • F  ragments of mature adipose tissue • Often myxoid background • Occasional multivacuolated lipoblast-like cells

Spindle Cell Lipoma • B  land and uniform spindle cells • Ropy collagen fibers • Mast cells 

Pleomorphic Lipoma • “ Floret” cells • Smudged chromatin

Spindle cell lipoma is characterized by a mixture of mature adipocytes and bland spindle cells in short fascicles (Fig. 17.3A). Hyaline, ropy collagen fibers and mast cells are seen (Fig. 17.3B).56 Tumors designated as pleomorphic lipoma show predominantly mature adipocytes admixed with large, atypical multinucleated stromal cells with multiple hyperchromatic nuclei arranged in a floret-type pattern (Fig. 17.4).29,57 Tumors with hybrid features of spindle cell and pleomorphic lipoma are quite common. DIFFERENTIAL DIAGNOSIS OF SPINDLE CELL/ PLEOMORPHIC LIPOMA • A  typical lipomatous tumor/well-differentiated liposarcoma • Schwannoma • Myxoid liposarcoma • Myxofibrosarcoma, low grade • Solitary fibrous tumor • Dermatofibrosarcoma protuberans

A myxoid background and multivacuolated lipoblastlike cells are well-recognized diagnostic pitfalls, particularly for pleomorphic lipoma, and can lead to an erroneous diagnosis of a myxoid or well-differentiated liposarcoma or other myxoid/spindle cell neoplasm.51,57 This is particularly likely in cases that lack the classic floret cells or mature adipose tissue component. Spindle cell/pleomorphic lipoma is distinguished from liposarcoma by its typical anatomic distribution and superficial location, along with supportive immunohistochemistry (CD34 positivity in spindle cells, loss of RB expression) and cytogenetic findings (chromosomal aberrations of 13q and 16q, see Table 17.1). The positivity for CD34 and lack of S-100 in the spindle cells also help to distinguish it from a schwannoma (Table 17.2). Myxoid liposarcoma and myxofibrosarcoma have more

prominent blood vessels and lack mature adipose tissue and collagen fibers. With a spindle-cell-predominant variant, the distinction from a dermatofibrosarcoma protuberans and a solitary fibrous tumor, especially the fat-forming variant, can be difficult because both mimics can have mature adipose tissue and CD34-positive spindle cells. Clinical correlation, immunohistochemistry, and cytogenetic findings are essential in difficult cases.29,56,58 

Atypical Lipomatous Tumor/WellDifferentiated Liposarcoma Liposarcoma is the most common soft tissue sarcoma of adults. There are three major subtypes that are biologically distinct: (1) well differentiated and dedifferentiated, (2) myxoid, and (3) pleomorphic. The terms well-differentiated liposarcoma and atypical lipomatous tumor are synonymous, and the terminology depends on local practice (namely, comprehension by treating clinicians). Atypical lipomatous tumor applies to lesions of the limbs and trunk that do not recur if adequately excised, whereas well-differentiated liposarcoma is appropriate for deep-seated lesions (e.g., retroperitoneum, spermatic cord, and mediastinum), where recurrence and local aggressiveness are possible given that complete resection is often not feasible. CYTOMORPHOLOGY OF ATYPICAL LIPOMATOUS TUMOR/WELL-DIFFERENTIATED LIPOSARCOMA • C  lusters of variably sized lipogenic cells with lipid vacuoles • Atypical stromal cell nuclei • Lipoblasts may be present • Occasional floret cells with hyperchromatic nuclei • Nonlipogenic pleomorphic or spindle-cell tissue fragments suggestive of dedifferentiation

In general, tissue fragments of large but variably sized, univacuolated adipocytes (Fig. 17.5) are admixed with blood and extruded lipid. Lipoblasts—smaller multivacuolated cells with atypical, scalloped nuclei—are present only in some cases (Fig. 17.6), but they are not required for the diagnosis. The nuclei of larger adipocytes range from small and compressed to large, round to oval, and hyperchromatic (Fig. 17.7). They are often multilobated or convoluted. Similar atypical nuclei are seen in fragments of collagenous stromal tissue.48,50,59,60 DIFFERENTIAL DIAGNOSIS OF ATYPICAL LIPOMATOUS TUMOR/WELL-DIFFERENTIATED LIPOSARCOMA • • • •

 ipoma with fat necrosis L Extruded lipid from normal fat Spindle cell/pleomorphic lipoma Hibernoma

CHAPTER 17  Soft Tissue

A

B • Fig. 17.3  Spindle Cell Lipoma.  (A) This tumor is characterized by a mixture of dispersed bland spindle

cells and mature adipocytes (Romanowsky stain). (B) After some searching, long, ropy collagen fibers are identified, embedded in a myxoid background (Romanowsky stain).

545

546 CHA P T ER 1 7    Soft Tissue

• Fig. 17.4  “Floret” Cells.  Multinucleated giant cells, some with nuclei in a wreathlike configuration (arrow),

are a characteristic but nonspecific finding in pleomorphic lipomas (Papanicolaou stain). (Courtesy Dr. Henryk Domanski, Lund University Hospital, Lund, Sweden.)

Extensive sampling of a large liposarcoma is necessary because diagnostic areas can be extremely focal.29,50,52,57 The first impression might be that of a lipoma, but the clinical presentation of lipomas and liposarcomas is generally so different that this rarely presents a significant dilemma. Nevertheless, careful examination of the specimen is important to identify the sometimes subtle nuclear and cellular alterations of a well-differentiated liposarcoma (Fig. 17.5). Benign lipid droplets entrapped in blood are a common finding whenever any fatty tissue, including normal adipose tissue, is sampled. Its nonspecific nature is revealed by the absence of nuclei. Fat necrosis is comprised of lipophages whose cytoplasm is foamy and filled with small vacuoles, whereas the cytoplasm of adipocytes and lipoblasts is optically clear. Some of the multilobated hyperchromatic cells of a liposarcoma can resemble the floret cells of a pleomorphic lipoma; clinical and radiographic correlation is the key to this distinction. Giant marker and ring chromosomes derived from amplification of 12q13-15 are characteristic of atypical lipomatous tumor/well-differentiated liposarcoma (see Table 17.1) and help to differentiate it from its benign mimics such as hibernoma and spindle cell/ pleomorphic lipoma, which each have their own characteristic cytogenetic alterations. With lipomatous tumors, however, one rarely gets enough material from an FNA for a conventional karyotype. They are more amenable to molecular diagnostic or FISH analysis. Overexpression of MDM2 and CDK4 from amplification of the MDM2 and CDK4 genes on chromosome 12q13-15, a characteristic genetic abnormality in atypical lipomatous tumor/well-differentiated liposarcoma and dedifferentiated liposarcoma, can be detected by immunohistochemistry and is diagnostically helpful.61 MDM2 expression is observed in other sarcomas, however, including other tumor types with MDM2 amplification (intimal sarcoma, low-grade central and

parosteal osteosarcoma) and tumors without MDM2 amplification (myxofibrosarcoma, radiation-associated sarcoma, and pleomorphic liposarcoma). The rare inflammatory subtype of atypical lipomatous tumor/well-differentiated liposarcoma yields moderately cellular smears with abundant small lymphocytes, larger lymphoid cells, and plasma cells. The scattered large, atypical cells with multiple or multilobated nuclei of this variant evoke the unusual differential diagnosis of Hodgkin lymphoma, anaplastic large cell lymphoma, inflammatory myofibroblastic tumor, and metastatic carcinoma.62 

Pleomorphic Liposarcoma Pleomorphic liposarcoma is a rare malignancy, accounting for about 5% of liposarcomas. It commonly occurs in the extremities of elderly adults and has a poor prognosis. It is distinguished from other high-grade pleomorphic sarcomas by the presence of multivacuolated, highly atypical lipoblasts. CYTOMORPHOLOGY OF PLEOMORPHIC LIPOSARCOMA • • • • •

 ypercellular smear H Dispersed cells and three-dimensional clusters Pleomorphic cells with marked nuclear atypia Variable number of atypical lipoblasts Mitoses and necrosis

Smears are moderately to highly cellular, with dispersed, markedly anaplastic and pleomorphic cells and threedimensional tumor cell clusters. The cells range in contour from polygonal to spindle-shaped. Nuclei are bizarre and

Tumor

SMA

Desmin

S-100

CD34

KIT/ DOG-1

CK/EMA

β-catenin (nuclear)

STAT6

TLE1

H3K27me 3

Spindle cell lipomaa

−

−

−

+ spindle cells

−

−

−

−

−

retained

Schwannoma

−

−

+ diffuse

−

−

−

−

−

−

retained

MPNST

−

−/+

+ focal

−/+

−

−/+

−

−

−/+

nuclear loss

Synovial sarcoma

−

−

−/+

−

−

+

−/+

−

+

−/+

GIST

−/+

−

−

+

+

−

−

−

−

retained

Leiomyoma and leiomyosarcoma

+ diffuse

+ diffuse

−

−

−

−/+

−

−

−

retained

Solitary fibrous tumor

−

−

−

+

−

−

−/+

+

−

retained

Desmoid fibromatosis

−/+

−

−

−

−

−

+

−

−

retained

aAlso

show loss of Rb expression. CK, Cytokeratin; EMA, epithelial membrane antigen; GIST, gastrointestinal stromal tumor; MPNST, malignant peripheral nerve sheath tumor; SMA, smooth muscle actin.

CHAPTER 17  Soft Tissue

Downloaded for hamdan naifeh ([email protected]) at McGill University from ClinicalKey.com by Elsevier on February 13, 2020. For personal use only. No other uses without permission. Copyright ©2020. Elsevier Inc. All rights reserved.

TABLE 17.2    Immunoprofile of Spindle Cell Neoplasms

547

548 CHA P T ER 1 7    Soft Tissue

occasionally scalloped, with coarse chromatin and prominent nucleoli. The abundant cytoplasm varies from homogeneous to multivacuolated, with vacuoles of varying sizes (Fig. 17.8). Mitoses and necrosis are usually present.60,63,64 Some examples may show myxoid stroma.64 Extensive sampling to identify unequivocal atypical lipoblasts is necessary to separate pleomorphic liposarcoma from a pleomorphic sarcoma of another lineage. Immunohistochemistry, although helpful in excluding some cytologic mimics (including dedifferentiated liposarcoma, myxofibrosarcoma, and poorly differentiated signet-ring cell adenocarcinoma), generally has a very limited role in establishing the diagnosis of pleomorphic liposarcoma. Cytogenetically, pleomorphic liposarcoma exhibits increased chromosome numbers with

complex rearrangements, a profile, unfortunately, of no diagnostic value.29,60,63 

• Fig. 17.5  Well-Differentiated Liposarcoma.  The much-touted varia-

•

tion in adipocyte size can be subtle and easily overlooked (compare with Fig. 17.1) (Papanicolaou stain).

Myxoid Neoplasms The diverse and mostly unrelated myxoid soft tissue lesions share one (often striking) morphologic feature: abundant, homogeneous myxoid matrix. In most instances, it appears as an amorphous, finely granular film that stains pale bluegreen with the Papanicolaou stain and a striking blue-violet with Romanowsky stains. A myxoid picture is produced by a very broad spectrum of lesions that includes nodular fasciitis, benign myxoid tumors, and myxoid sarcomas. Clinically, most benign lesions are superficially located (except for

Fig. 17.6  Lipoblast.  The enlarged, hyperchromatic nuclei of lipoblasts are scalloped because of multiple impinging cytoplasmic lipid droplets (Papanicolaou stain).

• Fig. 17.7  Atypical Lipomatous Tumor/Well-Differentiated Liposarcoma.  Atypical nuclei in deep-seated

fatty lesions denote malignancy, even when classic lipoblasts are not readily identified (Papanicolaou stain).

CHAPTER 17  Soft Tissue

549

• Fig. 17.8  Pleomorphic Liposarcoma.  Clusters of pleomorphic cells, including numerous atypical lipoblasts, are seen (Romanowsky stain).

intramuscular myxoma), whereas sarcomas are deep-seated (with the exception of myxofibrosarcoma). Morphologically, benign lesions generally have lower cellularity and less nuclear atypia than malignant neoplasms, but exceptions do occur. Limited sampling may yield hypocellular smears with abundant matrix and result in an erroneous low-grade assignment. Despite the challenges, myxoid lesions, particularly the lowgrade ones, can often be subtyped based on cytomorphology alone with accuracy rates up to 81%.65 Molecular analysis and immunohistochemistry is helpful in resolving the differential diagnosis of some of the malignant lesions, like myxoid liposarcoma, low-grade fibromyxoid sarcoma, and extraskeletal myxoid chondrosarcoma (see Table 17.1).9

Intramuscular Myxoma Intramuscular myxoma is a benign, painless, slowly growing, well-circumscribed but unencapsulated neoplasm of adults aged 40 to 70 years, usually involving the muscles of the thigh, shoulder, buttocks, and upper arm. Cellular myxoma is a morphologic variant that has increased cellularity and a vascular component, but lacks the nuclear atypia and hyperchromasia of low-grade myxofibrosarcoma. Myxomas have GNAS1 mutations and pose a small risk of local recurrence. The juxtaarticular myxoma is cytologically similar to a cellular myxoma but occurs adjacent to large joints, especially the knee, and lacks GNAS1 mutations. FNAs from most intramuscular myxomas are sparsely cellular, consisting in large part of abundant, myxoid matrix (Fig. 17.9A), but aspirates from a cellular myxoma can be surprisingly cellular (Fig. 17.9B). Fibroblast-like cells,

CYTOMORPHOLOGY OF INTRAMUSCULAR MYXOMA • • • • • • •

 ypocellular H A film of smooth or granular matrix material Absent or very scant vascular component Uniform cells with a bland nucleus Long, fibrillary cytoplasmic processes Multinucleated atrophic muscle fibers Macrophages with vacuolated cytoplasm

isolated and in clusters, are uniform in size and display a variety of shapes: round to oval, spindle-shaped, stellate, and polygonal. Their small, round to oval nucleus has fine, uniform chromatin, small nucleoli, and no atypia. The cytoplasm is finely granular with long, fibrillary cytoplasmic processes and occasional small vacuoles. Macrophages and mast cells can be present. The vascular component, if present, consists of very rare short vessels (Fig. 17.9B). Often, multinucleated skeletal muscle fibers, sometimes atrophic, are strewn about. Mitoses are virtually never observed.66-68 DIFFERENTIAL DIAGNOSIS OF INTRAMUSCULAR MYXOMA • • • • • • •

 anglion cyst G Nodular fasciitis Soft tissue perineurioma with prominent myxoid stroma Low-grade myxofibrosarcoma Low-grade fibromyxoid sarcoma Myxoid liposarcoma Extraskeletal myxoid chondrosarcoma

550 CHA P T ER 1 7    Soft Tissue

A

B •

Fig. 17.9  Intramuscular Myxoma.  (A) Many Intramuscular myxomas are paucicellular, comprised mostly of myxoid matrix. Rare macrophages and bland spindle cells are present (Papanicolaou stain). (B) Cellularity is more pronounced with a cellular myxoma. Stubby vessels can be seen, especially with the cellular myxoma (Romanowsky stain).

Aspirates from ganglion cysts contain only abundant granular myxoid material and occasional macrophages; the spindle-shaped, stellate, or polygonal myxoma cells are absent. Nodular fasciitis shows a more haphazard cellular arrangement of polymorphic myofibroblasts and ganglion-like cells, often with interspersed inflammatory cells. Soft tissue

perineurioma has more slender perineural cells and characteristically expresses epithelial membrane antigen (EMA) and claudin-1, but EMA can be focally positive in myxoma.69 Both myxofibrosarcoma and myxoid liposarcoma have a more prominent vascular component than the typical intramuscular myxoma. Although low-grade myxofibrosarcoma

CHAPTER 17  Soft Tissue

and low-grade fibromyxoid sarcoma have notable nuclear atypia, their distinction from cellular myxoma can be difficult. Adequate sampling from different parts of the mass is essential to avoid missing a low-grade fibromyxoid sarcoma, which has alternating areas of myxoid and collagenous stroma. The stroma in extraskeletal myxoid chondrosarcoma is more brightly magenta and fibrillar rather than blue-purple and smooth or granular as seen in a myxoma, and the lacelike arrangement of tumor cells is more uniform than that of myxoma cells (see Fig. 17.15). 

Soft Tissue Perineurioma Soft tissue perineurioma is a benign peripheral nerve sheath neoplasm composed of perineurial cells. It usually occurs in the subcutaneous tissue in the limbs of middle-aged adults. The distinctive histologic growth patterns, such as storiform, perivascular whorled, lamellar or reticular, are imperceptible in FNA smears. Nevertheless, the presence of slender cells with bipolar, long, thin cytoplasmic processes, characteristic of perineurial cells, is a reproducible finding in smears.70,71 Perineurial cells are positive for EMA, claudin-1, and CD34 (in two-thirds of cases) and negative for S-100, a useful immunoprofile to support the diagnosis. CYTOMORPHOLOGY OF SOFT TISSUE PERINEURIOMA • • • •

 ariable cellularity V Myxoid or collagenous stroma Absent or very scant vascular component Slender cells with a bland nucleus and bipolar, thin, long cytoplasmic processes

The aspirate cellularity is variable, as is the type of stroma. Prominent myxoid stroma is common, leading to the impression of a “low-grade myxoid spindle cell neoplasm.” The typical slender cells are either isolated or arranged in loose clusters. Stripped nuclei and strings of cytoplasmic processes are common. Nuclei are bland, with finely granular, evenly dispersed chromatin. DIFFERENTIAL DIAGNOSIS OF SOFT TISSUE PERINEURIOMA • • • • • •

 ermatofibrosarcoma protuberans D Intramuscular myxoma, cellular type Neurofibroma and schwannoma Low-grade fibromyxoid sarcoma Low-grade myxofibrosarcoma Low-grade malignant peripheral nerve sheath tumor

For superficial lesions, the main differential diagnosis is dermatofibrosarcoma protuberans, which can be distinguished from soft tissue perineurioma by the absence of expression of EMA and claudin-1. Cellular myxoma,

551

low-grade fibromyxoid sarcoma, and perineurioma with prominent myxoid stroma can be indistinguishable from each other in small biopsies without the aid of ancillary studies, although EMA has limited specificity in this context.69 Other benign peripheral nerve sheath tumors like neurofibroma and schwannoma show more diffuse immunoreactivity for S-100, although hybrid tumors with both perineurioma and schwannoma components exist.72 The tumor cells in low-grade sarcomas have more cytologic atypia and lack the long, thin cytoplasmic processes characteristic of perineurioma. 

Myxofibrosarcoma Myxofibrosarcoma is one of the most common sarcomas. It typically occurs in the extremities of elderly patients, principally during the sixth to eighth decades of life. Usually dermal or subcutaneous, it involves deeper soft tissues in one-third to one-half of cases. Tumors are graded as low, intermediate, or high grade. Up to 60% of cases recur locally regardless of grade, and they tend to become progressively higher grade with each recurrence. Higher-grade lesions have the potential to metastasize to lungs, bones, and lymph nodes.

CYTOMORPHOLOGY OF MYXOFIBROSARCOMA • V  ariable amounts of myxoid matrix • Short segments of curved, collagenized vessels • Mildly atypical spindle and stellate cells (low-grade tumors) • Marked nuclear pleomorphism, multinucleation, and necrosis (high-grade tumors) • Pseudolipoblasts

Myxofibrosarcomas are morphologically quite variable, ranging from low- to high-grade tumors. In general, the amount of myxoid material is inversely proportional to cellularity and grade. The curvilinear vessels that are characteristic of these tumors in histologic sections are absent from smears in many cases, but when present they appear as short segments of collagenized capillaries best seen in paucicellular areas (Fig. 17.10A). The neoplastic cells are randomly distributed and noncohesive. Occasional small aggregates occur, and higher-grade lesions contain large cell groups and tissue fragments. Most cells in a low-grade tumor are spindle-shaped, with occasional tadpole, stellate, and polygonal forms. Nuclear pleomorphism and atypia increase with grade. In higher-grade lesions, binucleation and multinucleation are more prominent. Irrespective of grade, tumor cells with vacuolated cytoplasm, resembling lipoblasts but containing acid mucins rather than lipid, are found (Fig. 17.10B).23,65,66,73,74 Mitotic activity is uncommon.

552 CHA P T ER 1 7    Soft Tissue

A

B • Fig. 17.10  Myxofibrosarcoma.  (A) The vascular component, when present, manifests itself as short curvilinear segments of thick-walled vessels with adherent matrix and pleomorphic tumor cells (Papanicolaou stain). (B) Pseudolipoblasts (arrow) contain acid mucins rather than lipid (Romanowsky stain).

DIFFERENTIAL DIAGNOSIS OF MYXOFIBROSARCOMA

CYTOMORPHOLOGY OF LOW-GRADE FIBROMYXOID SARCOMA

• • • • • • •

• A  bundant myxoid matrix • Uniform fibroblast-like oval to spindle cells with mild nuclear atypia • No significant vascularity or nuclear pleomorphism

 ellular myxoma C Nodular fasciitis Myxoid liposarcoma Low-grade fibromyxoid sarcoma Pleomorphic liposarcoma Dedifferentiated liposarcoma Other high-grade sarcomas with a clear line of differentiation

Cellular myxoma and nodular fasciitis lack the curvilinear vessels and nuclear atypia of myxofibrosarcoma. Myxoid liposarcoma contains delicate branching vessels and true lipoblasts and usually does not have much nuclear atypia or pleomorphism. The distinction between low-grade myxofibrosarcoma and the similarly named low-grade fibromyxoid sarcoma is difficult but possible, especially in conjunction with clinical and cytogenetic findings. Myxofibrosarcoma and pleomorphic liposarcoma show overlapping clinical and morphologic features and may be difficult to distinguish from one another on biopsy if the characteristic large atypical lipoblasts of pleomorphic liposarcoma are absent. Other high-grade sarcomas with myxoid change but with a clear line of differentiation, such as dedifferentiated liposarcoma, should be excluded in a deep-seated tumor. 

