Atlas of Fine Needle Aspiration Cytology [1 ed.] 9789386107640, 9789351521112

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Atlas of Fine Needle Aspiration Cytology

Atlas of Fine Needle Aspiration Cytology

Editor

Lester J Layfield MD FASCP Professor and Chairman Department of Pathology and Anatomical Sciences University of Missouri Columbia, Missouri, USA

®

JAYPEE BROTHERS MEDICAL PUBLISHERS (P) LTD New Delhi • London • Philadelphia • Panama

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Jaypee Brothers Medical Publishers (P) Ltd Headquarters Jaypee Brothers Medical Publishers (P) Ltd 4838/24, Ansari Road, Daryaganj New Delhi 110 002, India Phone: +91-11-43574357 Fax: +91-11-43574314 Email: [email protected] Overseas Offices J.P. Medical Ltd 83, Victoria Street, London SW1H 0HW (UK) Phone: +44-2031708910 Fax: +02-03-0086180 Email: [email protected]

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Website: www.jaypeebrothers.com Website: www.jaypeedigital.com © 2014, Jaypee Brothers Medical Publishers The views and opinions expressed in this book are solely those of the original contributor(s)/author(s) and do not necessarily represent those of editor(s) of the book. All rights reserved. No part of this publication may be reproduced, stored or transmitted in any form or by any means, electronic, mechanical, photocopying, recording or otherwise, without the prior permission in writing of the publishers. All brand names and product names used in this book are trade names, service marks, trademarks or registered trademarks of their respective owners. The publisher is not associated with any product or vendor mentioned in this book. Medical knowledge and practice change constantly. This book is designed to provide accurate, authoritative information about the subject matter in question. However, readers are advised to check the most current information available on procedures included and check information from the manufacturer of each product to be administered, to verify the recommended dose, formula, method and duration of administration, adverse effects and contraindications. It is the responsibility of the practitioner to take all appropriate safety precautions. Neither the publisher nor the author(s)/ editor(s) assume any liability for any injury and/or damage to persons or property arising from or related to use of material in this book. This book is sold on the understanding that the publisher is not engaged in providing professional medical services. If such advice or services are required, the services of a competent medical professional should be sought. Every effort has been made where necessary to contact holders of copyright to obtain permission to reproduce copyright material. If any have been inadvertently overlooked, the publisher will be pleased to make the necessary arrangements at the first opportunity. Inquiries for bulk sales may be solicited at: [email protected]

Atlas of Fine Needle Aspiration Cytology First Edition: 2014 ISBN 978-93-5152-111-2 Printed at:

Dedicated to My wife Julia Crim MD who has supported me throughout my academic career and Britt-Marie Ljung MD and Walter Coulson MD who introduced me to the discipline of fine needle aspiration cytology and mentored me during my early academic career.

Contributors Zubair W Baloch MD PhD Professor Department of Pathology and Laboratory Medicine Hospital of the University of Pennsylvania Philadelphia, Pennsylvania, USA

Hasan Ghaffar MD Baystate Medical Center Springfield, Massachusetts, USA Jerzy Klijanienko MD PhD MIAC Institute Curie, Paris, France

Barbara A Centeno MD Moffitt Cancer Center University of South Florida Tampa, Florida, USA

Lester J Layfield MD FASCP Professor and Chairman Department of Pathology and Anatomical Sciences University of Missouri Columbia, Missouri, USA

Harvey M Cramer MD FRCP(C) Associate Professor and Director of Cytopathology Indiana University Health Pathology Laboratory Indianapolis, Indiana

Shahla Masood MD Professor and Chairman University of Florida College of Medicine–Jacksonville Department of Pathology Medical Director, Shands Jacksonville Breast Health Center

Chief of Pathology and Laboratory Medicine, Shands Jacksonville Jacksonville, Florida, USA Martha Bishop Pitman MD Medical Director Cytopathology Laboratory Massachusetts General Hospital Associate Professor of Pathology Harvard Medical School Boston, Massachusetts, USA Marilin Rosa MD Assistant Professor and Director of Cytopathology University of Florida College of Medicine–Jacksonville Department of Pathology Jacksonville, Florida, USA

Preface Diagnostic approach and technical methodology for the diagnosis of neoplastic disease has undergone considerable advancement in the last decade. The rise of a multidisciplinary approach to the diagnosis and treatment of oncological diseases along with the development of so-called “personalized medicine” has markedly increased the use of small biopsy techniques, including fine needle aspiration. Advances in cytology, immunocytochemistry, and molecular techniques have allowed FNA and small core biopsies to yield diagnostic material along with sufficient material for prognostic and predictive studies. Hence, fine needle aspiration has become a popular and effective initial technique for the workup of most newly discovered nodules and masses suspected to be of neoplastic origin. Fine needle aspiration coupled with rapid on-site evaluation allows for triage of limited specimens to appropriate diagnostic, prognostic and predictive testing. The initial cytologic evaluation is paramount in this diagnostic approach. Fine needle aspiration cytology often coupled with mini-core needle biopsies prepared as cellblocks has become the standard of practice in the workup of potentially neoplastic lesions. Once the diagnosis of malignancy is obtained, material can be set aside for immunocytochemistry, electron microscopy, flow cytometry, fluorescence in situ hybridization analysis and molecular technologies often based on polymerase chain reaction methodologies. Using this approach, all necessary diagnostic and predictive data can be obtained with minimal morbidity for the patient and at a relatively low price. The present atlas is designed to illustrate all common neoplastic lesions samplable by fine needle aspiration throughout the body. These lesions are richly illustrated and described in the text. Key features are listed in tabular form and differential diagnoses are discussed. Additionally, a large number of uncommon or rare neoplasms are illustrated and discussed. For the more common lesions, ancillary diagnostic techniques are discussed, including molecular techniques when relevant. Each chapter approaches its organ system with a uniform format. When a given neoplasm occurs in more than one organ system, it is discussed and illustrated in the relevant chapters and crossreferenced. Every attempt has been made to abundantly illustrate the neoplasms most commonly encountered in a large fine needle aspiration cytology practice. Non-neoplastic entities are illustrated and discussed when they may present as mass lesions, enter the differential diagnosis of a neoplasm or may be encountered in oncologic patients. The atlas is designed to be useful for residents, fellows, cytotechnologists and pathologists working in both academic and community settings. Lester J Layfield

Acknowledgments I wish to thank Tammy Phanichkul and Leanna Wintch for transcribing the manuscript and aiding in assembling the text, tables and photomicrographs. I thank Howard H Wu MD for supplying some of the photomicrographs for chapters 8 and 10. I thank M/s Jaypee Brothers Medical Publishers (P) Ltd., New Delhi, India, for giving me the opportunity and encouragement to edit this book. I would specially like to thank Ms Chetna Malhotra Vohra (Senior Manager–Business Development) and Saima Rashid (Development Editor) for help in bringing this book to fruition.

Contents

xiii

Contents 1. Clinical Utility, Technique and Ancillary Studies

1

Lester J Layfield • • • • • • • • • • •

Guidance Techniques  1 Biopsy Equipment  1 Biopsy Procedure  2 Fixation and Staining  3 Ancillary Techniques  3 Culture for Infectious Organisms  4 Electron Microscopy  4 Immunocytochemistry  4 Flow Cytometry  5 Fluorescence in Situ Hybridization and Chromogenic in Situ Hybridization  6 Molecular Diagnostics  6

2. Head & Neck and Salivary Gland

11

Jerzy Klijanienko • • • • • • • • • • •

General Considerations  11 FNA as a Diagnostic Method in Head and Neck Tumors  11 FNA as a Diagnostic Method in Salivary Gland Tumors  17 FNA Classification of Salivary Gland Tumors  19 Tumors in Which Myoepithelial Cells Predominate  19 Tumors with Oncocytic Cell Predominance  23 Tumors with Basal Cell Predominance  25 Tumors with Squamous Cell Predominance  28 Tumors with Mucin Production  29 Tumors with Acinic Cell Predominance  32 Tumors with Nonspecific/Poorly Differentiated Cell Predominance  34

3. Fine Needle Aspiration of Thyroid

40

Zubair W Baloch • • • •

FNA Indications, Procedure, Specimen, Adequacy, and Classification  40 Cytomorphology of Thyroid Lesions  41 Rare Tumors of Thyroid Gland  55 Role of Special Studies in the Diagnosis of Thyroid Tumors in Cytologic Specimens  57

4. Mediastinum Lester J Layfield • Clinical Considerations  63 • Cytologic Findings in Common Mediastinal Lesions  64 • Malignant Neoplasms of the Mediastinum  69

63

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Atlas of Fine Needle Aspiration Cytology

5. Lung and Pleura

77

Lester J Layfield • • • • • •

Clinical Considerations and Technical Aspects  77 Cytologic Features of Normal Lung  78 Specific and Nonspecific Inflammatory Changes  80 Pulmonary Neoplasms  83 Metastatic Lesions to the Lung  96 Pleural Lesions  97

6. The Breast

104

Shahla Masood, Marilin Rosa • • • • • • • • • •

Inflammatory and Reactive Lesions  105 Nonproliferative Breast Changes  107 Fibroepithelial Tumors  108 Proliferative Breast Disease  110 Papillary Neoplasms  113 In Situ Lesions of the Breast  115 Invasive Carcinoma  116 Mesenchymal Tumors  121 Lymphoproliferative Disorders  123 Lesions of the Male Breast  124

7. Fine Needle Aspiration of Lymphoid Lesions

133

Hasan Ghaffar • Technical Considerations  134 • Ancillary Studies  134 • Diagnosis of Lymphoma  136

8. Liver

149

Harvey M Cramer • Clinical Considerations and Technical Aspects  149 • Localized Lesions and Lesions of Infectious Etiology  152 • Primary Hepatic Neoplasms  155

9. Pancreas Cytology Martha Bishop Pitman, Barbara A Centeno • • • • • • •

Specimen Preparation and Tissue Triage  174 Normal Pancreas and Gastrointestinal Contaminants  176 Pancreatitis  178 Non-neoplastic Cysts  181 Solid Neoplasms  183 Cystic Neoplasms  195 Metastatic Tumors  205

174

Contents

10. Kidney and Adrenal

xv

211

Harvey M Cramer, Lester J Layfield • • • • • • • • •

Kidney  211 Renal Cell Carcinoma, Clear Cell Type  215 Papillary Renal Cell Carcinoma  220 Chromophobe Renal Cell Carcinoma  222 Benign Renal Neoplasms  228 Adrenal  245 Non-Neoplastic Lesions of the Adrenal  246 Benign Adrenocortical Nodules Including Adenomas and Hyperplasia  246 Adrenocortical Carcinoma  247

11. Male and Female Genital Tracts

258

Lester J Layfield • • • • •

Clinical Considerations  258 Prostate  258 Non-neoplastic Testicular Lesions  262 Testicular Neoplasms  263 Lesions of the Ovary, Cervix and Uterine Fundus  268

12. Musculoskeletal System

282

Lester J Layfield • • • • • • • • •

Musculoskeletal Lesions with a Myxoid or Mucinous Change  283 Neoplasms of the Adipose Tissue  290 Giant Cell Containing Lesions of the Musculoskeletal System  297 Small Round Cell Tumors  302 Polygonal and Epithelioid Neoplasms  311 Pleomorphic Sarcomas  318 Neoplasms with Cartilaginous Differentiation  319 Bone Forming Lesions  323 Spindle Cell Lesions  325

13. Pediatric Tumors

338

Jerzy Klijanienko • • • • •

General Considerations  338 Lymphomas (Non-Hodgkin and Hodgkin Lymphomas)  339 Retroperitoneal Tumors  340 Round Cell Sarcomas and Other Connective Tissue Tumors  344 Other Tumors  349

14. Skin and Superficial Subcutaneous Tissue Lester J Layfield • General Considerations  354 • Diagnostic Accuracy  354 • Complications and Adverse Outcomes  355

354

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Atlas of Fine Needle Aspiration Cytology

• • • • • •

Index

Methodology  355 Non-Neoplastic Cutaneous Lesions  355 Cysts of the Skin and Superficial Subcutaneous Tissues  357 Benign Epithelial Cutaneous Tumors  359 Malignant Epithelial Tumors  361 Spindle Cell Lesions of Skin  367

371

Chapter

1

Clinical Utility, Technique and Ancillary Studies Lester J Layfield

„„ INTRODUCTION Fine needle aspiration (FNA) is a versatile technique that can be used for investigation of a wide spectrum of pathologic entities at diverse body sites. Few absolute contraindications exist for using the technique. Unanesthetized patients should be able to cooperate with the aspirator and follow simple directions. While needle tract implantations have been documented, they are rare and should not dissuade physicians from utilizing the technique.1-4 FNA is most effective for localized lesions and is not suitable when a well-defined target is not present. For superficial and palpable lesions, imaging guidance is usually unnece­ ssary, but for small palpable lesions within the thyroid ultrasound (US) guidance has been shown to improve diagnostic accuracy. The National Cancer Institute Thyroid Fine Needle Aspiration State of the Science Conference recommended US guidance for FNA sampling.5 While most patients can easily tolerate FNA as an office procedure, risk factors such as age, coagulation disorders, diabetes, and respiratory failure should be considered when evaluating a patient for potential FNA. „„ GUIDANCE

TECHNIQUES

A variety of guidance techniques are used for deeply located lesions. These include US, computerized axial tomography (CAT), endoscopic US guidance, fluoroscopy,

and even magnetic resonance imaging when special needles are utilized. US may be the most widely utilized guidance method due to its ‘real time’ ability to image the lesion of interest and guide the needle with immediate feedback. Instrumentation is relatively portable allowing clinic and inpatient room use.6 While US utility is limited at some sites (lung) because air blocks the transmission of images, the technique can be used at a wide variety of body sites including the abdomen, retroperitoneum, pancreas, thyroid, and the musculoskeletal system. Endoscopy and bronchoscope techniques are increasingly coupled with US or fluoroscopy allowing more direct access to pancreatic, pulmonary, and mediastinal lesions. These techniques are in part replacing older percutaneous FNA procedures. „„ BIOPSY

EQUIPMENT

A large variety of needle types and sizes are available for the performance of FNA cytology. For palpable lesions, standard disposable hypodermic or spinal needles are utilized with gauges between 27 and 22. These needles come in a variety of lengths and can be used with or without a syringe depending on whether the aspirator wishes to utilize suction or not. When suction is felt to be unnece­ ssary, the needle can be used alone. Potential advantages for this technique include reduced contamination of the specimen with blood and a reduced visibility of the needle

2

Atlas of Fine Needle Aspiration Cytology

and syringe to the patient.7,8 This may be particularly important in children where a large syringe and syringe holder may be frightening. Currently, 25 gauge needles are popular due to the reduced potential for patient discomfort and a reduction in potential hemorrhage. Use of these smaller gauge needles is associated with some degradation in the amount of material obtained, but usually sufficient material is aspirated to yield a diagnosis. Slightly larger gauged needles generally supply more tissue for cell block preparations useful in immunocytochemistry and molecular diagnostics. When suction is desired, a 10 cc syringe is optimal and a variety of syringe holders are commercially available (Fig. 1.1). These syringe holders give greater control for needle placement and allow the target nodule to be firmly fixed by one hand, while the other hand holds the syringe and draws back on the syringe plunger. For deeply situated lesions and lesions where image guidance is desirable, longer needles are utilized. These come in a variety of gauges from 25 to 18. Twenty-two or twenty-three gauge needles appear optimal. The standard needle for deep targets is the Chiba needle. More recently, a number of mini core biopsy needles have been developed and have the advantage of obtaining larger tissue fragments useful for cell block preparations (Fig. 1.2). The combined use of FNA and core needle biopsy can improve diagnostic accuracy, and in many circumstances the techniques are complimentary.9,10 Formalin-fixed, paraffin-embedded cell block material or mini core biopsy material is the preferred specimen for immunohistochemistry/cytochemistry.11,12

When FNA is performed in conjunction with imaging guidance or as part of an endoscopic or bronchoscopic procedure, rapid on-site evaluation (ROSE) should be considered. While not available at all institutions, its use can ensure diagnostic specimens and aid in specimen triage for special studies.13,14

Fig. 1.1: Three of the most common styles of syringe holders. The central holder minimizes size and visibility to the patient but retains the ability to use one hand to hold the syringe and draw back on the plunger

Fig. 1.2: The small needle at the bottom of the illustration has a beveled end that allows a cutting action. Except for its length, it is identical to common hypodermic needles and it comes in sizes of 20–24 gauge. The larger needle cuts a small core optimal for cell block preparation and histologic examination

„„ BIOPSY

PROCEDURE

The patient should be placed in a comfortable position that allows the aspirator clear access to the nodule. The skin over the nodule is cleaned by either alcohol or another form of topical disinfectant (betadine and chlorhexidine). The nodule of interest is then fixed between the index and middle fingers of the aspirator’s left hand, while the needle with or without syringe and syringe holder is brought to a position immediately above the skin overlying the nodule. After stabilization of the needle and syringe, the needle is passed quickly through the skin and into the nodule or mass of interest. If suction is used, the plunger of the syringe is brought back 2–3 cc creating a vacuum and multiple (7–10) rapid back and forth motions are made in the nodule with slight variations in direction. This allows sampling of a wide area of the nodule. Large lateral variations in direction cause pain and should not be performed. Immediately before withdrawal of the needle, the suction is released. When the aspirator selects not to use suction, the needle is simply moved rapidly to and fro within the target lesion and then withdrawn after 7–10 back and forth strokes. Using this method, the needle

Clinical Utility, Technique and Ancillary Studies

Fig. 1.3: Preparation technique for smears using two glass slides smears are made by rotating the top slide onto the lower slide and making a smooth single stroke away from the frosted end of the lower slide

is held directly between the fingertips that give excellent control for needle placement. The advantage of this technique is that there is generally less bleeding but cell yield may be reduced.7 Immediately following withdrawal of the needle, hemostasis is achieved by applying a cotton 2×2 with firm pressure. Following aspiration, the material obtained is rapidly expelled onto a glass slide and then smeared in a single motion by a second slide placed at right angles to the first (Fig. 1.3). A single smooth stroke is made evenly dispersing the material on the first slide. When material is desired for liquid-based preparations or cell blocks, the contents of the needle can be expelled into liquid-based media, RPMI, or formalin for cell block preparation. Liquid-based preparations can be used for FNA at a variety of sites and under conditions where expert smear preparation is not available. These preparation methods may have some advantages.15-19 Differences in preparation techniques can influence diagnostic criteria and specimen appearance.20 „„ FIXATION

AND STAINING

Three staining techniques are utilized for smeared FNA material. In the first method, the specimen is allowed to air-dry and is then stained by a Romanowsky technique. These are Giemsa-based stains and various formulations have different names including May-Grünwald-Giemsa (MGG), Wright stain, and Diff-Quik (Harleco, Philadelphia). The second popular method is the Papanicolaou method

3

that is performed on alcohol-fixed material. This material may be either direct smears or liquid-based preparations. The third method that may be performed on direct smears but is more commonly utilized for cell block material is the hematoxylin and eosin (H&E) method. Each staining technique has its advantages. Air-dried preparations can be immediately evaluated in the clinic area and are utilized for ROSE. These preparations optimally demonstrate mucosubstances and stromal materials. This is a great advantage in examination of salivary gland lesions as well as musculoskeletal neoplasms. Nuclear detail is not as well demonstrated in air-dried preparations as it is with the Papanicolaou and H&E methods. The Papanicolaou method is favored by classically trained gynecologic cytolo­ gists. It is unsurpassed for evaluating nuclear detail and is excellent for demonstrating squamous differentiation. Extracellular substances may not be as well demonstrated with Papanicolaou staining as in air-dried preparations. The H&E technique has the advantages of giving good nuclear detail along with good demonstration of many extracellular substances. It also allows individuals more familiar with histopathology to apply this knowledge base to cytologic preparations. The H&E stain is the preferred stain for cell block material. It should be remembered that relative cell sizes vary with the fixation methods. Air-drying causes the cell to flatten on the glass surface and appear larger than when the cell is fixed in either ethanol or formalin (Figs 1.4A and B). A variety of special stains can be used on both smear and cell block preparations. These stains can be performed on smeared material without a major change in technique.21-23 Special stains are most helpful in identifying infectious organisms but are also helpful in demonstrating both intra- and extracellular substances including melanin and a variety of mucins. „„ ANCILLARY

TECHNIQUES

Ancillary studies are becoming increasingly important for the complete workup of cytologic specimens. With the advent of ‘personalized medicine’ new horizons for ancillary testing of cytologic specimens are developing. Hence, molecular analysis for therapeutic drug selection is joining electron microscopy, immunocytochemistry, and flow cytometric techniques in the diagnostic arsenal of the cytopathologist. Appropriate utilization of these ancillary testing modalities in many cases will require the triage of limited cytologic specimens into appropriate fixatives and

4

Atlas of Fine Needle Aspiration Cytology

A

B

Figs 1.4A and B:  (A) Diff-Quik-stained air-dried smear of thyroid epithelium showing larger nuclear and cell size in comparison to material shown in figure (B), which is H&E stained. Both figures are shot at 1000x magnification. Note larger apparent size of nuclei in the air-dried specimen

transport media. ROSE is increasingly important to ensure appropriate triage of limited specimens. „„ CULTURE

FOR INFECTIOUS ORGANISMS

Submission of cytologic specimens into appropriate transport or culture media for microbacteriological culture is important and represents one of the first ancillary testing methodologies applied to cytologic specimens.23,24 Repeat biopsies or additional passes may be necessary to ensure adequate material for culture.23 Specific culture media for aerobic, anaerobic, and acid-fast bacteria can improve culture yield.24 „„ ELECTRON

MICROSCOPY

While ultramicroscopic examination of both histopathologic and cytopathologic material has largely been superseded by immunohistochemistry, the technique retains utility for the workup of some poorly differentiated malignancies as well as confirming the neuroendocrine nature of some neoplasms. Material is optimally fixed for electron microscopy by directly injecting an aspirate into a small tube containing glutaraldehyde. The material can be converted into a pellet by centrifugation and then further processed.25 In some cases, material from highly cellular aspirates can be directly placed on clean glass slides and immersed in glutaraldehyde and subsequently popped off the slide when needed. FNA with immediate glutaraldehyde fixation of the sample can yield excellent results using ultramicroscopy (electron microscopy).26-28

„„ IMMUNOCYTOCHEMISTRY Immunocytochemistry is widely used to determine the direction of differentiation of neoplastic cells. Hence, immunocytochemical studies can be of immense aid in the differential diagnosis of neoplasms. The technique is of increasing importance for selection of therapy, and immunocytochemistry is being utilized for demonstration of estrogen receptor, progesterone receptor, and HER-2/ neu status in adenocarcinoma of the breast.29-32 Recently, immunocytochemistry performed on FNA specimens for thyroid transcription factor-1 (TTF-1), napsin A, and p63 has become important for confirmation of tumor subtype in non-small cell carcinomas of the lung.33,34 While the vast majority of antibodies used in immunohistochemistry can be utilized in immunocytochemistry for the staining of smear preparations, cytospins, liquid-based preparations, and cell blocks, the antibodies may demonstrate different affinities and staining patterns in cytologic material than they do in formalinfixed, paraffin-embedded histopathologic materials.35-38 It should also be borne in mind that most laboratories do not possess a library of optimal controls for cytologic smear and liquid-based preparations, but standard formalin-fixed, paraffin-embedded controls for immunohistochemistry do represent appropriate controls for cell block preparations.39 Hence, careful attention to unexpected staining patterns and background characteristics is important in evaluating immunocytochemistry performed on smears and liquid-based preparations. When smear preparations and liquid-based preparations are utilized for immunocytochemistry, cultured cells may represent a viable control alternative.39

Clinical Utility, Technique and Ancillary Studies

„„ FLOW

CYTOMETRY

Currently, flow cytometry is utilized for two distinctly different analyses. The first utilizes the stoichiometric nature of binding of fluorochromes (feulgen stain) to DNA. This allows measurement of the amount of DNA present within a given cell population by the flow cytometry technique. Neoplasms can be evaluated for proliferation by measurement of the S-phase fraction40-42 and for ploidy. Determination of DNA ploidy has both diagnostic and prognostic value for a number of neoplasms. Histograms plotting DNA content can reveal neoplasms to be diploid or aneuploid aiding in their diagnosis and prediction of prognosis (Figs 1.5A and B).43,44 Aspirated material can be

5

expelled directly into RPMI for flow cytometric analysis, and FNA specimens when of adequate cellularity make excellent substrates for analysis. The second and more utilized application for flow cytometry is in the analysis of cell surface antigens for the diagnosis of lymphomas and leukemias. In this usage, flow cytome­ try can determine the clonality and specific phenotype of lymphoid populations (Fig. 1.6).45,46 Moreover, flow cytometry can segregate cells by size also aiding in classification of lymphomas. New multicolored flow cytometry instruments can classify lymphoid cells for multiple antigens simultaneously. The technique is easily adaptable to FNA specimens.46

A

B Figs 1.5A and B:  (A) Histogram of DNA content. This benign cell population demonstrates a large diploid DNA peak and a much small tetraploid peak representing cell division; (B) DNA histogram of a malignant cell population with a large diploid peak and a large peak representing an aneuploid cell population

6

Atlas of Fine Needle Aspiration Cytology

Fig. 1.6:  Flow cytometry scatter plot showing distribution of surface markers in a lymphoid cell population

to specific drug agents (Figs 1.7 and 1.8).48-53 While these techniques were initially developed for histopathologic specimens, subsequent studies have shown them to work on cell block preparations as well as smear preparations and thus can be utilized for FNA derived material.54 FISH is most commonly utilized for HER-2/neu amplification status in ductal carcinoma of the breast, N-myc amplification status in neuroblastoma, and for the diagnosis of a variety of sarcomas. More recently, FISH has been utilized for the detection of the EML-4–ALK inversion important in therapy selection for adenocarcinomas of the lung. Traditionally, FISH has been the most commonly used technique, but it suffers from the need for a fluorescence microscope and the nonarchivability of the stained specimens. More recently, CISH has been developed, and a dual-color modification appears to be equivalent to the FISH technique but has the advantage of requiring only a light microscope, and the test slides are archivable.