Low-Grade Fibromyxoid Sarcoma Low-grade fibromyxoid sarcoma (LGFMS), first described by Evans,75 affects mainly younger adults in the third to fifth decades and arises in the deep soft tissue of the thigh or trunk. Despite its deceptively bland morphology, up to 45% of cases eventually metastasize after a long indolent course.51 Histologically, LGFMS has a characteristic whorled arrangement of bland spindle cells with alternating areas of myxoid and collagenous stroma, but the cytologic findings,

described in only a few case reports and small series,76-79 are somewhat nonspecific. Often the myxoid, hypocellular areas are sampled, yielding a homogeneous population of fibroblast-like oval to spindle cells with slightly plump but bland, uniform nuclei in a myxoid background. Vascularity is variable depending on the area sampled (Fig. 17.11A). DIFFERENTIAL DIAGNOSIS OF LOW-GRADE FIBROMYXOID SARCOMA • • • • • • •

 ellular myxoma C Soft tissue perineurioma Low-grade myxofibrosarcoma Low-grade malignant peripheral nerve sheath tumor Extraskeletal myxoid chondrosarcoma Solitary fibrous tumor Desmoid fibromatosis

A cellular myxoma is usually less cellular and poorly vascularized. Perineuriomas have longer cytoplasmic processes. Low-grade myxofibrosarcoma and malignant peripheral nerve sheath tumors have more notable nuclear atypia. Clinically, low-grade myxofibrosarcoma tends to occur in the superficial soft tissues of elderly patients, whereas LGFMS is usually situated in the deep soft tissues of young adults. Like LGFMS, extraskeletal myxoid chondrosarcoma occurs in young to middle-aged adults and involves the deep soft tissue of the extremities. Also like LGFMS, extraskeletal myxoid chondrosarcoma yields smears with uniform oval- to

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A MUC4

B • Fig. 17.11  Low-Grade Fibromyxoid Sarcoma.  (A) Uniform, deceptively bland fibroblasts are embedded in a myxoid matrix (Papanicolaou stain). (B) Cytoplasmic MUC4 expression distinguishes low-grade fibromyxoid sarcoma from its mimics.

spindle-shaped cells in a myxoid background. Immunohistochemistry is helpful: MUC4 is a highly sensitive and specific marker of LGFMS (Fig. 17.11B).69,80 Cytogenetic testing is also helpful: LGFMS harbor a FUS-CREB3L2 fusion secondary to the translocation t(7;16)(q34;p11), and FUS FISH is a reliable diagnostic aid (see Table 17.1).81,82 

Myxoid Liposarcoma Myxoid liposarcoma occurs in slightly younger individuals than the other liposarcomas, primarily those in their fourth and fifth decades, with a predilection for the deep soft tissue of the lower limbs, especially the thigh. It is characterized by a FUS-DDIT3 rearrangement (see Table 17.1). Myxoid

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liposarcoma has prominent myxoid matrix, a characteristic branching vascular pattern, uniform round or ovoid tumor cells, and small lipoblasts. Tumors are graded based on cellularity as low-, intermediate-, or high-grade, and the grade is prognostically significant. High-grade tumors, previously known as “round-cell liposarcoma,” frequently show a predominantly round cell morphology.

Smears are moderately to markedly cellular, with a strikingly monotonous appearance. Lower-grade myxoid liposarcomas contain tissue fragments (Fig. 17.12A) with a prominent myxoid stroma, thin-walled vessels, and numerous round-to-oval tumor cells with scant cytoplasm, finely granular chromatin, and indistinct cell borders. The tumor cells are uniformly oval to fusiform, with monomorphic nuclei, evenly distributed, finely granular chromatin, and up to two inconspicuous nucleoli. Regardless of grade, myxoid liposarcomas have abundant myxoid matrix with arborizing, thin-walled capillaries of uniform caliber (Fig. 17.12B). The lipoblasts are variable in number from case to case and usually small, either univacuolated or bivacuolated, resembling signet-ring cells (“signet-ring cell”-like lipoblasts), often present along the capillaries (Fig. 17.12C). In contrast, higher-grade tumors have a greater component of isolated and clustered cells.24,29,50,52,65,66,74 Tumor cells are substantially larger and rounder, with more

CYTOMORPHOLOGY OF MYXOID LIPOSARCOMA • A  bundant granular myxoid matrix in tissue fragments • Delicate, branching, thin-walled capillaries • Uniform round-to-oval cells with occasional small cytoplasmic vacuoles • Univacuolated and bivacuolated lipoblasts (“signet-ring cell”-like lipoblasts), especially along capillaries • Increased number of round cells with scant cytoplasm and prominent nucleoli in high-grade tumors

A

C

B

D • Fig. 17.12  Myxoid Liposarcoma.  (A) Discrete tissue fragments contain myxoid matrix, vessels, and tumor

cells (Romanowsky stain). (B) Delicate branching “chicken-wire” capillaries are easily obtained by aspiration and are seen in abundance on smears (Papanicolaou stain). (C) The lipoblasts in myxoid liposarcomas resemble signet-ring cells and histiocytes and are often associated with the vessels (Romanowsky stain). (D) Fluorescence in situ hybridization is often performed with centromeric (red) and telomeric (green) probes that flank the CHOP (DDIT3) gene breakpoint. There is one normal chromosome (probes adjacent = yellow), but the split-apart green and red signals indicate a translocation on the other chromosome in the 4 tumor cells in this field. (Courtesy Dr. Paola Dal Cin, Brigham and Women’s Hospital, Boston, MA.)

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anisokaryosis of the centrally placed nucleus, vesicular chromatin, and multiple prominent nucleoli. The cytoplasm is scant, more densely staining, and rarely vacuolated. The prominent vessels are often obscured by densely packed tumor cells. Due to its prognostic significance, the presence of the hypercellular component in the biopsy should be documented. DIFFERENTIAL DIAGNOSIS OF MYXOID LIPOSARCOMA • • • • •

 ow-grade myxofibrosarcoma L Spindle cell lipoma with myxoid changes Low-grade myxofibrosarcoma Extraskeletal myxoid chondrosarcoma Myoepithelioma of soft tissue

The cytomorphology of myxoid liposarcomas is often distinctive and allows for a correct diagnosis. Immunohistochemistry is usually not necessary and of limited value, especially because myxoid liposarcoma lacks a specific immunophenotype. The detection of the typical chromosomal abnormalities t(12;16)(q13;p11) or t(12;22)(q13;q12) by either karyotype or FISH for DDIT3 rearrangement (Fig. 17.12D) can help distinguish it from other myxoid and round cell tumors (see Table 17.1).9,24,29,50,52,65,66 

Lipoblastoma Lipoblastoma is a rare benign tumor affecting mainly males 3 years of age or younger. It involves the superficial subcutaneous tissues of the limbs, especially the lower extremities, as a slowly growing circumscribed mass. More deeply seated lesions tend toward larger size and a diffusely infiltrative growth pattern (lipoblastomatosis). Local recurrence is uncommon and is primarily restricted to the infiltrative lesions. CYTOMORPHOLOGY OF LIPOBLASTOMA • M  yxoid matrix with lipid vacuoles • Prominent thin, branching capillaries • Three types of cells: bland lipoblasts, primitive spindle cells, and mature adipocytes

Smears are moderately cellular, with cohesive clusters and sheets of bland lipoblasts and primitive spindleshaped cells embedded in a myxoid background with fatty vacuoles. Myxoid material can be abundant or sparse and restricted to small- to medium-sized tissue fragments with irregular borders. Delicate branching capillaries are conspicuous. Bare nuclei can be widespread, and variable numbers of mature adipocytes are seen, depending on the degree of maturation. The lipoblasts are generally small, round, and uniform in appearance. Chromatin is delicate

555

or moderately coarse, and nucleoli are largely absent.50,83,84 Nuclear atypia is limited to focal minimal nuclear membrane irregularities; it is not as prominent as that seen with the lipoblasts of a liposarcoma. In addition to affecting a much younger population, lipoblastoma is distinguished from its primary morphologic mimic, myxoid liposarcoma, by absent atypia, less-abundant myxoid stroma and the presence of solid sheets of tumor cells. Cytogenetic analysis (FISH for a PLAG1 rearrangement) has high diagnostic utility (see Table 17.1).50,83-85 

Myxofibrosarcoma-Like Dedifferentiated Liposarcoma Dedifferentiated liposarcoma is a nonlipogenic spindle cell or pleomorphic sarcoma containing distinct areas of atypical lipomatous tumor/well-differentiated liposarcoma. Notably, dedifferentiation is not limited to liposarcomas (see “Dedifferentiated Sarcomas” below). Dedifferentiated liposarcoma has a variable morphologic picture, but most cases are nonlipogenic and resemble either undifferentiated pleomorphic sarcoma or myxofibrosarcoma. Dedifferentiated liposarcomas range from low- to high-grade tumors, and they may even be lipogenic and resemble pleomorphic liposarcoma (so-called “homologous dedifferentiation”).64,86,87 Whenever the typical morphology of a myxofibrosarcoma is encountered in the retroperitoneum, the diagnosis of a dedifferentiated liposarcoma should be the foremost diagnostic consideration.88,89 CYTOMORPHOLOGY OF MYXOFIBROSARCOMA-LIKE DEDIFFERENTIATED LIPOSARCOMA • A  bundant granular myxoid matrix • Thick-walled branching vessels • Spindle cells with mild to moderate nuclear atypia and sometimes, vacuolated cytoplasm • Occasional multinucleated tumor cells

Smears are moderately to highly cellular, with a prominent myxoid background. Dispersed spindled to stellate cells surround loosely cohesive tumor cell clusters arrayed around long vascular channels. The vessels show complex branching similar to the delicate capillaries of myxoid and round cell liposarcoma, but their walls are notably thicker (Fig. 17.13). Most cells have a round-to-oval, hyperchromatic nucleus with evenly distributed chromatin and an indistinct nucleolus. The cytoplasm is scant to moderate and sometimes vacuolated. Other cells have more abundant, stellate-shaped cytoplasm, slightly larger and irregular nuclei with smudgy chromatin, and a greater tendency toward binucleation or multinucleation. Rare diagnostic lipoblasts may be seen.64,90 The presence of heterologous osteosarcomatous or rhabdomyosarcomatous differentiation can be misleading in small biopsy specimens. Molecular analysis and clinical details are important for the correct diagnosis. Just as for atypical lipomatous tumor/well-differentiated

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•

Fig. 17.13  Dedifferentiated Liposarcoma, Myxofibrosarcoma-Like.  These tumors may be myxoid, with complex, branching, thick-walled vasculature, and resemble a myxofibrosarcoma. True lipoblasts, as depicted in this case, are a helpful feature for the distinction from other myxoid lesions, but most dedifferentiated liposarcomas are nonlipogenic (Romanowsky stain).

liposarcoma, demonstrating overexpression of MDM2 and CDK4 by immunohistochemistry (or amplification of MDM2 by FISH) is helpful. 

Myxoinflammatory Fibroblastic Sarcoma Myxoinflammatory fibroblastic sarcoma (MIFS) is a locally aggressive fibroblastic neoplasm of the distal extremities in middle-aged adults. When first described in 1998, it was called “inflammatory myxohyaline tumor of distal extremities with virocytes or Reed-Sternberg-like cells,” “acral myxoinflammatory fibroblastic sarcoma,” or “inflammatory myxoid tumor of the soft parts with bizarre giant cells.”1 Although most arise in the subcutaneous tissue of the feet and hands, proximal sites can be affected. The translocation t(1;10) involving TGFBR3 and MGEA5 is present in both MIFS and hemosiderotic fibrolipomatous tumor, linking these two rare entities to each other.91 CYTOMORPHOLOGY OF MYXOINFLAMMATORY FIBROBLASTIC SARCOMA • M  yxoid matrix • Mixed population of bland spindle cells and mononuclear epithelioid cells • Large atypical Reed-Sternberg-like cells with macronucleoli • Mixed inflammatory cells with hemosiderin and macrophages • Pseudolipoblasts

Typical cytologic features include a cellular smear with a prominent myxoid background, spindle cells with bipolar processes, mononuclear epithelioid cells, scattered large atypical ganglion-like or Reed-Sternberg-like cells with inclusion-like nucleoli, and mixed inflammatory cells (Fig. 17.14). The hypervacuolated epithelioid cells may resemble lipoblasts.92-95 Given the heterogeneity of the tumor, FNA samples may not contain all of the above elements, posing significant diagnostic challenges. DIFFERENTIAL DIAGNOSIS OF MYXOINFLAMMATORY FIBROBLASTIC SARCOMA • • • • • •

 odular fasciitis N Fibroinflammatory reactive lesion Myxofibrosarcoma Liposarcoma Inflammatory myofibroblastic tumor Hodgkin lymphoma

MIFS is a low-grade sarcoma with a risk for frequent local recurrence but infrequent distant metastases. It is important not to misinterpret the bland fibroblastic proliferation admixed with inflammation as a reactive process or nodular fasciitis. Conversely, the large atypical cells in a background of mixed inflammation should not be confused with a high-grade sarcoma96 or Hodgkin lymphoma. Finally, the pseudolipoblasts should not be construed as indicative of a myxoid liposarcoma or an inflammatory well-differentiated liposarcoma. Immunohistochemistry

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•

Fig. 17.14  Myxoinflammatory Fibroblastic Sarcoma.  The occasional large ganglion-like or ReedSternberg-like cells in a background of otherwise bland spindle cells, vacuolated epithelioid cells, and inflammatory cells may lead to confusion with a high-grade sarcoma or Hodgkin lymphoma (Romanowsky stain).

is of limited value in establishing the diagnosis of MIFS but is helpful in excluding some mimics such as Hodgkin lymphoma. 

Extraskeletal Myxoid Chondrosarcoma Extraskeletal myxoid chondrosarcoma (EMC) is a slowly growing malignant tumor of adults, with a median age at diagnosis of 50 years. It involves the deep soft tissues of the proximal limbs, especially the thigh and popliteal fossa. Despite the name chondrosarcoma, there is no evidence of cartilaginous differentiation. It is classified as a tumor of unknown differentiation by the World Health Organization (WHO).1 CYTOMORPHOLOGY OF EXTRASKELETAL MYXOID CHONDROSARCOMA • C  onsistent appearance from case to case • Bright magenta fibrillary stroma with Romanowskystained preparations • Variable degree of lacunar formation • Cords and lacelike arrangements • Uniform, bland epithelioid to spindled cells

Smears are moderately cellular, with a background of chondromyxoid matrix (Fig. 17.15A) and a monotonous population of plump epithelioid to spindle-shaped tumor cells arranged in a lacelike pattern of anastomosing, loosely cohesive cords and nests. The malignant cells are uniform and lack nuclear pleomorphism (Fig. 17.15B). Nuclei are round or oval and hyperchromatic, with finely stippled

chromatin. The single nucleolus is small and inconspicuous. Nuclear grooves and clefts are common. Cytoplasm is homogeneous, scant to moderately abundant, and often appears wispy and tapered, with well-defined cell borders.97,98 DIFFERENTIAL DIAGNOSIS OF EXTRASKELETAL MYXOID CHONDROSARCOMA • • • •

 ow-grade fibromyxoid sarcoma L Myoepithelial tumor of soft tissue High-grade myxoid liposarcoma Ossifying fibromyxoid tumor

Of all the myxoid soft tissue neoplasms, EMC shows the least morphologic variation from one tumor to another. The myxoid matrix has a fibrillary texture that is distinct from the usually granular appearance of most other myxoid lesions. It stains bright magenta with air-dried preparations (see Fig. 17.15A). Among the myxoid sarcomas, EMC has the fewest vascular structures. Chrondroblast-like lacunar formation can occur, but no definite hyaline cartilage differentiation has been described.98 There is no specific immunoprofile; less than 20% of cases show immunoreactivity for S-100. NR4A3 gene rearrangement with different fusion partners (including EWSR1) is considered a hallmark genetic event and is of diagnostic value (see Table 17.1).9,97,99 NR4A3 FISH is especially useful, given that some cytologic mimics also have EWSR1 rearrangements, including myoepithelial carcinomas of soft tissue and myxoid liposarcoma. 

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A

B • Fig. 17.15  Extraskeletal Myxoid Chondrosarcoma.  (A) The extraskeletal myxoid chondrosarcoma has a

distinctive fibrillary chondromyxoid matrix (Romanowsky stain). (B) Long cytoplasmic extensions link one cell to another, often conferring a lacelike appearance (Papanicolaou stain).

Spindle Cell Neoplasms The benign tumors in this group yield intact tissue fragments with few if any dispersed cells, whereas the malignant lesions feature more cell dissociation, greater cellularity, necrosis, and mitotic activity. An important task is determining whether the mass is of smooth muscle, nerve sheath,

myofibroblastic, or other mesenchymal origin. Although some characteristic cytomorphologic features suggest a specific entity or line of differentiation, definitive classification depends on immunohistochemical studies using a panel of antibodies (see Table 17.2).19,23,49,100 Spindle cell carcinoma and melanoma often need to be considered in the differential diagnosis as well.

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A

B •

Fig. 17.16  Leiomyosarcoma.  (A) Spindle cells are packed in cohesive tissue fragments with wellcircumscribed, rigid edges (Romanowsky stain). (B) Nuclei are hyperchromatic, with finely textured chromatin in lower-grade tumors and more coarsely clumped chromatin in the higher-grade neoplasms (Papanicolaou stain).

Leiomyosarcoma Soft tissue (nonuterine) leiomyosarcomas are encountered in several different settings. Intraabdominal tumors (40% to 45%) occur in older women in the retroperitoneum, mesentery, or omentum. Metastases to lung and liver are common. Subcutaneous and deep soft tissue tumors (20%–30%) involve the extremities, particularly the thigh, with a slight male predominance. Vascular leiomyosarcomas (5%) occur adjacent to muscular-walled blood vessels, particularly the inferior vena cava and large veins of the lower limbs in older adults. Given that they may recur if incompletely excised but rarely metastasize, most cutaneous tumors previously classified as cutaneous leiomyosarcoma are now called atypical intradermal smooth muscle neoplasm when completely confined to the dermis; because of their location they rarely undergo FNA.51 Leiomyosarcomas are immunoreactive for smooth muscle actin (SMA), desmin, and caldesmon. They are negative for S-100. About one-third of cases exhibit positivity for keratins and EMA. The cellular arrangement is variable; loosely cohesive clusters, closely packed tissue fragments with rigid edges (Fig. 17.16A), and short fascicles with elongated cells are seen. The percentage of isolated cells increases with tumor grade. In the

CYTOMORPHOLOGY OF LEIOMYOSARCOMA • • • • • •

 ascicular tissue fragments F Spindle-shaped cells “Cigar-shaped” nuclei, some with indentation Naked nuclei Abundant homogeneous, finely granular cytoplasm Mitoses

myxoid variant, occasional hyaline stromal fragments (with blood vessels) contain a diffuse, granular ground substance. A parallel, side-by-side arrangement of tumor cells is common. The centrally placed nucleus is generally oval to elongated, with blunted or rounded ends (“cigar-shaped”) that are often indented (Fig. 17.16B). Naked nuclei are common. Anisokaryosis, pleomorphism, and multinucleation increase with tumor grade and are prominent features of the pleomorphic variant. The cytoplasm is generally abundant, bipolar, and homogeneously granular. Mitoses are common, and atypical forms are seen in higher-grade lesions. The presence of even a single mitosis in a deep-seated smooth muscle tumor is highly suspicious for malignancy. Necrosis can be prominent in poorly differentiated tumors.19,23,49,101,102

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DIFFERENTIAL DIAGNOSIS OF LEIOMYOSARCOMA • • • • • •

 eiomyoma L Schwannoma Malignant peripheral nerve sheath tumor Gastrointestinal stromal tumor Solitary fibrous tumor Desmoid fibromatosis

Most leiomyosarcomas can be distinguished from a leiomyoma because the former has more generalized nuclear atypia, more mitotic activity, and necrosis. Unless attached to the uterus or the gastrointestinal tract, any deep-seated smooth muscle tumor should be considered malignant until proven otherwise. Nerve sheath tumors have wavy, “fishhook” nuclei and fibrillary cytoplasmic processes, with immunoreactivity for S-100. Gastrointestinal stromal tumor is characterized by a prominent vascular component, cellular clusters with irregular outlines, and spindle cells with delicate cytoplasmic processes (see Fig. 7.18A and B). Solitary fibrous tumor and desmoid fibromatosis are distinguished from leiomyosarcoma by their characteristic immunoprofiles (see Table 17.2) and by the absence of nuclear atypia.101-103 

Schwannoma Schwannomas are benign tumors of nerve sheath origin that primarily affect adults. They have a wide anatomic distribution but usually involve the limbs, head and neck, retroperitoneum, and posterior mediastinum. CYTOMORPHOLOGY OF SCHWANNOMA • • • • •

 arge, cohesive fragments L Wavy, “fishhook” nuclei Pointed nuclear ends Nuclear palisading Filamentous cytoplasm

Schwannomas often elicit pain during aspiration. Smears contain large tissue fragments that have irregular rather than sharp edges and vary greatly in their cellularity—they can be highly or only sparsely cellular (Fig. 17.17A). Antoni A and Antoni B areas can be hard to distinguish with smears. Verocay bodies (palisades of nuclei alternating with anucleate expanses of fibrillar cytoplasm) are present and are especially well seen on cell block preparations. The elongated, spindle-shaped cells, arranged in syncytium-like fragments, have minimally atypical nuclei that range in shape from oval to wavy to fishhook-like and taper to pointed ends, and cytoplasm is abundant and filamentous (Fig. 17.17B). In “ancient” schwannomas, nuclear pleomorphism is marked but focal and scattered. Mitotic figures are rarely seen. Aspirates from cystic, myxoid, or densely collagenous schwannomas yield few cells and are often nondiagnostic.19,49,101,104

DIFFERENTIAL DIAGNOSIS OF SCHWANNOMA • • • • • •

 pindle cell lipoma S Malignant peripheral nerve sheath tumor, low grade Leiomyosarcoma, low grade Solitary fibrous tumor Neurofibroma Desmoid fibromatosis

A schwannoma often raises the possibility of a malignancy because the rather thick aspirated tissue fragments appear very cellular on smears, particularly in the cellular variant. This erroneous impression of malignancy is heightened with an ancient schwannoma and its large, bizarre nuclei.105,106 Diffuse and strong immunoreactivity for S-100 and SOX10 is characteristic of schwannomas and can be helpful in distinguishing it from malignancies such as a low-grade malignant peripheral nerve sheath tumor. Except for the epithelioid variant, a malignant peripheral nerve sheath tumor shows only focal (if any) expression of S-100 and SOX10. A spindle cell lipoma can be distinguished from schwannoma because the former has mature adipose tissue and is immunoreactive for CD34 in the spindle cells. A solitary fibrous tumor has more complex cellular clusters, with ropy collagen fibers, bland spindle cells, and oval rather than wavy nuclei; it is almost invariably immunoreactive for CD34 and STAT6 and negative for S-100. A neurofibroma contains spindle-shaped fibroblasts and scattered axons in addition to wavy Schwann cells, and a myxoid background is a common finding. Desmoid fibromatosis yields less cellular smears and more abundant collagenous stroma.101,106,107 

Malignant Peripheral Nerve Sheath Tumor Malignant peripheral nerve sheath tumors (MPNSTs) occur sporadically and in patients with neurofibromatosis (NF-1). Often arising from a large peripheral nerve or a preexisting neurofibroma, they also occur in soft tissue and over a wide anatomic distribution. The limbs are most commonly affected, followed by the trunk and the head and neck. CYTOMORPHOLOGY OF MALIGNANT PERIPHERAL NERVE SHEATH TUMOR • M  oderate to high cellularity • Many dispersed cells • Spindle-shaped cells with comma-shaped nuclei and tapered, pointed, or rounded ends • Nuclear pleomorphism • Scant fibrillar cytoplasm • Round or polygonal cells (epithelioid variant) • Mitotic figures • Heterologous elements (bone, cartilage, glands, rhabdomyoblasts, angiosarcoma)

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A

B • Fig. 17.17  Schwannoma.  (A) Smears often contain thick tissue fragments that appear densely cellular, erroneously suggesting a sarcoma (Papanicolaou stain). (B) The neoplastic cells have abundant filamentous cytoplasm and an oval, curved, or wavy nucleus (Romanowsky stain).

Cytomorphology varies depending on the grade and subtype. Aspirates are moderately to highly cellular, with variably sized fascicles, isolated cells, and naked nuclei. Occasional rosette-like or pseudoglandular configurations are seen, but nuclear palisading is rare. Tumor cells are relatively large and predominantly spindled to oval, but can be pleomorphic, rounder, or both in higher-grade lesions. Mild to marked

nuclear pleomorphism, with occasional bizarre giant nuclei, hyperchromasia, and inconspicuous to prominent nucleoli, is seen. The nuclear-to-cytoplasmic ratio is increased. The cells have scant, tapering, and fibrillar cytoplasm, and wavy or comma-shaped nuclei (Fig. 17.18). Mitoses are usually readily identified. Necrosis and apoptosis can be present. MPNSTs, except for the epithelioid variant, show only focal

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• Fig. 17.18  Malignant Peripheral Nerve Sheath Tumor.  Although some of the elongated, wavy or fishhook nuclei of benign nerve sheath tumors are present, in general, they give way to plumper forms with less pointed ends, with hyperchromatic nuclei and greater atypia (Papanicolaou stain).

and patchy staining for S-100 in less than 50% cases and for GFAP in 20% to 30% cases. The cytomorphology, although not specific enough by itself for the initial (primary) diagnosis, usually displays sufficient features for documentation of recurrence or metastasis.108-111 A subset of MPNSTs has loss-of-function mutations in EED or SUZ12, which encode subunits of the polycomb repressor complex 2 (PRC2).112 PRC2 inactivation leads to loss of trimethylation of histone H3K27me3, which can be detected by immunohistochemistry,113 but H3K27me3 loss is not entirely sensitive or specific for MPNST. DIFFERENTIAL DIAGNOSIS OF MALIGNANT PERIPHERAL NERVE SHEATH TUMOR • • • • • • •

 chwannoma (especially with “ancient” change) S Synovial sarcoma Leiomyosarcoma Spindle cell carcinoma Spindle cell melanoma Malignant solitary fibrous tumor Dedifferentiated liposarcoma (retroperitoneum)

Densely cellular fascicles alternating with hypocellular myxoid zones, geographic necrosis, and heterologous differentiation, though favoring an MPNST on histology, are rarely seen with smears. Low-grade MPNSTs can be difficult to distinguish cytologically from benign peripheral nerve sheath tumors such as cellular schwannoma and neurofibroma. A traumatic neuroma after the excision of a

sarcoma can be mistaken for recurrent sarcoma. The spindle cell component of synovial sarcoma has less morphologic variability and nuclear pleomorphism, but up to 25% of MPNSTs express keratins. The nuclei of a leiomyosarcoma have blunted/truncated (rather than pointed) ends, and cytoplasm is denser. In general, the cytologic diagnosis of MPNST is extremely difficult, even with the help of ancillary studies, because of the lack of reliable specific makers for its line of differentiation, diverse histopathology, and complex karyotype. Clinical details are often as important (if not more so) for a correct diagnosis of MPNST.51,101,109 

Synovial Sarcoma Synovial sarcoma is a malignant tumor that occurs at any age but mainly in young adults (aged 10 to 35 years) and accounts for up to 10% of all sarcomas. It has a wide anatomic distribution and, despite its name, no particular relationship to synovium. Most are deep seated and arise in the lower limbs, especially the thigh. Others occur in the trunk and a wide variety of visceral organs. Radiologic evidence of calcification is common. Histologically, synovial sarcomas can be biphasic (both spindle cell and epithelial components present) or monophasic (spindle cell), and there is a poorly differentiated type that appears as a small round blue cell malignancy. Synovial sarcomas are immunoreactive for EMA and keratins, especially in areas of epithelial differentiation, but also focally in the spindle cell areas. Up to two-thirds of synovial sarcomas express CD99 (O13),

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and about 30% are positive for S-100. TLE1 has high sensitivity for synovial sarcoma but should be interpreted with caution given its modest specificity. All forms of synovial sarcoma share a reproducible, tumor-specific chromosomal translocation t(X;18)(p11.2;q11.2) that results in an SS18-SSX fusion gene. Because immunohistochemical studies may not be conclusive in difficult cases, FISH or RT-PCR are effective tools for definitive diagnosis (see Table 17.1).10,40,114,115 CYTOMORPHOLOGY OF SYNOVIAL SARCOMA • H  igh cellularity • Cell clusters alternating with dispersed cells • Cell clusters with shaggy edges and thin, branching capillaries • Extreme uniformity of cells • Bland, oval nucleus • Scant, delicate, tapering cytoplasm • Occasional epithelial elements • Mitoses • Mast cells

FNAs are moderately to markedly cellular, with occasional fragments of either myxoid or fibrous extracellular matrix. Irrespective of subtype, at low magnification there is a distinctive pattern of dispersed cells alternating with cohesive cell clusters that contain delicate branching capillaries (Fig. 17.19). The clusters can be large, with prominent fascicular and whorled growth patterns and shaggy edges. Epithelial structures (glands, acini, tubules, and papillae) can be seen in biphasic tumors, but often the epithelial differentiation is subtle (Fig. 17.20).116-119

Synovial sarcomas are remarkable for the striking uniformity of the cells (Fig. 17.21A). They are small to medium sized and round or fusiform. Epithelial differentiation, if present, confers a rounded, polygonal, or even cuboidal-to-columnar outline, and occasional epithelial cells are flattened as they stretch around three-dimensional cell clusters. The nucleus is bland, round to oval, with finely granular chromatin and a small nucleolus. Nucleoli are occasionally prominent in the epithelial component. In general, the cells have a high nuclear-tocytoplasmic ratio, with scant, delicate, tapering cytoplasm that is denser and more abundant in the epithelial cells. Mitoses and mast cells are present. Occasionally, necrosis is identified.116-119 DIFFERENTIAL DIAGNOSIS OF SYNOVIAL SARCOMA • • • • • • •

 eiomyosarcoma L Malignant peripheral nerve sheath tumor Solitary fibrous tumor Ewing sarcoma Metastatic carcinoma Ectopic hamartomatous thymoma Carcinosarcoma

The nuclei of a leiomyosarcoma have blunted/truncated (rather than rounded) ends, and cytoplasm is denser. Some nuclei of an MPNST are more curved and pointed, and there is greater nuclear pleomorphism. Relying too heavily on TLE1 immunohistochemistry to diagnose synovial sarcoma is a notable pitfall because one-third of MPNSTs express TLE1. A solitary fibrous tumor has fewer dispersed cells and more complex cellular clusters,

• Fig. 17.19  Synovial Sarcoma.  There is a distinctive pattern of dispersed cells alternating with cohesive cell clusters (Papanicolaou stain).

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with ropy collagen fibers and blood in the background. Immunohistochemistry can be helpful because a solitary fibrous tumor is usually immunoreactive for STAT6 and CD34 and negative for EMA. At least 20% of synovial sarcomas have a poorly differentiated round cell morphology resembling Ewing sarcoma (Fig. 17.22). In such cases (indeed, in most cases when a primary diagnosis is being rendered), immunohistochemistry and analysis by SS18 FISH are indispensable for confirming the diagnosis of synovial sarcoma (see Fig. 17.21B). Ectopic hamartomatous thymoma is a rare benign biphasic tumor composed of sheets of bland spindle cells, squamoid epithelial islands, and mature adipose tissue, showing more diffuse keratin positivity and focal CD34 positivity in small spindle cells. Finally, the cells of synovial sarcoma are much

more uniform than those of a metastatic sarcomatoid carcinoma or carcinosarcoma.116-120 

Solitary Fibrous Tumor Solitary fibrous tumor (SFT) is a fibroblastic neoplasm that involves the pleura, as well as a wide variety of extrapleural sites, including the peritoneum, mediastinum, retroperitoneum, upper respiratory tract, orbit, deep soft tissues, indeed, almost any human organ. Many SFTs were formerly called hemangiopericytomas. SFT affects adults and presents as a slowly growing mass. The majority are benign, but about 5% to 10% behave in a malignant fashion.121 SFTs are characterized by an NAB2STAT6 fusion,122,123 and immunohistochemical expression of STAT6 offers valuable support for the diagnosis of SFT.

• Fig. 17.20  Synovial Sarcoma, Biphasic.  Epithelial differentiation is recognized as only a vague palisad-

ing of nuclei along the edges of tissue fragments or as clusters of more epithelioid-appearing cells among the spindle cells (Papanicolaou stain).

SS18-t

SS18-c

SS18-18q11.2

A •

B

Fig. 17.21  Synovial Sarcoma, Monophasic.  (A) The oval to round nuclei are remarkably uniform, although rare elongated, blunt-ended, and bent forms are seen (Papanicolaou stain). (B) Fluorescence in situ hybridization is often performed with centromeric (SS18-c, green) and telomeric (SS18-t, red) probes that flank the SYT gene breakpoint. There is one normal chromosome (probes adjacent = yellow), but the split-apart green and red signals indicate a translocation on the other chromosome. (Courtesy Dr. Paola Dal Cin, Brigham and Women’s Hospital, Boston, MA.)

CHAPTER 17  Soft Tissue

565

•

Fig. 17.22  Synovial Sarcoma, Poorly Differentiated Round-Cell Type.  The tumor cells have a round cell morphology resembling that of a Ewing sarcoma. Mast cells are a characteristic feature (arrow) (Romanowsky stain). (Courtesy Dr. Bastiaan De Boer, Pathwest, QE II Medical Centre, Nedlands Perth, Australia.)

CYTOMORPHOLOGY OF SOLITARY FIBROUS TUMOR • • • • • •

 ariable cell yield and bloody background V A meshwork of irregular fascicles and individual cells Bland spindle cells with a fusiform nucleus Scant elongated cytoplasmic processes Ropy collagen Naked nuclei

The aspirate yields scant to moderate cellularity, with a bloody background that contains ropy collagen fibers. Cellular fragments with a meshwork of irregular fascicles alternate with isolated spindle-shaped cells (Fig. 17.23A and B). The cells are uniformly bland, with a fusiform nucleus, finely dispersed chromatin, an inconspicuous nucleolus, and scant elongated cytoplasm. Naked nuclei are common. Cell block preparations are very helpful in capturing the characteristic bland, monomorphic spindle-shaped cells insinuated between collagen bundles (Fig. 17.23C) and hemangiopericytoma-like (i.e., thin, branching, staghorn-like) vessels.2,124-126 Highly cellular clusters with nuclear pleomorphism, prominent nucleoli, necrosis, and noticeable mitotic activity should raise the suspicion of a malignant SFT.125-127 A fat-forming variant of SFT yields variably prominent adipocytes, even lipoblast-like cells, a diagnostic pitfall that can lead to the misinterpretation of the SFT as any of the benign and malignant lipomatous tumors.128 SFTs are almost invariably positive for CD34 and STAT6 (Fig. 17.23D).129,130 They may express CD99, but

they are negative for S-100, actin, desmin, and keratins (see Table 17.2). DIFFERENTIAL DIAGNOSIS OF SOLITARY FIBROUS TUMOR • • • • • •

 alignant peripheral nerve sheath tumor, low grade M Monophasic synovial sarcoma Desmoid fibromatosis Sarcomatoid mesothelioma Spindle cell thymoma Benign or malignant lipomatous tumor (for fat-forming variant)

MPNST has more nuclear pleomorphism and atypia. Synovial sarcoma exhibits even more striking uniformity in the cell population and cellular arrangements than SFT. Desmoid fibromatosis yields hypocellular smears, with more abundant collagenous stroma. Notably, all mesenchymal mimics are negative for STAT6. Sarcomatoid mesothelioma and spindle cell thymoma are positive for keratins and negative for CD34. 

Desmoid (Deep) Fibromatosis The fibromatoses are a broad spectrum of locally aggressive fibroblastic neoplasms that display an infiltrative growth pattern and can recur but never metastasize. Deep or desmoid fibromatosis presents as an abdominal, extraabdominal, or intraabdominal mass. In childhood and adolescence,

566 CHA P T ER 1 7    Soft Tissue

B

A

STAT6

C

D • Fig. 17.23  Solitary Fibrous Tumor.  (A) Cellular fragments with a meshwork of irregular fascicles alternate

with isolated spindle-shaped cells (Papanicolaou stain). (B) High magnification reveals collagen fibers coursing through the tumor (Papanicolaou stain). (C) Cell block preparations are very helpful for capturing the characteristic bland, monomorphic spindle-shaped cells insinuated between collagen bundles (hematoxylin and eosin stain). (D) Nuclear STAT6 expression is highly sensitive and specific for SFT.

desmoid fibromatosis can be the initial manifestation of familial adenomatous polyposis (characterized by APC mutations). CTNNB1 mutations are present in most sporadic desmoid tumors. Nuclear β-catenin expression secondary to CTNNB1 or APC mutations is seen in up to 75% of cases. Superficial fibromatoses (palmar, plantar, penile) are genetically distinct but also show locally aggressive growth and frequent recurrences, although up to half of all cases show nuclear β-catenin expression. CYTOMORPHOLOGY OF DESMOID FIBROMATOSIS • V  ariable, often low cellularity • Bland spindle-shaped cells, isolated or in slender fascicles • Fragments of dense collagenous stroma • Oval to elongated nuclei, frequently with crush artifact • Multinucleated degenerated skeletal muscle cells (if infiltrative or intramuscular) • Rare mitotic activity

FNAs have low but variable cellularity, with long fascicular clusters, isolated bland, spindle-shaped fibroblasts, and scattered degenerated skeletal muscle fibers (Fig 17.24A). They have an oval to elongated nucleus with bipolar ends and evenly dispersed chromatin, often with marked crush artifact when embedded in the collagenous stroma. Some have tapering cytoplasmic processes, whereas others are stellate. Rare inflammatory cells are present, but cytologic atypia is absent, and mitotic activity is rare. Fragments of dense collagenous stroma are common, and large long fascicular tissue fragments may be seen.26,131 Occasionally, myxoid stroma is present. As with the cytomorphology, the immunoprofile is not entirely specific. Positive staining for β-catenin (aberrant nuclear staining, Fig. 17.24B), variable SMA expression, and negative staining for CD34, desmin, and KIT support the diagnosis of a desmoid fibromatosis in the right clinical setting (see Table 17.2). Nodular fasciitis produces more cellular and pleomorphic smears with a haphazard cellular arrangement and ganglionlike cells. Low-grade fibromyxoid sarcoma has alternating myxoid and fibrous areas and is positive for MUC4. Scars

CHAPTER 17  Soft Tissue

567

beta-catenin

A

B • Fig. 17.24  Desmoid Fibromatosis.  (A) Long fascicular arrangements of spindle-shaped fibroblasts and

scattered degenerated skeletal muscle fibers are characteristic (Papanicolaou stain). (B) Aberrant nuclear staining for Β-catenin is seen in tumor cells but not in adjacent endothelial cells (arrow).

DIFFERENTIAL DIAGNOSIS OF DESMOID FIBROMATOSIS • • • • • • •

 odular fasciitis N Low-grade fibromyxoid sarcoma Scar tissue Nerve sheath tumor Smooth muscle tumor Solitary fibrous tumor Gastrointestinal stromal tumor

have less cellularity and greater cohesion. The nuclei in nerve sheath tumors are longer and wavier, those in smooth muscle tumors are blunted, whereas those of fibromatosis have pointed, bipolar ends. SFT and gastrointestinal stromal tumor should be considered in the differential diagnosis of an intraabdominal desmoid fibromatosis. These two mimics have more complex, irregular tissue fragments and a more prominent vascular component. Immunohistochemical studies are usually indispensable in classifying these spindle cell neoplasms (see Table 17.2). Because of its dense collagenous nature, desmoid fibromatosis yields paucicellular or nondiagnostic smears in more than half of cases; a larger needle/core biopsy is usually required to obtain a diagnostic sample.26,131 

Nodular Fasciitis Nodular fasciitis is a relatively common, self-limiting, fibroblastic and myofibroblastic pseudosarcomatous proliferation that typically occurs in subcutaneous tissue. It occurs in all age groups but is most common in young adults. The most common sites are the upper extremities, trunk, and head and neck (including salivary gland), but any part of the body can be affected. It has a typical clinical presentation: rapid growth over a relatively short period of time, usually no more than 2 months. The lesion usually measures 2 to 3 cm in greatest dimension and is rarely bigger than 5 cm. It can be sore,

tender, or both. Only a minority of patients report a history of trauma to the affected area (which is likely unrelated). Because it has recurrent gene rearrangements involving the USP6 locus, the traditional view of nodular fasciitis as a nonneoplastic, reactive lesion has been challenged.132 CYTOMORPHOLOGY OF NODULAR FASCIITIS • M  yxoid background (early phase) • Clusters and numerous dispersed polymorphic (e.g., spindle-shaped, stellate) myofibroblasts • No significant hyperchromasia • Uninucleated or binucleated ganglion-like cells with eccentric nuclei and prominent nucleoli • Inflammatory cells

The moderately cellular aspirates have a heterogeneous background of myxoid material, inflammatory cells, and delicate branching vessels (Fig. 17.25A). Prominent myxoid stroma is more commonly seen in early-phase lesions, whereas fragments of collagenous stroma are more common in long-standing lesions. There are numerous solitary spindle-shaped and stellate myofibroblastic cells (Fig. 17.25B–D), but some also form cohesive groups. Nuclei are uniform and lack significant atypia. They are small in the fusiform cells and somewhat larger in the plumper stellate cells. Their chromatin is delicate and open, and the inapparent nucleolus of the spindle-shaped cells is more distinct in the larger cells. Cytoplasm is generally abundant, but wispy with tapering ends and ill-defined borders. Mitotic figures can be alarmingly numerous (see Fig. 17.25D). Osteoclast-like giant cells are occasionally present. The myofibroblasts are positive for SMA and panmuscle actin, but negative for desmin and S-100, compatible with their myofibroblastic nature. The presence of uninucleated or binucleated ganglion-like cells is a characteristic finding in the conditions known as proliferative

568 CHA P T ER 1 7    Soft Tissue

A

B

C

D •

Fig. 17.25  Nodular Fasciitis.  (A) Smears can be cellular, with numerous myofibroblasts embedded in a myxoid matrix (Romanowsky stain). (B) Dispersed, polymorphic myofibroblasts are characteristic (Romanowsky stain). (C) The myxoid matrix is less conspicuous with the Papanicolaou stain. (D) Mitoses are not unusual (Romanowsky stain).

fasciitis and proliferative myositis, which do not have USP6 rearrangements. Nodular fasciitis is one of the most notorious mimics of malignancy among all the soft tissue lesions, and definitive diagnosis on cytology is challenging for most practitioners.133 The differential diagnosis includes a variety of sarcomas. The key to arriving at the correct (benign!) diagnosis is the correlation of clinical information with the FNA findings. The diagnosis is often suggested just by the clinical presentation (e.g., a rapidly growing subcutaneous mass smaller than 3 cm), and the typical FNA findings serve simply to confirm this clinical impression. 

neck region. Fibrosarcomatous progression occurs in 10% to 15% of cases. DFSP and the related giant cell fibroblastoma in children carry the characteristic COL1A1-PDGFB fusion gene.1

Dermatofibrosarcoma Protuberans

Aspirates yield moderately cellular smears composed of dense cellular clusters with a prominent storiform growth pattern. Occasional fragments of metachromatic fibrillary stroma are demonstrable with a Romanowsky stain. Adipose tissue entrapped within fascicles of spindle cells is a useful but inconsistent finding. Isolated fibroblast-like and stellate cells are minimally pleomorphic, with bland, oval nuclei. The chromatin is fine and homogeneous, and nuclear

Dermatofibrosarcoma protuberans (DFSP) is a slowly growing fibroblastic tumor of the dermis and subcutis of young adults, often present for 5 years or even decades before it is biopsied and diagnosed. It occurs mainly on the trunk, proximal extremities, and groin, but a small percentage occur on the scalp or elsewhere in the head and

CYTOMORPHOLOGY OF DERMATOFIBROSARCOMA PROTUBERANS • D  ense, storiform cell clusters in a collagenous stroma • Dispersed or loose clusters of spindle cells in a myxoid stroma (myxoid variant) • Mildly atypical spindle cells with minimal pleomorphism • Occasional entrapped adipose tissue

CHAPTER 17  Soft Tissue

membranes are smooth and round. Small, distinct nucleoli are usually present. The cytoplasm is moderate in amount, pale, and finely granular. Cell borders are ill-defined within tissue fragments, but bipolar cytoplasmic processes are prominent on dispersed cells. In the myxoid variant, dispersed or loose clusters of spindled to stellate cells are more prominent than the typical storiform clusters.134-136 Some cases show malignant progression (fibrosarcomatous DFSP) and appear as a nondistinct spindle cell sarcoma (see “Adult Fibrosarcoma”). Tumor cells are strongly and diffusely positive for CD34 and negative for S-100, EMA, and desmin. Detection of the COL1A1-PDGFB fusion gene by FISH or RT-PCR is helpful in difficult cases (see Table 17.1).9 DIFFERENTIAL DIAGNOSIS OF DERMATOFIBROSARCOMA PROTUBERANS • • • • • •

 enign fibrous histiocytoma B Scar Nodular fasciitis Soft tissue perineurioma Neurofibroma Low-grade fibromyxoid sarcoma

Clinical features such as the anatomic location and rate of growth distinguish benign fibrous histiocytoma (dermatofibroma) from DFSP, but the mitotic rate is variable and unhelpful. Scars have less cellularity, greater cohesion, and more angulated nuclei. Nodular fasciitis has a more haphazard cellular arrangement, spindle-shaped and ganglion-like cells, less rounded nuclei, and more prominent nucleoli, often with an inflammatory infiltrate. Nerve sheath tumors are excluded by the lack of neural nuclear features and the absence of immunoreactivity for S-100 and EMA. Low-grade fibromyxoid sarcoma may be confused with a myxoid DFSP, but the former is usually more deep-seated, negative for CD34, and positive for MUC4.100,134,135 