„„ FLUORESCENCE

„„ MOLECULAR

The techniques of fluorescence in situ hybridization (FISH) and chromogenic in situ hybridization (CISH) allow both assessment of cell morphology and direct microscopic visualization of probe specific intranuclear signals.47 These techniques can be utilized to demonstrate a number of amplifications, inversions, and translocations important in diagnosis, prognosis, and prediction of response

Molecular diagnostics has become increasingly important for the identification of therapeutically important mutations that signal potential response to tyrosine kinase inhibitor drugs. A variety of techniques exist based on polymerase chain reaction methodologies. Among these are Sanger sequencing, high-resolution amplicon melting analysis, and pyrosequencing. Sequencing techniques can be used for the evaluation of clinically important mutations including BRAF, EGFR, KRAS, and c-KIT

A

B

IN SITU HYBRIDIZATION AND CHROMOGENIC IN SITU HYBRIDIZATION

DIAGNOSTICS

Figs 1.7A and B:  (A) FISH stain for Her2/neu copy number status. This is a nonamplified cell population with an equal value of Her2/ neu probes and CEP17 probes; (B) FISH staining of a breast specimen with Her2/neu amplification. Note that most cells have two green probes (CEP17) and many red probes resulting in a ratio of red/green of over 4 to 1

Clinical Utility, Technique and Ancillary Studies

A

7

B

Figs 1.8A and B:  (A) EWSR1 FISH analysis showing two sets of fused probes indicating the absence of a translocation; (B) FISH specimen demonstrating one set of fused probes and a separate red and green probe (break apart) signaling a translocation indicative of a Ewing family tumor

(Fig. 1.9).55-60 High-resolution amplicon melting analysis is an alternate technique for rapid mutation detection and has been utilized for the analysis of gastrointestinal stromal tumors and malignant melanomas.57,58 These techniques are applicable for mutational analysis of cytologic specimens from a variety of solid malignancies61 as well as hematopoietic malignancies.62 Molecular diagnostics is playing an increasingly important role in selection of therapy for non-small cell carcinomas of the lung. Recognition that mutations in EGFR and ALK rearrangements were predictive of response to specific tyrosine kinases resulted in the need for molecular analysis of all non-small cell pulmonary carcinomas

believed to be adenocarcinomas.63 Because many nonsmall cell carcinomas of the lung are initially diagnosed by bronchoscopic-guided FNA, application of molecular techniques to these small cytologic samples has become therapeutically important. Diagnostic workup of pulmonary masses and nodules involves bronchoscopic biopsy and/or FNA with the determination of whether the malignancy is a small cell or non-small cell carcinoma. When a non-small cell carcinoma is identified, subclassification into adenocarcinoma or squamous cell carcinoma is necessary. Such classification is aided by application of immunocytochemistry with antibodies directed against napsin A, TTF-1, and p63. Once a carcinoma is identified

Fig. 1.9:  Automated sequencing analysis demonstrating the L576P mutation present in a tested melanoma. Note peak under ‘Y’ in lower register

8

Atlas of Fine Needle Aspiration Cytology

as an adenocarcinoma, the treating oncologist will often request mutational analysis for EGFR and then analysis for ALK. Some oncologists also require mutational analysis for KRAS.63 These analyses can be routinely performed on cytologic specimens. Mutational analysis is becoming increasingly important for the prediction of response to chemotherapeutic agents in patients with metastatic malignant melanoma.64 The most important recognized mutations for therapeutic decisions for melanoma are BRAF (V600e), c-KIT, and NRAS. The majority of melanomas harboring KIT mutations arise in the mucosae, in acral locations, and in sun-damaged skin. BRAF mutations predominate in non-sun-damaged skin melanomas. Ocular melanomas are also known to harbor KIT mutations.60 Documentation of mutations in these genes is helpful in guiding systemic therapy in patients with stage IV malignant melanoma.64 Other sites where mutational analysis appears important include the colorectal area,65 the thyroid,66 gastrointestinal stromal tumors,67 musculoskeletal neoplasms, and in the pancreaticobiliary system.68 „„ REFERENCES 1. Glasgow BJ, Brown HH, Zurgozu AM, et al. Quantitation of tumor seeding from fine-needle aspiration of ocular melanoma. Am J Ophthalmol. 1988;105:538-46. 2. Mighell AJ, High AS. Histologic identification of carcinoma in 21 gauge needle tracks after fine needle aspiration biopsy of head and neck carcinoma. J Clin Pathol. 1998;51:241-3. 3. Roussel F, Dalion J, Benozio M. The risk of tumoral seeding in needle biopsies. Acta Cytol. 1989;33:936-9. 4. Powers CN. Complications of fine needle aspiration biopsy: the reality behind the myths. In: Schmidt WA, (Ed). Cytopathology. Chicago: ASCP Press; 1996. P. 69-91. 5. Ciba ES, Alexander EK, Benson CB, et al. Indications for thyroid FNA and pre-FNA requirements: a synopsis of the National Cancer Institute Thyroid Fine-Needle Aspiration State of the Science Conference. Diagn Cytopathol. 2008;36(6):390-9. 6. Langlois S, Le P. Portable ultrasound on deployment. ADF Health. 2003;4(2):77-80. 7. Zajdela A, Zillhardt P, Voillemot N. Cytological diagnosis by fine needle sampling without aspiration. Cancer. 1987;59:1201-05. 8. Kate MS, Kamal MM, Bobhate SK, et al. Evaluation of fine needle capillary sampling in superficial and deep-seated lesions. An analysis of 670 cases. Acta Cytol. 1998;42:679-84. 9. Koscick RL. Petersilage CA, Makley JT, et al. CT-guided fine needle aspiration and needle core biopsy of skeletal lesions. Complementary diagnostic techniques. Acta Cytol. 1998;42:697-702. 10. Domanski HA, Akerman M, Carlen B, et al. Core-needle biopsy performed by the cytopathologist. A technique to

complement fine-needle aspiration of soft tissue and bone lesions. Cancer (Cytopathology). 2005; 105:229-39. 11. Fetsch PA, Simsir A, Brosky K, et al. Comparison of three commonly used cytologic preparations in effusion immunocytochemistry. Diagn Cytopathol. 2002;26(1):61-6. 12. Leong AS-Y. Immunostaining of cytologic specimens. Am J Clin Pathol. 1996;105:139-40. 13. Schmidt RL, Witt BL, Matynia AP, et al. Rapid one-site evaluation increases endoscopic ultrasound-guided fineneedle aspiration adequacy for pancreatic lesions. Diag Dis Sci. 2012. 14. Schmidt RL, Witt BL, Lopez-Calderon LE, et al. The influence of rapid onsite evaluation on the adequacy rate of fine-needle aspiration cytology: a systematic review and Meta-analysis. Am J Clin Pathol. 2013;139(3):300-8. 15. Komatsu K, Nakanishi Y, Seki T, et al. Application of liquidbased preparations to fine needle aspiration cytology in breast cancer. Acta Cytol. 2008;52:591-6. 16. Rossi ED, Larghi A, Verna EC, et al. Endoscopic ultrasoundguided fine-needle aspiration with liquid-based cytologic preparation in the diagnosis of primary pancreatic lymphoma. Pancreas. 2010;39(8):1299-302. 17. Tetikkurt US, Oz Puyan F, Oz F, et al. Diagnostic value of liquid-based (Liqui-PREP) preparations and interobserver reproducibility in fine needle aspiration cytology of the nodular thyroid lesions. Diagn Cytopathol. 2012;40(5):388-93. 18. Rossi, ED, Fadda G. Thin-layer liquid-based preparation of non-gynaecological exfoliative and fine-needle aspiration biopsy cytology. Diagn Histopathol. 2008;14(11):563-70. 19. Saleh HA, Hammoud J, Zakaria R, et al. Comparison of Thin-Prep and cell block preparation for the evaluation of Thyroid epithelial lesions on fine needle aspiration biopsy. Cytojournal. 2008;5:3. 20. Fischer AH, Clayton AC, Bentz JS, et al. Performance differences between conventional smears and liquid-based preparations of thyroid fine-needle aspiration samples: analysis of 47,076 responses in the College of American Pathologists Interlaboratory Comparison Program in Non-Gynecologic Cytology. Arch Pathol Lab Med. 2013;137(1):26-31. 21. Gupta PK, Baloch ZW. Cytohistology: essentials and basic concepts. Cambridge: Cambridge University Press; 2011. p. 164. 22. Sachdeva R, Kline TS. Aspiration biopsy cytology and special stains. Acta Cytol. 1981;25:678-83. 23. Krane JF, Renshaw AA. Relative value and cost-effectiveness of culture and special stains in fine needle aspirates of the lung. Acta Cytol. 1998;42:305-11. 24. Layfield LJ, Glasgow BJ, DuPuis MH. Fine needle aspiration of lymphadenopathy of suspected infectious etiology. Arch Pathol Lab Med. 1985;109:810-12. 25. Lazaro AV. Technical note: improved preparation of fine needle aspiration biopsies for transmission electron microscopy. Pathology. 1983;15:399-402. 26. Akhtar M, Bakry M, Al-Jeaid AS, et al. Electron microscopy of fine needle aspiration biopsy specimens: a brief review. Diagn Cytopathol. 1992;8:278-82.

Clinical Utility, Technique and Ancillary Studies 27. Dabbs DJ, Silverman JF. Selective use of electron microscopy in fine needle aspiration cytology. Acta Cytol. 1988;32:880-4. 28. Qiononez GE, Ravinsky E, Paraskevas M, et al. Contribution of transmission electron microscopy to fine-needle aspiration biopsy diagnosis: comparison of cytology and combined cytology and transmission electron microscopy with final histological diagnosis. Diagn Cytopathol. 1996;15:282-7. 29. Beatty BG, Bryant R, Wang W, et al. HER-2/neu detection in fine-needle aspirates of breast cancer: fluorescence in-situ hybridization and immunocytochemical analysis. Am J Clin Pathol. 2004;122(2):246-55. 30. Kapila K, Al-Awadhi S, Francis IM. Her-2 neu (Cerb-B2) expression in fine needle aspiration samples of breast carcinoma: a pilot study comparing FISH, CISH and immunocytochemistry. J Cytol. 2011;28(2):54-6. 31. Pegolo E, Machin P, Riosa F, et al. Hormone receptor and human epidermal growth factor receptor 2 status evaluation on ThinPrep specimens from breast carcinomas: correlation with histologic sections determination. Cancer Cytopathol. 2012;120(3):196-205. 32. Shabaik A, Lin G, Peterson M, et al. Reliability of Her2/neu, estrogen receptor and progesterone receptor testing by immunohistochemistry on cell block by FNA and serious effusions from patients with primary and metastatic breast carcinoma. Diagn Cytopathol. 2011;39(5):328-32. 33. Collins BT. Endobronchial ultrasound guided fine-needle aspiration of biopsy of pulmonary non-small cell carcinoma with subclassification by immunohistochemistry panel. Cancer Cytopathol. 2012 Aug 23 (epub ahead of print). 34. Sethi S, Geng L, Shidham VB, et al. Dual color multiplex TTF-1 + Napsin A and p63 + CK5 immunostaining for subcategorizing of poorly differentiated pulmonary non-small carcinomas into adenocarcinoma and squamous cell carcinoma in fine needle aspiration specimens. Cytojournal. 2012;9:10 doi 10.4103/1742-6413.94570. 35. Suthipintawong C, Leong AS-Y. Immunostaining of cell preparations: a comparative evaluation of common fixatives and protocols. Diagn Cytopathol. 1996;15:167-74. 36. Dabbs DJ, Abendroth CS, Grenko RT, et al. Immuno­ cytochemistry on the ThinPrep processor. Diagn Cyto­ pathol. 1997;17:388-92. 37. Polak J, van Noorden S. Introduction to immunocytochemistry. Current techniques and problems. 2nd edn. Berlin: Springer-Verlag; 1997. 38. Nadji M, Ganjei P. Special report. Immunocytochemistry in diagnostic cytology: a 12-year perspective. Am J Clin Pathol. 1990;94(4):470-5. 39. Kurtycz DFI, Logrono R, Leopando M, et al. Immuno­ cytochemistry controls using cell cultures. Diagn Cytopathol. 1997;17:74-9. 40. Vielh P, Carton M, Padoy E, et al. S-Phase fraction as an independent prognostic factor of long-term overall survival in patients with early stage or locally advanced invasive breast carcinoma. Cancer (Cytopathology). 2005;105:476-82.

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41. Davidson B, Dong HP, Berner A, et al. The diagnostic and research applications of flow cytometry in cytopathology. Diagn Cytopathol. 2012;40(6):525-35. 42. Hedley DW, Friedlander ML, Taylor IW, et al. Method for analysis of cellular DNA content of paraffin-embedded pathological material using flow cytometry. J Histochem Cytochem. 1983;31(11):1333-5. 43. Halme L, Arola J, Numminen K, et al. Biliary dysplasia in patients with primary sclerosing cholangitis: additional value of DNA ploidy. Liver Int. 2012;32(5):783-9. 44. Song T, Lee JW, Choi CH, et al. Ploidy and S-phase fraction are correlated with lymphovascular space invasion that is predictive of outcomes in endometrial cancer. Int J Clin Oncol. 2012;17(6):590-7. 45. Barrena S, Almeida J, Del Carmen García-Macias M, et al. Flow cytometry immunophenotyping of fine-needle aspiration specimens: utility in the diagnosis and classi­ fication of non-Hodgkin lymphomas. Histopathology. 2011;58(6):906-18. 46. Nicol TL, Silberman M, Rosenthal DL, Borowitz MJ. The accuracy of combined cytopathologic and flow cytometric analysis of fine-needle aspirates of lymph nodes. Am J Clin Pathol. 2000;114(1):18-28. 47. Fuller CE, Perry A. Fluorescence in situ hybridization (FISH) in diagnostic and investigative neuropathology. Brain Pathol. 2002;12(1):67-86. 48. Lal P, Salazar PA, Hudis CA, Ladanyi M, Chen B. HER-2 testing in breast cancer using immunohistochemical analysis and fluorescence in situ hybridization: a single-institution experience of 2,279 cases and comparison of dual-color and single-color scoring. Am J Clin Pathol. 2004;121(5):631-6. 49. Sartelet H, Grossi L, Pasquier D, et al. Detection of N-myc amplification by FISH in immature areas of fixed neuroblastomas: more efficient than Southern blot/PCR. J Pathol. 2002;198(1):83-91. 50. Yang P, Hirose T, Hasegawa T, Hizawa K, Sano T. Dualcolour fluorescence in situ hybridization analysis of synovial sarcoma. J Pathol. 1998 Jan;184(1):7-13. 51. McManus AP, O’Reilly MA, Jones KP, et al. Interphase fluorescence in situ hybridization detection of t(2;13)(q35;q14) in alveolar rhabdomyosarcoma—a diagnostic tool in minimally invasive biopsies. J Pathol. 1996;178(4):410-4. 52. Wallander ML, Geiersbach KB, Tripp SR, et al. Comparison of reverse transcription-polymerase chain reaction, immunohistochemistry, and fluorescence in situ hybridization methodologies for detection of echinoderm microtubuleassociated protein-like 4-anaplastic lymphoma kinase fusion-positive non-small cell lung carcinoma: implications for optimal clinical testing. Arch Pathol Lab Med. 2012;136(7):796-803. 53. Willmore-Payne C, Holden J, Turner KC, et al. Translocations and amplifications of chromosome 12 in liposarcoma demonstrated by the LSI CHOP breakapart rearrangement probe. Arch Pathol Lab Med. 2008;132(6):952-7.

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54. Bentz JS. Molecular testing in cytopathology: where are we, where do we go from here? CAP Today. 2006 (Feb). 55. Chang H, Lee H, Yoon SO, et al. BRAF(V600E) mutation analysis of liquid-based preparation-processed fine needle aspiration sample improves the diagnostic rate of papillary thyroid carcinoma. Hum Pathol. 2012;43(1):89-95. 56. Hasanovic A, Rekhtman N, Sigel CS, et al. Advances in fine needle aspiration cytology for the diagnosis of pulmonary carcinoma. Path Res Int. 2011 (2011) Article ID 897292, doi 10.4061/2011/897292. 57. Layfield LJ, Wallander ML. Diagnosis of gastrointestinal stromal tumors from minute specimens: cytomorphology, immunohistochemistry, and molecular diagnostic findings. Diagn Cytopathol. 2012;40(6):484-90. 58. Holden JA, Willmore-Payne C, Coppola D, et al. Highresolution melting amplicon analysis as a method to detect c-kit and platelet-derived growth factor receptor alpha activating mutations in gastrointestinal stromal tumors. Am J Clin Pathol. 2007;128(2):230-8. 59. Willmore-Payne C, Holden JA, Tripp S, et al. Human malignant melanoma: detection of BRAF- and c-kit-activating mutations by high-resolution amplicon melting analysis. Hum Pathol. 2005;36(5):486-93. 60. Wallander ML, Layfield LJ, Emerson LL, et al. KIT mutations in ocular melanoma: frequency and anatomic distribution. Mod Pathol. 2011;24(8):1031-5.

61. Aisner DL, Sams SB. The role of cytology specimens in molecular testing of solid tumors: techniques, limitations, and opportunities. Diagn Cytopathol. 2012;40(6):511-24. 62. Ochs RC, Bagg A. Molecular genetic characterization of lymphoma: application to cytology diagnosis. Diagn Cytopathol. 2012;40(6):542-55. 63. Witt BL, Wallander ML, Layfield LJ, et al. Respiratory cyto­ logy in the era of molecular diagnostics: a review. Diagn Cytopathol. 2012;40(6):556-63. 64. Grossmann AH, Grossmann KF, Wallander ML. Molecular testing in malignant melanoma. Diagn Cytopathol. 2012; 40(6):503-10. 65. Malapelle U, Bellevicine C, Russo A, et al. KRAS testing on colo-rectal carcinoma cytological imprints. Diagn Cytopathol. 2011;39(4):274-7. 66. Filie AC, Asa SL, Geisinger KR, et al. Utilization of ancillary studies in thyroid fine needle aspirates: a synopsis of the National Cancer Institute Thyroid Fine Needle Aspiration State of the Science Conference. Diagn Cytopathol. 2008;36(6):438-41. 67. Holden JA, Willmore-Payne C, Layfield LJ. Gastrointestinal stromal tumors: a guide to the diagnosis. Surgic Pathol Clin. 2010;3(2):241-76. 68. Chadwick BE. Beyond cytomorphology: expanding the diagnostic potential for biliary cytology. Diagn Cytopathol. 2012;40(6):536-41.

Chapter

2

Head & Neck and Salivary Gland Jerzy Klijanienko



GENERAL CONSIDERATIONS

The head and neck area is a complex anatomical structure where various tissues and organs are present in a narrow space. This variability gives rise to a large spectrum of different tumors arising from these tissues. Moreover, the presence of a rich blood and lymphatic vascularization associated with the presence of numerous groups of lymph nodes draining different areas adds an additional degree of difficulty in the diagnosis of head and neck tumors. Clearly, some anatomical sites and related tumor groups may be individualized, but their radiologic, clinical, and even morphologic presentation may overlap. These sites/ groups are head and neck mucosal tumors, skin tumors, orbital tumors, head and neck cavity tumors, salivary gland tumors, thyroid tumors, enlarged lymph nodes, soft tissue and bone tumors, and neck masses. The distinction between primary and metastatic tumors is an additional challenge in both the histologic and fine-needle aspiration (FNA) diagnosis. Close cooperation with the clinician and the radiologist is necessary to clarify the anatomical relationships of the target lesion, the nature of any previous lesion, and the details of prior therapy. FNA, being a minimally invasive technique, is particularly suitable in this sensitive area where an incisional biopsy may cause complications. FNA can obviate the need for surgery if the lesion is shown to be non-neoplastic or if it confirms suspected metastatic or recurrent tumor. In this chapter, we describe head and neck, and salivary gland tumors. Thyroid lesions are described in Chapter 3.



FNA AS A DIAGNOSTIC METHOD IN HEAD AND NECK TUMORS

Fine needle aspiration is not traditionally used in the diagnosis of the most common malignancy that is primary squamous cell carcinoma, but it has been successfully applied to its recurrences and metastases,1-7 as well as to enlarged lymph nodes, orbital and intraocular tumors,8-10 lymphomas,11-15 melanomas,16,17 secondary tumors, and lateral neck masses.18 In the following section, we describe the cytology of squamous cell carcinoma, nasopharyngeal carcinoma (NPC), paraganglioma, olfactory neuroblastoma, neck masses and orbital lesions.