Inflammatory Myofibroblastic Tumor Inflammatory myofibroblastic tumor (IMT) is a rare lowgrade neoplasm of children and adolescents that was once regarded as a reactive inflammatory pseudotumor. IMT has a local recurrence rate of 10% to 25%, and metastases occur in just under 5% of cases. It has a predilection for visceral organs including lung and liver, as well as the abdominal cavity. Most IMTs show fascicular proliferation of plump myofibroblasts with a prominent infiltrate of plasma cells and lymphocytes. A small subset of aggressive IMTs, with epithelioid cytomorphology, myxoid stroma, and a neutrophil-rich inflammatory infiltrate, are sometimes referred to as epithelioid inflammatory myofibroblastic sarcoma.137,138 Most IMTs yield highly cellular smears with a prominent inflammatory population composed mostly of plasma

569

CYTOMORPHOLOGY OF INFLAMMATORY MYOFIBROBLASTIC TUMOR • • • •

Inflammatory cells Mildly pleomorphic spindle cells Large polygonal cells Elongated cytoplasmic tails

cells and lymphocytes. Isolated spindle-shaped tumor cells are admixed with loose clusters and dense aggregates. The tumor cells exhibit typical myofibroblastic cytomorphology: slightly eccentrically placed, plump nuclei with finely granular chromatin and conspicuous small nucleoli. Fine cytoplasmic vacuolization and cytoplasmic tails or extensions are typical (Fig 17.26A). Large ganglion-like myofibroblasts with abundant cytoplasm, enlarged, sometimes binucleated, vesicular nuclei, and prominent nucleoli are also common.139-141 Like other myofibroblastic tumors, IMTs are positive for SMA (80%), desmin (60%), and keratins (30%). Although not entirely specific for IMT, and seen in only 50% of cases, immunoreactivity for anaplastic lymphoma receptor tyrosine kinase (ALK), secondary to an ALK gene rearrangement, can be diagnostically helpful (Fig 17.26B) (see Table 17.1).142 DIFFERENTIAL DIAGNOSIS OF INFLAMMATORY MYOFIBROBLASTIC TUMOR • • • • • • •

Inflammatory pseudotumor Inflammatory leiomyosarcoma Desmoid fibromatosis Gastrointestinal stromal tumor Dedifferentiated liposarcoma Follicular dendritic cell sarcoma Angiomatoid fibrous histocytoma

Inflammatory pseudotumor is usually associated with a clinical history of trauma, a surgical procedure, or infection. Most other morphologic mimics can be distinguished from IMT by immunohistochemistry: the mimics lack immunoreactivity for ALK and have instead their own characteristic immunoprofile: desmoid fibromatosis (β-catenin), GIST (KIT and DOG1), dedifferentiated liposarcoma (MDM2 and CDK4), follicular dendritic cell sarcoma (CD21 and CD35), and leiomyosarcoma (SMA, desmin, and caldesmon). Demonstration of an EWSR1 gene rearrangement by either FISH or RT-PCR is helpful to separate angiomatoid fibrous histiocytoma from an ALK-negative IMT.143 

Adult Fibrosarcoma Adult fibrosarcoma is best regarded as a diagnosis of exclusion. Most of the cases diagnosed before the era of immunohistochemistry and electron microscopy would be reclassified today as a MPNST or monophasic synovial sarcoma. The true fibrosarcomas that remain are the infantile

570 CHA P T ER 1 7    Soft Tissue

ALK

A

B • Fig. 17.26  Inflammatory Myofibroblastic Tumor.  (A) Spindle cells with plump nuclei, prominent nucleoli, and frequent cytoplasmic tails are arranged in loose clusters (Papanicolaou stain). (B) Up to half of all cases show cytoplasmic ALK expression, secondary to an ALK gene rearrangement.

type (which have an ETV6-NTRK3 fusion) and the fibrosarcomatous variant of DFSP in adults. Fibrosarcoma cannot reliably diagnosed by FNA.1,144 

Fibrohistiocytoid Neoplasms The tumors in this morphologic group are bound by histiocytoid and multinucleated giant cell morphology. Only a few tumor types show true fibrohistiocytic differentiation, mainly tenosynovial giant cell tumor (localized and diffuse types) and giant cell tumor of soft tissue. Angiomatoid fibrous histiocytoma, a tumor of uncertain differentiation, may show overlapping features and is discussed in this section.

Tenosynovial Giant Cell Tumor, Localized and Diffuse Types The localized and diffuse types of tenosynovial giant cell tumor share similar cellular compositions and cytomorphology but differ in their growth pattern, clinical features, and biologic behavior. The localized type (also known as giant cell tumor of tendon sheath) most commonly presents as a slowly growing, circumscribed, and at least partially encapsulated nodule in close proximity to the synovium of the tendon sheaths of the hands and feet. It occurs more commonly in females between the ages of 30 and 50 years. Diffuse-type tenosynovial giant cell tumor (formerly known as pigmented villonodular synovitis), is a locally destructive lesion with an infiltrative growth pattern, occurring at intraarticular and extraarticular sites. This infiltrative tumor tends to affect younger patients than its localized counterpart, and affected patients are prone to recurrence. Intraarticular lesions affect predominantly the knee (75% of cases), followed by the hip (15%), ankle, elbow, and shoulder. The extraarticular lesions are usually located in periarticular soft tissue with or without involvement of the adjacent joint. Both have a COL6A3-CSF1 gene fusion in a small subset of tumor cells, mainly the larger epithelioid cells.

CYTOMORPHOLOGY OF TENOSYNOVIAL GIANT CELL TUMOR • M  ononuclear histiocytoid cells of varying shapes (ovoid, polygonal, and spindle-shaped) • Foamy histiocytes • Osteoclast-type giant cells • Extracellular and intracytoplasmic hemosiderin (“ladybug cells”) • Moderate anisocytosis and minimal anisokaryosis

Aspirations from localized and diffuse-type tumors have moderate to high cellularity. The predominant cells—mononuclear histiocytoid cells with folded nuclei and foamy cytoplasm, and larger epithelioid cells with eccentric nuclei—are dispersed as isolated cells and arranged in irregular clusters (Fig. 17.27A and B). The nuclei show minimal variation. They are ovoid, with smooth contours, finely granular chromatin, inconspicuous nucleoli, frequent grooves, and occasional intranuclear inclusions. Binucleation is not uncommon. Although mitotic figures are occasionally present, nuclear atypia and necrosis are very rare. Variable numbers of foamy histiocytes and osteoclasttype giant cells (see Fig. 17.27B) are admixed with the mononuclear histiocytoid cells. The giant cells vary widely in size and in the number of nuclei (3 to 50 or more).145,146 The absence of giant cells does not exclude the diagnosis, especially for the diffuse-type tumors. Hemosiderin deposition in the mononuclear cells and macrophages is inevitably present, and the resulting speckled pattern has led to the fanciful description of these cells as ladybug cells (“ladybird cells” in Britain) (see Fig. 17.27A).147 DIFFERENTIAL DIAGNOSIS OF TENOSYNOVIAL GIANT CELL TUMOR • • • •

 outy tophi G Chronic synovitis with synovial hyperplasia Metastatic melanoma Giant cell-rich sarcoma

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571

A

B • Fig. 17.27  Tenosynovial Giant Cell Tumor.  (A) Mononuclear histiocytoid cells are arranged in clusters. There is prominent intracytoplasmic hemosiderin, imparting a speckled, ladybug-like appearance to the cells (Papanicolaou stain). (B) Multinucleated giant cells are a prominent feature (Romanowsky stain).

Gouty tophi can have many giant cells, but the lack of mononuclear cells and the presence of birefringent needleshaped crystals on polarizing microscopy (Figs. 17.28A and B) is diagnostic. Chronic synovitis has more inflammation and sometimes necrosis, but giant cells and hemosiderin deposition are usually not prominent. Metastatic melanoma and giant cell-rich sarcoma have more pleomorphism, nuclear atypia, and necrosis. In difficult cases, immunohistochemistry

can be helpful: the mononuclear cells and the giant cells are both positive for the histiocytic markers CD163 and PU.1, and a subset may show desmin staining. 

Giant Cell Tumor of Soft Tissue Giant cell tumor of soft tissue commonly arises as a superficial lesion in the limbs of adults, although a wide patient age

572 CHA P T ER 1 7    Soft Tissue

A

B • Fig. 17.28  Gout.  (A) The sample is comprised of innumerable needle-shaped crystals in a background of granular debris (Papanicolaou stain). (B) Examination under polarized light demonstrates the crystals more clearly.

range is observed. Histologically, tumors show a multinodular growth pattern, with fibrous septae separating tumor nodules comprised of ovoid and round mononuclear cells, osteoclastlike giant cells, and hemosiderin-laden macrophages within a richly vascularized stroma. Tumors may show local recurrence but rarely metastasize. Despite shared morphologic features, giant cell tumor of soft tissue is genetically distinct from giant cell tumor of bone, which has H3.3 mutations.148

CYTOMORPHOLOGY OF GIANT CELL TUMOR OF SOFT TISSUE • • • •

 istiocytoid spindle-to-ovoid cells with bland nuclei H Multinucleated osteoclast-like giant cells Loose clusters and dispersed cells Hemorrhagic background

CHAPTER 17  Soft Tissue

573

• Fig. 17.29  Giant Cell Tumor of Soft Tissue.  Mononuclear histiocytoid cells, often with vacuolated cytoplasm, may look plasmacytoid, unipolar, or bipolar. They are accompanied by large and often bizarrely shaped multinucleated giant cells (Romanowsky stain).

Giant cell tumor of soft tissue has been described only in case reports.149-151 It shows a mixture of histiocytoid cells with spindle or ovoid nuclei admixed with multinucleated giant cells, variably arranged in loose clusters or isolated cells (Fig 17.29). The mononuclear cells have moderate cytoplasm that is frequently vacuolated and appear plasmacytoid, unipolar, or bipolar. The multinucleated giant cells have similar nuclei to the mononuclear cells, often containing numerous (greater than 10) nuclei and may be large and bizarrely shaped. A hemorrhagic background is common. Although at least half of all cases show metaplastic bone formation on surgical resection, this has not yet been noted in descriptions of its cytomorphology. DIFFERENTIAL DIAGNOSIS OF GIANT CELL TUMOR OF SOFT TISSUE • • • •

 olid aneurysmal bone cyst S Tenosynovial giant cell tumor Nodular fasciitis Giant cell-rich sarcoma

Giant cell tumor of soft tissue may be difficult to distinguish morphologically from an extraosseous solid aneurysmal bone cyst, but the latter has UPS6 rearrangements. Tenosynovial giant cell tumors show a more heterogeneous population of cells, including foamy histiocytes and a morphologically varied mononuclear component that includes spindle, ovoid, and polygonal

shapes. Nodular fasciitis may have osteoclast-like giant cells but typically has myofibroblastic morphology and harbors USP6 fusions. Giant cell-rich sarcomas show overt features of malignancy, including pleomorphism and necrosis. Although most giant cell tumors of soft tissue arise in superficial sites, clinical and imaging correlation may be required to exclude an osseous primary. 

Angiomatoid Fibrous Histiocytoma Angiomatoid fibrous histiocytoma (AFH), formerly known as angiomatoid malignant fibrous histiocytoma, is a distinctive mesenchymal neoplasm of uncertain lineage. It is a slowly growing tumor in the deep dermis and subcutis of the extremities in children, adolescents, and young adults, but it may also occur in middle-aged patients and rarely in other sites such as the lung and bone. It has an indolent behavior: local recurrences occur in 2% to 10% and metastases in less than 1% of patients. Histologically AFH is characterized by nodules and sheets of histiocytoid cells arranged in illdefined fascicles or whorls; cystic spaces with hemorrhage; and a peripheral, thick fibrous pseudocapsule containing a lymphoplasmacytic infiltrate. Most aspirate smears are at least moderately cellular. Occasionally, hypocellular, bloody smears are encountered as a result of sampling from the cystic pseudovascular spaces. The tumor cells, isolated or in clusters, have a histiocytoid appearance: ovoid to spindled in shape, ill-defined cell borders, wispy cytoplasm, often folded nuclei with finely granular chromatin and inconspicuous nucleoli. Some cellular

574 CHA P T ER 1 7    Soft Tissue

•

Fig. 17.30  Angiomatoid Fibrous Histiocytoma.  Ball-like clusters of histiocytoid cells are commonly associated with capillaries lined by spindle-shaped endothelial cells.

CYTOMORPHOLOGY OF ANGIOMATOID FIBROUS HISTIOCYTOMA • • • • •

 t least moderately cellular A Dispersed cells or clusters Whorled arrangement of tumor cells Ovoid to spindled histiocytoid cells Capillaries with spindled endothelial cells in cellular clusters • Bloody background and hemosiderin granules • Occasional lymphocytes and plasma cells

clusters contain capillaries with spindled endothelial cells and a whorled arrangement of tumor cells (Fig. 17.30). A bloody background and hemosiderin granules are common, whereas the lymphoplasmacytic infiltrate (at the periphery of the lesion) is rarely seen in FNA samples. Occasional nuclear atypia, pleomorphism, and mitoses are present, but these lack prognostic implications.143,152,153 DIFFERENTIAL DIAGNOSIS OF ANGIOMATOID FIBROUS HISTIOCYTOMA • B  enign fibrous histiocytoma (especially the aneurysmal type) • Nodular fasciitis • Inflammatory myofibroblastic tumor • Follicular dendritic cell sarcoma

fasciitis and IMT are myofibroblasts, which have plumper nuclei and more distinctive cytoplasmic tails/extensions. A bloody background, hemosiderin, and capillary-rich smears favor AFH over follicular dendritic cell sarcoma. In addition, AFH expresses EMA, desmin, CD68, and CD99 in about 50% cases and lacks reactivity for keratin, S-100, CD34, and follicular dendritic cell markers (CD21, CD35). FISH to detect an EWSR1 or FUS rearrangement is valuable in confirming the diagnosis of AFH in the right setting (see Table 17.1).143 

Round Cell Neoplasms The round cell neoplasms are primarily tumors of young patients and account for the majority of solid malignancies in the pediatric age group. Most are high-grade malignancies. Great diagnostic success is attainable in this group of tumors using FNA in conjunction with ancillary studies. The sensitivity and specificity of FNA for the round cell neoplasms exceed 90%, and, with an adequate specimen, a diagnosis of a specific histologic subtype is accurately rendered in 93% of cases. Accuracy is even higher with the use of ancillary techniques (see Table 17.1 for genetic alterations).18,34,35,49,154 Immunohistochemistry is extremely helpful in resolving the differential diagnosis and prompting confirmatory molecular testing (see Table 17.3).

Neuroblastoma Benign fibrous histiocytoma, especially the aneurysmal type, has more variable cytomorphology and Toutonlike multinucleated cells. The neoplastic cells of nodular

Neuroblastoma is the third-most common malignant tumor of childhood and the most common malignancy in the neonate. Most cases are diagnosed before the age of 5 years.

Tumor

CD99

FLI-1

NKX2.2

Desmin

Myogenin

EMA / keratin

TLE1

WT1

ETV4

CCNB3

S-100

NSE

PHOX2B

Neuroblastoma

−

−

−/+

−

−

−

−

−

−

−

−/+

+

+

Ewing sarcoma

+++ diffuse

+

+

−

−

−/+

−

−

−

−

−

−

−

DSRCTa

−/+

−

−

+ Dotlike

−

+

−

−

−

−

−

+

−

Embryonal RMS

−

−

−

+

+

−/+

−

−

−

−

−

−

−

Alveolar RMS

−

−

−

+

+++ diffuse

−/+

−

−

−

−

−

−

−

Myxoid lipoarcoma, high grade

−

−

−

−

−

−

−

−

−

−

+/−

−

−

Synovial sarcoma, poorly differentiated

+/−

−

−/+

−

−

+

+

−

−

−

−

−

−

CIC-rearranged sarcoma

−/+

−

−

−

−

−

−

+

+

−

−

−

−

BCOR-CCNB3 sarcomab

+/−

−

−

−

−

−

+

−

−

+

−

−

−

aAlso

positive for WT-1 (using polyclonal antibodies to C-terminus). positive for BCOR, SATB2, and cyclin D1. EMA, Epithelial membrane antigen. bAlso

CHAPTER 17  Soft Tissue

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TABLE 17.3    Immunoprofile of Round Cell Sarcomas

575

576 CHA P T ER 1 7    Soft Tissue

Most cases arise in the adrenal gland or along the intraabdominal portion of the sympathetic chain, but they can occur anywhere along the sympathetic chain.

CYTOMORPHOLOGY OF NEUROBLASTOMA • • • • •

 ibrillary matrix F Mostly noncohesive, small undifferentiated cells Rare neurogenic rosettes “Salt-and-pepper” chromatin Rare ganglion-like cells

Smears are cellular, with variable amounts of fibrillary, metachromatic matrix (neuropil) in the background and between the cells. Necrotic debris and dystrophic calcifications are sometimes observed. Tumor cells are predominantly isolated; cohesive aggregates are less common. Homer-Wright rosettes are present. Most cells are uniformly small and undifferentiated in appearance. Those undergoing differentiation are larger and have a moderate amount of cytoplasm. In better differentiated lesions, Schwannian-type spindle cells are present. The nuclei are round to oval, with only slight irregularities, finely granular (“salt-and-pepper”) chromatin, and small nucleoli. Occasional nuclear molding is noted. In better differentiated lesions, binucleated and multinucleated ganglion-like cells have a more coarsely granular chromatin, prominent nucleoli, and a moderate amount of cytoplasm. Neuroblastomas are invariably positive for neuron specific enolase and show occasional staining for neurofilament protein, synaptophysin, and GFAP. Tumors are consistently positive for GATA3,155 and PHOX2B distinguishes neuroblastoma from it mimics (see Table 17.3).156 In cases with Schwannian differentiation, immunoreactivity for S-100 is seen. Cytogenetic analysis is essential for prognosis and clinical management (see Table 17.1).34,35,49,154,157 

Ewing Sarcoma Ewing sarcoma (ES) is a primitive neoplasm showing varying degrees of neuroectodermal differentiation. It is the secondmost common sarcoma of bone in children and young adults, after osteosarcoma. Extraskeletal ES accounts for 10% to 20% of cases and presents as a rapidly enlarging, often painful mass of the deep soft tissues, with a predilection for the thigh, pelvis, paravertebral region of the trunk, and foot. ES is characterized by an EWSR1-FLI1 (85%) or EWSR1-ERG (10%) gene fusion, or fusions between EWSR1 and other members of the ETS family of transcription facters.1 The highly cellular smears often have numerous naked nuclei and a “tigroid” background (a spotted or striped appearance caused by innumerable small cytoplasmic fragments). The cells are predominantly isolated, with scattered

CYTOMORPHOLOGY OF EWING SARCOMA • “ Tigroid” background • Hypercellular smears with dispersed isolated round cells • “Light” cells and “dark” cells • Nuclear molding • Vacuolated cytoplasm

small cohesive clusters (Fig. 17.31A). Pseudorosettes are a fairly specific but rare finding. The cells of ES are approximately two to three times the size of a small lymphocyte and have a high nuclear-to-cytoplasmic ratio. Nuclear molding is prominent.18,34,35,49,158 Traditionally, two distinct cell types have been described, but these just represent a mixture of viable and dying tumor cells. The “light” (viable) cells have paler nuclei with finely granular, evenly dispersed chromatin and distinct nuclear membranes with slight contour irregularities. One or two small nucleoli or chromocenters are readily identifiable, as is an increased amount of pale cytoplasm. The “dark” (dying) cells are smaller, with dark, irregularly distributed, and smudged chromatin and unappreciable nucleoli. In both cell types, the nucleus is round to oval. No multinucleated or ganglion-type cells are present. A thin rim of cytoplasm contains small intracytoplasmic vacuoles that contain glycogen. Perinuclear cytoplasmic clearing is seen in some cases. Cell borders are typically ill defined, and fine, wispy cytoplasmic tags can sometimes be seen on Papanicolaoustained smears.18,34,158,159 DIFFERENTIAL DIAGNOSIS OF EWING SARCOMA • • • • • • • •

 lveolar rhabdomyosarcoma A Poorly differentiated synovial sarcoma Desmoplastic small round cell tumor Precursor lymphoblastic leukemia/lymphoma Neuroblastoma Small cell osteosarcoma Mesenchymal chondrosarcoma “Molecular subsets” of undifferentiated round cell sarcomas

The differential diagnosis includes all small round cell neoplasms, but most importantly alveolar rhabdomyosarcoma, poorly differentiated synovial sarcoma, desmoplastic small round cell tumor, neuroblastoma, and precursor lymphoblastic leukemia/lymphoma. Immunohistochemistry is paramount in addressing the differential diagnosis (see Table 17.3). Rhabdomyosarcoma usually has larger rhabdomyoblasts with more abundant, denser cytoplasm, and multinucleated and spindle-shaped cells in addition to small round cells; desmin and myogenin (myf-4) are positive. Positive staining for TLE1, EMA, or keratins along with cytogenetic analysis aids in the correct recognition of

CHAPTER 17  Soft Tissue

577

A EWSR1 22q12 3’ green (t) 5’ red (c)

B •

Fig. 17.31  Ewing Sarcoma.  (A) The tigroid background, characteristic light and dark cells, and the occasional cytoplasmic vacuoles are better appreciated with air-dried Romanowsky-stained preparations (Romanowsky stain). (B) Fluorescence in situ hybridization (FISH) with centromeric (red) and telomeric (green) probes that flank the EWS gene breakpoint show one normal chromosome (adjacent probes = yellow), but the split-apart green and red signals indicate a translocation on the other chromosome in all three cells. FISH cannot distinguish the EWS gene rearrangements in Ewing sarcoma, desmoplastic small round cell tumor, clear cell sarcoma, angiomatoid fibrous histiocytoma, and extraskeletal myxoid chondrosarcoma (as well as other tumors), but these tumors are morphologically and immunophenotypically distinct. (Courtesy Dr. Paola Dal Cin, Brigham and Women’s Hospital, Boston, MA.)

the poorly differentiated synovial sarcoma (see Fig. l7.22). Desmoplastic small round cell tumor has smaller and more cohesive cells and exhibits a polyphenotypic immunoprofile. Small cell osteosarcomas show more nuclear pleomorphism and hyperchromasia. The cells of precursor lymphoblastic leukemia/lymphoma are accompanied by lymphoglandular bodies and are immunoreactive for lymphoid markers.158,160 Molecular testing is required to identify the newly recognized subsets of undifferentiated round cell sarcomas, which include CIC-rearranged sarcoma and BCOR-CCNB3

sarcoma; tumors show overlapping features with ES but more variable (i.e., often negative or patchy) CD99 (O13) staining. ES shows diffuse membranous staining with CD99 (O13), but this pattern is not entirely specific for ES. CD99 is expressed by other small round cell tumors such as mesenchymal chondrosarcoma and lymphoblastic lymphoma/ leukemia and thus poses significant diagnostic hazards. The anti-FLI1 antibody is more specific, but both CD99 and FLI1 can be positive in lymphoblastic leukemia/lymphoma.