Squamous Cell Carcinoma Squamous cell carcinoma is the most common malignancy of head and neck area. Because it arises in the mucosa of deep cavities like the oral cavity, pharynx, or larynx, it is not accessible to cytologic diagnosis and the optimal diagnostic method remains tissue biopsy. In contrast, the most common indication of FNA in patients with squamous cell carcinoma is investigation of suspected local recurrence or nodal metastasis of previously diagnosed and treated cancer. FNA is of great assistance in the management of patients with suspected metastatic or recurrent disease, since therapeutic decisions can be made earlier and without the need for further diagnostic surgery. It is usually not difficult in nonirradiated tissue to distinguish

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Atlas of Fine Needle Aspiration Cytology

Fig. 2.1: Squamous cell carcinoma. Spindle-shaped and keratinized cells, May-Grunwald-Giemsa (MGG)

Fig. 2.2: Poorly differentiated squamous cell carcinoma. Absence keratinizing cells (MGG)

between tumor recurrence and inflammation or scarring.19 Depending on its differentiation, squamous cell carcinomas are composed of atypical cells varying in shape from polygonal to “spindle” cells (Figs 2.1 to 2.4). Nuclei are enlarged, hyperchromatic, and have irregular nuclear membranes. Keratinization is usually present and appears orange (Papanicolaou stain) or waxy blue (Romanowsky stain). Frequently aspirates consist grossly of yellowish fluid.

type II), and undifferentiated carcinoma of nasopharyngeal type (UCNT, WHO type III). UCNT is distinguished by its particular histology, geographic distribution, relationship to Epstein–Barr virus, and the absence of an alcohol or tobacco etiologic relationship.20 Cells from UCNT are anaplastic, roundish, or “spindle” (Figs 2.5 to 2.7). They form loose clusters with no specific architectural pattern. Nuclei are large with chromatin clearing and one or two prominent nucleoli. Cytoplasm is poorly delineated or not seen. The background usually contains lymphoid cells, macrophages and plasma cells.21-23 The distinction from Hodgkin disease or large cell non-Hodgkin lymphoma may be difficult. Immunostaining for cytokeratin and CD45 is usually helpful. Epstein–Barr virus-associated

Nasopharyngeal Carcinoma Nasopharyngeal carcinoma consists of three distinctive entities: keratinized squamous cell carcinoma (WHO type I), nonkeratinizing squamous cell carcinoma (WHO

Fig. 2.3: Squamous cell carcinoma (Papanicolaou stain)

Fig. 2.4: Metastatic squamous cell carcinoma. Predominant necrotic and inflammatory background and isolated keratinizing cells (MGG)

Head & Neck and Salivary Gland

13

Fig. 2.5: Undifferentiated carcinoma of nasopharyngeal type. Strongly nucleated cells with badly seen cytoplasmic margins. Background is inflammatory (MGG)

Fig. 2.6: Undifferentiated carcinoma of nasopharyngeal type, anaplastic cells (MGG)

nuclear antigen is demonstrable by anticomplement immunofluorescence in undifferentiated tumors. Patients with NPC classically present with a lymph node metastasis in the neck without a known primary. Patients are often of Mediterranean, African, or southern Chinese origin. Cytologic recognition of UCNT is important, since the primary tumor is often clinically occult.

primary tumor shows roundish or “spindle” cells with fine red cytoplasmic granulation lying as isolated cells, or in follicular arrangements.24-27 The cells show anisonucleosis. A follicular arrangement of the tumor cells may suggest a follicular thyroid carcinoma, but the characteristic anisokaryosis and the presence of spindle cells with red cytoplasmic granules closely resemble medullary carcinoma which is the main differential diagnosis (Figs 2.8 to 2.10). Tumors with a predominant spindle cell pattern can be confused with other spindle cell tumors in the neck (spindle cell medullary carcinoma of thyroid and soft tissue tumors). Marked nuclear pleomorphism in some paragangliomas may suggest malignancy. Immunocytochemistry may help since paraganglioma cells stain positively for neuroendocrine markers and are nonreactive for calcitonin. Intranuclear vacuoles (cytoplasmic pseudoinclusions), as in papillary carcinomas of the thyroid, may be found in paragangliomas; thus, knowledge of the exact anatomic site is important. Finally, paragangliomas are extremely vascular lesions, and aspirates often appear to be pure blood. If this is the case, smears may be nondiagnostic but diagnostic tissue fragments may be found in a cell block preparation.19

Paraganglioma Paraganglioma is a neuroendocrine tumor and its cytologic pattern suggests its neuroendocrine origin. A correct cytologic diagnosis may be made when a cervical

Olfactory Neuroblastoma Fig. 2.7: Undifferentiated carcinoma of nasopharyngeal type, spindle cells (MGG)

This rare neoplasm occurs in the upper nasal cavity and may cause nasal obstruction. It may present as an enlarged cervical lymph node. Several cases diagnosed by FNA

14

Atlas of Fine Needle Aspiration Cytology

Fig. 2.8: Paraganglioma. Large neuroendocrine cells with eosinophilic cytoplasm. Prominent anisonucleosis (MGG)

Fig. 2.9: Paraganglioma. Roundish, neuroendocrine cells (MGG)

have been reported.28-30 The tumor cells are usually round with scant cytoplasm. The pseudorosettes formed by the tumor cells may be mistaken for microacini of an adenocarcinoma, but the nuclear morphology is relatively bland and finely fibrillar material may be seen in the center of the rosettes similar to the common neuroblastoma (Fig. 2.11). Immunostaining for neuroendocrine and epithelial markers is helpful in the differential diagnosis.

in most cases, clinical data are important for diagnosis (Figs 2.12 and 2.13). There is a clear clinical distinction between cystic lesions in children and adults. Cystic lesions in children may be classified as nasolabial/nasoalveolar, thyroglossal, periauricular/cervical lateral (1st–3rd arch origin), thymic cyst and vascular malformations. In adults, cystic lesions may be classified as midline or as lateral.31-34 Midline cysts are usually thyroglossal cysts. The content of a thyroglossal cyst can be cytologically indistinguishable from that of a branchial or other type of cyst. The differential diagnosis is predominately based

Cystic Neck Masses Cystic neck masses are a common target for FNA. Because aspirates show nonspecific fluid and squamous material

Fig. 2.10: Paraganglioma, atypical cells (MGG)

Fig. 2.11: Olfactory neuroblastoma; note the similar aspect to neuroblastoma (MGG)

Head & Neck and Salivary Gland

15

Fig. 2.12: Fine needle aspiration of a neck lateral, cystic mass

Fig. 2.13: Aspirate from lateral neck cyst. Note amorphic material (MGG)

on the anatomical site of the lesion. The content may be mucinous with mucin secreting and/or ciliated columnar epithelial cells being found in the smears. Thyroid epithelial cells are rarely present. Lateral cysts in young adults are usually branchial cysts. They develop relatively rapidly as a firm mass of significant size in the lateral neck. These cysts are most often seen in young adults but may become clinically apparent at any age. Lateral cysts in adults after 40 years of age are always suspicious for carcinoma and should be verified by surgery. Lateral metastases are from conventional squamous cell carcinoma, bronchogenic squamous cell carcinomas, or metastatic papillary thyroid carcinomas.35 A lymph node metastasis of well-differentiated squamous cell carcinoma with liquefactive necrosis is a very important and difficult differential diagnosis. Material sampled from necrotic carcinoma is more obviously necrotic than inflammatory; smears usually contain at least a few squamous epithelial cells with malignant nuclear features or abnormal keratinized cells with bizarre, globoid shapes and condensed orangeophilic (Papanicolaou) cytoplasm. Conversely, atypical reactive and metaplastic squamous cells in smears from an inflamed branchial cyst can be of concern. Because many cystic metastases arise for oropharyngeal primaries, human papillomavirus testing may aid in confirmation of the malignant nature of the specimen. When a metastasis is diagnosed with no evidence of a primary tumor, systematic biopsies of the homolateral tonsil should be performed. However, salivary gland tumors like pleomorphic adenoma, Warthin’s

tumor, basal cell adenoma, acinic cell carcinoma and low-grade mucoepidermoid carcinoma may yield cyst fluid36,37 (Figs 2.14 to 2.17).

Tumors of the Orbit Orbital and intraocular tumors may be diagnosed by FNA8-15 (Figs 2.18 to 2.20). The most common lesion of diagnostic importance is a lymphoproliferative process. Cytologic diagnosis may be difficult and requires the use of ancillary techniques. Tumors of the lacrimal gland resemble primary salivary gland tumors, and the same diagnostic criteria (see below) apply. Distinction of primary from metastatic neoplasms in this site is necessary.38

Fig. 2.14: Histologic section of lymphoepithelial cyst, Hematoxylin and Eosin Stain (HES)

16

Atlas of Fine Needle Aspiration Cytology

Fig. 2.15: Histologic section of an epidermal cyst (HES)

Fig. 2.16: Histologic section of pleomorphic adenoma with central cyst (HES)

Fig. 2.17: Presumed cystic lesion of the scalp. Fine needle aspiration is strongly in favor of Langerhans cell histiocytosis (MGG)

Fig. 2.18: Fine needle aspiration of an orbital tumor

Fig. 2.19: Intraocular aspiration. Malignant melanoma (MGG)

Fig. 2.20: Intraorbital aspiration. Retinoblastoma (MGG)

Head & Neck and Salivary Gland

The most common lacrimal gland tumors are pleomorphic adenoma, carcinoma ex pleomorphic adenoma and salivary duct carcinoma. Lacrimal ducts can give rise to neoplasms similar to transitional and squamous cell carcinomas. Sarcomas also arise in the orbit and include rhabdomyosarcoma, osteosarcoma, and Ewing sarcoma/ peripheral primitive neuroectodermal tumor. The orbit is also a target of metastatic tumors, mainly mammary adenocarcinoma, pulmonary small cell neuroendocrine carcinoma and malignant melanoma. Fine needle aspiration of intraocular tumors has not gained wide acceptance among ophthalmologists despite the negligible risk of tumor cell dissemination and other complications like hematoma. FNA offers a means of distinguishing between primary and metastatic intraocular malignancies as well as between malignant and benign primary neoplasms, which is necessary for clinical management. Indications of intraocular FNA include: (1) the patient refuses enucleation; (2) a definitive diagnosis cannot be made by classical and ancillary ophthalmologic techniques; and (3) metastatic tumor is suspected in the absence of a known primary site.39 Malignant melanoma, retinoblastoma, medulloepithelioma and metastatic neoplasms are the most common intraocular lesions. FNA smears of intraocular melanoma are hypercellular and are usually rich in pigment. The cells may be epithelioid, spindle-shaped, or both. In amelanotic melanomas, immunocytochemistry (HMB-45 and Melan-A) aids greatly in diagnosis. Aspirates from melanoma should be routinely analyzed for genetic alterations. The most important genetic alteration associated with a poor prognosis is inactivation of BAP1, which most often occurs through mutation of one allele and subsequent loss of an entire copy of chromosome 3 to unmask the mutant copy. Retinoblastoma has a characteristic age distribution, family history, radiologic findings and fundoscopic appearance. It is rarely a target of FNA. Smears from metastatic retinoblastomas are hypercellular with poorly differentiated round cells similar to neuroblastoma.40-42 The cells are small with hyperchromatic nuclei and scant basophilic cytoplasm. Rosette-like structures, nuclear molding and predominant necrosis are frequently seen. 

FNA AS A DIAGNOSTIC METHOD IN SALIVARY GLAND TUMORS

The diagnosis of salivary gland tumors may be challenging. Difficulties are numerous but the most challenging

17

is accurate classification that includes 9 types of benign and 18 types of malignant neoplasms.43 Salivary glands are localized in different and complex anatomical areas, where the distinction of putative origin may be unclear, even when modern diagnostic imaging methods are used. Numerous pseudotumoral processes may be present in these anatomical areas. Finally, the constantly growing knowledge about salivary gland neoplasms is reflected in the constant modification of histologic classifications.44 Because salivary neoplasms are often localized in the superficial body tissues, they constitute an easy target for FNA evaluation. Indications of preoperative use of FNA are well recognized, but this technique is not universally accepted.37 The facts favoring its application are as follows: • High specificity and accuracy of FNA in experienced hands • Cytologic diagnosis is equally accurate as frozen section diagnosis • Pretherapeutic diagnosis allows optimal patient management and “prepared” surgery • Excellent application of cytologic diagnostic parameters to recognition of malignant versus benign • Excellent distinction between salivary primary tumor and metastases to the salivary glands • Excellent recognition of lymphoproliferative disorders at the salivary localization • Excellent recognition on pseudotumoral processes that contraindicate surgery • Possibility to perform a cell block or to collect cellular material for ancillary studies • Possibility to follow up patients with benign tumors • Low risk of false-negative, false-suspicious, and falsepositive diagnoses • Extremely low risk of complications • Possibility to sample major and minor salivary glands (Figs 2.21 and 2.22) • Economical technique • Low use of immunohistochemistry in the diagnosis of salivary gland tumors. The unfamiliarity of pathologist and his/her lack of experience is a limitation of FNA usage as a diagnostic method in salivary gland tumors. Numerous studies have analyzed FNA performance as a diagnostic technique. It is well recognized that high-grade salivary carcinomas are easily diagnosed as malignant and adenomas are diagnosed as benign. Several

18

Atlas of Fine Needle Aspiration Cytology

Fig. 2.21: Fine needle aspiration of a lacrimal gland tumor

Fig. 2.22: Fine needle aspiration of an intraoral tumor

types of carcinomas of low-grade malignancy may result in false-negative diagnoses. Low-grade mucoepidermoid carcinoma and carcinoma in pleomorphic adenoma are frequently underdiagnosed. However, some benign tumors are associated with false-positive diagnoses. Warthin’s tumor is a classic example and must be distinguished from squamous cell and mucoepidermoid carcinomas. Pseudotumoral processes like necrotizing sialometaplasia and Küttner tumor also require separation from squamous cell and mucoepidermoid carcinomas. The methodology for the diagnosis of salivary gland tumors differs between oncology centers. Some hospitals prefer the use of frozen sections as a diagnostic modality. It was reported that, even in hospitals with a large salivary gland tumor experience, the intraoperative rate of accurate diagnosis is good but not totally satisfactory. As shown in a recent literature review,45 the overall accuracy of frozen section was acceptable (90% sensitivity, 99% specificity) and somewhat higher than that of FNA. It could be used to confirm a diagnosis for patients referred for surgery following a negative FNA where there is concern about the low sensitivity of FNA. It is evident that the use of FNA for diagnosis may substantially improve patient management, since preoperative knowledge of the salivary gland diagnosis allows for informing the patient of treatment modalities and the hospital to program bed occupancy and surgical planning. The description of principal cytologic aspects of particular groups of salivary gland neoplasms allows for their cytologic diagnosis in a large percentage of cases.37 Previously described cytology classifications

allow for categorization of tumors into benign, low-grade malignant, high-grade malignant and metastatic. In this categorization scheme, misclassification is possible, but it should not be clinically significant because the therapeutic implications are similar. It is not harmful to the patient to misdiagnose acinic cell carcinoma as low-grade mucoepidermoid carcinoma, or adenoid cystic carcinoma as epimyoepithelial carcinoma. However, it is important to accurately recognize Warthin’s tumor or pleomorphic adenoma and exclude primary or metastatic squamous cell carcinoma or adenoid cystic carcinoma, respectively. The distinction between “salivary primary” and “metastatic to salivary” is of great clinical value. Salivary glands are a target of metastases and a large majority of metastases are from the scalp and, head and neck areas. The presence of a parotid tumor in a patient with a known cutaneous malignant melanoma is not automatically a metastasis from the malignant melanoma. The accurate diagnosis of such a tumor may significantly change the clinical approach. Similarly to solid tumors, lymphoproliferative disorders may be diagnosed by cytology. Cytology material is of excellent quality for flow cytometry and molecular and genetic techniques. The accurate diagnosis of a pseudotumoral process is of great clinical value and will contraindicate surgery.37 Pseudotumors like Küttner tumor, nonspecifically enlarged periparotid lymph nodes, salivary cysts, and specific inflammatory lesions (tuberculosis, sarcoidosis) may closely imitate salivary gland neoplasms. Their accurate diagnosis may substantially modify therapeutic strategies.

Head & Neck and Salivary Gland

Patients suffering from nonsalivary malignancies may develop benign salivary tumors. The cytologic diagnosis of a benign salivary neoplasm eliminates a metastasis and may allow only clinical follow-up. The value of FNA in the setting of Warthin’s tumor misdiagnosed radiologically and clinically as a metastasis was recently reported.46 In summary, pretherapeutic FNA diagnosis of salivary gland tumors is highly useful. The cytologic diagnosis of salivary gland tumors is based mainly on classical morphologic parameters with little use of immunohistochemistry or other ancillary techniques. FNA is a minimally invasive and an inexpensive method characterized by a low rate of false-negative, false-suspicious, and falsepositive diagnoses.47-54 

FNA CLASSIFICATION OF SALIVARY GLAND TUMORS

Smears from salivary tumors are usually cell-rich and stroma-rich. The correct recognition of both, cellular and stromal tumor components, is fundamental for accurate tumor diagnosis. To reach the level of high accuracy, the pathologist should use a simplified cytologic classification of tumors as previously published.37 This classification is based on the recognition of: • Myoepithelial • Oncocytic • Basal cell • Squamous • Mucus secreting • Acinar cell, and • Nonspecific/poorly differentiated cells When this method is applied, the risk of diagnostic error is substantially reduced. In this chapter, we present salivary tumors according to the cytologic classification reported previously where malignant, benign and pseudotumoral conditions show similar morphologies. 

TUMORS IN WHICH MYOEPITHELIAL CELLS PREDOMINATE

Pleomorphic adenoma, adenoid cystic carcinoma, polymorphous low-grade adenocarcinoma and epimyoepithelial carcinoma belong to this group of tumors. Cytology smears are cell-rich and stroma-rich and composed mainly of myoepithelial cells. Pleomorphic adenomas have distinctive morphologic characteristics, whereas all three carcinomas may exhibit overlapping

19

patterns. Intracapsular carcinoma in pleomorphic adenoma (in situ carcinoma ex pleomorphic adenoma) is difficult to detect and is a source of false-negative results.

Pleomorphic Adenoma/Myoepithelioma Pleomorphic adenoma is one of the most common salivary gland neoplasms. It accounts for 60–70% of all major and minor salivary gland tumors. Classically, it arises in the major salivary glands in young subjects with a slight predilection for women. Multifocal localization is common. Clinically, pleomorphic adenoma exhibits benign behavior, but incompletely excised tumors can recur. Rarely, some tumors may give rise to metastases (metastasizing pleomorphic adenoma). Cytologically, pleomorphic adenoma is easy to diagnose if two principal components are found.55-60 The first component is myoepithelial plasma-like cells, and the second is chondromyxoid stroma. Plasma-like myoepithelial cells are large, with eccentric nuclei and well-delimited cytoplasm. Some cells may be irregular and show a coarse chromatin, but these anomalies should not be interpreted as a sign of malignancy. Mitotic figures are absent. Chondromyxoid stroma is characteristic and composed of fibrillary matrix with poorly delimited margins (important in differential diagnosis!). It stains magenta using the Romanowsky method. The chondromyxoid subtype of pleomorphic adenoma is stroma-rich and cellpoor. The cellular subtype is cell-rich and stroma-poor. Myoepitheliomas (plasmacytoid or spindle cell subtypes) are cell-rich, and the stroma is extremely scant or absent. In some tumors, epithelial cells are also seen. Epithelial cells may be detected when Papanicolaou stain is used or if metaplasia of epithelial cells is present. In such instances, oncocytes, squamous cells, sebaceous cells, or mucus may be found. Tyrosine crystalloids, pseudonecrosis, mucus, and pinkish osteoid may be occasionally observed. A diagnostic difficulty is the presence of hyaline globules similar to those seen in myoepithelial cell malignancies (adenoid cystic carcinoma, polymorphous low-grade carcinoma, and epimyoepithelial carcinoma) (Figs 2.23 to 2.27).

Adenoid Cystic Carcinoma Adenoid cystic carcinoma is one of the three most common primary salivary gland malignancies. It constitutes around 10% of all salivary glands tumors. It mainly arises in patients in the fifth to seventh decades of life. Clinical behavior is unpredictable with a high incidence of local recurrence. Distant metastases, when present, are mainly located in the lungs, lymph nodes and bones.

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Atlas of Fine Needle Aspiration Cytology

Fig. 2.23: Pleomorphic adenoma. Chondromyxoid matrix and myoepithelial cells (MGG)

Fig. 2.24: Pleomorphic adenoma, predominance of myoepithelial cells (MGG)

Fig. 2.25: Epithelial cells are well evidenced using Papanicolaou stain

Fig. 2.26: Oncocytic metaplastic cells in pleomorphic adenoma

Fig. 2.27: Pleomorphic adenoma with a mucoid background that should be differentiated from mucoepidermoid carcinoma (MGG)

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Fig. 2.28: Adenoid cystic carcinoma. Hyaline globules (MGG)

Fig. 2.29: Adenoid cystic carcinoma. Hyaline globules (Papanicolaou stain)

Adenoid cystic carcinoma exhibits characteristic cytologic patterns that may overlap with other carcinomas of this group.61-63 Hyaline globules, tubular structures, finger-like structures, and dark cells with scant cytoplasm are characteristics of this entity. Conversely to pleomorphic adenoma, cells of adenoid cystic carcinoma are smaller, darker, and do not exhibit a plasmacytoid pattern. Sometimes they present as naked nuclei. Pinkish stroma is well delimited at the periphery (compare with fibrillary matrix with poorly delimited margins in pleomorphic adenoma) and often forms cylindrical structures and hyaline globules. Necrosis or mitotic figures may be occasionally seen (Figs 2.28 to 2.33).

Polymorphous Low-grade Adenocarcinoma

Fig. 2.30: Adenoid cystic carcinoma. Tubular structures, characteristic pattern (MGG)

Polymorphous low-grade adenocarcinoma is a low-grade malignancy arising mainly in the minor salivary glands. These neoplasms occur predominately in the oral cavity, palate and upper lips. The palate is the characteristic localization. It represents approximately 5% of all salivary gland tumors. The age of onset ranges from the third to ninth decades of life. Clinically, it is a low-grade malignancy with only exceptional recurrences if surgical treatment was complete. Bone invasion of the hard palate is not a prognostic factor. Smears are cell-rich and stroma-rich.64-66 The cellular material is composed of epithelial cells. Myoepithelial

Fig. 2.31: Adenoid cystic carcinoma. Basal, dark cells (MGG)

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Fig. 2.32: Adenoid cystic carcinoma. Basal dark cells in aggregates with hyaline globules (MGG)

Fig. 2.33: Adenoid cystic carcinoma. Anastomising tubular structures (MGG)

cells are not distinguishable from the epithelial component. The cells form cords, sheets, or pseudopapillae. Cells are polyhedral, oval, or polygonal with characteristic elongated nuclei. Nuclear pleomorphism is slight. Because the chromatin is fine and dusty, nuclei have a clarified appearance. Mitotic figures are rare. Stroma consists of hyaline globules similar to those of adenoid cystic carcinoma as well as nonspecific stromal cores, and branched pseudopapillae or true papillae with central vessels. Primary salivary papillary cystadenocarcinoma may be a variant of polymorphous low-grade carcinoma (Figs 2.34 and 2.35).

Epimyoepithelial Carcinoma

Fig. 2.34: Polymorphous low-grade adenocarcinoma. Delicate, clarified and elongated nuclei. Distinctive pattern from adenoid cystic carcinoma (MGG)

Fig. 2.35: Polymorphous low-grade adenocarcinoma, structures, and spindle nuclei (Papanicolaou stain)

Epimyoepithelial carcinoma belongs to a group of highgrade malignancies. The distinction between pure clear cell carcinoma and epimyoepithelial carcinoma appears academic, since these two subtypes belong to the same entity with different degrees of differentiation. It is a rare primary salivary gland malignancy usually located in a major salivary gland. Morphologically, epimyoepithelial carcinoma shows clear myoepithelial cells.67-73 Some cases may exhibit a double epithelial (darker) and myoepithelial (clear) cell population, best demonstrated on Papanicolaou staining.

tubular

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TUMORS WITH ONCOCYTIC CELL PREDOMINANCE

When oncocytic cells are predominant on smears, the diagnosis of a benign salivary tumor should be rendered.37 Two benign entities should be considered: Warthin’s tumor and oncocytic adenoma (oncocytoma). Some malignant entities like poorly differentiated acinic cell carcinoma and mucoepidermoid carcinoma may show epithelial cells with abundant cytoplasm mimicking true oncocytes.

Warthin’s Tumor Fig. 2.36: Epimyoepithelial carcinoma. Similar pattern to adenoid cystic carcinoma, but hyaline globules are less rigid (Diff-Quik)

Cytonuclear atypia, mitotic figures and necrosis are rare. Stroma is usually abundant and composed of round hyaline globules similar to those in adenoid cystic carcinoma as well as nonspecific connective tissue cores (Fig. 2.36).

Myoepithelial Carcinoma Myoepithelial carcinoma is an extremely rare malignancy. Cytology smears demonstrate a nonspecific carcinoma with vesicular nuclei.37

Carcinoma Ex Pleomorphic Adenoma Carcinoma arising in pleomorphic adenoma may be unrecognized in cytology smears especially when the carcinoma is intracapsular. In infiltrative (extra-capsular) forms, the cytology is similar to that of well-defined mucoepidermoid or adenoid cystic carcinomas (underlying pleomorphic adenoma is usually not detectable).74-77

Malignant Mixed Tumor This tumor has the characteristic appearance of a true carcinosarcoma [composed of both stromal (chondrosarcoma or osteosarcoma) and epithelial malignancies].78,79

Important Points and Differential Diagnosis in Tumors where Myoepithelial Cells Predominate This is heterogeneous group of neoplasms that exhibit a similar cytomorphology but are sufficiently distinctive to allow accurate diagnosis. The most important clues to the differential diagnosis are summarized in Table 2.1.

Warthin’s tumor is a common adenoma accounting for 2–10% of all parotid tumors. It arises in the major salivary glands. Frequently, Warthin’s tumor is multifocal and/ or bilateral. Three distinctive components are present in the smear: oncocytes, polymorphous lymphoid cells, and a pseudonecrotic background. Oncocytes are either isolated, clustered, or in pseudopapillary formations. Mast cells within the clusters are common. The lymphoid component resembles smears from a reactive lymph node. Finally, the pseudonecrotic background shows amorphous material with inflammatory cells and crystals. Some necrotic debris may resemble mucus from low-grade mucoepidermoid carcinoma. A few cases may show squamous or sebaceous cells. The diagnosis is straightforward when all three components are present.80-82 The diagnosis is more difficult when the smears show only one or two components. The presence of only pseudonecrosis may be a source of a false-positive or false-suspicious diagnosis (in favor of low-grade mucoepidermoid carcinoma or squamous cell carcinoma). The exclusive presence of polymorphous lymphocytes may result in the diagnosis of an inflammatory lymph node. Finally, the exclusive presence of oncocytes may be misdiagnosed as an oncocytic adenoma (Figs 2.37 to 2.41).

Oncocytic Adenoma/Adenosis Oncocytic adenoma/adenosis is an unusual salivary tumor/pseudotumor.37,83 Smears are composed exclusively of oncocytes without a polymorphous lymphoid population or a pseudonecrotic background.

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Table 2.1: The most important clues to the differential diagnosis of tumors with myoepithelial cell predominance Pattern

What to do

•

Plasmacytoid cells

Characteristic of pleomorphic adenoma; exclude other malignancies

•

Chondromyxoid stroma or fibrillary matrix with poorly delimited margins

Characteristic of pleomorphic adenoma; exclude other malignancies

•

Plasmacytoid cells and chondromyxoid stroma or fibrillary matrix with poorly delimited margins associated with mucus

Possible pattern in classical pleomorphic adenoma; exclude possibility of mucoepidermoid carcinoma arising in pleomorphic adenoma. If this carcinoma remains underdiagnosed, there are no clinical consequences to the patients (quasi-benign behavior of “intracapsular” carcinomas)

• Hyaline globules

Hyaline globules are not only seen in adenoid cystic carcinoma but are also commonly seen in other myoepithelial-predominant neoplasms (polymorphous low-grade adenocarcinoma and epimyoepithelial carcinoma) as well as in basaloid tumors. In polymorphous low-grade adenocarcinoma, the nuclei are larger, rounder, and more uniform and usually are clearer. Adenoid cystic carcinoma shows characteristic tubular and finger-like structures that are absent in polymorphous low-grade adenocarcinoma. Epimyoepithelial carcinoma, when well differentiated (clear cell carcinoma), shows the presence of a double cell population: darker (ductal) and clear (myoepithelial) cells. However, hyaline globules may be occasionally seen in cellular pleomorphic adenoma—source of false-positive diagnoses. They are also common in extra-salivary malignancies such as basaloid squamous cell carcinoma and skin appendage tumors

•

Cylindrical structures, finger-like structures, hyaline globules associated with darker cells

Strongly suggestive of adenoid cystic carcinoma

•

Elongated and clear nuclei

Strongly suggestive of polymorphous low-grade adenocarcinoma

• Hard palate tumor

Clinically suggestive of polymorphous low-grade adenocarcinoma or pleomorphic adenoma

•

Possibility of carcinosarcoma

Patterns of pleomorphic adenoma and signs of sarcoma

• Patterns of adenoid cystic carcinoma with clarified cells

Possibility of epimyoepithelial carcinoma

Fig. 2.37: Warthin’s tumor. Typical aspect: clustered oncocytic cells and lymphocytic and necrotic background (MGG)

Fig. 2.38: Warthin’s tumor. Oncocytic cells and dirty background (Papanicolaou stain)

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Fig. 2.39: Warthin’s tumor. Only necrotic component present. It should be differentiated from necrotic carcinoma or salivary cyst (MGG)

Fig. 2.40: Warthin’s tumor. Mast cells in oncocytic cluster, well seen as dark nuclei (MGG)

Important Points and Differential Diagnosis in Tumors where Oncocytic Cells Predominate

salivary carcinoma, and extra-salivary basaloid carcinomas. Despite the variability of clinical presentation and variable behavior, the common feature of these tumors is the presence of basaloid roundish dark cells. The morphologic similarity of basaloid tumors complicates histologic separation. Surprisingly, differentiation of basaloid tumors seems to be simpler on cytologic smears than on surgical biopsies. In fact, only canalicular and basal cell adenomas are morphologically similar; the other entities differ significantly in smears. Extra-salivary basaloid carcinomas, such as basaloid squamous cell carcinoma or basal cell carcinoma of the skin, may be excluded on the basis of clinical presentation.

Well-differentiated oncocytic cells are markers of a benign salivary gland tumor.37 The most important clues to the differential diagnosis are summarized in Table 2.2. 

TUMORS WITH BASAL CELL PREDOMINANCE

This is a heterogeneous group of neoplasms. Seven different entities belong to this category: basal cell and canalicular adenomas, basal cell adenocarcinoma, solid variant of adenoid cystic carcinoma, neuroendocrine (small cell)

Fig. 2.41: Warthin’s tumor. Presence of mucus differentiated from mucoepidermoid carcinoma (MGG)

should

be

Basal Cell Adenomas (Canalicular and Basal Cell Adenoma) Canalicular/basal cell adenomas represent less than 2% of all salivary neoplasms. Canalicular adenoma occurs in older patients, usually in females and mainly arises in the upper lip. Basal cell adenoma usually occurs in younger patients and has a significant predilection for the major salivary glands. A variant of membranous basal cell adenoma is thought to be a precursor of adenoid cystic carcinoma. Cytologically, both entities are indistinguishable and composed of clustered or isolated dark basaloid cells, naked nuclei, and nonspecific connective fragments.84-87 Hyaline globules with an intense eosinophilic appearance (Romanowsky stain) may be observed. Peripheral palisading is usually seen. In larger clusters, focal squamous metaplasia may be found. There is neither cytonuclear atypia nor mitoses (Figs 2.42 to 2.46).

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Table 2.2: The most important clues to the differential diagnosis of tumors with oncocytic cell predominance Pattern

What to do

•

Oncocytic cells

Characteristic of Warthin’s tumor and oncocytic adenoma; excludes any malignancy

•

Mast cells within oncocytic clusters

Characteristic of Warthin’s tumor, but may be also seen in mucoepidermoid carcinoma

•

Abundant lymphocytic background

Characteristic of Warthin’s tumor. When oncocytic cells are absent, possible misdiagnosis with stimulated lymph node

•

Abundant pseudonecrotic background

Characteristic of Warthin’s tumor. When oncocytic cells are absent, possible misdiagnosis with low-grade mucoepidermoid carcinoma, necrotic squamous cell carcinoma, or necrotic lymphadenitis. Some cysts may also exhibit similar morphology

• Oncocytic cells without lymphocytes or pseudonecrosis

Strongly suggestive of oncocytic adenoma or oncocytosis

•

Strongly suggestive of metaplastic pleomorphic adenoma

Oncocytic cells and chondromyxoid background

Because of their benign behavior, surgical removal with negative margins is optimal treatment. The membranous subtype of basal cell adenoma has a tendency to recur (Table 2.3).

Basal Cell Adenocarcinoma Basal cell adenocarcinoma is the malignant counterpart of basal cell adenoma and represents approximately 1% of all salivary neoplasms. It occurs in young adults and older patients. There is no sex predilection, and tumor is limited exclusively to the major salivary glands. Cytologically, basal cell adenocarcinomas have characteristic features and are composed of three-dimensional clusters of malignant appearing dark and roundish cells

Fig. 2.42: Basal cell adenoma. Small basaloid cells and not specific connective debris (MGG)

with irregular nuclei and a granulated chromatin.87 Glandular and rosette-like patterns are common. Isolated cells are often present. In some cases, mitotic figures are seen. Nuclei are often poorly preserved during smearing. Hyaline globules are frequently seen within the epithelial clusters. Some cases may show areas of keratin debris or squamous cells (Figs 2.47 to 2.49).

Solid Variant of Adenoid Cystic Carcinoma In poorly differentiated variants, a solid pattern predominates and these adenoid cystic carcinomas may be difficult to accurately diagnose. Solid adenoid cystic carcinoma occurs most often in older adults.

Fig. 2.43: Basal cell adenoma. Peripheral palisading (MGG)

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Table 2.3: The most important clues to the differential diagnosis of basal cell adenomas Pattern

What to do

•

Basal cells, scant cytoplasm, three-dimensional clusters, palisading, naked nuclei and nonspecific connective tissue debris

Characteristic of basal cell adenomas

•

Squamous cells

Usual histologic pattern; sometimes seen on smears

•

Hyaline globules

Usual pattern, but differentiate with adenoid cystic carcinoma

•

Necrosis, mitotic figures

Against adenoma, favoring basal cell adenocarcinoma

Fig. 2.44: Basal cell adenoma. Three-dimensional clusters (MGG)

Fig. 2.45: Basal cell adenoma. Three-dimensional clusters (note preserved microarchitecture) (Papanicolaou stain)

Fig. 2.46: Basal cell adenoma. Squamous cells, a rare feature (MGG)

Fig. 2.47: Basal cell adenocarcinoma, basaloid malignant cells (MGG)

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Fig. 2.48: Basal cell adenocarcinoma, basaloid cells, necrosis, not specific connective debris (MGG)

Fig. 2.49: Basal cell adenocarcinoma, hyaline globules (MGG)

Cytologically, solid adenoid cystic carcinoma is composed of bland dark basaloid cells. Nuclei are irregular with small nucleoli. Mitotic figures are frequent. Cells show scant, dark basophilic cytoplasm. Background is necrotic. Hyaline globules that stain intensely red using the Romanowsky method are frequent.



Primary Poorly Differentiated Neuroendocrine (Small Cell) Carcinoma These malignancies have identical morphology to their pulmonary counterpart. This entity is rare and represents less than 10% of all salivary carcinomas. Cytologically, poorly differentiated neuroendocrine (small cell) carcinoma is composed of anaplastic, fragile cells with an abundant necrotic background and nuclear smearing artifact; rosette formations and nuclear molding suggest its neuroendocrine origin. Its diagnosis is possible only after exclusion of a primary elsewhere88 (Fig. 2.50).

TUMORS WITH SQUAMOUS CELL PREDOMINANCE

All neoplasms showing a predominance of squamous cells are malignant. Separation of primary and metastatic squamous cell carcinoma is clinically important. Occasionally, benign squamous metaplastic cells occur in pleomorphic adenomas, Warthin’s tumors, or basal cell tumors, but this component is not predominant. Rare squamous cells are present in high-grade mucoepidermoid carcinoma.

Important Points and Differential Diagnosis in Tumors with Basaloid Cell Predominance Basaloid tumors represent a heterogeneous group of neoplasms with different putative origins. Because of morphologic similarities, they are challenging to accurately diagnose. The most important clues to the differential diagnosis are summarized in Table 2.4.

Fig. 2.50: Primary neuroendocrine carcinoma. Note similarity to its pulmonary counterpart (MGG)

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Table 2.4: The most important clues to the differential diagnosis of basal cell malignancies Pattern

What to do

•

Basal cells, scant cytoplasm, three-dimensional clusters, palisading, naked nuclei, nonspecific background debris

Characteristic of basal cell adenocarcinoma if necrosis and mitotic figures are found. Necrosis and mitotic figures are absent in basal cell adenomas

•

Squamous cells

Differentiate from basaloid squamous cell carcinoma of head and neck area or with squamous component of basal cell adenocarcinoma/basal cell adenoma

• Hyaline globules, tubular and finger-like structures

Usual pattern in adenoid cystic carcinoma. Basal cell adenocarcinomas do not show tubular or finger-like structures

•

Sign of malignancy. Predominant in neuroendocrine carcinoma

Necrosis, mitotic figures

Primary Squamous Cell Carcinoma Primary squamous cell carcinoma is relatively infrequent and represents up to 5% of salivary malignancies. The tumors are histologically identical to squamous cell carcinomas of the head and neck mucosal surfaces.89 Its diagnosis is possible only after exclusion of potential metastatic sites from primaries in the scalp, eyelid, or head and neck area (Figs 2.51 and 2.52).

carcinoma mimics poorly differentiated carcinoma or squamous cell carcinoma37 (Figs 2.53 to 2.56).

Important Points and Differential Diagnosis in Tumors with Squamous Cell Predominance Squamous cells are markers of malignancy in salivary gland tumors. The most important clues to the differential diagnosis are summarized in Table 2.5.

High-grade Mucoepidermoid Carcinoma



High-grade mucoepidermoid carcinoma is one of the most common entities of the salivary glands. It represents up to 30% of salivary gland malignancies. Usually, smears are extremely cellular and composed of intermediate cells, squamous cells, and background mucin.90 Rare mucinsecreting cells may also be present. The accurate diagnosis may be difficult, since high-grade mucoepidermoid

Mucin is common in aspirates from salivary glands tumors. When abundant, it is indicative of mucoepidermoid carcinoma. Occasionally, mucin may be seen in pleomorphic adenoma, Warthin’s tumor and salivary cysts. Inflammatory conditions may also show mucin. It is important to differentiate mucin from chondromyxoid stroma and necrosis.

Fig. 2.51: Primary squamous cell carcinoma. Keratinizing cells (MGG)

Fig. 2.52: Primary squamous cell carcinoma, spindle-shaped cells (MGG)

TUMORS WITH MUCIN PRODUCTION

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Fig. 2.53: High-grade mucoepidermoid carcinoma. Intermediate cells (MGG)

Fig. 2.54: High-grade mucoepidermoid carcinoma. Intermediate cells (Papanicolaou stain)

Fig. 2.55: High-grade mucoepidermoid carcinoma. Intermediate cells and mucus; the diagnosis is evident (MGG)

Fig. 2.56: High-grade mucoepidermoid carcinoma. Atypical nonspecific cells (MGG)

Table 2.5: The most important clues to the differential diagnosis of tumors with squamous cell predominance Pattern

What to do

•

Malignant squamous cells without background mucin

Characteristic of squamous cell carcinoma or high-grade mucoepidermoid carcinoma

•

Malignant squamous cells with background mucin

Characteristic of high-grade mucoepidermoid carcinoma

•

Cell resembling oncocytes

May be a pattern of high-grade mucoepidermoid carcinoma

• Abundant necrotic background

Characteristic of squamous cell carcinoma or high-grade mucoepidermoid carcinoma

•

Malignant squamous cells without background mucin

Exclusion of primary site on the scalp, eyelid, or head and neck area

•

Squamous or keratin debris associated with chondromyxoid stroma, myoepithelial or basal cells

Suggestive of metaplastic pleomorphic adenoma, squamous metaplasia in basal cell tumors, squamous metaplasia in Warthin’s tumor

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Fig. 2.57: Low-grade mucoepidermoid carcinoma. Vegetal-like mucussecreting cells (MGG)

Fig. 2.58: Low-grade mucoepidermoid carcinoma. Mucoid background with low cellularity (MGG)

Low-grade Mucoepidermoid Carcinoma

background, and mucin (Fig. 2.59). Special attention is required when a diagnosis of low-grade mucoepidermoid carcinoma arising in a submandibular gland is considered.37,93,94 Sialadenitis may be seen in patients affected by HIV. Lymphoepithelial lesion may simulate an inflammatory lymph node or a salivary gland cyst.95,96

Low-grade mucoepidermoid carcinoma is composed of intermediate cells, mucus-secreting cells, and abundant background mucin90-92 (Figs 2.57 and 2.58). Welldifferentiated squamous cells are rare. Mucin-secreting cells may present as clear cells, goblet cells, or clarified, plant-like cells. Mucin-secreting cells in some tumors may appear identical to microvacuolated macrophages. The accurate diagnosis may be difficult, since many low-grade mucoepidermoid carcinomas produce smears composed only of mucin and “microvacuolated macrophages.” These smears may mimic a salivary cyst. This pattern is a reason for the elevated rate of false-negative diagnoses reported in the literature. A necrotic mucin-rich background should be differentiated from the background debris of a Warthin’s tumor. Finally, morphologic signs of a chondromyxoid background associated with classical patterns of low-grade mucoepidermoid carcinoma should be differentiated from a mucoepidermoid carcinoma arising in pleomorphic adenoma.

Salivary Cysts Usually salivary cysts contain clear or yellowish fluid similar to saliva, but some may contain a viscous fluid resembling mucin (Figs 2.60 to 2.63). The search for intermediate cells and squamous cells aids in the differential diagnosis with low-grade mucoepidermoid carcinoma.

Mucinous Carcinoma This is an extremely rare primary salivary malignancy morphologically identical with other mucinous carcinomas of the digestive tract.37

Sclerosing Chronic Sialadenitis, Sialadenitis-NOS Sclerosing chronic sialadenitis (Küttner tumor) may be cytologically difficult to recognize when the smears are composed of dark basaloid cells, inflammatory

Fig. 2.59: Sclerosing chronic sialadenitis; basal cells, mucin and inflammatory cells (MGG)

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Fig. 2.60: Salivary cyst. Surgical specimen

Fig. 2.61: Salivary cyst. Same case as Figure 2.60 (H&E)

“Microvacuolated macrophages” should always be differentiated with mucin-secreting cells. Recently, cystic lesions have been described in patients affected by HIV.90,97-101

Sialadenosis

Important Points and Differential Diagnosis in Tumors with Mucin Secretion The most important clues to the differential diagnosis of tumors with mucosecretion are summarized in Table 2.6. 

TUMORS WITH ACINIC CELL PREDOMINANCE

Acinic cells are present on smears in two distinctive settings: in sialadenosis and in well-differentiated acinic cell carcinoma.

Fig. 2.62: Salivary cyst. Dirty background mimicking tumoral necrosis or mucin (MGG)

This bilateral salivary gland enlargement is usually present in clinical conditions like diabetes and autoimmune disease. It is a benign pseudotumoral condition. Acinic cells are well differentiated, with small nuclei, and acinic cells lie in small clusters.102-105

Acinic Cell Carcinoma Acinic cell carcinoma is one of the most frequent salivary gland malignancies. Depending on its differentiation, the smear pattern varies.106,107 In well-differentiated carcinomas, smears are similar to those seen in sialadenosis, but are usually hypercellular. Cells are regular, without cytonuclear atypia. Cytoplasm is abundant and

Fig. 2.63: Salivary cyst. Crystalloids (MGG)

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Table 2.6: The most important clues to the differential diagnosis of tumors with mucosecretion Pattern

What to do

•

Malignant squamous cells with background mucin

Characteristic of mucoepidermoid carcinoma

•

Intermediated cells with abundant background mucin

Characteristic of mucoepidermoid carcinoma

•

Oncocytic or clear cells

May be present in low-grade mucoepidermoid carcinoma

•

Necrotic background with mucin

Differentiate from Warthin’s tumor and Küttner tumor (at submandibular localization). Possibility of diagnostic error!

•

Background mucin and numerous “microvacuolated macrophages”

Possibility of low-grade mucoepidermoid carcinoma. True salivary cysts rarely contain macrophages or mucin. Major source of diagnostic error!

•

Mucin and pleomorphic adenoma patterns

Two possibilities: mucoepidermoid carcinoma arising in pleomorphic adenoma or pleomorphic adenoma exhibiting mucus secretion. Histologic control may be necessary

microvacuolized or clarified. Nuclei are small and regular. A variable number of naked nuclei are present. In less well-differentiated carcinomas, cells are more atypical and less characteristic, but in general cytoplasmic microvacuolization is present. The differential diagnosis consists of sialadenosis for well-differentiated acinic cell carcinomas and with other carcinomas for less-differentiated acinic cell carcinomas (Figs 2.64 to 2.66).

Important Points and Differential Diagnosis in Tumors with Acinic Cell Predominance The most important clues to the differential diagnosis of tumors with acinic cell predominance are summarized in Table 2.7.

Fig. 2.64: Acinic cell carcinoma. Well-differentiated microvacuolated cells (MGG)

Fig. 2.65: Acinic cell carcinoma. Moderately differentiated cells (MGG)

Fig. 2.66: Acinic cell carcinoma. Intracytoplasmic granules (MGG)

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Atlas of Fine Needle Aspiration Cytology

Table 2.7: The most important clues to the differential diagnosis of tumors with acinic cell predominance Pattern

What to do

•

Well-differentiated acinic cells

Common pattern for sialadenosis and well-differentiated acinic cell carcinoma. Cells are small and clustered in sialadenosis and isolated or clustered in carcinoma

•

Atypical microvacuolated cells

Usually seen in less-differentiated acinic cell carcinomas, or in other adenocarcinomas

•

Oncocytic or clear cells

May be present in acinic cell carcinoma

•

Numerous naked nuclei

In favor of acinic cell carcinoma

•

Bilateral lesion in patients with systemic disease

In favor of sialadenosis

•

Pediatric age

In favor of acinic cell carcinoma



TUMORS WITH NONSPECIFIC/ POORLY DIFFERENTIATED CELL PREDOMINANCE

Salivary Duct Carcinoma Salivary duct carcinoma is a high-grade malignancy related morphologically to mammary ductal carcinoma. This entity represents approximately 3% of all salivary tumors. These carcinomas occur in the parotid gland with a male predominance in the sixth and seventh decades of life. Clinical presentation is characteristic for a high-grade malignancy. Cytologically, salivary duct carcinoma is similar to invasive ductal carcinoma of the breast and is composed of an admixture of plasmacytoid cells, atypical cells, and/ or malignant oncocytic-like cells.108-113 Cells are frequently isolated, but also clustered. If clustered, tubular, glandular

Fig. 2.67: Salivary duct carcinoma similar to its breast counterpart (MGG)

and papillary formations are observed. Some tumors may show extensive comedonecrosis associated with apoptotic debris, macrophages, and even calcifications. The main differential diagnosis of salivary duct carcinoma is a high-grade mucoepidermoid carcinoma, knowing that background mucin may be scant and misdiagnosed as comedonecrosis. Salivary duct carcinomas rich in oncocytic cells should be differentiated with malignant oncocytoma or metastatic breast carcinoma. The diagnosis of salivary duct carcinoma is possible after exclusion of a breast primary (Fig. 2.67).

Undifferentiated Carcinoma Undifferentiated carcinoma is a high-grade carcinoma with morphology similar to UCNT occurring in Epstein– Barr virus endemic areas. At a salivary gland localization, this tumor is relatively common in Greenland114,115 (Fig. 2.68).

Fig. 2.68: Large cell carcinoma. Atypical malignant cells (MGG)

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35

Fig. 2.69: Parotid metastasis from epithelioid amelanotic melanoma from the intraocular primary site (MGG)

Fig. 2.70: Parotid metastasis from breast primary epithelioid angiosarcoma (MGG)

Metastatic Tumors

Particular problems are represented by synchronous or metachronous lacrimal gland and parotid gland tumors.

Metastatic tumors to the major salivary glands are common.116-118 They represent the most frequent salivary malignancy at the author’s institute. The most common primary sites are malignant melanoma, cutaneous squamous cell carcinoma, breast adenocarcinoma and pulmonary small cell carcinoma. In patients with a history of previous malignant disease, metastatic tumors are easily recognized on cytologic smears (Figs 2.69 and 2.70).

Important Points and Differential Diagnosis in Tumors with Nonspecific/Poorly Differentiated Cell Predominance The most important clues to the differential diagnosis of tumors with nonspecific/poorly differentiated cell predominance are shown in Table 2.8.