578 CHA P T ER 1 7    Soft Tissue

NKX2.2 shows high sensitivity for ES, but may be positive in several mimics.161 Up to 30% of ES show some reactivity for keratins and neuroendocrine markers (synaptophysin and chromogranin), but ES is nonreactive for muscle markers, leukocyte common antigen, and κ and λ light chains. Molecular genetic analysis is the most important and reliable ancillary study for confirming the diagnosis. Detection of the gene fusion transcript EWSR1-FLI1 or another variant gene fusion, not just an EWSR1 rearrangement by FISH, is diagnostically important for ES (see Table 17.1 and Fig. 17.31B).18,162  NEOPLASMS WITH EWSR1 REARRANGEMENTS • • • • • • • • • • •

 wing sarcoma E Desmoplastic small round cell tumor Extraskeletal myxoid chondrosarcoma Clear cell sarcoma of soft tissue Clear cell sarcoma-like tumor of gastrointestinal tract Angiomatoid fibrous histiocytoma Myxoid liposarcoma (small subset) Low-grade fibromyxoid sarcoma (small subset) Myoepithelial tumor of soft tissue Clear-cell carcinoma of salivary gland Primary pulmonary myxoid sarcoma

Desmoplastic Small Round Cell Tumor Desmoplastic small round cell tumor (DSRCT) is an aggressive malignant neoplasm of uncertain histogenesis and a striking predilection for the serosal surfaces. Most cases are intraabdominal, but the tumor can involve the pelvis, retroperitoneum, scrotum, and pleura. DSRCT affects mainly male adolescents and young adults between the ages of 15 and 35 years. DSRCT can arise in unusual sites and does occur in females; such unusual presentations can pose a diagnostic challenge. CYTOMORPHOLOGY OF DESMOPLASTIC SMALL ROUND CELL TUMOR • • • • •

 heets and clusters of small- to intermediate-sized cells S Fragments of desmoplastic stroma Uniformly round, oval, or slightly angulated cells Nuclear molding Vague, small acinar structures

Smears are variably cellular, with sheets and clusters of loosely cohesive, small- to intermediate-sized cells (Fig. 17.32A). The groups often recapitulate the irregular shapes of the nests and islands seen in histologic sections. Occasional isolated cells are present among fragments of variably cellular and collagenous (desmoplastic) stroma (Fig. 17.32B). The tumor cells are uniformly undifferentiated and predominantly round to oval, with rare polygonal or spindled forms. Nuclei are hyperchromatic, with finely granular chromatin and inconspicuous nucleoli. Nuclear molding can be striking,

particularly at the edges of groups. The scant cytoplasm is pale and amphophilic to eosinophilic, and occasional rhabdoid cells are present. Tumors in postchemotherapy cases can have isolated larger cells with conspicuous nucleoli.163,164 The differential diagnosis is similar to that for ES. DSRCT cells demonstrate a peculiar but characteristic polyphenotypic differentiation, with positive immunoreactivity for low molecular weight keratins, EMA, neuroendocrine markers, and desmin, the latter in a characteristic dotlike pattern (see Table 17.3). They are also positive for WT-1 (with an antibody against C-terminal of WT-1) and variably positive for CD99 (cytoplasmic). DSRCT harbors a specific cytogenetic abnormality: a t(11;22)(p13;q12) translocation involving the EWS gene on 22q12 and the WT1 gene on 11p13 (see Table 17.1).35,163,165,166 

Embryonal Rhabdomyosarcoma Four main subtypes of rhabdomyosarcoma (RMS) are recognized in the current WHO classification: embryonal, alveolar, pleomorphic, and spindle cell/sclerosing.1 The embryonal and alveolar forms occur mainly in children, whereas pleomorphic RMSs occur almost exclusively in adults, and the spindle cell/sclerosing RMSs affect both children and adults. RMS is the most common sarcoma of childhood, and embryonal RMS accounts for the majority in children (60%). The most common sites are the head and neck region (including the orbit and meninges), the genitourinary tract, and the trunk. Because the distinction between its two principal forms, embryonal and alveolar, has significant prognostic implications, and cytomorphology is usually insufficient for this distinction, ancillary diagnostic methods, especially cytogenetic/molecular studies, are the standard of care.2,167 CYTOMORPHOLOGY OF EMBRYONAL RHABDOMYOSARCOMA • • • • •

 redominantly isolated cells P Round or spindle-shaped cells Cellular pleomorphism Variable number of rhabdomyoblasts Occasional inclusion-like cytoplasmic condensation (myogenic differentiation)

Aspirates are moderately to highly cellular, composed predominantly of isolated cells, with occasional loose clusters and, uncommonly, a frothy “tigroid” or myxoid background. Tumor cells vary from small to intermediate sized and are round, polygonal, or spindle shaped. Larger cells show rhabdomyoblastic differentiation, with elongated, strap- or tadpole-shaped cytoplasm and eccentric nuclei. Anisonucleosis and cellular pleomorphism are especially pronounced among the larger cells. Nuclei have dense hyperchromatic chromatin and irregular membranes. Occasional cells have inclusion-like cytoplasmic condensations indicating

CHAPTER 17  Soft Tissue

579

A

B •

Fig. 17.32  Desmoplastic Small Round Cell Tumor.  (A) This tumor differs morphologically from other round cell neoplasms because its cells retain some cohesiveness and are rarely dispersed as isolated cells (Papanicolaou stain). (B) Cell block sections reveal the desmoplastic stroma that surrounds nests of neoplastic cells (hematoxylin and eosin stain).

myogenic differentiation. Cytoplasmic cross-striations are rarely seen.35,167-171 Embryonal RMS cells show myogenic differentiation manifested by positive immunoreactivity for desmin and muscle-specific actin, and multifocal expression of the protein products of the specific myogenic nuclear transcription factors myo-D1 and myogenin (myf-4). 

Alveolar Rhabdomyosarcoma Alveolar RMS, a subtype with unfavorable prognosis, is a tumor that occurs most frequently in adolescents. The limbs, head and neck region, and trunk are the most common sites.

580 CHA P T ER 1 7    Soft Tissue

A

B •

Fig. 17.33  Alveolar Rhabdomyosarcoma.  (A) In This field, the cells are relatively monomorphic (Romanowsky stain). (B) The cells are dispersed and generally larger and more uniformly round to polygonal than those seen in embryonal rhabdomyosarcoma (Romanowsky stain).

CYTOMORPHOLOGY OF ALVEOLAR RHABDOMYOSARCOMA • • • • •

 arger cells than in embryonal rhabdomyosarcoma L Uniformly round to polygonal cells Variable number of rhabdomyoblasts Multinucleated tumor giant cells with wreathlike nuclei Mitoses

Aspirates are highly cellular and infrequently have a “tigroid” background. Most cells are undifferentiated, with uniformly round to polygonal outlines (Fig. 17.33). Compared to the tumor cells of the embryonal variant, alveolar RMS cells are rounder, with larger and more irregular nuclei. Rhabdomyoblasts and multinucleated giant tumor cells, with or without wreathlike nuclei, are helpful diagnostic features when present. Mitoses are common.2,167,169,171

CHAPTER 17  Soft Tissue

DIFFERENTIAL DIAGNOSIS OF ALVEOLAR RHABDOMYOSARCOMA • • • • •

 wing sarcoma E Neuroblastoma Poorly differentiated synovial sarcoma Precursor lymphoblastic lymphoma/leukemia Extrarenal malignant rhabdoid tumor

Like its embryonal cousin, alveolar RMS is immunoreactive for desmin, muscle-specific actin, myo-D1, and myogenin. Diffuse nuclear expression of myo-D1 and myogenin is characteristic of alveolar RMS and distinguishes it from other RMS subtypes.172 Cytogenetic or molecular genetic detection of PAX3-FOXO1 or PAX7-FOXO1 is essential for confirming and refining the diagnosis of RMS (see Table 17.1).9,167 

581

more elongated, spindled, penicillate, or angulated nuclei.176 The cytoplasm varies from scant-to-moderate in quantity, with occasional vacuolization. Mitotic figures are easily found. Traversing delicate vessels and myxoid stroma are common in large clusters. Necrosis and single-cell apoptosis are also present. CIC-rearranged sarcoma shows morphologic overlap with ES but more variable CD99 staining, ranging from negative, patchy (Fig. 17.34B), to (occasionally) diffuse. Most tumors show WT1 and ETV4 nuclear expression.177,178 DIFFERENTIAL DIAGNOSIS OF CICREARRANGED SARCOMA • • • • •

 wing sarcoma E Alveolar rhabdomyosarcoma Poorly differentiated synovial sarcoma Myoepithelial neoplasms of soft tissue Precursor lymphoblastic lymphoma/leukemia

Undifferentiated Round Cell Sarcomas, including CIC-Rearranged Sarcoma Among undifferentiated non-Ewing round cell sarcomas, newly recognized subsets with novel fusions are increasing in number; examples include CIC-rearranged sarcoma and BCOR-CCNB3 sarcoma (the latter arises almost exclusively in bone). These tumors show overlapping morphologic features with ES and more variable, often negative or patchy, CD99 (O13) staining. In comparison to ES, CIC-rearranged sarcoma typically shows greater cytologic atypia, increased amphophilic cytoplasm, myxoid stroma, and necrosis. It affects patients across all ages (median age ∼26 years) with an equal sex distribution. CIC-rearranged sarcomas are most common in the somatic soft tissue of the extremities and trunk. They follow a highly aggressive clinical course, with frequent metastases and death, and are resistant to Ewing-specific drug regiments. CIC-DUX4 is the most common fusion gene, with a small subset having a CIC-FOXO4 fusion173-175 (see Table 17.1). CYTOMORPHOLOGY OF CIC-REARRANGED SARCOMA • R  ound cells with mild atypia and pleomorphism • Enlarged hyperchromatic nuclei with irregular membranes and large nucleoli • Scant or moderately abundant cytoplasm, occasionally vacuolated • Cohesive groups with myxoid stroma and traversing vessels • Single cell apoptosis and necrosis, frequent mitotic figures

Smears of CIC-rearranged sarcomas are cellular and show a population of round tumor cells, some singly dispersed and others arranged in large groups. The tumor cells exhibit appreciable pleomorphism, atypia, and anisonucleosis (Fig. 17.34A). The nuclei are hyperchromatic, with irregular nuclear contours and frequent large nucleoli; some tumor cells have

The differential diagnosis is challenging, and definitive diagnosis of a CIC-rearranged sarcoma and other “molecular subsets” ultimately requires molecular testing. Nevertheless, immunohistochemistry helps exclude other (and more likely) round cell sarcomas (see Table 17.3) and lymphoproliferative disorders (with TdT and LCA), and it identifies cases for molecular testing. 

Epithelioid Neoplasms Epithelioid soft tissue neoplasms are tumors composed of medium-sized or large, round or polygonal cells with ample cytoplasm. Metastatic carcinoma and melanoma are always in the differential diagnosis. Epithelioid neoplasms yield cellular smears with cohesive tumor cell aggregates, as well as numerous individually dispersed neoplastic cells. This group of tumors includes the epithelioid variants of many benign and malignant soft tissue neoplasms such as epithelioid schwannoma and epithelioid angiosarcoma, as well as specific, well-defined entities such as granular cell tumor, epithelioid sarcoma, and alveolar soft part sarcoma. Immunohistochemical studies play a decisive role in establishing the diagnosis of most epithelioid soft tissue tumors and excluding metastatic carcinoma, melanoma, and even large-cell lymphoma (see Table 17.4).

Epithelioid Sarcoma Epithelioid sarcoma is a rare malignant mesenchymal neoplasm with an epithelioid cytomorphology and immunophenotype. It affects the distal extremities (especially the hands and wrists) of young adults, with a predilection for males. The tumors are generally superficial, involving the dermis and subcutis, or tendo-aponeurotic. They present as slowly growing masses often of long duration. Pain and ulceration are frequent. A subset of epithelioid sarcomas are termed proximal type; they tend

582 CHA P T ER 1 7    Soft Tissue

A CD99

B •

Fig. 17.34  CIC-Rearranged Sarcoma.  (A) Although the cells resemble those of Ewing sarcoma, they are more irregular and variable (Romanowsky stain). (B) Whereas Ewing sarcoma is usually strongly and diffusely positive for CD99, this sarcoma is often either negative or only weakly or focally reactive for CD99.

to affect the deep soft tissue of the pelvis and perineum and are more aggressive than the classical (distal) form. Both types have aberrations of the SMARCB1 (INI1) gene on 22q, which results in the nuclear loss of INI1 protein expression in most cases.179 Smears are dominated by isolated cells admixed with small, loosely cohesive clusters of cells associated with

central fibrillary matrix. The tumor cells are round, polygonal, or spindle shaped, with mild to moderate nuclear pleomorphism. The nuclei are large, round, and eccentrically placed, with vesicular, occasionally clumped chromatin and one or more small nucleoli. Binucleation and multinucleation are common. The moderate to abundant cytoplasm is dense and can contain vacuoles

CHAPTER 17  Soft Tissue

(Fig. 17.35). Large atypical epithelioid cells with a rhabdoid appearance are common in the proximal type, and so are necrosis and mitoses (normal or atypical). The vaguely pseudogranulomatous growth pattern on histology is imperceptible on smears.180-183 CYTOMORPHOLOGY OF EPITHELIOID SARCOMA • N  umerous dispersed round, polygonal, or spindleshaped cells • Loosely cohesive clusters of spindle-shaped cells associated with central fibrillary matrix • Eccentrically placed nuclei with vesicular chromatin and small nucleoli • Dense cytoplasm with small vacuoles • Well-defined cell borders • Rhabdoid cells (proximal type) • Binucleation and multinucleation • Necroinflammatory debris

DIFFERENTIAL DIAGNOSIS OF EPITHELIOID SARCOMA • • • • • • •

 etastatic carcinoma and melanoma M Epithelioid hemangioendothelioma Pseudomyogenic hemangioendothelioma Epithelioid angiosarcoma Epithelioid malignant peripheral nerve sheath tumor Myoepithelial carcinoma of soft tissue Extrarenal malignant rhabdoid tumor

Even with the help of immunohistochemistry, epithelioid sarcoma is difficult to distinguish from a metastatic carcinoma because it is often diffusely positive for EMA and keratins. The clinical presentation of an extremity mass in a young patient is an important clue to the correct diagnosis. Although epithelioid sarcoma is negative for vascular markers such as CD31 and ERG, it is often positive for CD34, a potential problem in distinguishing it from an epithelioid hemangioendothelioma (see Fig. 4.18), pseudomyogenic

TABLE 17.4    Immunoprofile of Epithelioid Neoplasms

Tumor

CK/EMA

S-100 HMB-45 Desmin

CD34

CD31 and ERG CAMTA1

FOSB

INI1

TFE3

Alveolar soft part sarcoma

−

−/+

−

−/+

−

−

−

−

Retained

+

Carcinoma and mesothelioma

+

−

−

−

−

−

−

−

Retained

−

CCS and melanoma

−

+

+

−

−

−

−

−

Retained

−

CCS-like tumor of the GI tract

−

+

−

−

−

−

−

−

Retained

−

Chordomaa

+

+

−

−

−

−

−

−

Retained

−

Epithelioid sarcoma

+

−

−

−

+

−

−

−

Lost (90%)

−

Epithelioid hemangioendothelioma

−/+

−

−

−

+

+

+

−

Retained

−

Pseudomyogenic hemangioendothelioma

+ (AE1/3)

−

−

−

−

+

−

+

Retained

−

Epithelioid angiosarcoma

−/+

−

−

−

+

+

−

−

Retained

−

Epithelioid MPNST

−

+

−

−

−

−

−

−

Lost (50%)

−

Myoepithelial carcinoma

+

+

−

−

−

−

−

−

Lost (10– 40%)

−

PEComa

−

−

+

+

−

−

−

−

Retained

−

aAlso

583

positive for brachyury. CCS, Clear cell sarcoma; GI, gastrointestinal; MPNST, malignant peripheral nerve sheath tumor; PEComa, perivascular epithelioid cell tumor.

584 CHA P T ER 1 7    Soft Tissue

hemangioendothelioma, and epithelioid angiosarcoma (see Fig. 2.44A and B). CAMTA1 immunohistochemistry identifies epithelioid hemangioendothelioma,184 and FOSB distinguishes pseudomyogenic hemangioendothelioma from its mimics. Positive staining for S-100 favors metastatic melanoma, epithelioid MPNST, and myoepithelial carcinoma of soft tissue. Loss of nuclear INI1 expression favors epithelioid sarcoma (both types) and excludes most mimics, except for the epithelioid MPNST and extrarenal malignant rhabdoid tumor, which lose INI1 expression in 50% and 98% of cases, respectively. Extrarenal malignant rhabdoid tumor occurs in younger patients, is CD34-negative, and has mutations in SMARCB1 gene (see Table 17.3).179 

Clear Cell Sarcoma of Soft Tissue Clear cell sarcoma is a rare sarcoma with melanocytic differentiation. The tumor affects primarily adolescents and young adults. Presenting as a slowly growing, often painful

A

mass of the deep tissues of the extremities (foot/ankle), it is usually closely associated with fascia, tendons, or aponeuroses. Most lesions are relatively small (less than 5.0 cm). It is characterized by the presence of a recurrent EWSR1-ATF1 fusion gene (and rarely, EWSR1-CREB1). CYTOMORPHOLOGY OF CLEAR CELL SARCOMA • • • • • •

 ispersed round, polygonal, or spindle-shaped cells D Prominent nucleolus Wreathlike multinucleated giant cells Intranuclear cytoplasmic pseudoinclusions Clear or pale cytoplasm Tigroid background

Cytologic preparations are moderately to highly cellular, with a remarkably clean or tigroid background. The cells are mostly isolated, with occasional small clusters. They vary in size and shape and can be round, polygonal, and

B EMA

C

INI1

D • Fig. 17.35  Epithelioid Sarcoma.  (A) The dispersed epithelioid cells have eccentrically placed nuclei and

dense cytoplasm with sharply defined cell borders (Romanowsky stain). (B) The cells are polygonal, round, or spindle shaped, and some have cytoplasmic vacuoles (Romanowsky stain). (C) Tumor cells are epithelial membrane antigen positive. (D) There is loss of nuclear INI1 expression, which is characteristic of epithelioid sarcoma. Note that the admixed normal endothelial cells and inflammatory cells have retained nuclear expression.

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585

• Fig. 17.36  Clear Cell Sarcoma of Soft Tissue.  Cell borders are clearly defined. Binucleation and prominent nucleoli are present (Papanicolaou stain).

fusiform. Most nuclei are uniform in size, large, round to oval, and eccentrically placed (Fig. 17.36). They are slightly hyperchromatic, with finely granular, irregularly distributed, vesicular chromatin. The nucleolus is usually central, prominent, and solitary, but two or three smaller nucleoli can be seen. Intranuclear cytoplasmic pseudoinclusions are almost invariably present. Binucleated cells are ubiquitous, and wreathlike multinucleated giant cells are rare but diagnostically important. The moderately abundant cytoplasm is clear and pale, even imperceptible in some cells, and rarely finely vacuolated. Most of the scant intracytoplasmic melanin is found in macrophages. Occasional mitoses are seen, but necrosis is not a typical feature. Most cases are strongly positive for S-100 and HMB-45. Clinical and molecular findings (see Table 17.1 and Fig. 17.35B) help distinguish it from metastatic cutaneous melanoma.185,186 Clear cell sarcoma-like tumor of the gastrointestinal tract harbors the same gene fusions EWSR1-CREB1 and EWSR1-ATF1; although positive for S-100, it is negative for HMB-45. 

Alveolar Soft Part Sarcoma Accounting for less than 1% of all sarcomas, alveolar soft part sarcoma (ASPS) is a tumor of older adolescents and young adults, with a slight female predominance. It is a slowly growing, deep-seated, painless mass most common in the lower extremities or limb girdle, especially the anterior thigh. In children, the head and neck region is mainly affected. These tumors are negative for keratins, EMA, chromogranin, and synaptophysin. ASPS has a characteristic

genetic anomaly, an unbalanced (X;17) translocation (see Table 17.1) that results in an ASPSCR1-TFE3 fusion gene and nuclear overexpression of TFE3 protein.

CYTOMORPHOLOGY OF ALVEOLAR SOFT PART SARCOMA • • • •

 aked nuclei N Large, round to polygonal cells Prominent nucleolus Abundant granular and fragile cytoplasm

Aspirates show slight to moderate cellularity, with numerous naked nuclei. Often, a background of disrupted cytoplasmic contents and capillary networks devoid of tumor cells is seen. The cells are largely noncohesive but sometimes aggregate in large groups. Pseudoalveolar arrangements surrounded by a thin vascular structure, though a characteristic feature on histology, is a very rare finding on smears. The cells are uniformly round to polygonal, with some variation in size. The mildly pleomorphic, hyperchromatic, large nuclei are round to oval, with vesicular, finely stippled chromatin, smooth nuclear contours, and a central, prominent, round nucleolus. The fragile cytoplasm is abundant and finely granular (Fig. 17.37). Intracytoplasmic and extracellular periodic acid–Schiff-positive rhomboid crystals are a helpful feature but are rarely identified. Mitotic activity is low.187,188

586 CHA P T ER 1 7    Soft Tissue

•

Fig. 17.37  Alveolar Soft Part Sarcoma.  Large polygonal cells have abundant granular cytoplasm, large nuclei, vesicular chromatin, smooth nuclear contours, and a prominent central, round nucleolus (Papanicolaou stain).

DIFFERENTIAL DIAGNOSIS OF ALVEOLAR SOFT PART SARCOMA

CYTOMORPHOLOGY OF EPITHELIOID HEMANGIOENDOTHELIOMA

• • • • • •

• R  ound to polygonal plasmacytoid cells • Only slight nuclear pleomorphism and frequent binucleation • Frequent nuclear grooves and pseudoinclusions • Dense cytoplasm with occasional intracytoplasmic lumina • Hyaline stroma • Few mitoses

 ranular cell tumor G Melanoma Rhabdomyoma Paraganglioma Renal cell carcinoma PEComa

Renal cell carcinoma, paraganglioma, rhabdomyoma, melanoma, granular cell tumor, and PEComa resemble ASPS, but all lack rhomboid crystals, and all have different immunoprofiles.188 ASPS tumor cells show strong nuclear staining with TFE3 and variable staining with desmin and S-100, and are negative for keratins, EMA, chromogranin, and HMB-45. A specific diagnosis can usually be made on the basis of its distinctive cytomorphology, immunoprofile, and characteristic chromosomal translocation/gene fusion. 