Table 2.8: The most important clues to the differential diagnosis of tumors with nonspecific/poorly differentiated cell predominance Pattern

What to do

•

High-grade carcinoma pattern

The most pertinent clue is to differentiate between salivary primary highgrade malignancy and a metastatic tumor. The exact diagnosis may be difficult, and a diagnosis of high-grade carcinoma is usually sufficient. The presence of squamous cells and mucin-secreting cells is strongly in favor of high-grade mucoepidermoid carcinoma. Some high-grade mucoepidermoid carcinomas may not be characteristic and mimic salivary duct carcinoma or metastatic mammary carcinoma

•

Oncocytic malignant cells

Differentiate between mucoepidermoid carcinoma, poorly differentiated acinic cell carcinoma, metastatic carcinoma and oncocytic primary salivary carcinoma

• Comedonecrosis

The differential diagnosis may be difficult. Abundant granular cytoplasm, nuclear atypia, and coarse chromatin are usually seen in oncocytic carcinoma and necrosis is usually absent or scant. Comedonecrosis is common in salivary duct carcinoma

• Known history of previous malignant disease

Metastatic tumor should always be differentiated from a primary salivary carcinoma

36 

Atlas of Fine Needle Aspiration Cytology

REFERENCES

1. McLean NR, Harrop-Griffiths K, Shaw HJ, et al. Fine needle aspiration cytology in the head and neck region. Br J Plast Surg. 1989;42:447-51. 2. Cramer H, Lampe H, Downing P. Intraoral and transoral fine needle aspiration. A review of 25 cases. Acta Cytol. 1995;39:683-8. 3. Domanski H, Åkerman M. Fine-needle aspiration cytology of tongue swellings. A study of 75 cases. Diagn Cytopathol. 1998;18:387-92. 4. Castelli M, Gattuso P, Reyes C, et al. Fine needle aspiration biopsy of intraoral and pharyngeal lesions. Acta Cytol. 1993;37:448-50. 5. Mondal A, Raychoudhuri BK. Peroral fine needle aspiration cytology of parapharyngeal lesions. Acta Cytol. 1993;37:694-8. 6. Helsel JC, Bardales RH, Mukunyadzi P. Fine-needle aspiration cytology of malignant neoplasms of the sinonasal tract. A review of 22 primary and metastatic tumors. Cancer. 2003;99:105-12. 7. Engzell U, Zajicek J. Aspiration biopsy of tumours of the neck. I. Aspiration biopsy and cytologic findings in 100 cases of congenital cysts. Acta Cytol. 1970;14:51-7. 8. Zajdela A, Vielh P, Schlienger P, et al. Fine-needle cytology of 292 palpable orbital and eyelid tumors. Am J Clin Pathol. 1990;93:100-4. 9. Font RL, Laucirica R, Ramzy I. Cytologic evaluation of tumors of the orbit and ocular adnexa: an analysis of 84 cases studied by the “squash” technique. Diagn Cytopathol. 1994;10:135-42. 10. Sturgis CD, Silverman JF, Kennerdell JS, et al. Fine-needle aspiration for the diagnosis of primary epithelial tumors of the lacrimal gland and ocular adnexa. Diagn Cytopathol. 2001;24:86-9. 11. Ljung B-M, Chan D, Miller TR, et al. Intraocular lymphoma. Cytologic diagnosis and the role of immunologic markers. Acta Cytol. 1988;32:840-7. 12. Medeiros LJ, Harris NL. Lymphoid infiltrates of the orbit and conjunctiva. A morphologic and immunophenotypic study of 99 cases. Am J Surg Pathol. 1989;13:459-71. 13. Jeffrey PB, Cartwright D, Atwater SK, et al. Lacrimal gland lymphoma. A cytomorphologic and immunophenotypic study. Diagn Cytopathol. 1995;12:215-22. 14. Laucirica R, Font RL. Cytologic evaluation of lymphoproliferative lesions of the orbit/ocular adnexa: an analysis of 46 cases. Diagn Cytopathol. 1996;15:241-5. 15. Nassar DL, Raab SS, Silverman JF, et al. Fine-needle aspiration for the diagnosis of orbital hematolymphoid lesions. Diagn Cytopathol. 2000;23:314-7. 16. Chan DH, Miller TR, Ljung BM, et al. Fine needle aspiration biopsy in uveal melanoma. Acta Cytol. 1989;33:599-05. 17. Dávila RM, Miranda MC, Smith ME. Role of cytopathology in the diagnosis of ocular malignancies. Acta Cytol. 1998;42:362-6.

18. Augsburger JJ, Shields JA, Folberg R, et al. Fine needle aspiration biopsy in the diagnosis of intraocular cancer. Cytologic-histologic correlations. Ophthalmology. 1985;92: 39-9. 19. Orell SR, Klijanienko J. Head and neck; salivary glands. In: Orell SR, Sterrett GF, Houston MJ, (Eds). Fine Needle Aspiration Cytology. 5th edition. New York: Elsevier Churchill Livingstone; 2012. pp. 38-77. 20. Micheau C. What’s new in histological classification and recognition of nasopharyngeal carcinoma (NPC). Path Res Pract. 1986;181:249-53. 21. Chan MK, McGuire LJ, Lee JC. Fine needle aspiration cytodiagnosis of nasopharyngeal carcinoma in cervical lymph nodes. A study of 40 cases. Acta Cytol. 1989;33:344-50. 22. Jayaram G, Swain M, Khanijow V, et al. Fine-needle aspiration cytology of metastatic nasopharyngeal carcinoma. Diagn Cytopathol. 1998;19:168-72. 23. Kollur SM, El Hag IA. Fine-needle aspiration cytology of metastatic nasopharyngeal carcinoma in cervical lymph nodes: comparison with metastatic squamous cell carcinoma, and Hodgkin’s and non-Hodgkin’s lymphoma. Diagn Cytopathol. 2003;28:18-22. 24. Engzell U, Franzen S, Zajicek J. Aspiration biopsy of tumours of the neck. II. Cytologic findings in 13 cases of carotid body tumour. Acta Cytol. 1971;15:25-30. 25. Lack EE, Cubilla AL, Woodruff JM. Paragangliomas of the head and neck region. A pathologic study of tumours from 71 patients. Hum Pathol. 1979;10:191-218. 26. Gonzales-Campora R, Otal-Salaversi C, Panea-Flores P, et al. Fine needle aspiration cytology of paraganglionic tumors. Acta Cytol. 1988;32:386-90. 27. Chen LT, Hwang W-S. Fine needle aspiration of carotid body paraganglioma. Acta Cytol. 1989;33:681-2. 28. Jelen M, Wozniak Z, Rak J. Cytologic appearance of esthesioneuroblastoma in a fine needle aspirate. Acta Cytol. 1988;32:377-80. 29. Collins BT, Cramer HM, Hearn SA. Fine needle aspiration cytology of metastatic olfactory neuroblastoma. Acta Cytol. 1997;41:802-10. 30. Logrono R, Futoran RM, Hartig G, et al. Olfactory neuroblastoma (esthesioneuroblastoma): appearance on fine needle aspiration: report of a case. Diagn Cytopathol. 1997;17:205-8. 31. Burgess KL, Hartwick R J, Bedard YC. Metastatic squamous carcinoma presenting as a neck cyst. Differential diagnosis from inflamed branchial cleft cyst in fine needle aspirates. Acta Cytol. 1993;37:494-8. 32. Üstün M, Risberg B, Davidson B, et al. Cystic change in metastatic lymph nodes: a common diagnostic pitfall in fine needle aspiration cytology. Diagn Cytopathol. 2002;27:387-92. 33. Gertner R, Podoshin L, Fradis M. Accuracy of fine needle aspiration biopsy in neck masses. Laryngoscope. 1984;94:1370-1. 34. Warson F, Blommaert D, DeRoy G. Inflamed branchial cyst; a pitfall in aspiration cytology. Acta Cytol. 1986;30:201-2.

Head & Neck and Salivary Gland 35. Micheau C, Klijanienko J, Luboinski B, et al. So-called branchiogenic carcinoma is actually cystic metastases in the neck from a tonsillar primary. Laryngoscope. 1990;100:878-83. 36. Nettle WJ, Orell SR. Fine needle aspiration in the diagnosis of salivary gland lesions. Aust NZ J Surg. 1989;59:47-51. 37. Klijanienko J, Vielh P, Batsakis JD, et al. Monographs in clinical cytology. Vol. 15. Salivary Gland Tumours. Basel, Switzerland: Karger; 2000. 38. Klijanienko J, El-Naggar AK, Servois V, et al. Histologically similar synchronous or metachronous lacrimal salivarytype and parotid gland tumors: a series of 11 cases. Head Neck. 1999;21:512-6. 39. Czerniak B, Woyke S, Domagala W, et al. Fine-needle aspiration cytology of intraocular malignant melanoma. Acta Cytol. 1983;28:171-4. 40. Chan DH, Miller TR, Descheues J. Fine needle biopsy of retinoblastoma. Am J Ophthalmol. 1984;97:686-90. 41. Decaussin M, Boran M D-S, Salle M, et al. Cytological aspiration of intraocular retinoblastoma in an 11-year-old boy. Diagn Cytopathol. 1998;19:190-3. 42. Sen S, Singha U,Kumar H, et al. Diagnostic intraocular fine needle aspiration biopsy—an experience in three cases of retinoblastoma. Diagn Cytopathol. 1999;21:331-4. 43. Seifert G, Sobin LH. Histological typing of salivary gland tumours. World Health Organization. International histological classification of tumours. 2nd ed. Berlin: SpringerVerlag; 1991. 44. Ellis GL, Auclair PL, Gnepp DR. Surgical pathology of the salivary glands. Philadelphia: Saunders; 1991. 45. Schmidt RL, Hunt JP, Hall BJ, et al. A systematic review and meta-analysis of the diagnostic accuracy of frozen section for parotid gland lesions. Am J Clin Pathol. 2011;136:729-38. 46. Klijanienko J, Petras S, De Bosschere L, et al. False positive FDG PET/CT uptake in Warthin tumor in head and neck oncological patients confirmed by a fine needle aspiration. Diagn Cytopathol. 2012;40:282-4. 47. Atuta T, Grenman R, Laippala P, et al. Fine-needle aspiration biopsy in the diagnosis of parotid gland lesions: evaluation of 438 biopsies. Diagn Cytopathol. 1996;15:185-90. 48. Al-Khafaji B, Nestok BR, Katz RL. Fine-needle aspiration of 154 parotid masses with histologic correlation. Ten-year experience at the University of Texas M.D. Anderson Cancer Center. Cancer (Cancer Cytopathol). 1998;84:153-9. 49. Stewart CJR, MacKenzie K, McGarry GW, et al. Fine needle aspiration cytology of salivary gland: a review of 341 cases. Diagn Cytopathol. 2000;22:139-46. 50. Young JA. Diagnostic problems in fine needle aspiration cytopathology of the salivary glands. J Clin Pathol. 1994;47:193-8. 51. Cramer H, Layfield L, Lampe H. Fine needle aspiration of salivary gland lesions. In: Schmidt WA, (Ed). Cytopathology Annual. Baltimore: Williams and Wilkins; 1993. p. 181-206. 52. MacLeod CB, Frable WJ. Fine needle aspiration biopsy cytology of the salivary glands: problem cases. Diagn Cytopathol. 1993;9:216-25.

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53. Orell SR. Diagnostic difficulties in the interpretation of fine needle aspirates of salivary gland lesions: the problem revisited. Cytopathology. 1995;6:285-300. 54. Batsakis JG, Sneige N, El-Naggar AK. Fine-needle aspiration of salivary glands: its utility and tissue effects. Ann Otol Rhinol Laryngol. 1992;101:185-8. 55. Klijanienko J, Vielh P. Fine-needle sampling of salivary gland lesions I. Cytology and histology correlation of 412 cases of pleomorphic adenoma. Diagn Cytopathol. 1996;14:195-200. 56. Viguer JM, Vicandi B, Jiménez-Hefferman JA, et al. Fine needle aspiration cytology of pleomorphic adenoma. An analysis of 212 cases. Acta Cytol. 1997;41:786-94. 57. Bottles K, Ferrell LD, Miller TR. Tyrosine crystals in fine needle aspirates of a pleomorphic adenoma of the parotid gland. Acta Cytol. 1984;28:490-2. 58. Kawahara A, Harada H, Kage M, et al. Characterization of the epithelial components in pleomorphic adenoma of salivary gland. Acta Cytol. 2002;46:1095-100. 59. Dodd LG, Caraway NP, Luna MA, et al. Myoepithelioma of the parotid. Report of a case initially examined by fine needle aspiration biopsy. Acta Cytol. 1994;38:417-21. 60. Chhieng DC, Cohen J-M, Cangiarella JF. Fine-needle aspiration of spindle cell and mesenchymal lesions of the salivary glands. Diagn Cytopathol. 2000;23:253-9. 61. Klijanienko J, Vielh P. Fine needle sampling of salivary gland lesions III. Cytology and histology correlation of 75 cases of adenoid cystic carcinoma. Review and experience at the Institute Curie with emphasis on cytologic pitfalls. Diagn Cytopathol. 1997;17:36-41. 62. Nagel H, Hotze HJ, Laskawi R, et al. Cytologic diagnosis of adenoid cystic carcinoma of salivary glands. Diagn Cytopathol. 1999;20:358-66. 63. Mohan H, Handa U, Amanjit, et al. Adenoid cystic carcinoma of the external auditory canal. A case report with diagnosis by fine needle aspiration. Acta Cytol. 2003;47:792-4. 64. Frierson HF Jr, Covell JL, Mills SE. Fine needle aspiration cytology of terminal duct carcinoma of minor salivary gland. Diagn Cytopathol. 1987;3:159-62. 65. Klijanienko J, Vielh P. Salivary carcinomas with papillae: cytology and histology analysis of polymorphous lowgrade adenocarcinoma and papillary cystadenocarcinoma. Diagn Cytopathol. 1998;19:244-9. 66. Gibbons D, Saboorian MH, Vuitch F, et al. Fine-needle aspiration findings in patients with polymorphous lowgrade adenocarcinoma of the salivary glands. Cancer. 1999;87:31-6. 67. Carrillo R, Poblet E, Rocamora A, et al. Epithelialmyoepithelial carcinoma of the salivary gland. Fine needle aspiration cytologic findings. Acta Cytol. 1990;34:243-7. 68. Kocjan G, Milroy C, Fisher EW, et al. Cytologic features of epithelial-myoepithelial carcinoma of salivary gland: potential pitfalls in diagnosis. Cytopathology. 1993;4:173-80. 69. Klijanienko J, Vielh P. Fine needle sampling of salivary gland lesions VII. Cytology and histology correlation of 5 cases of epithelial-myoepithelial carcinoma. Diagn Cytopathol. 1998;19:405-9.

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Atlas of Fine Needle Aspiration Cytology

70. Ng WK, Choy C, Ip P, et al. Fine needle aspiration cytology of epithelial-myoepithelial carcinoma of salivary glands. A report of three cases. Acta Cytol. 1999;43:675-80. 71. Miliauskas J, Orell SR. Fine-needle aspiration cytological findings in five cases of epithelial/myoepithelial carcinoma of salivary glands. Diagn Cytopathol. 2003;28:163-7. 72. Layfield LJ, Glasgow BJ. Aspiration cytology of clear-cell lesions of the parotid gland: morphologic features and differential diagnosis. Diagn Cytopathol. 1993;9:705-12. 73. Wax T, Layfield L. Epithelial-myoepithelial cell carcinoma of the parotid gland: a case report and comparison of cytologic features with other stromal, epithelial, and myoepithelial cell containing lesions of the salivary glands. Diagn Cytopathol. 1996;14:298-304. 74. Jacobs JC. Low-grade mucoepidermoid carcinoma ex pleomorphic adenoma. A diagnostic problem in fine needle aspiration biopsy. Acta Cytol. 1994;38:93-7. 75. Miliauskas J, Orell SR. Acinic cell carcinoma arising in pleomorphic adenoma of parotid gland. Cytopathology. 2000;11:356-9. 76. Klijanienko J, El-Naggar AK, Servois V, et al. Mucoepidermoid carcinoma ex pleomorphic adenoma. Nonspecific preoperative cytologic findings in six cases. Cancer. 1998;84:231-4. 77. Klijanienko J, El-Naggar AK, Vielh P. Fine-needle sampling findings in 26 carcinomas ex pleomorphic adenomas: diagnostic pitfalls and clinical considerations. Diagn Cytopathol. 1999;21:163-6. 78. Klijanienko J, El-Naggar AK, Servois V, et al. Clinically aggressive metastasizing pleomorphic adenoma: report of two cases. Head Neck. 1997;19:629-33. 79. Klijanienko J, Servois V, Jammet P, et al. Pleomorphic adenoma. Am J Surgi Pathol. 1998;22:772-3. 80. Klijanienko J, Vielh P. Fine needle sampling of salivary gland lesions II. Cytology and histology correlation of 71 cases of Warthin’s tumor (adenolymphoma). Diagn Cytopathol. 1997;16:221-5. 81. Ballo MS, Shin HJC, Sneige N. Sources of diagnostic error in the fine needle aspiration diagnosis of Warthin’s tumor and clues to a correct diagnosis. Diagn Cytopathol. 1997;17:230-4. 82. Parwani AV, Ali SZ. Diagnostic accuracy and pitfalls in fine needle aspiration interpretation of Warthin’s tumor. Cancer. 2003;99:166-71. 83. O’Dwyer P, Farrar WB, James AG, et al. Needle aspiration biopsy of major salivary gland tumors: Its value. Cancer. 1986;57:554-7. 84. Hood I, Qizilbash AH, Salama SS, et al. Basal cell adenoma of parotid. Difficulty of differentiation from adenoid cystic carcinoma on aspiration biopsy. Acta Cytol. 1983;27:515-20. 85. Stanley MW, Horwitz CA, Rollins SD, et al. Basal cell (monomorphic) and minimally pleomorphic adenomas of the salivary glands. Distinction from the solid (anaplastic) type of adenoid cystic carcinoma in fine-needle aspiration. Am J Clin Pathol. 1996;106:35-1.

86. Sparrow SA, Frost FA. Salivary monomorphic adenoma of dermal analogue type: report of two cases. Diagn Cytopathol. 1993;9:300-3. 87. Klijanienko J, El-Naggar AK, Vielh P. Comparative cytological and histological study of 15 salivary basal cell tumors; diagnostic and differential diagnostic considerations. Diagn Cytopathol. 1999;21:30-4. 88. Lussier C, Klijanienko J, Vielh P. Fine-needle sampling of metastatic non-lymphomatous tumors to the major salivary glands. A clinico-pathologic study of 40 cases cytologically diagnosed and histologically correlated. Cancer. 2000;90:350-6. 89. Klijanienko J, Vielh P. Fine needle sampling of salivary gland lesions VI. Cytological review of 44 cases of primary salivary squamous-cell carcinoma with histologic correlation. Diagn Cytopathol. 1998;18:174-8. 90. Klijanienko J, Vielh P. Fine needle sampling of salivary gland lesions IV. Review of 50 cases of mucoepidermoid carcinoma with histologic correlation. Diagn Cytopathol. 1997;17:92-8. 91. Cohen MB, Fisher PE, Holly EA, et al. Fine needle aspiration biopsy diagnosis of mucoepidermoid carcinoma. Acta Cytol. 1990;34:43-9. 92. Kumar N, Kapila K, Verma K. Fine needle aspiration cytology of mucoepidermoid carcinoma: a diagnostic problem. Acta Cytol. 1991;35:357-9. 93. Cheuk W, Chan JKC. Küttner tumor of the submandibular gland. Fine-needle aspiration cytologic findings of seven cases. Am J Clin Pathol. 2002;117:103-8. 94. Williams SB, Foss RD, Ellis GL. Inflammatory pseudotumors of the major salivary glands. Clinicopathologic and immunohistochemical analysis of six cases. Am J Surg Pathol. 1992;16:896-902. 95. Finfer MD, Gallo I, Perchick A, et al. Fine needle aspiration biopsy of cystic benign lymphoepithelial lesion of the parotid gland in patients at risk for the acquired immune deficiency syndrome. Acta Cytol. 1990;34:821-6. 96. Casiano RR, Cooper JD, Gould E, et al. Value of needle biopsy in directing management of parotid lesions in HIV-positive patients. Head Neck. 1991;13:411-4. 97. Elliott JN, Oertel YC. Lymphoepithelial cysts of the salivary glands. Histological and cytologic features. Am J Clin Pathol. 1990;93:39-43. 98. Layfield LJ, Gopez EV. Cystic lesions of the salivary glands: cytologic features in fine-needle aspiration biopsies. Diagn Cytopathol. 2002;27:197-204. 99. Jayaram G, Khurana N, Basu S. Crystalloids in a cystic lesion of parotid salivary gland. Diagn Cytopathol. 1993;9:70-1. 100. Granter SR, Renshaw AA, Cibas ES. Nontyrosine crystalloids in fine-needle aspiration specimens of the parotid gland: a report of two cases and review of the literature. Diagn Cytopathol. 1999;20:44-6. 101. Chhieng DC, Argosino R, McKenna B, et al. Utility of fine needle aspiration in the diagnosis of salivary gland lesions in patients infected with human immunodeficiency virus. Diagn Cytopathol. 1999;21:260-4.

Head & Neck and Salivary Gland 102. Ascoli V, Albedi FM, De Blasiis R, et al. Sialadenosis of the parotid gland: report of four cases diagnosed by fine-needle aspiration cytology. Diagn Cytopathol. 1993;9:151-5. 103. Henry-Stanley MJ, Bencke J, Bardales RH, et al. Fine needle aspiration of normal tissue from enlarged salivary glands: sialosis or missed target? Diagn Cytopathol. 1995;13: 300-3. 104. Gupta S, Sodhani P. Sialadenosis of parotid gland: a cytomorphologic and morphometric study of four cases. Analyt Quant Cytol Histol. 1998;20:225-8. 105. Wax TD, Layfield LJ, Zaleski S, et al. Cytomegalovirus sialadenitis in patients with the acquired immunodeficiency syndrome: a potential diagnostic pitfall with fine-needle aspiration cytology. Diagn Cytopathol. 1994;10:169-74. 106. Klijanienko J, Vielh P. Fine needle sampling of salivary gland lesions V. Cytology of 22 cases of acinic cell carcinoma with histologic correlation. Diagn Cytopathol. 1997;17:347-52. 107. Nagel H, Laskawi R, Büter JJ, et al. Cytologic diagnosis of acinic-cell carcinoma of salivary glands. Diagn Cytopathol. 1997;16:402-12. 108. Klijanienko J, Vielh P. Cytologic characteristics and histomorphologic correlations of 21 salivary duct carcinomas. Diagn Cytopathol. 1998;19:333-7. 109. Fyrat P, Cramer H, Feczko J, et al. Fine needle aspiration biopsy of salivary duct carcinoma: report of five cases. Diagn Cytopathol. 1997;16:526-30. 110. Khurana KK, Pitman MB, Powers CN, et al. Diagnostic pitfalls of aspiration cytology of salivary duct carcinoma. Cancer. 1997;81:373-8.

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111. Klijanienko J, Vielh P. Cytologic characteristics and histomorphologic correlations of 21 salivary duct carcinomas. Diagn Cytopathol. 1998;19:333-7. 112. Garcia-Bonafé M, Catala I, Tarragona J, et al. Cytologic diagnosis of salivary duct carcinoma: a review of seven cases. Diagn Cytopathol. 1998;19:120–3. 113. Fyrat P, Cramer H, Feczko J, et al. Fine needle aspiration biopsy of salivary duct carcinoma: report of five cases. Diagn Cytopathol. 1997;16:526-30. 114. Moore JG, Bocklage T. Fine-needle aspiration biopsy of large cell undifferentiated carcinoma of the salivary glands: presentation of two cases, literature review, and differential cytodiagnosis of high-grade salivary gland malignancies. Diagn Cytopathol. 1998;19:44-50. 115. Safneck JR, Ravinsky E, Yadzi HM, et al. Fine needle aspiration biopsy findings in lymphoepithelial carcinoma of salivary gland. Acta Cytol. 1997;41:1023-30. 116. Lussier C, Klijanienko J, Vielh P. Fine-needle sampling of metastatic non-lymphomatous tumors to the major salivary glands. A clinico-pathologic study of 40 cases cytologically diagnosed and histologically correlated. Cancer. 2000;90:350-6. 117. Zhang C, Cohen J-M, Cangiarella JF, et al. Fine-needle aspiration of secondary neoplasms involving the salivary glands. A report of 36 cases. Am J Clin Pathol. 2000;113:21-8. 118. Mandreker S, Pinto RW, Usgaonkar U. Sebaceous carcinoma of the eyelid with metastasis to the parotid region. Diagnosis by fine needle aspiration cytology. Acta Cytol. 1997;41:1636-7.

Chapter

3

Fine Needle Aspiration of Thyroid Zubair W Baloch

Thyroid nodules are common and affect up to 7% of US population; they are often seen in women and a great majority is benign. Fine needle aspiration (FNA) is considered an essential tool in providing a rational approach to the clinical management of thyroid nodules. The results of FNA can determine whether a thyroid nodule should be followed clinically or undergo surgical excision.1,2 

FNA INDICATIONS, PROCEDURE, SPECIMEN, ADEQUACY, AND CLASSIFICATION

Every patient with a palpable thyroid nodule is a candidate for FNA and should undergo further evaluation to determine if an FNA is required. Thyroid nodules measuring at least 1.0 cm in dimension can be detected by palpation and are therefore clinically significant. However, many thyroid nodules even though measuring more than 1.0 cm may not be readily palpated due to their location in the thyroid gland.3 These nodules and those measuring less than 1.0 cm are usually found during radiologic examination of the head and neck for nonthyroidal lesions. Thyroid nodules can be biopsied by palpation or under ultrasound. The latter technique is becoming the method of choice since it provides precise information regarding the location, size, and structure (solid vs. cystic) of the nodule and is highly effective in getting an adequate sample for cytologic interpretation.4

Guidelines published by both clinical and radiologic societies have shown that in the absence of aggressive behavior (invasion into surrounding structures and presence of metastatic lymph nodes) no single ultrasonographic feature can be employed to detect thyroid malignancy.5 Ultrasonographic features that are frequently observed in the most common thyroid malignancy, papillary carcinoma, include marked hypoechogenecity, microcalcifications, coarse or macrocalcifications, infiltrative margins, marked vascularity, and taller than wide shape.5,6 Similarly, it has been shown that most benign nodules on ultrasound are characterized by an equal mix of solid and cystic components, the so-called “spongiform” appearance.7 The widespread use of ultrasound has led to the detection of many incidental thyroid malignancies known as microcarcinoma; measuring less than or equal to 1.0 cm. A majority of these cases are biologically indolent.8 The other factors that increase the risk of thyroid cancer and are taken into account for selecting a thyroid nodule for biopsy include patients’ exposure to whole body and neck irradiation during childhood and history of hereditary syndromes. Thyroid FNA can be performed in many clinical settings. It is preferable that one or a group of physicians with interest and clinical experience in thyroid FNA and its interpretation should carry out the aspiration and interpretation. In the current era of widespread availability of ultrasound equipment, most thyroid nodules are biopsied under ultrasound guidance; however, larger palpable lesions

Fine Needle Aspiration of Thyroid

can be aspirated manually or under radiologic guidance. Both manual and ultrasound-guided aspiration requires experience on the part of aspirator. It has been shown that ultrasound guidance can significantly improve the diagnostic efficacy of thyroid FNA. The aspiration technique is similar to FNA of a superficially palpable nodule located in other organs. The number of needle passes in the nodule should be kept to a minimum (preferably < 4) to avoid bleeding.9,10 FNA specimens can be prepared by making air-dried and alcohol-fixed smears for staining with Romanowsky (Diff-Quik, Wright–Geimsa stains) and Papanicolaou stains, respectively. The smears can be processed alone or with a liquid-based preparation or cell block.11 The Romanowsky staining method is one of the best methods available for immediate evaluation of FNA specimens. In thyroid FNA, this stain can highlight the background watery colloid and cell architecture (papillary, monolayer sheets, and macro- and microfollicles), and distinguish between cell types (follicular, Hürthle, lymphocytes, and macrophages). Liquid-based preparations can be utilized either alone or as an adjunct to smears.11 The leading cause of a nondiagnostic thyroid FNA specimen is failure to procure a sufficient number of cells required to render a definite diagnosis. Several studies have proposed different numbers of cell groups as criteria for specimen adequacy. The presence of six follicular groups on two slides with at least 10–20 cells in each group is considered to be the best criterion. This number falls within the range provided by previous studies.12,13 Thyroid FNA specimens are usually classified by employing a tiered system (3–6 diagnostic categories). Several classification schemes have been proposed by various authors based on personal/institutional experience and clinical organizations.1,3

41

In 2007, the National Cancer Institute sponsored the “Thyroid Fine Needle Aspiration State of the Science Conference” to develop criteria for consensus cytology classification, called the Bethesda system. A key contribution of this system was to define three specific categories for indeterminate readings, each correlating with a more narrow range of malignancy risk, resulting in a six-tiered cytology classification (Table 3.1). 

CYTOMORPHOLOGY OF THYROID LESIONS

Tables 3.2 and 3.3 list the cytomorphologic features of the common benign and malignant lesions of the thyroid.

Nodular Goiter The term goiter encompasses both nodular and diffuse enlargement of the thyroid, and clinically can be divided into toxic and nontoxic variants based on the clinical symptoms and thyroid function tests (hypothyroid, euthyroid, or hyperthyroid).14,15 The cytology specimen from a goitrous nodule shows (depending on the preparation method) copious watery colloid, small, round to oval-shaped follicular cells with dark nuclei arranged in monolayer sheets, groups with follicle formation, or as single cells (Figs 3.1A to E).16 The cytoplasm is usually scant in follicular cells and can show numerous, small blue-black granules (lysosomes containing hemosiderin and lipofuscin pigments).16 Macrophages are usually present and their number depends on the presence or absence of degenerative changes or a cystic component.16,17 The aspirates from a hyperplastic/adenomatoid nodule tend to be more cellular and contain an admixture of follicular cells and oncocytic cells arranged in monolayer sheets in a background of watery colloid and macrophages.16-18

Table 3.1: Bethesda scheme for classifying thyroid FNA specimens The Bethesda System for Reporting Thyroid Cytopathology: Implied Risk of Malignancy and Recommended Clinical Management Diagnostic category •

Risk of malignancy (%)

Nondiagnostic or unsatisfactory

Usual management Repeat FNA with ultrasound guidance

•

Benign

•

Atypia of undetermined significance or follicular lesion of undetermined significance

~ 5–15%

0–3%

Clinical follow-up Repeat FNA

•

Follicular neoplasm or suspicious for a follicular neoplasm

15–30%

Surgical lobectomy

•

Suspicious for malignancy

60–75%

Near-total thyroidectomy or surgical lobectomy

•

Malignant

97–99%

Near-total thyroidectomy

42

Atlas of Fine Needle Aspiration Cytology

Table 3.2: Key cytomorphologic features of common non-neoplastic thyroid lesions Diagnostic entity

Cytomorphologic features

Nodular goiter

•

•

Papillary hyperplastic nodule

•

•

Chronic lymphocytic thyroiditis

•

•

Background: – Watery colloid with minimal or no thick colloid, macrophages with or without hemosiderin (dependent on degenerative changes in the nodule), giant cells and calcified debris – Focal random nuclear atypia in the form of nuclear enlargement and hyperchromasia (usually encountered in long standing goiter) Cells: – Admixture of follicular cells and oncocytic cells arranged in loosely cohesive groups, monolayer sheets and occasional microfollicles – Spindle shaped cells with prominent round to oval nucleus and nucleoli (indicative of reparative changes commonly seen in cystic nodules) Background: – Watery colloid with macrophages with or without hemosiderin – Multinucleated giant cells and few inflammatory cells – Hypocellular stromal fragment closely associated with cell groups Cells: – Follicular cells, flame cell and oncocytes arranged in papillary fragments with transgressing vessels – Prominent cytoplasmic vacuolation in some cases – Random nuclear enlargement, intranuclear grooves and lack of other diagnostic nuclear features of papillary thyroid carcinoma Background: – Minimal or no watery colloid (highly dependent on duration of hypothyroidism) – Round to oval deposits of thick colloid (AKA luminal colloid) – Mixed population of lymphocytes, plasma cells, lymphoid follicles, giant cells and lymphohistiocytic aggregates – Psammoma bodies, very rare – Stromal tissue fragments (dependent upon the severity of disease) Cells: – Predominantly oncocytic follicular cells (Hurthle cells) arranged in loosely cohesive groups and as single cells, few follicular cells (amount highly dependent upon duration of hypothyroidism) – Infiltration of cell groups by lymphocytes which usually appear as crushed cells (AKA lymphocytic tangles) – Random nuclear atypia of oncocytic cells in the form nuclear enlargement and prominent nucleoli – Intranuclear grooves commonly seen in oncocytic cells – Multinucleated cells and giant cells

Solitary papillary hyperplastic nodules frequently occur in children and teenagers. A radionuclide scan might show these nodules as hyperfunctioning. By histologic examination, these lesions are encapsulated and often demonstrate central cystification, with the tips of the papillae pointing to the center of the cyst. The cells lining the papillae demonstrate oncocytic cytoplasm and lack nuclear features of papillary thyroid carcinoma (PTC).19,20 The FNA specimens of solitary papillary hyperplastic

nodules demonstrate cellular smears, transgressing vessels, papillary clusters, nuclear atypia and pleomorphism, nuclear grooves, multinucleated giant cells, and cells with vacuolated cytoplasm (Figs 3.2A to C). In view of these features, some of these cases could be misclassified as “suspicious of” or consistent with papillary carcinoma. The cytologic features that seemed to be useful for differentiating solitary papillary hyperplastic nodules from papillary carcinoma included flame cell change, watery or

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Table 3.3: Key cytomorphologic features of common malignant thyroid lesions Diagnostic entity

Cytomorphologic features

Papillary carcinoma, classic variant

•

•

•

Papillary carcinoma, follicular variant

•

•

Medullary carcinoma

•

•

Background: – Dense “bubble gum” colloid. Quantity of watery colloid dependent upon cystic degeneration – Inflammatory or prominent lymphocytic component in tumors arising in chronic lymphocytic thyroiditis – Multinucleated giant cells – Irregular calcified deposits and/or psammoma bodies Cells: General features – Hypercellular smears with tissue fragments (papillary structures and/or follicles and/or monolayer sheets) – Numerous isolated atypical cells in the background, isolated cell groups with squamoid morphology – Absence of necrosis, cystic background in cystic variant and scant cellularity in desmoplastic variant – Cellular swirls Cells: Morphology – Monotonous, larger than normal follicular cells, cuboidal and/or columnar in shape – Eosinophilic to amphophilic cytoplasm – Can demonstrate wide variation in size and shape – Nuclei are large, oval shaped, overlapping, irregular nuclear membranes with multiple indentations, grooves, pseudo-inclusions; clear and/or pale nuclear chromatin and eccentric small nucleoli Background: – Watery (usually feature of macrofollicles within the tumor) and thick colloid – Few macrophages and inflammatory cells Cells: – Hypercellular smears with cells arranged microfollicles, monolayer sheets, rosettes and/or tubules – Vaguely oncocytic cytoplasm (best appreciated on Romanowsky stained preparations) – Round to oval nuclei with smooth nuclear membranes, delicate intranuclear grooves (less prominent than classic variant) – Pale nuclear chromatin, eccentric indistinct nucleoli – Intranuclear inclusions are less frequent than classic variant Background: – Colloid absent – Stromal fragment – Amyloid – Irregular calcified deposits – Giant cells Cells: – Prominent population singly scattered cells; rare loosely cohesive fragments – Round, oval or spindle cell morphology. – Plasmacytoid cells – Abundant granular cytoplasm (prominent cytoplasmic granules in Romanowsky preparations) – Salt and pepper nuclear chromatin – Indistinct nucleoli – Binucleation – Intranuclear inclusions – Calcitonin positive, TTF-1 positive and thyroglobulin negative

Contd...

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Contd... Diagnostic entity

Cytomorphologic features

Poorly differentiated carcinoma

•

Background: – Usually clean and hemorrhagic, foci of necrosis can be seen in rare cases • Cells: – Small and monotonous cells arranged in cohesive cell groups, insulae or nests – Endothelial cells can be seen at the periphery of cohesive cell groups – Round nuclei with even chromatin pattern and small indistinct nucleoli – Mitoses present – Cells with nuclear features of papillary thyroid carcinoma can be seen in some cases – TTF-1 and thyroglobulin positive, calcitonin negative

Anaplastic carcinoma

•

•

Background: – Necrosis – Stromal fragments – Naked nuclei Cells: – Variably sized round to oval and spindle shaped cells with scant cytoplasm – Usually single scattered cells, rare loosely cohesive cell groups – Squamoid cells – Marked nuclear pleomorphism – Prominent nucleoli – Bizarre cells – Multinucleated cells (rarely osteoclast type giant cells) – Few cases may demonstrate a component of cells with nuclear features of papillary carcinoma or oncocytic follicular neoplasm – Thyroglobulin and calcitonin negative, focally positive for cytokeratin and TTF-1

inspissated colloid, short nonbranching papillae, and lack of well-formed intranuclear inclusions in the examples of hyperplasia.21

Diffuse Toxic Goiter (Graves Disease) Patients with Graves disease usually do not undergo FNA; only patients with solitary nodules, which are hypofunctioning (cold) on radioiodine scan in a background of hyperfunctioning tissue, are selected for FNA. The specimens from Graves disease are usually cellular, show features similar to hyperplastic goiter, and may contain lymphocytes and oncocytic cells. Rarely the follicular cells may display focal nuclear chromatin clearing and rare nuclear grooves; however, other diagnostic nuclear features of papillary carcinoma are not observed.22-25

Autoimmune Thyroiditis/Chronic Lymphocytic Thyroiditis Hashimoto first described this condition in 1912.26 It is characterized by follicular atrophy with oncocytic (Hürthle cell) metaplasia and diffuse lymphocytic infiltration.27 It is

more common in women, with a female to male ratio of approximately 10:1. The patients usually have circulating antibodies to thyroglobulin, thyroid peroxidase (microsomal antigen), colloid antigen, and thyroid hormones. In addition, there is an increased prevalence of HLA-DR5 and a strong family history of autoimmune disorders, especially Graves disease.28,29 Fine needle aspiration is usually performed in patients with thyroiditis who present with distinct nodules, which are cold on thyroid scan. The specimens usually show scant colloid, oncocytes (Hürthle), follicular cells, lymphocytes, and a few plasma cells. The lymphocytes are usually seen in the background, percolating between cell groups, and in some cases one may see an intact lymphoid follicle (Figs 3.3A to C).27 The oncocytic follicular (Hürthle) cells may display nuclear atypia and similarly follicular cells may show some chromatin clearing and nuclear grooves; however, one should refrain from interpreting these changes as malignant.30,31 Papillary carcinoma arising in the background of thyroiditis is seen as a separate population, devoid of a lymphocytic infiltrate and with appropriate nuclear features.20

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A

B

C

D

E

Figs 3.1A to E: A case of nodular goiter demonstrating background watery colloid (A—Diff-Quik Stain), macrophages (B—Diff-Quik Stain). The alcohol fixed smear from the same case demonstrates follicular cells with small round nuclei arranged in flat sheets (C—Papanicolaou Stained Smear). Thin-Prep® preparation shows colloid and small sized follicular cells in cohesive groups (D). The histology of nodular goiter demonstrates macrofollicles filled with watery colloid (E)

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A

C In some specimens of lymphocytic thyroiditis, there may be a preponderance of oncocytic follicular (Hürthle) cells, leading to a diagnosis of Hürthle cell neoplasm. This diagnosis should only be entertained in cases, which display a separate monotonous population of Hürthle cells devoid of a lymphocytic infiltrate. Similarly, an extensive lymphocytic infiltrate that may appear monotonous can be mistaken for malignant lymphoma arising in lymphocytic thyroiditis. If one suspects lymphoma, it is advisable that an aliquot of specimen be submitted for flow cytometry to confirm the morphologic suspicion.32,33

Follicular Neoplasm/Suspicious for Follicular Neoplasm Thyroid (FNA) cannot distinguish between benign and malignant follicular patterned lesions. Both follicular adenoma and carcinoma demonstrate similar

B

Figs 3.2A to C: A case of hyperplastic nodule with papillary architecture demonstrating vague papillary configuration consisting of follicular cells intermixed with stromal cells (A—Papanicolaou stain). The cells lining the periphery of papillary groups demonstrate “picket fence” arrangement without any considerable nuclear overlapping and crowding (B—Papanicolaou stain). The histology of this lesion show papillary groups with hypocellular edematous core (C—Hematoxylin and Eosin stain)

cytomorphology.34 The cytologic diagnosis of “neoplasm” reflects the limitations of thyroid cytology.35,36 The diagnosis of follicular carcinoma is based only on the demonstration of capsular and/or vascular invasion.37-39 Several authors have shown that, at most, 20–30% of cases diagnosed as “follicular neoplasm” are diagnosed as malignant on histologic examination and the rest are either follicular adenomas or cellular adenomatoid nodules, that is, benign.40,41 Interestingly, half of the malignant cases are diagnosed as follicular variant of papillary thyroid carcinoma (FVPTC). The FNA of a follicular neoplasm is usually hypercellular and shows a monotonous population of follicular cells with minimal or absent background watery colloid. The colloid is usually seen as round dense eosinophilic deposits on Romanowsky stain with or without surrounding cells.42,43 The edges of dense colloid aggregates usually

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A

B

C

Figs 3.3A to C: Cytomorphology of chronic lymphocytic thyroiditis demonstrating oncocytic follicular cells (AKA Hurthle cells) arranged in loosely cohesive groups infiltrated by crushed lymphocytes (A—Diff-Quik stain, B—Papanicolaou stain). Histology of such cases demonstrates oncocytic metaplasia of follicular epithelium and presence of lymphoid germinal centers (C—Hemotoaxylin and Eosin stain)

demonstrate cracks/splits (vertical to the center) and can mimic psammoma bodies; however, on close inspection it lacks the typical “calcified rings.” The lesional cells can be seen as three-dimensional groups or microfollicles with prominent nuclear overlapping and crowding (Figs 3.4A to C).39,43

Oncocytic Follicular (AKA Hürthle Cell) Neoplasm/Suspicious for Oncocytic Follicular (AKA Hürthle Cell) Neoplasm The oncocytic cells (oxyphil, Askanazy cells and Hürthle cells) are usually larger than follicular cells and have distinct cell borders, voluminous granular eosinophilic cytoplasm, and eccentrically or centrally placed round nuclei with a prominent nucleolus. By electron microscopy, the cytoplasm is filled with enlarged mitochondria. They are commonly seen in long-standing Graves disease, autoimmune

thyroiditis, thyroids affected by radiation, follicular-derived neoplasms, and some cases of adenomatoid goiter.44,45 A majority of oncocytic follicular/Hürthle cell neoplasms of the thyroid are solitary mass lesions that show complete or partial encapsulation. On gross pathologic examination, they are distinguished from the surrounding thyroid by their distinctive brown to mahogany color. Oncocytic follicular neoplasms are defined as being composed of at least 75% oncocytic cells. These can be divided into benign and malignant categories based on the same pathologic criteria as applied in the diagnosis of follicular carcinoma, which is, the identification of capsular and/or vascular invasion.44,46 Fine needle aspiration specimens obtained from an oncocytic follicular neoplasm usually demonstrate a monotonous population of oncocytic cells arranged in cohesive groups/tissue fragments and as single cells. The

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A

B

C

Figs 3.4A to C: A case diagnosed as follicular neoplasm/ suspicious for follicular neoplasm demonstrating a monotonous population of follicular cells arranged in follicular patterned groups (A—Papanicolaou stain). The ThinPrep® demonstrates a large cellular group of follicular cells (B). On surgical excision this case showed an encapsulated follicular patterned nodule without any evidence of invasive characteristics; diagnosed as follicular adenoma (C—Hematoxylin and Eosin stain)

tissue fragments usually occur as follicles, monolayer sheets, and as two to three cell thick cords (reminiscent of trabecular growth pattern seen in histology specimens) with minimal nuclear overlapping and crowding as compared with cells seen in aspirates of non-oncocytic lesions (Figs 3.5A to C).47,48 Neoplastic oncocytic follicular cells, that is, obtained from either adenoma or carcinoma are usually large and round to oval or polygonal in shape with well-defined cell borders.37,49 Random nuclear atypia is commonly observed in oncocytic follicular lesions; this can be seen in the form of nuclear enlargement, multinucleation, cellular pleomorphism, and prominent “cherry red” nucleoli. Intranuclear grooves are common in nonpapillary oncocytic follicular lesions; however, the nuclei maintain a round shape with prominent nucleoli and other major diagnostic features of papillary carcinoma

are not seen.50 The nuclear features of oncocytic follicular cells are less pronounced in monolayer preparations.51,52 The FNA specimens of oncocytic neoplasms usually contain thick colloid that occurs in the form of circular deposits representing the lumen of thyroid follicles. It has been shown that the presence of intracytoplasmic lumens and transgressing vessels are common in FNA specimens of neoplastic oncocytic follicular lesions (Fig. 3.5C).53 It has been suggested that aspirates of oncocytic lesions demonstrating nuclear pleomorphism, macro-nucleoli, bi- to multinucleation, or necrosis are more indicative of oncocytic follicular carcinoma.54,55 One must be aware that similar atypical nuclear features can also be seen in aspirates of non-neoplastic oncocytic lesions seen in longstanding goiter, toxic nodular goiter, and chronic lymphocytic thyroiditis.

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A

C

The differential diagnosis of oncocytic follicular neoplasm includes other tumors of the thyroid gland with oncocytic cytoplasm. These include variants of PTC such as oncocytic variant, Warthin-like variant, and tall cell variant of papillary carcinoma, oncocytic variant of medullary thyroid carcinoma (MTC), and granular cell tumor of the thyroid. An occasional intrathyroidal oncocytic parathyroid adenoma can also mimic an oncocytic follicular neoplasm.