Epithelioid Hemangioendothelioma Epithelioid hemangioendothelioma (EHE) is a rare malignant vascular neoplasm, affecting adults over the age of 20 years. It occurs in the extremities, head and neck region, trunk, mediastinum, bone, and visceral organs such as lung and liver. About half arise from a large or medium-sized vein (angiocentric). Multicentricity is frequent. EHE is characterized by cords of epithelioid endothelial cells embedded in a myxohyaline stroma. A WWTR1-CAMTA1 gene fusion is identified in most cases.1

Smears are variably cellular, with fragments of metachromatic, hyaline or chondroid stroma. Although predominantly isolated, the cells can form small, loose aggregates and rosette-like, pseudoacinar arrangements. The cells are monomorphic, with round, oval, or polygonal contours, a plasmacytoid appearance, and moderate amounts of dense cytoplasm (see Fig. 4.18). Rare sharply demarcated intracytoplasmic vacuoles distort the nucleus. Nuclei are round to oval with frequent binucleation and minimal nuclear pleomorphism. The chromatin is unevenly distributed, and one or two small nucleoli are seen. Occasional larger cells have hyperchromatic, irregular nuclei with nuclear membrane clefts and protrusions. Mitoses are seldom seen.189,190 Tumor cells are strongly and diffusely positive for the vascular markers CD31, CD34, and ERG. About 20% to 30% are immunoreactive for keratin, but they are negative for EMA. Nuclear CAMTA1 expression by immunohistochemistry is highly sensitive and specific for EHE.184

CHAPTER 17  Soft Tissue

DIFFERENTIAL DIAGNOSIS OF EPITHELIOID HEMANGIOENDOTHELIOMA

CYTOMORPHOLOGY OF PSEUDOMYOGENIC HEMANGIOENDOTHELIOMA

• • • • • • •

• N  oncohesive or loosely cohesive plump spindle and epithelioid cells • Round vesicular nuclei • Abundant cytoplasm with cytoplasmic extensions and vacuoles • Ganglion-like cells • Neutrophils may be present

 etastatic carcinoma M Melanoma Epithelioid sarcoma Epithelioid angiosarcoma Pseudomyogenic hemangioendothelioma Mesothelioma Epithelioid hemangioma

EHE is distinguished cytologically from metastatic carcinoma, melanoma, epithelioid sarcoma, and angiosarcoma by its lower degree of nuclear atypia and mitotic activity. EHEs of the lung have a predilection for spreading to the pleural surfaces, clinically mimicking a mesothelioma and not infrequently presenting with a pleural effusion. The similarity extends to morphologic evaluation: EHEs strongly resemble epithelioid mesothelioma. The distinction rests with immunohistochemistry: tumor cells of epithelioid mesothelioma are positive for mesothelial cell markers (calretinin, WT-1) whereas those of EHE are reactive for endothelial cell markers (CD31, CD34, ERG) and CAMTA1. Epithelioid hemangioma is more superficially located (and rarely sampled by FNA) and more obviously vasoformative. It has histiocytoid endothelial cells and lacks hyaline or chondroid stroma and is often associated with an inflammatory infiltrate. 

Pseudomyogenic Hemangioendothelioma Pseudomyogenic hemangioendothelioma (also known as epithelioid sarcoma-like hemangioendothelioma) is a newly recognized vascular neoplasm of intermediate biologic potential.191,192 These rare tumors typically arise in young adults with a marked male predilection; the lower limb is the most frequent site. The clinical features are distinctive: affected patients develop numerous nodules involving multiple tissue planes, with dermal, subcutaneous, subfascial, intramuscular, and osseous lesions. Despite the striking clinical presentation, most patients are treated with conservative management aimed at local surgical control. Pseudomyogenic hemangioendothelioma follows an overall indolent course, with frequent local recurrences but only rare reports of distant metastases and disease-related death. Microscopically, the infiltrative tumor nodules are comprised of sheets and fascicles of spindle cells with round, vesicular nuclei and abundant eosinophilic cytoplasm (hence, “pseudomyogenic”). A neutrophilic infiltrate in the absence of necrosis is common. SERPINE-FOSB fusion is characteristic.193 The cytomorphologic features have not yet been systematically described, but this tumor inevitably enters the differential diagnosis of epithelioid neoplasms in the extremities of young adults. The tumor cells of pseudomyogenic hemangioendothelioma show noncohesive or loosely cohesive, plump spindle and epithelioid cells,

587

resembling nodular fasciitis. The tumor cells have abundant cytoplasm with cytoplasmic extensions and vacuoles. Nuclei are round and vesicular with frequent nucleoli (Fig 17.38). Immunohistochemistry is necessary for definitive diagnosis: tumors are positive for the vascular markers CD31, FLI1, and ERG (but not for CD34) and show expression of keratin AE1/AE3. FOSB is an established, useful diagnostic marker that distinguishes pseudomyogenic hemangioendothelioma from its mimics194 (see Table 17.4). DIFFERENTIAL DIAGNOSIS OF PSEUDOMYOGENIC HEMANGIOENDOTHELIOMA • • • • • •

 etastatic carcinoma M Epithelioid hemangioendothelioma Epithelioid angiosarcoma Epithelioid sarcoma Epithelioid variant of other sarcomas Nodular fasciitis

The distinctive clinical presentation of pseudomyogenic hemangioendothelioma is helpful; imaging studies may show the pathognomonic multifocal lesions (which are PET-avid) across tissue planes. By contrast, patients with nodular fasciitis usually present with a rapidly growing mass in the upper extremities. Metastatic carcinoma is negative for vascular markers. FOSB is helpful in excluding most other mesenchymal mimics, including EHE (which is positive for CAMTA1), epithelioid angiosarcoma, and epithelioid sarcoma (which is CD34-positive and loses nuclear INI1 expression). 

Epithelioid Angiosarcoma Epithelioid angiosarcoma, a rare but highly aggressive malignant vascular tumor, has a marked predilection for males, usually adults in mid- to late life. It is a rapidly growing tumor of deep soft tissues, but it can be encountered in a variety of other sites, including the lower extremities, retroperitoneum, and abdominal cavity.195 Smears are variably cellular, depending on the degree of dilution by blood. The mostly noncohesive cells infrequently aggregate into small clusters and sometimes

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• Fig. 17.38  Pseudomyogenic Hemangioendothelioma.  Plump spindle and epithelioid cells have round

nuclei with vesicular chromatin and abundant cytoplasm, with cytoplasmic extensions and vacuoles resembling nodular fasciitis (Diff-Quik stain). (Courtesy Dr. Henryk Domanski, Lund University Hospital, Lund, Sweden.)

CYTOMORPHOLOGY OF EPITHELIOID ANGIOSARCOMA

DIFFERENTIAL DIAGNOSIS OF EPITHELIOID ANGIOSARCOMA

• • • • •

• • • • • • • •

 xtensively bloody background E Large, noncohesive epithelioid cells Moderate to marked nuclear pleomorphism Prominent nucleolus Occasional vasoformative structures: pseudoacini with central erythrocytes or intracytoplasmic lumina containing erythrocytes • Numerous mitoses

even form pseudoacini. They are large, rounded epithelioid cells with moderately to markedly pleomorphic nuclei and smooth nuclear contours (see Fig. 2.44A and B). Binucleation and especially multinucleation are rare. Mitoses are frequent. The nuclei are eccentrically placed within an abundant, finely granular cytoplasm. Intracytoplasmic hemosiderin is detected in many cases. Vasoformative structures (e.g., pseudoacini with central erythrocytes; intracytoplasmic vacuoles containing intact or fragmented erythrocytes) are rare but very useful findings.196-198 Epithelioid angiosarcoma should be considered in the differential diagnosis of any poorly differentiated neoplasm with epithelioid cytomorphology, especially in the abdominal cavity. Epithelioid angiosarcoma cells stain with endothelial markers CD31, CD34, ERG, and FLI1, and with keratins in up to 50% cases (see Table17.4). 

 etastatic carcinoma M Metastatic melanoma Malignant mesothelioma Epithelioid hemangioendothelioma Large cell lymphoma Germ cell tumor Epithelioid sarcoma Epithelioid variant of other sarcomas

Granular Cell Tumor Granular cell tumors are relatively common, slowly growing neoplasms with neuroectodermal differentiation. With rare exceptions, they are benign. They occur most commonly in the head and neck region, including the tongue of middle-aged adults. Other common sites include the extremities, gastrointestinal tract, respiratory tree, thyroid, and breast.199

CYTOMORPHOLOGY OF GRANULAR CELL TUMOR • • • •

 are nuclei in a granular background B Uniform cellular appearance Small nuclei Abundant granular cytoplasm

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• Fig. 17.39  Granular Cell Tumor.  Small nuclei within abundant granular cytoplasm result in a low nuclearto-cytoplasmic ratio (Papanicolaou stain).

Smears show numerous naked nuclei and a finely granular background derived from the abundant, fragile cytoplasm of the tumor cells. Intact cells are uniform and occur as syncytial groups and isolated cells (Fig. 17.39). The small, round to oval nuclei are minimally pleomorphic, with finely granular chromatin and a small but conspicuous nucleolus. Very rare intranuclear cytoplasmic invaginations are seen, and cell borders are typically ill defined. Mitoses and necrosis are not seen in aspirates of benign lesions.199 Malignant granular cell tumors are exceedingly rare, and it is difficult to definitively separate them from their benign counterparts. The cytologic features that are suggestive of malignancy include spindle cell morphology, cellular pleomorphism, enlarged nucleoli, and mitoses.200,201 Granular cell tumors show strong cytoplasmic staining for S-100 and SOX10. They also express NKI/C3, owing to their lysozyme-rich cytoplasm. Interestingly, they are also positive for CD68, TFE3 (but lack a TFE gene rearrangement), inhibin, and calretinin. They are negative for muscle markers, GFAP, HMB-45, and keratins. DIFFERENTIAL DIAGNOSIS OF GRANULAR CELL TUMOR • • • • •

 at necrosis F Whipple disease Rhabdomyoma Alveolar soft part sarcoma Renal cell carcinoma

Fat necrosis has bean-shaped or oval histiocytes with foamy cytoplasm, multinucleated giant cells, and other inflammatory cells. A similarly mixed cell population is typical of Whipple disease. Rhabdomyoma may contain

multinucleated cells and is negative for S-100. Alveolar softpart sarcoma has prominent nucleoli, frequent binucleation and multinucleation, and pseudoacinar or pseudoalveolar arrangements; TFE3 FISH confirms a TFE3 rearrangement. Renal cell carcinomas usually have some cells with clear cytoplasm, and they are positive for PAX8. 

Pleomorphic Neoplasms Many pleomorphic soft tissue tumors are poorly differentiated or dedifferentiated forms of entities that have a clear line of differentiation (e.g., adipocytic, skeletal muscle, etc.). Pleomorphic liposarcoma, dedifferentiated liposarcoma, and high-grade myxofibrosarcoma have been discussed above, and pleomorphic RMS is discussed in this section. Pleomorphic soft tissue sarcomas are aggressive and considered high grade for management purposes. With wide sampling and immunoprofiling, most pleomorphic sarcomas prove to show a definitive line of differentiation (e.g., pleomorphic osteosarcoma, leiomyosarcoma, RMS, liposarcoma, or high-grade myxofibrosarcoma). Undifferentiated pleomorphic sarcoma is a diagnosis of exclusion and reserved for cases in which all efforts fail to reveal a line of differentiation. It is a diagnosis best deferred to the surgical resection.1

Undifferentiated Pleomorphic Sarcoma Undifferentiated pleomorphic sarcomas are considered part of the morphologic spectrum of all undifferentiated soft tissue sarcomas (USTSs).1 USTSs include undifferentiated sarcomas with round cell (discussed above), spindle cell, pleomorphic, epithelioid, or “not otherwise specified”

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• Fig. 17.40  Undifferentiated High-Grade Pleomorphic Sarcoma.  The bizarre tumor cells and necrotic background are nonspecific findings seen with any pleomorphic soft tissue neoplasm (Papanicolaou stain).

cytomorphology. Undifferentiated pleomorphic sarcomas probably account for 5% to 10% of sarcomas in adults over age 40 and occur mostly in the extremities and trunk; some are radiation associated. They show no identifiable line of differentiation by morphologic, immunohistochemical, or genetic grounds. It is a diagnosis of exclusion and best deferred to surgical resection, unless presenting as a recurrence or metastasis. Of note, the term “malignant fibrous histiocytoma” is now obsolete, although occasionally still erroneously propagated as a synonym for undifferentiated pleomorphic sarcoma. CYTOMORPHOLOGY OF UNDIFFERENTIATED PLEOMORPHIC SARCOMA • H  ighly cellular smears • Variable proportions of cellular clusters and dispersed cells • Marked cellular and nuclear pleomorphism and anaplasia • Giant cells with solitary or multiple nuclei • Numerous mitoses including atypical forms • Necrosis

The highly cellular smears contain variable proportions of cellular clusters and dispersed cells in a necrotic background. All the tumors in this group share impressive morphologic features that include markedly pleomorphic, spindle-shaped, or epithelioid cells with abundant cytoplasm; giant cells with solitary or multiple nuclei; prominent necrosis; and brisk mitotic activity with atypical forms

(Fig. 17.40). It is difficult to identify any cytomorphologic clues to suggest a line of differentiation in these tumors. The tumor cells can be focally positive for keratin, actin, desmin, or EMA, but they are usually negative for all other specific immunomarkers. Cytogenetics usually reveals a highly complex karyotype with marked intratumoral heterogeneity. DIFFERENTIAL DIAGNOSIS OF UNDIFFERENTIATED PLEOMORPHIC SARCOMA • • • • •

 arcomatoid carcinoma S Sarcomatoid mesothelioma Melanoma Anaplastic large cell lymphoma Pleomorphic sarcoma with a specific line of differentiation (e.g., rhabdomyosarcoma, liposarcoma) • Dedifferentiated sarcoma

Sarcomatoid carcinoma, sarcomatoid mesothelioma, malignant melanoma, and anaplastic large-cell lymphoma (ALCL) can have the same pleomorphism, including a mixture of highly atypical spindle cells, epithelioid cells, and giant cells. Immunohistochemical studies are usually necessary to make the distinction. Sarcomatoid carcinoma and mesothelioma should have at least focal positivity for one or more keratins, and mesothelioma is additionally positive for mesothelial cell markers (calretinin, WT-1, and D2-40). ALCL shows large, anaplastic, round cells with an embryo-like nucleus (see Fig. 12.25). ALCL cells are positive for CD30 and in some cases for ALK as well.

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•

Fig. 17.41  Pleomorphic Rhabdomyosarcoma.  Numerous dispersed rhabdoid cells have eccentrically located nuclei, prominent nucleoli, abundant cytoplasm, and a perinuclear cytoplasmic density (Romanowsky stain).

Melanoma often has melanin pigment in tumor cells and in adjacent macrophages, and the diagnosis can be confirmed by immunoreactivity for melanocytic markers. A differentiated pleomorphic sarcoma, such as pleomorphic liposarcoma (see Fig. 17.8), high-grade myxofibrosarcoma, or pleomorphic RMS (Fig. 17.41), is confirmed by demonstrating a clear line of differentiation morphologically or with ancillary techniques. Clinical history and the presence of a differentiated area are essential for the diagnosis of a specific dedifferentiated sarcoma, and in many cases definitive classification is deferred to surgical resection. Finally, the diagnosis of a radiation-induced high-grade sarcoma should be considered when an undifferentiated high-grade pleomorphic sarcoma occurs at the site of prior radiation therapy. Because an FNA represents only a portion of the neoplasm, excluding specific pleomorphic sarcomas, carcinoma, mesothelioma, melanoma, and lymphoma might not be possible because of sampling error. A pleomorphic neoplasm that is negative for differentiation markers is best reported as “pleomorphic malignant neoplasm, cannot classify further.” 

Pleomorphic Rhabdomyosarcoma Pleomorphic RMS is a high-grade sarcoma showing large polygonal cell cytomorphology and skeletal muscle differentiation. It most commonly occurs in the lower extremities of elderly patients.

CYTOMORPHOLOGY OF PLEOMORPHIC RHABDOMYOSARCOMA • H  ypercellular smears with predominantly dispersed cells • Marked cellular and nuclear pleomorphism • L  arge rhabdoid cells: eccentrically located nuclei, prominent nucleoli, abundant cytoplasm with a perinuclear density • Frequent binucleation and multinucleation • Numerous mitoses including atypical forms • Necrosis

Smears are hypercellular, with numerous dispersed cells and occasional small clusters of polygonal cells. Cellular and nuclear pleomorphism is marked with frequent, very large cells. Most cells exhibit a typical rhabdoid cytomorphology: eccentrically located nuclei with prominent nucleoli and abundant cytoplasm with a perinuclear globoid density. Binucleation and multinucleation are very common. Mitoses and necrosis are also present (see Fig. 17.41).

DIFFERENTIAL DIAGNOSIS OF PLEOMORPHIC RHABDOMYOSARCOMA • • • • •

 eterologous rhabdoid differentiation of other sarcomas H Pleomorphic leiomyosarcoma Proximal-type epithelioid sarcoma Metastatic melanoma Metastatic carcinoma

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The rhabdoid cytomorphology is non-specific and can be seen focally in many nonmyogenic tumors, including melanoma, carcinoma (e.g., renal cell carcinoma), and sarcomas such as the proximal type of epithelioid sarcoma. Demonstration of nuclear expression of myogenin (myf4) in additional to diffuse desmin positivity is critical for establishing rhabdomyoblastic differentiation. In a small-needle biopsy, a heterologous rhabdoid component of other sarcomas like malignant peripheral nerve sheath tumor and dedifferentiated liposarcoma should be given serious consideration before rendering a diagnosis of pleomorphic RMS. 

of life. The typical lesion shows bilateral periureteral fibrosis at the level of the L4 vertebra, often encasing the aorta.202 A subset of IRF cases are associated with IgG4-related disease.203

CYTOMORPHOLOGY OF IDIOPATHIC RETROPERITONEAL FIBROSIS • • • •

 ixed inflammatory infiltrate M Prominent crush artifact Fragments of fibrous tissue Spindle-shaped fibroblastic cells

Dedifferentiated Sarcomas Three different soft tissue tumors undergo dedifferentiation with relative frequency: well-differentiated liposarcoma, well-differentiated chondrosarcoma, and chordoma; overall dedifferentiation across soft tissue tumors is rare. Recent rapid growth of a previously stable mass is typical of these dedifferentiated tumors, and the correct classification of each hinges on identifying an adjacent well-differentiated component. For this reason, cytologists must boldly request additional, wider sampling when confronted with an undifferentiated pleomorphic or giant cell lesion during ROSE. Dedifferentiation occurs with approximately 10% of all chondrosarcomas, usually in patients about 10 years older than those with a conventional chondrosarcoma. Dedifferentiated chordoma is an exceedingly rare event. Dedifferentiated liposarcoma is the most common of the three and accounts for up to 10% of liposarcomas. It is usually retroperitoneal or intraabdominal. Its morphology is widely variable, but most manifest as a nonlipogenic sarcoma. Although many resemble an undifferentiated pleomorphic sarcoma or myxofibrosarcoma (see Fig. 17.10), some cases show low-grade morphology. The transition between the dedifferentiated and well-differentiated components is usually abrupt, but in rare cases it can be a gradual intermingling. Rarely, tumors show “homologous” dedifferentiation and mimic pleomorphic liposarcoma, with lipoblasts in otherwise nonlipogenic areas.90 Cytogenetic analysis can assist with the diagnosis because dedifferentiated liposarcoma, in addition to complex aberrations, retains the same giant marker and ring chromosomes as its well-differentiated counterpart. Immunohistochemistry for MDM2 and CDK4 is helpful in identifying most cases, and detection of MDM2 gene amplifications by molecular cytogenetic approaches (FISH, PCR, or both) can be of great diagnostic value.9,61 

Nonneoplastic Soft Tissue Lesions Idiopathic Retroperitoneal Fibrosis Idiopathic retroperitoneal fibrosis (IRF) is an enigmatic lesion affecting mostly men in their fourth to fifth decades

The moderately cellular smears contain rare fragments of fibrous tissue and inflammatory cells, including small lymphocytes, plasma cells, histiocytes, rare eosinophils, and mast cells. Neutrophils are also occasionally present. The inflammatory cells are partially obscured by prominent crush artifact. Sparse, benign-appearing, spindle-shaped fibroblastic cells are visible in thicker fragments of the fibrous tissue. Nuclei lack atypia, nucleoli are inconspicuous, and mitoses and necrosis are absent. The inflammatory infiltrate has a slight predominance of B cells over T cells (60% vs 40%).202,204,205

DIFFERENTIAL DIAGNOSIS OF IDIOPATHIC RETROPERITONEAL FIBROSIS • • • • • •

 odgkin disease H Non-Hodgkin lymphoma Myelolipoma and extramedullary hematopoiesis Well-differentiated inflammatory liposarcoma Metastatic carcinoma with desmoplastic stroma Sclerosing mesenteritis

The differential diagnosis includes any lesion with prominent fibrosis and inflammation, as well as myelolipoma. The nodular sclerosis variant of Hodgkin lymphoma has distinguishing Reed-Sternberg cells, lacunar cells, and associated clinical features. The lymphoid component of non-Hodgkin lymphoma is usually more monomorphic. Careful examination is required for the trilineage hematopoietic elements of a myelolipoma, a diagnosis supported by the identification of megakaryocytes (confirmed by immunohistochemistry for CD61). Well-differentiated inflammatory liposarcoma has cytologic atypia and immunoreactivity for MDM2 and CDK4. The desmoplastic stroma of a metastatic carcinoma can resemble the fibrotic areas of IRF, and thus immunocytochemistry for keratins and EMA are helpful in excluding this possibility. Sclerosing mesenteritis usually involves the small bowel mesentery, with more prominent fat necrosis. 

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Fig. 17.42  Elastofibroma.  Sharply defined, hypocellular fragments contain rare bland fibroblasts with curvilinear, rodlike elastic fibers (hematoxylin and eosin stain). (Courtesy Dr. Henryk Domanski, Lund University Hospital, Lund, Sweden.)

Elastofibroma

Amyloidoma (Tumoral Amyloidosis)

Elastofibroma is a relatively uncommon lesion thought to represent a reactive process, possibly from repeated trauma to the tissues between the scapulae and the chest wall. Elderly individuals, particularly women, are most commonly affected. It presents as a slowly growing subscapular mass attached to the rib periosteum.

An amyloidoma is an uncommon mass lesion caused by extensive amyloid deposition. Some but not all cases are associated with systemic amyloidosis. It can occur in many visceral organs and extremely rarely in bone and soft tissues.