Malignant Neoplasms Papillary Thyroid Carcinoma and Its Variants Well-differentiated thyroid carcinomas are the most common form of malignant thyroid tumors; they behave in an indolent manner and have an excellent prognosis. They are commonly seen in young adults, whereas, the

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B

Figs 3.5A to C: Oncocytic follicular neoplasm (AKA Hurthle cell neoplasm) shows oncocytic follicular cells arranged in loosely cohesive groups without nuclear overlapping and crowding (A and B). In some cases one can easily identify the transgressing blood vessels among the oncocytic follicular groups (C)

less-differentiated and anaplastic tumors of the thyroid are prevalent in an older age group. Papillary carcinoma is the most common form of thyroid malignancy and makes up to 80% of thyroid malignancies diagnosed in nonendemic goiter regions. It usually presents before the age of 40 years and is more frequent in women than in men. It can range in size from less than or equal to a centimeter, defined as microcarcinoma, to a large tumor mass with infiltration into surrounding neck structures. Classic PTC behaves in an indolent fashion; however, some histologic variants of papillary carcinoma behave in an aggressive manner with distant metastasis and even patient death. PTC most commonly metastasizes via lymphatics; however, vascular invasion can also be seen.14 The classic variant of papillary carcinoma predominantly consists of true papillae, that is, fingerlike projection

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with cores containing vessel(s) and connective tissue surrounded by tumor cells. The papillae are of different sizes and can display a complex branching pattern. It is not uncommon to encounter a few follicles intermixed with papillae in this variant of PTC. Minor cystic changes are common. By light microscopy, the tumor cells are larger and are cuboidal to low columnar and contain amphophilic to slightly eosinophilic cytoplasm. Nuclei are relatively large and oval in shape and show nuclear indentations/grooves and round intranuclear cytoplasmic pseudoinclusions. Nucleoli are usually small and situated close to the nuclear membranes (eccentric), along with the heterochromatin, thereby causing the nuclear membrane to appear “thick” and much of the interior of the nucleus to be “pale”, “empty”, “clear”, or “ground glass” in appearance. Follicles may be colloid filled or empty and occur as micro or macrofollicles.56 The cytodiagnosis of PTC is based on major and minor cytologic criteria. The major diagnostic feature is the characteristic nuclear morphology, regardless of cytoplasmic features, growth pattern, special stains, and immunohistochemical markers. This holds true for a majority of cases of PTC; however, some variants of PTC may be difficult to diagnose due to lack of some of the nuclear features.57,58 The FNA specimen of PTC is usually cellular and shows tumor cells arranged in papillary groups, (Figs 3.6A and B) three-dimensional clusters or as single cells in a background of watery or thick “ropy” colloid (AKA chewing gum colloid), nuclear or calcific debris, macrophages, and stromal fragments. The cell groups may show a typical concentric arrangement of lesional cells described as “cellular swirls”.13,59 The individual tumor cells are enlarged, elongated, that is, oval in shape with eosinophilic cytoplasm (cytoplasmic eosinophilia is common in Romanowsky-stained preparations but is usually indistinct in alcohol-fixed Papanicolaou-stained preparations; this also holds true for monolayer preparations). The nuclei show elongation, membrane thickening, chromatin clearing, grooves, and inclusions (Figs 3.6C to E). The nucleoli are usually small and eccentric. Intranuclear grooves and inclusions can be seen in other benign and malignant conditions of the thyroid. These include Hashimoto thyroiditis, nodular goiter, hyalinizing trabecular neoplasm (HTN), Hürthle cell tumors, and medullary carcinoma.20,60 Psammoma bodies occur in about 20% of cases of PTC. These are lamellated round to oval calcified structures that represent the “ghosts” of dead papillae. They are

more prominent in Papanicolaou-stained preparations and demonstrate basophilic, golden brown, or lavender staining. Multinucleated histiocytes are common in FNA specimens of PTC.61 These can be variable in size, shape, and number of nuclei. Squamous metaplasia can be seen in FNA specimens of PTC; however, it is more common in cases with cystic degeneration.62 The cytologic features of PTC may not be readily evident in monolayer preparations.63 Typically, these preparations demonstrate variable-sized tissue fragments with cellular crowding, overlapping, and nuclear molding giving rise to a “jigsaw puzzle” appearance. Intranuclear pseudoinclusions are not frequently found as compared with traditional cytologic preparations. The FVPTC is the second most common variant of PTC. Its diagnosis may be made when more than 70% of the tumor consists of neoplastic follicles lined by cells demonstrating diagnostic nuclear morphology of PTC. Three distinct types of follicular variant exist represented by the infiltrative type, the diffuse follicular variant, and the encapsulated follicular variant. The encapsulated FVPTC is characterized by the presence of a capsule around the lesion and is associated with an excellent prognosis. In some cases, the diagnosis of this particular variant of papillary carcinoma can be difficult due to the presence of multifocal rather than a diffuse distribution of nuclear features of PTC. Because of this peculiar morphologic presentation, these tumors can be misdiagnosed as adenomatoid nodule or follicular adenoma. Some authors have suggested that these tumors should be classified as “tumors of undetermined malignant potential” due to their excellent prognosis. However, others have shown that some cases belonging in this category can lead to distant metastases. The cytologic interpretation of FVPTC can be challenging due to a paucity of diagnostic nuclear features.64 The cytologic samples from FVPTC usually show enlarged follicular cells arranged in monolayer sheets and follicular groups in a background of thin and thick colloid (Figs 3.7A and B). The individual tumor cells show nuclear elongation, chromatin clearing, and thick nuclear membranes. The intranuclear grooves in FVPTC are delicate and do not traverse the entire length of nucleus; however, nuclear grooves and inclusions are very scarce. Thus, a majority of cytologic samples of FVPTC are diagnosed as suspicious for papillary carcinoma.65,66 Cystic PTC is often encapsulated and the cystic component usually ranges from half to occupying most of the

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A

B

C

D

E

Figs 3.6A to E: A case of classic variant of papillary thyroid carcinoma showing a cellular specimen with lesional cells arranged in complex papillary configurations (A—Papanicolaou stain). The tumor cells demonstrate enlarged oval nuclei (B—Papanicolaou stain), intranuclear grooves, eccentric nucleoli (C—Papanicolaou stain) and intranuclear inclusions-arrowhead (D—Papanicolaou stain). The histology demonstrates well formed papillae with vascular core lined by cells with nuclear features of papillary carcinoma (E—Hematoxylin and Eosin stain)

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A

B

Figs 3.7A and B: In cytology preparations the follicular variant of papillary thyroid carcinoma shows lesional cells arranged in follicular groups and tubular structures. The nuclear features of papillary carcinoma can be readily evident except for well-formed intranuclear inclusions (A—Papanicolaou Stain). The histology shows variably sized follicles containing thick colloid and lined by cells demonstrating diagnostic nuclear features

lesion. The papillary fronds lining the cyst wall may be visible on gross examination. The FNA specimens of this tumor contain large numbers of hemosiderin-laden histiocytes and considerable cellular debris. Sheets of intact follicular cells may be seen, which resemble those from cellular adenomatoid nodules; however, these will demonstrate nuclear cytology suspicious or diagnostic of PTC. In a benign cystic lesion, follicular cells are usually shrunken and demonstrate nuclear degeneration, and the presence of apparently well-preserved cells is therefore a warning of a possible PTC. These cells are slightly larger than normal follicular cells; their cytoplasm is denser; they lack paravacuolar granules; and their nuclei are larger than normal nuclei, not pyknotic, and demonstrate at least some intranuclear grooves and/or intranuclear inclusions. Some groups of larger cells with clear cytoplasm and dense, convex cellular borders are also seen. The edges of these cellular clusters have a scalloped appearance, giving rise to small papillary fragments. Squamous metaplasia has been reported in cases of cystic PTCs. Dense globules of pink-staining colloid (“pink balls”) may be present.67-69 Tall cell variant of PTC is an aggressive form of PTC and can lead to multiple local recurrences, distant metastases, and even death. This tumor was initially described as a papillary tumor containing oncocytic cells that are at least twice as long (tall) as they are wide; however, this has been modified to tumor cells being three times as tall as their width.70 The cytologic samples from this tumor contain elongated cells with sharp cytoplasmic borders, granular

eosinophilic cytoplasm, and variably sized nuclei with nuclear features of papillary carcinoma.71,72 The diagnostic nuclear features of PTC are readily found in aspirates of this variant. The intranuclear inclusions can be multiple within the same nucleus, giving rise to a “soap-bubblelike” appearance.72 Warthin-like variant of PTC morphologically resembles “Warthin’s tumor” of the salivary glands. These tumors can show papillary fragments or cellular groups of oncocytic cells with PTC nuclei infiltrated by lymphocytes and plasma cells. Aspirates from this form of PTC can be mistaken for chronic lymphocytic thyroiditis. However, the tumor cells have distinct PTC nuclei that do not resemble oncocytic follicular (Hürthle) cells.73

Diffuse Sclerosis Variant of PTC The diffuse sclerosis variant of papillary thyroid carcinoma (DSV-PTC) is rare, representing only approximately 3% of all papillary carcinomas. The tumor, which most often affects children and young adults, may present as a bilateral goiter. In histologic section, the tumor cells permeate the gland outlining the intraglandular lymphatics. Tumor papillae have associated areas of squamous metaplasia. Numerous psammoma bodies are found; lymphocytic infiltrates are also found around the tumor foci. This particular feature gives rise to a “snow storm” appearance on ultrasound examination. Cytologic preparations show tumor cells with nuclear features of papillary carcinoma arranged in nests and numerous psammoma bodies.

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Some cases may also demonstrate a brisk lymphocytic infiltrate around the tumor cell groups and in the background. Squamous metaplasia is commonly seen in aspirates of DSV-PTC.74-76 Columnar-cell variant of papillary thyroid carcinoma (CCV-PTC) is rare, occurring in adults of all ages. It is characterized by tall, slender, and columnar cells arranged in papillae and trabeculae. The cytoplasm is usually clear, sometimes eosinophilic or amphophilic. Nuclei are hyperchromatic and elongated in the tall cylindrical cells. Intranuclear grooves and intranuclear cytoplasmic inclusions are rare. These elongated nuclei differ sufficiently in their positions in the cells to produce a stratified or pseudostratified appearance. Cytologic preparations of this tumor demonstrate cohesive cell fragments with a prominent papillary architecture. The tumor cells appear columnar in shape with pale cytoplasm that tapers at one end. Nuclear palisading and stratification are prominent at the periphery of papillary fragments. Intranuclear grooves and intranuclear inclusions are rare as are psammoma bodies and multinucleated tumor giant cells. Because of the scarcity or lack of diagnostic nuclear features, the aspirates of CCV-PTC can be mistaken for medullary carcinoma or metastasis (especially from colon) to thyroid gland.74,77,78

Medullary Thyroid Carcinoma Medullary thyroid carcinoma (MTC) arises from the C-cells of the thyroid and constitutes about 10% of all malignant thyroid neoplasms. This thyroid tumor is peculiar among thyroid neoplasms due to its clinical presentation, familial incidence, association with lesions of other endocrine organs, and variable morphology.14 In the absence of genetic predisposition, no epidemiologic factors have been identified for medullary cancer. Germ line mutations lead to C-cell hyperplasia, which has been implicated as the precursor/in situ lesions of the medullary carcinoma in this genetically determined subset of cases.79 Medullary carcinoma usually presents as a firm painless thyroid nodule. Approximately 50% of patients show lymph node metastases and up to 15% can show distant metastases at presentation (commonly bone and liver). Typically, in histologic sections, MTC consists of sheets and solid nests of round cells with granular eosinophilic cytoplasm and a prominent eccentric nucleus with evenly dispersed chromatin (Fig. 3.8D). The tumor nests can contain randomly distributed pleomorphic nuclei or even multinucleated tumor cells. In some cases, the tumor

53

cells can show prominent nuclear grooves and even inclusions. The tumor cells may exhibit marked nuclear pleomorphism, prominent central nucleoli, and eosinophilic granular cytoplasm; such cases can be mistaken for oncocytic follicular neoplasms. MTC also exhibits a spindle cell morphology and mimics either primary or metastatic mesenchymal tumors of the thyroid, anaplastic carcinoma, or tumors of thymic origin. Several other histologic patterns of this tumor have been described, which include small cell, oncocytic and squamous cell, giant cell, pseudopapillary, carcinoid-like, insular, and mixed medullary and papillary. Amyloid can be seen in up to 80% of medullary cancers; therefore, Congo-red staining and polarized light microscopy are used to diagnose these tumors. The gold standard for the diagnosis of MTC is the immunostain for calcitonin that stains both the tumor cells and the amyloid. However, calcitonin-negative MTC has been reported in the familial setting. Another marker, which can be used to highlight MTC, is carcinoembryonic antigen (CEA). Increased levels of serum CEA, along with calcitonin values, are often used for follow-up in patients at risk of recurrent or metastatic disease. Lymph node metastases involving regional and mediastinal nodes can be seen in up to 50% of MTC cases. These tumors also show a propensity toward extrathyroidal extension and involvement of the contralateral lobe. The common sites for distant metastasis include lung, bone, liver, and adrenals.80,81 Fine needle aspiration specimens of MTC display a spectrum of morphologic patterns similar to surgical pathology specimens. The majority of MTC FNA specimens are cellular consisting of round to oval cells arranged mainly as single cells or loosely cohesive groups. The tumor cells show ample granular cytoplasm with eccentric nuclei imparting a plasmacytoid appearance to the cells. The nuclear chromatin is similar to that seen in neuroendocrine tumors; intranuclear inclusions (Figs 3.8A and B), and multinucleated cells may be seen. Marked nuclear pleomorphism is uncommon; however, when present the cases are indistinguishable from aspirates of anaplastic thyroid carcinoma. The neoplastic cells can assume a “spindle shape” and appear mesenchymal in origin (Fig. 3.8C). Amyloid may be observed as acellular material and can be distinguished from the thick colloid of papillary carcinoma by performing a Congo-red stain. The diagnosis of MTC can be confirmed by performing immunostains for calcitonin and thyroglobulin.82-84

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A

B

C

D

Figs 3.8A to D: Medullary carcinoma can consists of plasmacytoid cells (A—Diff-Quik Stain) with neuroendocrine chromatin (B—Papanicolaou stain) or spindle shaped cells (C—Papanicolaou stain). The histology shows nests of tumor cells embedded in sclerotic stroma containing amyloid (D—Hematoxylin and Eosin stain)

The cytomorphologic diagnosis of MTC can be challenging due to morphologic variability. The differential diagnosis of MTC includes HTN, oncocytic follicular neoplasm (AKA Hürthle cell neoplasm), PTC, follicular neoplasm with solid and trabecular growth pattern, poorly differentiated carcinoma/insular carcinoma, anaplastic carcinoma, plasmacytoma, and metastatic tumors to the thyroid especially melanoma.

Poorly Differentiated Carcinoma According to World Health Organization (WHO), the entity of poorly differentiated thyroid carcinoma (PDTC) is a follicular cell-derived neoplasm that shows limited evidence of structural follicular cell architecture and

occupies both morphologically and behaviorally an intermediate position between differentiated and undifferentiated (anaplastic) carcinomas.70 Insular growth pattern with areas of coagulative necrosis, nuclear pleomorphism, and more than 3 mitoses per 10 high-power field is considered by many experts as diagnostic features of PDTC. In histologic sections, these tumors usually demonstrate an epicenter of well-differentiated thyroid carcinoma, that is, PTC or follicular carcinoma. Lymphovascular invasion, metastases to regional lymph nodes, lung, and bones are commonly associated with PDTC.85-87 Aspirates of these tumors are cellular and demonstrate a monotonous population of cells arranged in large solid groups with cell crowding and overlapping,

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A

B

C

Figs 3.9A to C: Poorly differentiated carcinoma of the thyroid in cytologic preparations demonstrates a monotonous population of cells arranged in cohesive groups with nuclear overlapping and crowding (A—Diff-Quik Stain, B—Papanicolaou Stain). The histology shows lesional cells arranged in insulae/nests with a prominent intratumoral vascular network (C—Hematoxylin and Eosin stain)

mitoses, and apoptotic bodies. On high-power examination, nuclear pleomorphism is readily evident (Figs 3.9A and B). Endothelial wrapping of the tissue fragment can be seen in some cases (Fig. 3.9C). Since this growth pattern can also be encountered in MTC and secondary tumors of the thyroid such as metastatic neuroendocrine carcinoma; it is prudent to confirm the diagnosis of PDTC by performing immunostains for transcription factor 1 (TTF-1), thyroglobulin, and calcitonin.88,89

Anaplastic Carcinoma Anaplastic carcinoma of the thyroid is one of the most aggressive and fatal human tumors. It usually presents in older individuals and is more common in regions of endemic goiter. The aspirates from anaplastic carcinoma usually do not pose any diagnostic difficulties; they can be readily classified as malignant due to extreme cellular

pleomorphism and obvious malignant features.15 Various morphologic patterns have been described including giant cell/osteoclastoma type,90 spindle cell/sarcomatous type,91,92 squamous cell variant,92 angiomatoid type,93 and paucicellular variant94 (Figs 3.10A and B). The latter shows few malignant cells embedded in densely sclerotic stroma resembling Riedel disease; this variant can give rise to nondiagnostic specimens on FNAB.95,96 By immunohistochemistry, anaplastic carcinomas usually do not show thyroglobulin expression, and TTF-1 expression occurs focally in 0–50% of cases.92,97-99 A majority of anaplastic carcinomas will stain for cytokeratin.100 

RARE TUMORS OF THYROID GLAND

Hyalinizing trabecular neoplasm is a rare tumor of the thyroid. Aspirates of HTN that show cohesive lesional cells

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A

B

Figs 3.10A and B: Anaplastic carcinoma is characterized by spindle shaped tumor cells with extremes of nuclear pleomorphism and bizarre cells (A—Papanicolaou stain). The histology shows the pleomorphic spindle shaped tumor cells which mimic a sarcoma (B—Hematoxylin and Eosin stain)

with easily identifiable, well-formed intranuclear inclusions and grooves embedded or closely associated with an acellular matrix.101 Mucoepidermoid carcinoma (MEC) and sclerosing mucoepidermoid carcinoma (SMEC) of the thyroid show distinct morphologies and behave in an indolent fashion.102-105 MEC is characterized by foci of epidermoid/ squamous differentiation and duct-like elements. SMEC is usually seen in a background of lymphocytic thyroiditis as a poorly circumscribed mass showing tumor cells arranged in small sheets, anastomosing trabeculae, and narrow strands. Squamous differentiation is a prominent feature. Mucinous differentiation can also be seen in some tumor nests. These tumors show a prominent sclerohyaline stroma, which is heavily infiltrated by eosinophils.105 FNAB specimens of both MEC and SMEC show epidermoid and glandular elements with stromal fragments. Eosinophils can be seen in aspirates of SMEC. The squamous elements can be mistaken for a primary or secondary squamous carcinoma of the thyroid.104-107 Primary thyroid lymphoma is a rare but well-recognized thyroid neoplasm.108-110 It virtually always arises in a gland that is immunologically abnormal, and most lymphomas occur in the background of chronic lymphocytic thyroiditis.111,112 The thyroid gland is recognized as a MALT organ, and many of the primary lymphomas are MALTOMAS. These tumors show the morphologic and immunologic features of MALT lymphoma: they have plasmacytoid tumor cells and demonstrate lymphoepithelial

lesions.109 Less common are follicular center cell lymphomas, which may be small cell or more frequently large cell in morphology.109,113,114 The diagnosis can be made by FNA of the mass, and if lymphoma is suspected, the material can be sent for special studies including flow cytometry and molecular analysis in order to characterize the subtype of lymphoma.115 Rare examples of other hematopoietic tumors have been reported to involve the thyroid and may be the presenting symptom of a systemic hematologic disease including Langerhans cell histiocytosis,116,117 mycosis fungoides, Rosai–Dorfman disease,118 multiple myeloma,119,120 and various leukemias.121 The incidence of secondary tumors of thyroid ranges from 1.25% to 25%. Breast, lung, and kidney (mostly renal cell carcinoma) represent the most common primary sites that can give rise to metastases to thyroid.122-126 In some cases, secondary tumors can present as solitary nodules and may be mistaken for a primary thyroid tumor. Similarly on histology and FNA, some metastatic tumors can also be difficult to differentiate from primary thyroid neoplasms; clear cell carcinoma of kidney versus follicular carcinoma or adenoma with clear cell change, metastatic neuroendocrine carcinoma versus MTC, and poorly differentiated lung carcinoma versus anaplastic or PDTC. However, immunohistochemistry and a detailed history are always helpful for differentiation between primary and secondary tumors of thyroid.125,127

Fine Needle Aspiration of Thyroid



ROLE OF SPECIAL STUDIES IN THE DIAGNOSIS OF THYROID TUMORS IN CYTOLOGIC SPECIMENS

Immunohistochemistry All follicular-derived thyroid lesions both benign and malignant express TTF-1 and thyroglobulin. This immunopanel is helpful in differentiating primary versus secondary tumors of the thyroid. The diagnosis of medullary carcinoma can be established in FNA specimens by performing immunostains for calcitonin and calcitonin gene-related peptide. Medullary carcinoma also stains positively for CEA, chromogranin, and synaptophysin.82 Several reports have been published regarding the use of various immunohistochemical markers that can differentiate papillary carcinoma from other follicular epithelial-derived lesions of the thyroid. From an extensive list of these markers, the ones that have shown some promise include cytokeratin-19, HBME-1, and galectin-3. However, none of these have proven to be specific since all can be expressed in some benign lesions of thyroid. In addition, spurious staining of benign thyroid epithelium in chronic lymphocytic thyroiditis can lead to a false-positive diagnosis of malignancy.128 Therefore, it is suggested that if the confirmation of a cytologic diagnosis of papillary cancer requires use of immunohistochemistry, then it should be carried out by employing an immunopanel consisting of markers mentioned above in a sample containing enough cells or in cell block preparations.

Molecular Genetics/Diagnosis In the past decade, the literature on the thyroid gland has been focused mainly on the role of various biologic events and genetic determinants in the pathogenesis of thyroid tumors. Molecular biology/diagnosis of PTC: Several gene mutations and translocations have been described in PTC. The most commonly discussed are BRAF and RAS mutations; RET gene rearrangements and PAX8-PPAR gamma translocations (usually in FVPTC).129 Rearrangements of RET gene, known as RET/PTC, have been identified in papillary carcinoma of thyroid. The RET proto-oncogene is normally expressed in cells of neural crest origin and is located on chromosome 10q11.2 and codes for a cell membrane receptor tyrosine kinase. In normal thyroid, wild-type RET is only expressed in C-cells and not follicular cells. RET/ PTC seen in papillary carcinomas occurs due to fusion of tyrosine kinase domain of RET to the 5’ portion of the

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various genes. To date more than 10 novel types of rearrangements have been described in papillary carcinoma. RET/PTC 1 and 3 are the most common forms that occur in sporadic papillary carcinoma. The prevalence of RET/ PTC in papillary carcinoma varies significantly among various geographic regions; in the United States, it ranges from 11% to 43%. In sporadic tumors, RET/PTC1 is the most common form of rearrangement (60–70%) followed by RET/PTC3 (20–30%).129,130 It is now well-known that the RAF/MEK/ERK pathway is a significant contributor to the malignant phenotype associated with deregulated RAS signaling. BRAF activating mutations in thyroid cancer are most commonly the BRAF V600E mutation, and have been found in 29–69% of papillary thyroid cancers, 13% of poorly differentiated cancers, and 10% of anaplastic cancers. Interestingly, presence of BRAFV600E correlates with variants of PTC. Studies consisting of large cohorts of patients have shown a strong correlation of BRAF mutation with nonfavorable clinicopathologic features. BRAF mutations are independent of RET/PTC translocations and RAS mutations.131 Interestingly, molecular analysis has shown that FVPTC is a hybrid neoplasm of follicular carcinoma and PTC. RAS gene mutations and PAX8-PPAR-gamma translocation, abnormalities seen in follicular adenoma and carcinoma, are exclusively seen in FVPTC and not in classical PTC. Similarly, RET gene translocations and BRAF mutations that are common in classic PTC are rare in cases of FVPTC. Therefore, in view of morphologic features, clinical behavior and molecular analysis encapsulated FVPTC most likely is a hybrid of papillary carcinoma and follicular adenoma or carcinoma.131 Molecular analysis of FNA samples for BRAF and RAS mutations and RET/PTC translocations is recommended in the preoperative diagnosis of PTC in cases diagnosed as atypia of undetermined significance/follicular lesion of undetermined significance, follicular neoplasm/suspicious for follicular neoplasm, or suspicious for malignancy.132,133 Most molecular analyses performed on thyroid FNA specimens demonstrate a high-positive predictive value; however, recently a molecular test comprising a 162 gene panel has been developed to offer a negative predictive value of more than 90%.134 DNA microarray analysis: Recently, DNA microarray analysis of thyroid FNA samples has been shown to successfully distinguish between the majority of benign and malignant thyroid lesions. Lubitz et al. evaluated

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22 FNA samples by unsupervised hierarchical cluster analysis using a list of 25 differentially expressed genes. These authors found that their FNA cohort could be separated into three clusters: malignant, benign, and indeterminate. The benign and malignant groups were in complete concordance with histologic follow-up except in one case. Interestingly, in the indeterminate group, two cases were FVPTC on histologic examination.135 

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Fine Needle Aspiration of Thyroid 98. Fabbro D, Di Loreto C, Beltrami CA, et al. Expression of thyroid-specific transcription factors TTF-1 and PAX-8 in human thyroid neoplasms. Cancer Res. 1994;54(17):4744-9. 99. Bejarano PA, Nikiforov YE, Swenson ES, et al. Thyroid transcription factor-1, thyroglobulin, cytokeratin 7, and cytokeratin 20 in thyroid neoplasms. Appl Immunohistochem Mol Morphol. 2000;8(3):189-94. 100. LiVolsi VA, Brooks JJ, Arendash-Durand B. Anaplastic thyroid tumors. Immunohistology. Am J Clin Pathol. 1987; 87(4):434-42. 101. Bishop JA, Ali SZ. Hyalinizing trabecular adenoma of the thyroid gland. Diagn Cytopathol. 2011;39(4):306-10. 102. Cameselle-Teijeiro J. Mucoepidermoid carcinoma and solid cell nests of the thyroid. Hum Pathol. 1996;27(8):861-3. 103. Chan JK, Albores-Saavedra J, Battifora H, et al. Sclerosing mucoepidermoid thyroid carcinoma with eosinophilia. A distinctive low-grade malignancy arising from the metaplastic follicles of Hashimoto’s thyroiditis. Am J Surg Pathol. 1991;15(5):438-48. 104. Baloch ZW, Solomon AC, LiVolsi VA. Primary mucoepidermoid carcinoma and sclerosing mucoepidermoid carcinoma with eosinophilia of the thyroid gland: a report of nine cases. Mod Pathol. 2000;13(7):802-7. 105. Solomon AC, Baloch ZW, Salhany KE, et al. Thyroid sclerosing mucoepidermoid carcinoma with eosinophilia: mimic of Hodgkin disease in nodal metastases. Arch Pathol Lab Med. 2000;124(3):446-9. 106. Bondeson L, Bondeson AG. Cytologic features in fineneedle aspirates from a sclerosing mucoepidermoid thyroid carcinoma with eosinophilia. Diagn Cytopathol. 1996;15(4):301-5. 107. Cameselle-Teijeiro J, Febles-Perez C, Sobrinho-Simoes M. Cytologic features of fine needle aspirates of papillary and mucoepidermoid carcinoma of the thyroid with anaplastic transformation. A case report. Acta Cytol. 1997;41 4 Suppl:1356-60. 108. Hamburger JI, Miller JM, Kini SR. Lymphoma of the thyroid. Ann Intern Med. 1983;99(5):685-93. 109. Kossev P, Livolsi V. Lymphoid lesions of the thyroid: review in light of the revised European-American lymphoma classification and upcoming World Health Organization classification. Thyroid. 1999;9(12):1273-80. 110. Straus DJ. Primary thyroid lymphoma, a rare disease with a good treatment outcome. J Surg Oncol. 2010;101(7):543-4. 111. Sarinah B, Hisham AN. Primary lymphoma of the thyroid: diagnostic and therapeutic considerations. Asian J Surg. 2010;33(1):20-4. 112. Graff-Baker A, Sosa JA, Roman SA. Primary thyroid lymphoma: a review of recent developments in diagnosis and histology-driven treatment. Curr Opin Oncol. 2010; 22(1):17-22. 113. Das DK, Gupta SK, Francis IM, et al. Fine-needle aspiration cytology diagnosis of non-Hodgkin lymphoma of thyroid: a report of four cases. Diagn Cytopathol. 1993; 9(6):639-45.