CYTOMORPHOLOGY OF ELASTOFIBROMA

CYTOMORPHOLOGY OF AMYLOIDOMA

• • • •

• F  ragments of waxy, amorphous material with sharply defined edges and embedded fibroblasts and capillaries • Multinucleated giant cells • Scant inflammatory cells including plasma cells

 ypocellular, waxy matrix H Linear, dentate or serrated, rodlike structures Serrated globular bodies Scant uniform bland fibroblasts   

Cytologic preparations are remarkably hypocellular. Rare fibroblasts with bland nuclear features are seen within a homogeneous, waxy, hyalinized matrix. These stromal fragments have sharp borders. Linear, slightly curved or angulated, rodlike elastic fibers of varying sizes, lengths, and widths typically have dentations or serrations along the long edge and should not be mistaken for degenerated fungi (Fig 17.42). The globular bodies also have serrated borders and vary in diameter. Most are solitary, but occasional loose or tight clusters are found. Definitive diagnosis is possible if the elastic fibers and globular bodies are noted.206 

  

Cytologic preparations are hypocellular and contain abundant fragments of acellular, waxy, amorphous material with sharply defined edges and bland mesenchymal cells (Fig. 17.43). It is a metachromatic blue-purple with Romanowsky stains and pink, orange, or green-blue with the Papanicolaou stain. Scattered multinucleated giant cells are common. Inflammatory cells including plasma cells may or may not be present. A nonspecific diagnosis is avoided only if the amyloid deposition is confirmed by a Congo red stain, sulfated Alcian blue stain, or mass spectrometry analysis.207,208

594 CHA P T ER 1 7    Soft Tissue

•

Fig. 17.43  Amyloidoma.  Fragments of waxy, amorphous material have sharply defined edges with embedded fibroblasts and capillaries (Papanicolaou stain).

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166. Crapanzano JP, Cardillo M, Lin O, Zakowski MF. Cytology of desmoplastic small round cell tumor. Cancer. 2002;96(1): 21–31. 167. Klijanienko J, Caillaud JM, Orbach D, et al. Cyto-histological correlations in primary, recurrent and metastatic rhabdomyosarcoma: the Institut Curie’s experience. Diagn Cytopathol. 2007;35(8):482–487. 168. Seidal T, Walaas L, Kindblom LG, Angervall L. Cytology of embryonal rhabdomyosarcoma: a cytologic, light microscopic, electron microscopic, and immunohistochemical study of seven cases. Diagn Cytopathol. 1988;4(4):292–299. 169. Pohar-Marinsek Z, Bracko M. Rhabdomyosarcoma. Cytomorphology, subtyping and differential diagnostic dilemmas. Acta Cytol. 2000;44(4):524–532. 170. Layfield LJ, Liu K, Dodge RK. Logistic regression analysis of small round cell neoplasms: a cytologic study. Diagn Cytopathol. 1999;20(5):271–277. 171. Akhtar M, Ali MA, Bakry M, Hug M, Sackey K. Fine-needle aspiration biopsy diagnosis of rhabdomyosarcoma: cytologic, histologic, and ultrastructural correlations. Diagn Cytopathol. 1992;8(5):465–474. 172. Dias P, Chen B, Dilday B, et  al. Strong immunostaining for myogenin in rhabdomyosarcoma is significantly associated with tumors of the alveolar subclass. Am J Pathol. 2000;156(2):399– 408. 173. Choi EY, Thomas DG, McHugh JB, et  al. Undifferentiated small round cell sarcoma with t(4;19)(q35;q13.1) CIC-DUX4 fusion: a novel highly aggressive soft tissue tumor with distinctive histopathology. Am J Surg Pathol. 2013;37(9):1379–1386. 174. Italiano A, Sung YS, Zhang L, et  al. High prevalence of CIC fusion with double-homeobox (DUX4) transcription factors in EWSR1-negative undifferentiated small blue round cell sarcomas. Genes Chromosomes Cancer. 2012;51(3):207–218. 175. Sugita S, Arai Y, Tonooka A, et al. A novel CIC-FOXO4 gene fusion in undifferentiated small round cell sarcoma: a genetically distinct variant of Ewing-like sarcoma. Am J Surg Pathol. 2014;38(11):1571–1576. 176. Chebib I, Jo VY. Round cell sarcoma with CIC-DUX4 gene fusion: Discussion of the distinctive cytomorphologic, immunohistochemical, and molecular features in the differential diagnosis of round cell tumors. Cancer Cytopathol. 2016;124(5):350–361. 177. Specht K, Sung YS, Zhang L, Richter GH, Fletcher CD, Antonescu CR. Distinct transcriptional signature and immunoprofile of CIC-DUX4 fusion–positive round cell tumors compared to EWSR1-rearranged Ewing sarcomas: further evidence toward distinct pathologic entities. Genes Chromosomes Cancer. 2014;53(7):622–633. 178. Hung YP, Fletcher CD, Hornick JL. Evaluation of ETV4 and WT1 expression in CIC-rearranged sarcomas and histologic mimics. Mod Pathol. 2016;29(11):1324–1334. 179. Hollmann TJ, Hornick JL. INI1-deficient tumors: diagnostic features and molecular genetics. Am J Surg Pathol. 2011;35(10):e47–63. 180. Lemos MM, Chaves P, Mendonca ME. Is preoperative cytologic diagnosis of epithelioid sarcoma possible? Diagn Cytopathol. 2008;36(11):780–786. 181. Pohar-Marinsek Z, Zidar A. Epithelioid sarcoma in FNAB smears. Diagn Cytopathol. 1994;11(4):367–372. 182. Gonzalez-Peramato P, Jimenez-Heffernan JA, Cuevas J. Fineneedle aspiration cytology of “proximal-type” epithelioid sarcoma. Diagn Cytopathol. 2001;25(2):122–125.

599

183. Ikeda K, Tate G, Suzuki T, Mitsuya T. Fine needle aspiration cytology of primary proximal-type epithelioid sarcoma of the perineum: a case report. Acta Cytol. 2005;49(3):314–318. 184. Doyle LA, Fletcher CD, Hornick JL. Nuclear expression of CAMTA1 distinguishes epithelioid hemangioendothelioma from histologic mimics. Am J Surg Pathol. 2016;40(1):94–102. 185. Rau AR, Kini H, Verghese R. Tigroid background in fine-needle aspiration cytology of clear cell sarcoma. Diagn Cytopathol. 2006;34(5):355–357. 186. Thway K, Fisher C. Tumors with EWSR1-CREB1 and EWSR1-ATF1 fusions: the current status. Am J Surg Pathol. 2012;36(7):e1–e11. 187. Wakely Jr PE, McDermott JE, Ali SZ. Cytopathology of alveolar soft part sarcoma: a report of 10 cases. Cancer. 2009;117(6):500–507. 188. Lopez-Ferrer P, Jimenez-Heffernan JA, Vicandi B, GonzalezPeramato P, Viguer JM. Cytologic features of alveolar soft part sarcoma: report of three cases. Diagn Cytopathol. 2002;27(2):115–119. 189. Kilpatrick SE, Koplyay PD, Ward WG, Richards 2nd F. Epithelioid hemangioendothelioma of bone and soft tissue: a fine-needle aspiration biopsy study with histologic and immunohistochemical confirmation. Diagn Cytopathol. 1998;19(1):38–43. 190. Murali R, Zarka MA, Ocal IT, Tazelaar HD. Cytologic features of epithelioid hemangioendothelioma. Am J Clin Pathol. 2011;136(5):739–746. 191. Hornick JL, Fletcher CD. Pseudomyogenic hemangioendothelioma: a distinctive, often multicentric tumor with indolent behavior. Am J Surg Pathol. 2011;35(2):190–201. 192. Billings SD, Folpe AL, Weiss SW. Epithelioid sarcoma–like hemangioendothelioma. Am J Surg Pathol. 2003;27(1):48–57. 193. Walther C, Tayebwa J, Lilljebjorn H, et al. A novel SERPINE1FOSB fusion gene results in transcriptional up-regulation of FOSB in pseudomyogenic haemangioendothelioma. J Pathol. 2014;232(5):534–540. 194. Hung YP, Fletcher CD, Hornick JL. FOSB is a useful diagnostic marker for pseudomyogenic hemangioendothelioma. Am J Surg Pathol. 2017;41(5):596–606. 195. Hart J, Mandavilli S. Epithelioid angiosarcoma: a brief diagnostic review and differential diagnosis. Arch Pathol Lab Med. 2011;135(2):268–272. 196. Wakely Jr PE, Frable WJ, Kneisl JS. Aspiration cytopathology of epithelioid angiosarcoma. Cancer. 2000;90(4):245–251. 197. Pohar-Marinsek Z, Lamovec J. Angiosarcoma in FNA smears: diagnostic accuracy, morphology, immunocytochemistry and differential diagnoses. Cytopathol. 2010;21(5):311–319. 198. Klijanienko J, Caillaud JM, Lagace R, Vielh P. Cytohistologic correlations in angiosarcoma including classic and epithelioid variants: Institut Curie’s experience. Diagn Cytopathol. 2003;29(3):140–145. 199. Gibbons D, Leitch M, Coscia J, et  al. Fine needle aspiration cytology and histologic findings of granular cell tumor of the breast: review of 19 cases with clinical/radiologic correlation. Breast J. 2000;6(1):27–30. 200. Wieczorek TJ, Krane JF, Domanski HA, et al. Cytologic findings in granular cell tumors, with emphasis on the diagnosis of malignant granular cell tumor by fine-needle aspiration biopsy. Cancer. 2001;93(6):398–408. 201. Cimino-Mathews A, Illei PB, Ali SZ. Atypical granular cell tumor of the thyroid: cytomorphologic features on fine needle aspiration. Diagn Cytopathol. 2011;39(8):608–611.

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202. Corradi D, Maestri R, Palmisano A, et al. Idiopathic retroperitoneal fibrosis: clinicopathologic features and differential diagnosis. Kidney International. 2007;72(6):742–753. 203. Zen Y, Onodera M, Inoue D, et al. Retroperitoneal fibrosis: a clinicopathologic study with respect to immunoglobulin G4. Am J Surg Pathol. 2009;33(12):1833–1839. 204. Stein AL, Bardawil RG, Silverman SG, Cibas ES. Fine needle aspiration biopsy of idiopathic retroperitoneal fibrosis. Acta Cytol. 1997;41(2):461–466. 205. Dash RC, Liu K, Sheafor DH, Dodd LG. Fine-needle aspiration findings in idiopathic retroperitoneal fibrosis. Diagn Cytopathol. 1999;21(1):22–26.

206. Domanski HA, Carlen B, Sloth M, Rydholm A. Elastofibroma dorsi has distinct cytomorphologic features, making diagnostic surgical biopsy unnecessary: cytomorphologic study with clinical, radiologic, and electron microscopic correlations. Diagn Cytopathol. 2003;29(6):327–333. 207. Bardin RL, Barnes CE, Stanton CA, Geisinger KR. Soft tissue amyloidoma of the extremities: a case report and review of the literature. Arch Pathol Lab Med. 2004;128(11):1270–1273. 208. Elkins CT, Scharschmidt TJ, Wakely Jr PE. Amyloidomas of soft parts: diagnosis by fine-needle aspiration. Diagn Cytopathol. 2012;40(suppl 2):E126–E130.

18

Bone XIAOHUA QI AN

Introduction Primary bone tumors are relatively common, and the majority are benign and clinically silent. Bone lesions that have a characteristic radiologic appearance or are predominantly sclerotic, such as osteochondroma and fibrous dysplasia, are monitored clinically and radiologically and are not usually amenable to needle biopsy.1 Malignant bone tumors are rare, accounting for only 0.2% of all neoplasms.2 The cortical destruction and soft tissue extension of malignant bone tumors make them suitable targets for needle sampling. Fine-needle aspiration (FNA) for the evaluation of bone tumors was first introduced in the early 1930s.3,4 Reported accuracy rates of FNA range from 54% to 100%, depending on the era of the study, the availability of radiologic correlation, and the diagnostic terminology used.5-10 Although earlier studies confirmed the value of FNA for diagnosing metastatic malignancy in bone,11,12 FNA for primary bone tumors was limited by a high insufficient rate, the inability to reach intramedullary lesions when the cortex is intact, and the concern for sampling and interpretation errors in the evaluation of tumors with heterogeneous/biphasic histology.13 Recent years have seen an increasing use of minimally invasive biopsy techniques—FNA and core needle biopsy (CNB)—to investigate mass-forming or radiologically ambiguous small bone lesions.14 This approach emerged during the past decade, accompanied by advances in imageguided biopsy techniques, the discovery of the molecular genetic alterations of many primary bone tumors, and the development of ancillary diagnostic tests (Table 18.1).15 There is good concordance between FNA and CNB for osseous lesions, and the two techniques are complementary.14,16 Nevertheless, diagnosing and subclassifying primary bone tumors by needle biopsy remains one of the most challenging areas in surgical pathology and cytopathology.15,17 Clinical practice guidelines recommend that all patients with a suspected primary malignant bone tumor be referred to a cancer center/network with a bone sarcoma multidisciplinary team.18,19 The biopsy approach and interpretation demand a high level of collaboration among the members of a multidisciplinary team.13

The importance of correlation with clinical and radiologic findings cannot be overemphasized. Radiologic studies, including plain X-ray, ultrasonography, computed tomography, magnetic resonance imaging, and positron emission tomography, provide valuable information regarding the location (epiphysis vs metaphysis), size, pattern of bone destruction (lytic vs blastic), pattern of mineralization, periosteal reaction, the extent of bone or soft tissue involvement, content (cystic vs solid), and relationship to important structures (e.g., spinal cord).20 ADVANTAGES OF FINE-NEEDLE ASPIRATION FOR BONE LESIONS • S  afe for difficult sites such as vertebrae and pelvic bone • Allows for rapid on-site evaluation, tissue triage for ancillary studies, and initiation of treatment in lifethreatening scenarios • On-site pathologic-radiologic correlation • Some diagnostic features are more prominent on smears • Superior cellular material for molecular testing   

INDICATIONS FOR FINE-NEEDLE ASPIRATION OF BONE • • • • •

 ytic lesion with cortical thinning or destruction L Limited differential diagnosis, likely metastasis Small lesion, not amenable to CNB Suspect small round cell tumor Difficult site for CNB: ribs, sternum, clavicle, phalanges, vertebrae, and pelvic bone   

Specimen collection and preparation for routine cytologic analysis and ancillary testing are similar to those described for soft tissue FNA (see Chapter 17). Because regular decalcification with harsh acid solutions (e.g., nitric acid, hydrochloric acid) damages nucleic acids and compromises most molecular tests, including sequencing and fluorescence in situ hybridization (FISH), FNA material (nondecalcified) or bone biopsy cores treated with an ethylenediamine tetraacetic acid (EDTA)–based decalcification solution are more suitable for collecting samples for molecular studies.21  601

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TABLE 18.1    Useful Markers and Principal Molecular Alterations in Selected Primary Bone Tumors

Tumor/lesion

Markers

Principle molecular alterations

Adamantinoma

keratin, EMA, p63, D2-40

EPHB4-MARCH10 fusion

Aneurysmal bone cyst

N/A

CDH11-USP6 fusiona

Chondroblastoma

H3K36M,b S-100, SOX-9

H3F3B (K36M) mutation

Chondromyxoid fibroma

SMA, EMA, S-100, SATB2

GRM1 rearrangementa

Chondrosarcoma

S-100

IDH1 and IDH2 mutations

Mesenchymal chondrosarcoma

CD99 (cytoplasmic), S-100

HEY1-NCOA2 fusiona

Clear cell chondrosarcoma

S-100

N/A

S-100, keratin, brachyury

Germline duplication/somatic amplification of T SMARCB1 deletionsa

Chondrosarcoma variants

Chordoma pediatric/poorly differentiated

INI (loss of expression)

Ewing sarcomac

CD99, FLI-1, NKX2.2

EWSR1-FLI1 fusiona EWSR1-ERG fusiona

Fibrous dysplasiad

SATB2

GNAS mutations

Giant cell tumor of bone

H3G34Wb

H3F3A (G34W/V/R/L) mutations

Langerhans cell histiocytosis

S-100, CD1a, Langerin, cyclinD1

BRAF V600E

Osteoblastoma

FOSb

FOS and FOSB rearrangementsa

Osteosarcoma, conventional

SATB2

multiple complex rearrangements; TP53 and RB1 mutations

Osteosarcoma, low-grade

MDM2, CDK4

12q13-15 amplificationa

Angiosarcoma

CD31, CD34, Fli-1, ERG

Complex cytogenetic abnormality

Epithelioid hemangioendothelioma

CD31, CD34, ERG, keratin, CAMTA1

WWTR1-CAMTA1 fusiona

CD138, kappa/lambda, MUM1, CD56

complex cytogenetic abnormality

Vascular tumorsc

Plasmacytoma/myeloma aCan

be detected by fluorescence in situ hybridization. antibody. cSee also Chapter 17. dFNA is not recommended. EMA, Epithelial membrane antigen; FNA, fine-needle aspiration; GRM1, glutamate receptor gene; IDH, isocitrate dehydrogenase; N/A, not available bMutation-specific

Cartilaginous Tumors

Chondroblastoma

Cartilage-producing tumors form a wide clinicopathologic spectrum, ranging from benign tumors such as enchondroma to tumors with intermediate malignant potential such as atypical cartilaginous tumor/chondrosarcoma grade 1; to malignancies (conventional chondrosarcoma, grade 2 to 3, and dedifferentiated chondrosarcoma). Remarkable advances have been made in defining the molecular genetic alterations of most tumors in this group.21,22 The collaborative effort of a multidisciplinary team is the fundamental approach to the diagnostic workup of a cartilaginous tumor, especially with FNA or CNB samples.15,23

Chondroblastoma is a low-grade cartilage-forming tumor, occurring most often in children and adolescents. Most patients at the time of diagnosis are between the ages 10 and 25 years, and the male-to-female ratio is 2:1. The most common site is the epiphysis of long bones around knee, but the small tubular bones of hands and feet and flat bones such as the calcaneus and patella can also be affected. Chondroblastoma in adults tends to involve unusual sites, such as the skull base and temporal bone. Chondroblastoma usually presents as a solitary painful lesion. The radiographic features are classic: the mass is intramedullary, lytic, and small,

CHAPTER 18 Bone

A

B

C

D

603

H3K36M

•

Fig. 18.1  Chondroblastoma.  (A) The MRI shows a well-defined lytic lesion in the epiphyseal area of the distal femur. (B) Chondroblasts are uniform in size, with a round or polygonal shape. The nucleus is round to oval, with finely textured chromatin and frequent nuclear grooves (arrows) (Papanicolaou stain). (C) The chondroid matrix, embedded chondroblasts, and scattered osteoclast-like giant cells are nicely appreciated with cell block sections (H&E stain). (D) H3K36M is positive in the nuclei of the mononuclear chondroblasts but not the osteoclast-like giant cells.

occupying less than half of the epiphysis. It is well defined, with or without a sclerotic rim. Internal mineralization is a helpful feature if present (Fig. 18.1A). Most patients are treated with curettage, with a recurrence rate of 14% to 18%.24,25 CYTOMORPHOLOGY OF CHONDROBLASTOMA • • • • • • •

 ften hypercellular O Uniform chondroblasts Round to oval nucleus and prominent nuclear grooves Chondroid matrix Multinucleated osteoclast-like giant cells Occasional cytologic atypia and mitoses Absence of inflammatory cells   

FNA smears are often cellular and morphologically distin­ ctive, sufficient for a definitive diagnosis of chondroblastoma in cases with a classic clinical and radiologic presentation.26 The smears are composed of uniform chondroblasts, some in clusters, admixed with scattered multinucleated osteoclastlike giant cells (Fig. 18.1B and C). The characteristic chondroblast has a central or eccentrically located, round to oval nucleus with prominent grooves and a well-defined cytoplasmic membrane. Fragments of chondroid matrix material may or may not be present. Mitoses are uncommon. Chondroblastomas in unusual locations, in older adults, or complicated by secondary aneurysmal bone cyst (ABC) changes pose significant diagnostic challenges.25 They are often mistaken for ABC if only the secondary ABC component is sampled.22,27,28 The mixture of mononuclear cells and multinucleated giant cells in a chondroblastoma with

604 CHA P T ER 1 8   Bone

A

B • Fig. 18.2  Chondromyxoid Fibroma.  (A) Chondromyxoid fragments with embedded stellate to spindled

cells are a typical feature (Romanowsky stain). (B) Micronodules of chondromyxoid tissue have a central hypocellular, myxomatous area (upper right) and peripheral hypercellular condensation (cell block, hematoxylin and eosin stain).

DIFFERENTIAL DIAGNOSIS OF CHONDROBLASTOMA • • • • • •

 neurysmal bone cyst A Tenosynovial giant cell tumor Giant cell tumor of bone Clear cell chondrosarcoma Chondromyxoid fibroma Chondroblastoma-like osteosarcoma   

intraarticular involvement can be confused for a tenosynovial giant cell tumor.28 Chondroblastoma is a member of the triad of bone tumors that preferentially arise in the epiphysis, the other two being giant cell tumor of bone and clear cell chondrosarcoma. A large chondroblastoma can be difficult to distinguish from a giant cell tumor of bone or a clear cell chondrosarcoma. When chondroid matrix or cytologic atypia are conspicuous, a chondroblastoma can be confused with a chondromyxoid fibroma or a chondroblastic-like osteosarcoma, respectively.25 Recently, somatic driver mutations in H3F3B (majority) or H3F3A at codon K36M have been found in 95% of chondroblastomas, and H3F3A mutations in most giant cell tumors of bone.29,30 H3F3B mutations and the resulting mutant H3K36M protein are present in chondroblasts and not in osteoclastlike giant cells (Fig. 18.1D). The detection of an H3F3B mutation by sequencing or the mutant H3K36M protein by mutation-specific immunohistochemistry is extremely helpful in distinguishing chondroblastoma from its benign and malignant mimics.27,31,32 

Chondromyxoid Fibroma Chondromyxoid fibroma is a rare benign cartilage-forming tumor, occurring in young adults in the second and third

decades of life, with a slight male predominance. Chondromyxoid fibroma arises in a variety of bones, but most often in the metaphyseal region of the long tubular bones around knee, especially the proximal tibia.33 The radiographic presentation includes an eccentrically located, metaphyseal, lobulated, lucent lesion that is well defined but has features of cortical thinning or destruction.34 Most patients are treated with curettage or conservative resection, with a recurrence rate of 20%. CYTOMORPHOLOGY OF CHONDROMYXOID FIBROMA • • • • •

 sually sparsely cellular U Chondromyxoid fragments Stellate to spindle cells embedded in matrix Cytologic atypia not uncommon No true hyaline cartilage   

Most smears are sparsely cellular and composed of chondromyxoid fragments of variable sizes; isolated cells are seen in cases with hypercellularity.35 The stellate to spindled cells are embedded in the chondromyxoid matrix (Fig. 18.2A). Nuclei range from small to plump and chromatin from smudgy to vesicular. Cell block sections with tissue fragments usually capture the characteristic histologic features: micronodules of chondromyxoid tissue with central hypocellular, myxomatous areas and peripheral hypercellular condensation (Fig. 18.2B). DIFFERENTIAL DIAGNOSIS OF CHONDROMYXOID FIBROMA • • • •

 hondroblastoma C Enchondroma Low-grade chondrosarcoma Chondromyxoid fibroma-like osteosarcoma   

CHAPTER 18 Bone

A chondroblastoma yields more cellular smears, with more uniform, dispersed chondroblasts, and it arises in the epiphysis, not the metaphysis. True hyaline cartilage with lacunar spaces occupied by chondrocytes, as seen in enchondroma and low-grade chondrosarcoma, is not present in chondromyxoid fibroma.35 The cytologic atypia and hypercellularity of a subset of chondromyxoid fibromas can lead to a mistaken diagnosis of malignancy such as osteosarcoma or metastatic carcinoma. Although the recombination of the glutamate receptor gene (GRM1) with several 5′ partner genes has been identified in chondromyxoid fibroma, an immunohistochemical test to detect GRM1 expression is not yet available.36 SATB2, an osteoblastic marker used to identify osteosarcoma, can be positive in chondromyxoid fibroma and is therefore not helpful in distinguishing chondromyxoid fibroma from osteosarcoma.37 

Enchondroma Enchondroma is a common benign cartilaginous tumor that occurs within the medullary cavity of the small tubular bones, humerus, and femur. Although it can arise at any age, patients between the ages of 10 and 30 years are most affected. Most enchondromas are solitary; multiple lesions are associated with syndromes such as Ollier disease and Maffucci syndrome.23 Radiographically, enchondromas show a well-defined, lobulated, expansile, and lytic lesion in the metaphyseal region.38,39 In most cases, the radiographic features are sufficiently characteristic for the diagnosis, and symptom-free patients are followed up clinically without a confirmatory biopsy.23 Only radiologically ambiguous lesions are biopsied.17 CYTOMORPHOLOGY OF ENCHONDROMA • • • •

 ragments of mature cartilage F Small, uniform chondrocytes within lacunar spaces No significant myxoid or watery background Occasional cytologic atypia and pleomorphism   

Smears are composed of fragments of mature cartilage with small, uniform chondrocytes in lacunae, without a significant myxoid or watery background. The chondroid nature is best appreciated with air-dried, Romanowskystained smears (Fig. 18.3). Focal cytologic atypia and pleomorphism, typically in enchondromas of small peripheral bones, should not be overinterpreted as a sign of malignancy.13 Because enchondroma and low-grade chondrosarcoma are cytologically indistinguishable, demonstrating the infiltrative growth pattern of a low-grade chondrosarcoma with a core biopsy can be helpful for the distinction.40,41 

Chondrosarcoma and Chondrosarcoma Variants Chondrosarcomas are a heterogeneous group of malignant cartilaginous tumors, encompassing conventional chon­ drosarcoma, dedifferentiated chondrosarcoma, clear cell chondrosarcoma, and mesenchymal chondrosarcoma. Con­ ventional chondrosarcomas are further categorized as either

605

• Fig. 18.3  Enchondroma.  Fragments of mature cartilage with small, uniform chondrocytes in lacunae are well appreciated with air-dried, Romanowsky-stained smears.

primary or secondary, and either central or periosteal types, based on their clinical and radiologic presentation.23 Primary central chondrosarcoma, the third-most common primary malignant bone tumor in adults (after plasma cell myeloma and osteosarcoma), accounts for approximately 85% of all chondrosarcomas. It can arise at any age but affects mainly older patients in the fifth to seventh decades of life. The bones of the trunk (pelvis, ribs, scapula) and hip and shoulder girdles are most commonly involved.2 In the 2013 World Health Organization classification, chondrosarcoma has two International Classification of Diseases (ICD) codes, which reflect the different prognosis of a chondrosarcoma depending on its grade.5 Grade 1 chondrosarcoma is distinct from grade 2 and grade 3 chondrosarcomas and has the synonym “atypical cartilaginous tumor” to reflect its relatively indolent clinical course.42 Patients with atypical cartilaginous tumor/grade 1 chondrosarcoma in the long bones are treated nowadays with curettage and local adjuvants, with a 10% recurrence rate and an 83% 5-year survival rate. By contrast, patients with a grade 2 or 3 chondrosarcoma are treated with wide surgical resection and have a 5-year survival rate of 53%.23,42,43 Radiographically, low-grade chondrosarcomas show lytic erosion with intratumoral, endosteal scalloping and cortical thickening. High-grade chondrosarcomas show cortical destruction and soft tissue extension (Fig. 18.4A), making them easy targets for FNA, CNB, or both.44 The radiographic features are usually sufficient for the diagnosis of chondrosarcoma. In many scenarios, aggressive radiologic features trump a bland cytology appearance in establishing a final diagnosis.40 CYTOMORPHOLOGY OF CONVENTIONAL CHONDROSARCOMA • • • • •

 bundant chondroid matrix A Chondrocytes in lacunae or dispersed as single cells Cellularity and degree of atypia correlate with grade Binucleation and multinucleation Abundant vacuolated cytoplasm   

606 CHA P T ER 1 8   Bone

A

B

C

D • Fig. 18.4  Atypical Cartilaginous Tumor/Grade 1 Chondrosarcoma.  (A) The proximal humerus shows

an ill-defined lytic lesion with cortical destruction, soft tissue extension, and internal calcifications. (B) Extracellular chondroid material from an atypical cartilaginous tumor/grade 1 chondrosarcoma is indistinguishable from that seen in enchondromas (Romanowsky stain). (C) Fragments of chondroid matrix and sparse cellularity are characteristic (cell block, hematoxylin and eosin [H&E] stain). (D) A permeative/infiltrative growth pattern helps distinguish a low-grade chondrosarcoma from an enchondroma and is better seen on core biopsy samples (H&E-stained core biopsy).

Aspirates are rich in fragments of extracellular chondroid material (Fig. 18.4B and C), which is a spectacular magenta with air-dried, Romanowsky-stained smears and pale-green with alcohol-fixed, Papanicolaou-stained smears. An atypical cartilaginous tumor/grade 1 chondrosarcoma is cytomorphologically indistinguishable from an enchondroma. Demonstrating a permeating/infiltrative growth pattern (Fig. 18.4D), particularly with a CNB, is helpful for establishing the diagnosis of a low-grade chondrosarcoma. High-grade chondrosarcoma (grade 2 or 3) yields more cellular smears, more dispersed atypical

chondrocytes, and more prominent myxoid (rather than chondroid) matrix (Fig. 18.5A and B). Cellular and nuclear pleomorphism is conspicuous, and so are mitoses. Aspirates from recurrent chondrosarcomas are often very sparsely cellular and composed of watery/thin chondromyxoid material that should be distinguished from a postsurgical seroma. Dedifferentiated chondrosarcoma occurs as a component of approximately 10% to 15% of chondrosarcomas and carries a very poor prognosis. It is a biphasic tumor with two components in variable proportions:

CHAPTER 18 Bone

A

607

B •

Fig. 18.5  Grade 2 Chondrosarcoma.  (A) Grade 2 and 3 chondrosarcomas yield cellular smears and prominent myxoid matrix (Romanowsky stain). (B) Cytologic atypia is apparent. The chondromyxoid matrix is paler and less conspicuous with alcohol-fixed smears (Papanicolaou stain).

DIFFERENTIAL DIAGNOSIS OF CHONDROSARCOMA (BY SUBTYPE)

Low-Grade Chondrosarcoma • E  nchondroma • Chondromyxoid fibroma • Chondroblastoma 

High-Grade Chondrosarcoma • C  hondroblastic osteosarcoma • Chordoma 

Dedifferentiated Chondrosarcoma • U  ndifferentiated pleomorphic sarcoma • Leiomyosarcoma  • Metastatic sarcomatoid carcinoma 

Clear-Cell Chondrosarcoma

• Fig. 18.6  Dedifferentiated Chondrosarcoma. Dedifferentiated chon-

• L  ow-grade conventional chondrosarcoma • Metastatic clear-cell renal cell carcinoma 

drosarcoma has a sarcomatous component that is indistinguishable from a spindle-cell/pleomorphic sarcoma. The scant chondromyxoid matrix seen here is relatively nonspecific but might spur a search for better differentiated areas (Romanowsky stain).

Mesenchymal Chondrosarcoma • • • • •

 wing sarcoma E Small cell osteosarcoma Undifferentiated round cell sarcoma Lymphoma Metastatic neuroendocrine carcinoma   

a chondrosarcoma grade 1 component, juxtaposed to a high-grade noncartilaginous sarcomatous component (Fig. 18.6). Sampling only one of the components is a common pitfall.45 Clear cell chondrosarcoma is a rare variant of chondrosarcoma that is often seen in the femoral head. It is composed of sheets of clear cells with areas resembling conventional low-grade chondrosarcoma.46 Cytomorphologic features of clear cell chondrosarcoma, described in a few case reports,47,48 include cellular smears with dispersed large epithelioid tumor cells with abundant finely

vacuolated cytoplasm, a central to slightly eccentric round nucleus, and a prominent nucleolus. Fragments of chondromyxoid matrix and scattered osteoclast-like giant cells are also present. Mesenchymal chondrosarcoma is a rare high-grade malignant tumor with a distinct bimorphic pattern composed of islands of low-grade hyaline cartilage and areas of primitive small round cells.46 The cytologic features of mesenchymal chondrosarcoma have been described in a few case reports.49-51 As with other bimorphic sarcomas, sampling only one of the components is a pitfall. The small round-cell component mimics Ewing sarcoma, small-cell osteosarcoma, lymphoma, and the more recently described round-cell sarcomas with a CIC or BCOR-CCNB3 rearrangement.52 The low-grade cartilaginous component is easily mistaken for a more common entity such as low-grade chondrosarcoma or chondroblastic osteosarcoma.

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The recent identification of a HEY1-NCOA2 fusion facilitates definitive diagnosis of mesenchymal chondrosarcoma in difficult cases by FISH using HEY1 or NCOA2 probes or RT-PCR to detect the fusion transcript.53 Point mutations in isocitrate dehydrogenase (IDH1/2) genes occur in a variety of benign and malignant cartilage-forming tumors. IDH1/2 genes encode for the metabolic enzymes IDH1 and IDH2; these gain of function mutations have been linked to the pathogenesis of cartilage-forming tumors.54 The prevalence of IDH1/2 mutations is extremely variable: enchondroma (about 40% in solitary and 87% in syndromic lesions), conventional central chondrosarcoma, grades 1, 2, and 3 (38%–70%), secondary central chondrosarcoma (86%), periosteal chondrosarcoma (100%), and dedifferentiated chondrosarcoma (50%–60%).55 Mutational analysis is thus of little value for grading chondrosarcoma or distinguishing atypical cartilaginous tumor/grade 1 chondrosarcoma from benign cartilage-forming tumors. In addition, only 20% of the IDH1 mutations can be detected by mutation-specific immunohistochemistry using the IDH R132H antibody.55 Mutational

analysis to detect an IDH1/2 mutation is, however, helpful in distinguishing dedifferentiated chondrosarcoma from other pleomorphic/spindle cell sarcomas.56 And because IDH1/2 mutations are not seen in clear cell chondrosarcoma or mesenchymal chondrosarcoma, finding a mutation can be useful in distinguishing these chondrosarcoma variants from conventional chondrosarcoma.55 

Giant Cell–Rich Tumors Giant cell–rich tumors of bone represent a large group of tumors and tumor-like lesions, including giant cell tumor of bone, ABC, chondroblastoma, chondromyxoid fibroma, giant cell tumor of Paget’s disease, nonossifying fibroma, giant cell lesion of the small bones (giant cell reparative granuloma), brown tumor of hyperparathyroidism, and giant cell–rich osteosarcoma.24 They are characterized morphologically by the presence of numerous osteoclasts or osteoclast-like giant cells. The differential diagnosis of common giant cell–rich tumors is summarized in Table 18.2.

TABLE 18.2    Differential Diagnosis of Common Giant Cell-Rich Bone Neoplasms

Imaging features

Cytomorphology

Ancillary tests/ molecular alterations

Metaphysis of long bones

Cystic, multiloculated, soft tissue septae, fluid levels

Blood, spindle cells, few GCs; reactive bone

FISH for USP6 rearrangementa

10–25 M:F=2:1

Epiphysis of long bones

Well-defined, scalloped borders, sclerotic rim, internal mineralization

Chondroblasts with nuclear grooves; few GCs, chondroid matrix

Mutation-specific IHC H3K36M DOG1, SOX9

Giant cell tumor

20–40; M:F=2:1

Epiphysismetaphysis of long bones; sacrum

Large, pure lytic lesion with sharp borders, no mineralization

Dual cell population: mononuclear spindled or ovoid cells and numerous GCs; absence of matrix

Mutation-specific IHC H3G34W/ L/V/R

Osteoblastoma

10–30 M:F=2:1

Metadiaphyseal region of tubular bones; spine; sacrum

Expansile, welldefined mixed lytic and blastic lesion, 2-5 cm

Mono- and binucleated osteoblasts, scattered GCs, scant stroma

FISH for FOS and FOSB rearrangementsa

Osteosarcoma, giant-cell rich

10–20 and >50 M:F=1.3:1

Metaphysis of long bones

Destructive, illdefined; soft tissue component with matrix mineralization

Epithelioid to pleomorphic cells, tumor GCs and benign GCs, necrosis, mitoses, strands of osteoid matrix

N/A

Nonossifying fibroma

10–15; M:F=2:1

Metaphysis of long bones in lower extremities

Lytic and lobulated lesion with a sclerotic rim

Spindled fibroblasts, few GCs, foam cells; peripheral reactive bone on CNB

N/A

Diagnosis

Age (yrs.), M:F

Common location

Aneurysmal bone cyst

2 cm), well-defined lesion with mixed lytic and blastic features and sharp sclerotic margins (Fig. 18.9A). The distinctive clinical and radiographic features of osteoblastoma are diagnostic in most cases, obviating a needle biopsy; it is investigated by FNA and CNB only when there are ambiguous radiologic features. Most patients are treated with curettage or en bloc resection, with a recurrence rate of 20%. Recurrent rearrangements of FOS and FOSB are found in osteoblastoma and osteoid osteoma.70 CYTOMORPHOLOGY OF OSTEOBLASTOMA • S  mall clusters or dispersed osteoblasts • Variable number of osteoclast-like giant cells • Scant osteoid, hyaline cartilage matrix, or both   

Aspirates have at least moderate cellularity, with monomorphic osteoblasts, either as dispersed cells or small clusters of cells (Fig. 18.9B). The neoplastic osteoblasts are round or triangular cells with an eccentrically located nucleus, abundant cytoplasm, and a perinuclear cytoplasmic clear area (“hof ”). The nuclei are round to ovoid, with a central nucleolus; some cells are binucleated. Osteoid-like

 steosarcoma O Fracture callus Osteoid osteoma Aneurysmal bone cyst   

If 75% or more of the osteoblasts have a large, plump, epithelioid morphology (defined as osteoblasts at least twice the size of those seen in a conventional osteoblastoma), the osteoblastoma is an aggressive osteoblastoma. This minor subset of osteoblastoma is easily confused with osteosarcoma, especially the osteoblastoma-like osteosarcoma.72 Detection of FOS rearrangments by FISH or FOS mutations by mutation-specific immunohistochemistry might be diagnostically helpful in difficult cases.70 

Conventional Osteosarcoma and Osteosarcoma Variants Osteosarcoma, the most common sarcoma of bone, is composed of neoplastic cells that have an osteoblastic phenotype and deposit neoplastic bone or osteoid. Most osteosarcomas arise de novo, whereas others arise as a secondary malignancy in patients with a history of radiation exposure, Paget disease, chemotherapy, and foreign bodies (e.g., orthopedic implants). The age distribution is bimodal: the first peak is at 10 to 20 years of age, and the second peak is after 50 years of age. Osteosarcoma can affect any bone but most commonly the metaphyseal regions of the long tubular bones, specifically (in decreasing order of frequency) the distal femur, proximal tibia, and proximal humerus. In older

612 CHA P T ER 1 8   Bone

A

B • Fig. 18.9  Osteoblastoma.  (A) A computed tomography scan shows a well-defined, expansile, and lytic

lesion with internal ossification involving pedicle and lamina of a vertebra. (B) Uniform osteoblasts are round or triangular cells with an eccentrically located nucleus and abundant cytoplasm. (Courtesy Dr. Henryk Domanski, Lund University Hospital, Lund, Sweden.)

patients, the pelvis and axial skeleton are more commonly involved. The classic radiologic appearance of osteosarcoma is an ill-defined intramedullary lytic lesion with cortical destruction, periosteal reaction, and focal mineralization. Most patients are treated with preoperative chemotherapy followed by limb salvage complete excision.1 CYTOMORPHOLOGY OF CONVENTIONAL OSTEOSARCOMA

Osteoblastic • • • • • • • •

 ariable cellularity V Single cells and cell clusters Epithelioid to pleomorphic neoplastic cells Osteoblastic differentiation: eccentric nucleus and cytoplasmic “hof” Strands of osteoid Mitoses, including atypical forms Osteoclast-like giant cells Necrosis 

osteosarcoma resembles high-grade chondrosarcoma (Fig. 18.10B).76,78 Fibroblastic osteosarcoma shows predominantly atypical spindled to pleomorphic cells, resembling a spindle cell sarcoma, not otherwise specified. In addition, variants of osteosarcoma (telangiectatic, small cell, giant cell–rich, and osteoblastoma-like) yield very diverse cytomorphology. Telangiectatic osteosarcoma may show only scattered atypical osteoblastic cells, easily overlooked, in a background of blood. Giant cell–rich osteosarcoma produces abundant osteoclast-like giant cells in addition to malignant osteoblasts (Fig. 18.10C).79 Small-cell osteosarcoma has small osteoblasts and very scant osteoid, mimicking a small round-cell sarcoma such as Ewing sarcoma.80 DIFFERENTIAL DIAGNOSIS OF OSTEOSARCOMA

Chondroblastic • C  hondromyxoid matrix • Atypical mononucleated or binucleated chondroblasts • Focal osteoblastic differentiation   

Because of the range of its subtypes (osteoblastic, chondroblastic, and fibroblastic), conventional osteosarcoma embraces a broad spectrum of cytomorphology.73-76 Smears from a classic osteoblastic osteosarcoma show epithelioid to pleomorphic malignant cells, with admixed strands of osteoid and occasionally osteoblast-like tumor cells (Fig. 18.10A). Necrosis and numerous mitoses are present. The amount of osteoid is highly variable, and differentiating osteoid from collagenous matrix is difficult.74,77 Chondroblastic

• • • • • • • • • •

 racture callus F Myositis ossificans Osteoblastoma Giant cell tumor of bone High-grade chondrosarcoma Aneurysmal bone cyst Undifferentiated pleomorphic sarcoma/leiomyosarcoma Metastatic poorly differentatiated carcinoma Large cell lymphoma Ewing sarcoma or other round cell sarcoma   

Benign osteoblastic lesions/tumors like fracture callus, myositis ossificans, and osteoblastoma are the most important entities to consider in the differential diagnoses of osteosarcoma. Clinical and radiologic correlation is essential. Different subtypes of osteosarcoma evoke different sets of differential diagnosis. Chondroblastic osteosarcoma should be distinguished from a high-grade chondrosarcoma

CHAPTER 18 Bone

A

B

C

D

613

• Fig. 18.10  Osteosarcoma.  (A) Osteoblastic Osteosarcoma. Cellular smears contain epithelioid or pleo-

morphic malignant cells admixed with strands of osteoid (arrow) and occasional multinucleated tumor cells (Romanowsky stain). (B) Chondroblastic Osteosarcoma resembles high-grade chondrosarcoma. In this field, there is chondroid matrix (arrows) but no osteoid (Papanicolaou stain). (Courtesy Dr. Lisa Teot, Boston Children’s Hospital, Boston, MA.) (C) Giant–cell Rich Osteosarcoma. Abundant osteoclast-like giant cells are admixed with malignant osteoblasts (Papanicolaou stain). (D) Low-grade Osteosarcomas yield scant cellularity, with scattered clusters of low-grade spindle cells (Romanowsky stain).

because they have very different treatment strategies and prognoses. Telangiectatic osteosarcoma resembles an ABC. The giant cells in giant cell–rich osteosarcoma can be numerous, mimicking a GCT of bone. Malignant osteoblasts can appear epithelioid, resembling the cells of metastatic carcinoma, an especially problematic scenario in the setting of a secondary osteosarcoma due to radiation therapy for advanced epithelial malignancy such as prostatic carcinoma. The diagnosis of extraskeletal osteosarcoma, a very rare soft tissue sarcoma, can only be established with the exclusion of its benign and malignant mimics such as myositis ossificans, radiation-associated sarcoma, and heterologous osteosarcomatous differentiation of a sarcoma with another line of differentiation, most commonly dedifferentiated liposarcoma. SATB2, an osteoblastic marker, is sensitive but not specific for osteosarcoma.37,81 It can be positive in a variety of benign and malignant bone tumors, including fibrous dysplasia, chondromyxoid fibroma, and BCOR-CCNB3

fusion-positive sarcomas.37,82,83 SATB2 positivity should be interpreted in the context of clinical, radiologic, and cytomorphologic findings.84 

Low-Grade Osteosarsarcoma Low-grade surface and central osteosarcomas, namely parosteal osteosarcoma and low-grade central osteosarcoma, are low-grade osteosarcomas. Both are characterized by the presence of parallel-arranged trabeculae of woven or lamellar bone surrounded by a moderately cellular infiltrate of fibroblast-like spindle cells. Some parosteal osteosarcomas have a cartilaginous component, present as a cap covering the periphery of the tumor.85 Because of its bland cytomorphology, low-grade osteosarcomas are easily mistaken for benign mimics such as fibrous dysplasia, myositis ossificans, and rarely osteochondroma, especially in needle biopsy specimens. The yield of FNA is usually very poor for

614 CHA P T ER 1 8   Bone

low-grade osteosarcomas, showing only scattered clusters of low-grade spindle cells (Fig. 18. 10D). For this reason, a CNB, together with correlation of characteristic radiologic findings, is the key to a successful preoperative diagnosis. Like some lipomatous tumors of soft tissue (see Chapter 17), low-grade osteosarcomas have amplification of chromosome 12q13-15 via supernumerary ring chromosomes, resulting in gain or amplification of MDM2 and CDK4 genes.86 Detection of MDM2 amplification by either FISH or immunohistochemistry for MDM2 and CDK4 is useful in distinguishing low-grade osteosarcoma from benign fibroosseous lesions.15,87 

Notochordal Tumors • Fig. 18.11  Chordoma.  Clusters of large, round to cuboidal “physallif-

Benign Notochodal Cell Tumor Benign notochordal cell tumor (BNCT), likely a precursor lesion of chordoma, was introduced as an entity by the World Health Organization in 2013.2,88 BNCT is defined as an intraosseous, slow-growing notochordal proliferation with an indolent clinical course. BNCTs are usually small (