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114. Skacel M, Ross CW, Hsi ED. A reassessment of primary thyroid lymphoma: high-grade MALT-type lymphoma as a distinct subtype of diffuse large B-cell lymphoma. Histopathology. 2000;37(1):10-8. 115. Morgen EK, Geddie W, Boerner S, et al. The role of fineneedle aspiration in the diagnosis of thyroid lymphoma: a retrospective study of nine cases and review of published series. J Clin Pathol. 2010;63(2):129-33. 116. Behrens RJ, Levi AW, Westra WH, et al. Langerhans cell histiocytosis of the thyroid: a report of two cases and review of the literature. Thyroid. 2001;11(7):697-705. 117. Chong VF. Langerhans cell histiocytosis with thyroid involvement. Eur J Radiol. 1996;22(2):155-7. 118. Tamouridis N, Deladetsima JK, Kastanias I, et al. Cold thyroid nodule as the sole manifestation of Rosai-Dorfman disease with mild lymphadenopathy, coexisting with chronic autoimmune thyroiditis. J Endocrinol Invest. 1999; 22(11):866-70. 119. Patel R, Bayliss E, Trotter J. Thyroid involvement in multiple myeloma. Aust N Z J Med. 1992;22(2):139-41. 120. Stewart JM, Krishnamurthy S. Fine-needle aspiration cytology of a case of HIV-associated anaplastic myeloma. Diagn Cytopathol. 2002;27(4):218-22. 121. Solivetti FM, Thorel MF, Ferraro C, et al. [Thyroid gland involvement in acute leukemia. Ultrasonographic aspects of a case]. Radiol Med (Torino). 2000;100(5):389-91. 122. Hughes JH, Jensen CS, Donnelly AD, et al. The role of fineneedle aspiration cytology in the evaluation of metastatic clear cell tumors. Cancer. 1999;87(6):380-9. 123. el Hag IA, Chiedozi LC, al Reyees FA, et al. Fine needle aspiration cytology of head and neck masses. Seven years’ experience in a secondary care hospital. Acta Cytol. 2003;47(3):387-92. 124. Owens CL, Basaria S, Nicol TL. Metastatic breast carcinoma involving the thyroid gland diagnosed by fine-needle aspiration: a case report. Diagn Cytopathol. 2005;33(2):110-5. 125. Bula G, Waler J, Niemiec A, et al. Diagnosis of metastatic tumours to the thyroid gland by fine needle aspiration biopsy. Endokrynol Pol. 2010;61(5):427-9. 126. Cozzolino I, Malapelle U, Carlomagno C, et al. Metastasis of colon cancer to the thyroid gland: a case diagnosed on fine-needle aspirate by a combined cytological, immunocytochemical, and molecular approach. Diagn Cytopathol. 2010;38(12):932-5. 127. Lam KY, Lo CY. Metastatic tumors of the thyroid gland: a study of 79 cases in Chinese patients. Arch Pathol Lab Med. 1998;122(1):37-41. 128. Barroeta JE, Baloch ZW, Lal P, et al. Diagnostic value of differential expression of CK19, Galectin-3, HBME-1, ERK, RET, and p16 in benign and malignant follicular-derived lesions of the thyroid: an immunohistochemical tissue microarray analysis. Endocr Pathol. 2006;17(3):225-34. 129. Nikiforov YE. Molecular diagnostics of thyroid tumors. Arch Pathol Lab Med. 2011;135(5):569-77.

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130. Fusco A, Chiappetta G, Hui P, et al. Assessment of RET/ PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer. Am J Pathol. 2002;160(6):2157-67. 131. Nikiforova MN, Nikiforov YE. Molecular diagnostics and predictors in thyroid cancer. Thyroid. 2009;19(12):1351-61. 132. Nikiforov YE, Steward DL, Robinson-Smith TM, et al. Molecular testing for mutations in improving the fineneedle aspiration diagnosis of thyroid nodules. J Clin Endocrinol Metab. 2009;94(6):2092-8.

133. Ohori NP, Nikiforova MN, Schoedel KE, et al. Contribution of molecular testing to thyroid fine-needle aspiration cytology of “follicular lesion of undetermined significance/ atypia of undetermined significance”. Cancer Cytopathol. 2010;118(1):17-23. 134. Alexander EK, Kennedy GC, Baloch ZW, et al. Preoperative diagnosis of benign thyroid nodules with indeterminate cytology. New Eng J Med. 2012;367(8):705-15. 135. Lubitz CC, Ugras SK, Kazam JJ, et al. Microarray analysis of thyroid nodule fine-needle aspirates accurately classifies benign and malignant lesions. J Mol Diagn.2006;8(4):490-8.

Chapter

4

Mediastinum

Lester J Layield



CLINICAL CONSIDERATIONS

The mediastinum is a complex anatomic compartment located between the pleural cavities and extending superior to inferior from the thoracic inlet to diaphragm and anterior to posterior from sternum to spine. Within this space are numerous structures and organs each potentially giving rise to a number of congenital cysts, primary benign or malignant tumors, and sites of deposition for metastatic disease. To aid in the development of a strategy for differential diagnosis of lesions arising in this complex space, the mediastinum can be divided into superior, anterior, middle and posterior compartments.1 Lesions in the superior mediastinum are most often thymomas, thymic cysts, lymphomas, parathyroid adenomas and abnormalities of the thyroid gland. Pathologic entities arising in the anterior mediastinum include thymomas, thymic cysts, germ cell tumors, lesions of the thyroid and parathyroid glands, and malignant lymphomas including Hodgkin disease. The middle mediastinum may contain pericardial and bronchial cysts as well as malignant lymphomas. Finally, the posterior mediastinum is dominated by neoplasms showing neurogenous differentiation including schwannomas, neurofibromas, ganglioneuromas, ganglioneuroblastomas and neuroblastomas. This area is also the site of gastroenteric cysts.2-5 Traditionally, lesions of the mediastinum have been cytologically investigated by transthoracic fine needle aspiration (FNA) and more recently, endoscopic

ultrasound (EUS) or endobronchial ultrasound (EBUS)guided FNA techniques. With the use of these techniques, the cytologic study of mediastinal lesions has expanded to include staging of primary carcinomas of the lung.6-8 EUS-guided FNAs have also been shown to be helpful in the diagnosis of mediastinal lymphadenopathy9,10 and the workup of mediastinal masses of unknown etiology.11,12 In many cases, clinical and laboratory findings are necessary for the interpretation of cytologic material derived by FNA. Complete clinical history including prior neoplastic disease is helpful in classifying metastatic lesions involving mediastinal lymph nodes. Ancillary testing has increasing importance in the workup of primary mediastinal lesions and metastatic disease causing mediastinal lymphadenopathy. Specific molecular analysis of cell block material is of aid in guiding selection of therapy. Molecular diagnostics and immunohistochemical analysis is most accurately performed on cell block material. Immunohistochemistry is helpful in the identification of various types of germ cell tumors as well as in the workup of neuroendocrine neoplasms (carcinoids). Flow cytometry is helpful in the specific diagnosis of malignant lymphoma. Whenever possible, material should be obtained for cell block preparations and set aside for molecular testing and immunohistochemistry. The incidence of complications for mediastinal FNA is similar to that seen in lung.13-15 Accurate needle placement is helpful in avoiding complications. Rare complications include cardiac tamponade and local hemorrhage.

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Diagnostic Accuracy Diagnostic sensitivity and specificity are high for the FNA diagnosis of mediastinal neoplasms. Sensitivity of approximately 87% has been reported.16 A number of diagnostic pitfalls exist often resulting in a correct diagnosis of benign or malignant but failing to accurately establish a precise histologic type. Small cell carcinomas may be misinterpreted as malignant lymphoma and non-Hodgkin lymphomas may be confused with Hodgkin lymphoma.17 Thymoma may be misinterpreted as large cell lymphoma, while germ cell neoplasms may be mistaken for adenocarcinomas or even lymphoma.18 Sparse specimen cellularity is often a contributing factor in misdiagnosis and may result in the confusion of metastatic disease with thymic carcinoma or even thymoma. 

CYTOLOGIC FINDINGS IN COMMON MEDIASTINAL LESIONS

Primary Thymic Neoplasms Thymoma Thymomas are neoplasms of thymic epithelial cells and the designation is independent of the number of lymphocytes present.19 A number of classifications for thymoma have been proposed including the Müller-Hermelink classification, Suster and Moran classification, and the current WHO classification.20-22 The Müller-Hermelink system attempted to classify thymomas on the basis of their resemblance to cortical or medullary areas of the thymus. The WHO classification uses similar patterns and divisions but did not assign them into cortical, medullary, or mixed subtypes but rather used an alphanumeric terminology. The classification of Suster and Moran is simpler and divides thymomas into thymoma, atypical thymoma, and thymic carcinoma. Their system includes the type A, type AB, type B1, and type B2 neoplasms under the term thymoma. The WHO classification system has the advantage of correlation between thymoma type and stage. In all classifications, the neoplastic component is thymic epithelium, while the lymphoid cells are reactive. The epithelial cells usually appear as large polygonal cells with syncytial cytoplasm. These cells often have pale staining cytoplasm contrasting with the darker surrounding lymphocytes. The individual epithelial cells have round or oval regular nuclei with a vesicular appearance. Nucleoli are inconspicuous or small. The epithelial cells are arranged in sheets, clusters, pseudorosettes, or even ribbons. In the spindle cell form, fascicular or storiform patterns occur.

Type A thymomas are characterized by spindled epithelial cells arranged in a fascicular or storiform pattern. The nuclei are frequently bland appearing with dense chromatin and indistinct nucleoli. Mitotic figures are either absent or very rare. In most cases, there is focal glandular differentiation or microcyst formation. Type AB thymomas demonstrate well-formed lobules. These neoplasms are a mixture of lymphocyte, poor type A lymphocyte and rich type B components. The components may be present in variable proportions and may be either discrete or intermixed. The lymphocytes of the type B component are usually immature T cells, while those of the type A component are mostly mature T cells. Type B1 thymomas histologically closely resemble normal thymus. They are characterized by well-formed lobules separated by sclerotic stroma. The epithelial component may be inconspicuous and appear only as interspersed oval cells in a background of lymphocytes. The epithelial component does not form cellular nests or sheets. Type B2 thymomas are lobulated, but the thymic epithelial cell component may form clusters of large ovoid cells with vesicular nuclei and conspicuous nucleoli. The epithelial component may display a palisaded architecture around perivascular spaces and along fibrous septae. Lymphocytes are abundant. Type B3 thymomas are composed of lobules of polygonal epithelial cells separated by thick sclerotic septae. The epithelial cells often exhibit a squamoid appearance and cells palisade around perivascular spaces and along fibrous septae. The nuclei of the epithelial component often have complex folds and clefts but nucleoli are inconspicuous. Focally clear or eosinophilic cytoplasm is common. In contrast to the aforementioned thymoma types, type B3 thymomas display moderate nuclear atypia and mitotic figures in the epithelial component.

Cytologic Features of Thymoma The cytologic features of thymoma partially correlate with the histopathologic subtypes. Tao cytologically divided thymomas into small cell, intermediate, and large cell types along with spindle cell and pleomorphic forms.23,24 The relevant proportion of lymphocytes to epithelial cells varies according to type of thymoma. Most aspirates obtained from thymomas contain clusters of epithelial cells of varying size and smears and are moderately to highly cellular.25-27 The cell clusters may contain fibrovascular cores.26 At low power, the epithelial cells may be indistinct and are of round, oval, or spindle shape (Fig. 4.1). The cell borders are usually indistinct giving a syncytial appearance to the cell groups. The nuclei are

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Fig. 4.1: Low power view of material aspirated from a thymoma. The cells lie singly and in clusters. The individual epithelial cells vary in shape from round to oval or “spindle” shape (Diff-Quik)

Fig. 4.2: Cells groups obtained from thymomas have a syncytial appearance and are composed of cells with indistinct cell borders and small to moderate amounts of pale cytoplasm. The nuclei may have folds or cleaves in the membranes (Diff-Quik)

separated by small-to-moderate amounts of pale cytoplasm (Fig. 4.2). The nuclei are characterized by slightly irregular nuclear membranes that may have folds or cleaves (Fig. 4.3). The nuclear chromatin is usually homogeneous and often finely granular. Nucleoli are small or indistinct. The lymphocytes appear small and mature with inconspicuous nucleoli.26 Lymphoglandular bodies and tangible body macrophages are variably present.26-29 There appears to be some correlation between cytologic appearance and histologic subtype.26,27,29 Smears with a high lymphocyte to epithelial cell ratio tend to correspond to B and AB subtypes. Smears containing atypical cells correspond to the B3 subtype. Nuclear atypia of the epithelial component tends to correlate with aggressive behavior.26

tumors (carcinoids), lymphoma including Hodgkin lymphoma, and germ cell tumors especially seminoma. Primary and metastatic carcinomas usually show greater degrees of nuclear atypia than thymomas and lack the prominent lymphoid component. Thymic carcinoids lack the lymphoid component, and the epithelial component may form rosettes and trabecular cell clusters. The nuclei of carcinoid tumors have a salt and pepper chromatin. Immunocytochemistry will demonstrate reactivity for chromogranin and/or synaptophysin in neuroendocrine tumors.

Diagnostic Criteria • • • • • •

Variable but frequently abundant lymphoid component in the background Epithelial cells that may be of round, oval, or spindle shape Epithelial cell nuclei often slightly irregular with folds and cleaves Nuclear chromatin is homogeneous and finely granular Nucleoli either absent or small Epithelioid cells form clusters often with a syncytial appearance.

Differential Diagnostic Concerns The differential diagnosis of thymoma includes carcinomas both primary and metastatic, neuroendocrine

Fig. 4.3: The nuclei of cells obtained by FNA from thymomas have slightly irregular nuclear membranes with folds and cleaves. The nuclear chromatin is homogenous or finely granular. (Hematoxylin and Eosin)

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Lymphomas lack the epithelial component present in thymoma, and flow cytometry will demonstrate monoclonality in B-cell lymphomas. T-cell lymphomas are characterized by gene rearrangements. Seminomas are particularly difficult to separate from thymomas as both can contain a prominent lymphoid component. The nuclei of seminomas are larger often rounder than those of thymomas and have a vesicular chromatin pattern and large nucleoli. The background of seminoma also displays a “tigroid” appearance best seen in air-dried material.

Thymic Cysts and “Thymic Follicular Hyperplasia” Two distinct varieties of cysts occur within the thymus. Unilocular thymic cysts appear to arise from the third branchial pouch. They are small and more often not arise within the neck rather than the mediastinum. Aspiration of these cysts yields clear yellow fluid that may contain histiocytes, a mixed lymphoid population, and rarely flattened or cuboidal epithelial cells. Multilocular thymic cysts are acquired lesions and are accompanied by inflammation and fibrosis.30 These lesions can present as a large tumor-like mass composed of multiple individual cysts lined by flat, cuboidal, ciliated columnar, or most often squamous epithelium. This epithelium may undergo reactive change resulting in reactive atypia of the epithelial cells. Aspirates from multilocular thymic cysts contain a prominent population of mixed lymphocytes, histiocytes, and often a small number of epithelial cells usually of squamous differentiation. These squamous

Fig. 4.4: Aspirates obtained from thymic follicular hyperplasia are often characterized by a bland mixed lymphoid population (Diff-Quik)

cells may have slightly enlarged hyperchromatic nuclei, resulting in a differential diagnosis with well-differentiated squamous cell carcinoma. Distinction is possible by recognizing the small number of atypical squamous cells present in benign cysts as well as the lack of large sheets or clusters of epithelium and the minor degrees of nuclear atypia present. Thymic follicular hyperplasia may result in an enlarged thymus resulting in FNA. Histologically, thymic follicular hyperplasia is characterized by a prominent number of lymphoid follicles with germinal center formation. When these areas of follicular hyperplasia are numerous, needle aspirates yield a mixed lymphoid population (Fig. 4.4). Germinal centers may occasionally be found. The lymphoid component is mixed and polyclonal excluding a non-Hodgkin-type lymphoma. Reed–Sternberg cells are absent.

Langerhans Cell Histiocytosis Langerhans cell histiocytosis may present as a thymic mass.31 Cytologic findings in Langerhans cell histiocytosis of the thymus are essentially identical to the findings in bone and other sites. Aspirate smears are highly cellular with the cells dispersed singly or in loose aggregates (Fig. 4.5). The background contains a mixture of lymphocytes, plasma cells, and often a prominent number of bilobed eosinophils. The diagnostic cell is the Langerhanstype histiocyte that is of an oval or round shape and has a reniform nucleus.32,33 The reniform nucleus is often characterized by a well-formed nuclear groove best seen

Fig. 4.5: Smears of material aspirated from examples of Langerhans cell histiocytosis are characterized by high cellularity and a noncohesive cell pattern. The cell population is a mixture of lymphocytes, plasma cells, histiocytes and a variable number of eosinophilis (Hematoxylin and Eosin)

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Neuroendocrine Tumors

with Papanicolaou or hematoxylin and eosin (H&E) staining (Fig. 4.6). The Langerhans cells are discohesive and contain pale cytoplasm (Fig. 4.7). The nuclei may be either centrally or eccentrically located within the nucleus. These cells do not contain phagocytized debris, and mitotic activity is rare or absent. Electron microscopy can identify the characteristic Birbeck granules. Langerhans cell histiocytosis must be distinguished from monocytoid B-cell lymphomas and malignant histiocytosis. Occasionally, other childhood malignancies including rhabdomyosarcoma can be mistaken for examples of Langerhans cell histiocytosis.

The thymus is home to a variety of neuroendocrine neoplasms similar to those seen within the lung.37 Most neuroendocrine neoplasms of the thymus have similar morphologic and clinical features as atypical carcinoids of pulmonary origin. These neoplasms cytomorphologically demonstrate moderate nuclear pleomorphism with a fine stippled chromatin pattern. In air-dried preparations, the individual cells demonstrate moderate-to-abundant reddish granular cytoplasm. The overall appearance is intermediate between a classic benign carcinoid tumor and small cell carcinoma of the lung.

Ectopic Thyroid and Thyroid Neoplasms

Neurogenic Tumors

Ectopic thyroid and parathyroid tissue occurs with some frequency within the upper mediastinum. Ectopic thyroid tissue usually comes to clinical recognition due to nodular hyperplasia but may be found incidentally as part of a workup for other lesions.34,35 Material aspirated from ectopic thyroid may show all the pathologic changes seen in ectopic thyroid tissue. Nodular goiter is the most common finding, and the reader is referred to the chapter on thyroid for cytomorphologic features of benign and malignant thyroid lesions.

The posterior mediastinum is home to a number of neoplasms showing neurogenous differentiation. These neoplasms are divided into two broad categories: sympathetic nervous system tumors and tumors of the peripheral nerve sheath.38 Most neoplasms occurring in children belong to the sympathetic nervous system group that includes neuroblastoma, ganglioneuroblastoma and ganglioneuromas.38,39

Neuroblastoma

Between 7% and 10% of parathyroid adenomas occur within the anterior portion of the mediastinum.36 Parathyroid cysts may also be present within the mediastinum and yield the characteristic water clear fluid seen in the ectopic organ.

Neuroblastomas cytomorphologically demonstrate a classic small round cell malignancy smear pattern (Fig. 4.8). Smears are usually hypercellular and composed of numerous individually scattered small cells admixed with occasional cohesive groups.40,41 The individual cells have dark hyperchromatic nuclei and scant cytoplasm. Nuclear molding is common. Occasional larger cells may

Fig. 4.6: In smear preparations, the Langerhans cell has an oval or round shape and contains a reniform nucleus with a nuclear groove (Hematoxylin and Eosin)

Fig. 4.7: The Langerhans cells are discohesive with moderate to abundant amounts of pale cytoplasm surrounding a reniform nucleus (Hematoxylin and Eosin)

Hyperplastic Parathyroid Glands and Adenomas

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Fig. 4.8: Cytologically, neuroblastomas have the features of a small round cell malignancy. Smears are often hypercellular with a mixture of individual cells and cohesive cell groups (Diff-Quik)

Fig. 4.9: The individual cells of neuroblastoma have hyperchromatic round or oval nuclei surrounded by a thin cytoplasmic rim. Nuclear molding is common in the cell groups (Diff-Quik)

be seen that possess moderate amounts of cytoplasm and conform to the differentiating component of neuroblasts. Binucleated and multinucleated ganglion-type cells are seen in maturing neuroblastomas. Homer-Wright rosettes may be seen but are infrequent or often absent. On highpower examination, the nuclei of neuroblastoma cells are oval to slightly irregular in shape and have a finely dispersed dark granular chromatin (Fig. 4.9). Nucleoli are inconspicuous and usually absent. A characteristic and diagnostically important component of neuroblastoma is the presence of neuropil in the background. This material has a fibrillary appearance and represents tangled neuritic processes. Mitotic figures and karyorrhectic cells

are present in variable numbers and have importance in prediction of prognosis.

Fig. 4.10: Ganglioneuroblastomas contain multinucleated ganglion cells in addition to the small round cell component. The smear background often contains as filamentous component (Hematoxylin and Eosin)

Ganglioneuroblastoma Ganglioneuroblastomas are distinguished from neuroblastomas by the presence of many multinucleated ganglion cells (Fig. 4.10). The neoplastic cells of ganglioneuroblastomas are enmeshed in tissue fragments containing syncytial or fibrous cytoplasm and stroma (Fig. 4.11). The presence of a discreet population of ganglion cells and bland spindle-shaped cells enmeshed in a pale fibrous stroma is characteristic of ganglioneuroblastoma (Fig. 4.11).42

Fig. 4.11: The tissue fragments obtained from ganglioneuroblastomas have a syncytial or finely filamentous appearance (Diff-Quik)

Mediastinum

Schwannoma The posterior mediastinum can give rise to schwannomas, neurofibromas and malignant peripheral nerve sheath tumors. Schwannomas appear as well-encapsulated soft tissue nodules or may have a dumbbell shape as they arise from the vertebral column. Schwannomas are of variable cellularity and may contain atypical degenerative cells. Despite nuclear atypia and even a low mitotic index, these neoplasms are clinically benign. Smears obtained from schwannomas are of variable cellularity with the majority of cells forming small groups and clusters. These cells have a spindle shape, but the cytoplasm often forms a syncytial aggregate of filamentous material (Fig. 4.12). The cellularity of the aspirated fragments is variable, but these tissue fragments are characterized by a fibrillar appearance.43,44 While infrequently seen, nuclear palisading may occur giving rise to the “Verocay” body. The nuclei of the neoplastic cells are long and slender with pointed ends. The nuclei often are bent, curved, or have a fishtail-like appearance. In occasional cases, significant degenerative nuclear atypia is seen but does not impact prognosis. Microscopic descriptions of the cytologic features of neurofibroma and malignant peripheral nerve sheath tumor are given in the chapter on soft tissue and bone lesions. 

MALIGNANT NEOPLASMS OF THE MEDIASTINUM

Thymic Carcinoma Thymic carcinomas are neoplasms of the thymic epithelium exhibiting clear-cut cytologic features of

Fig. 4.12: Smears obtained from schwannomas are characterized by cell groups. These groups are composed of spindle shaped cells with bipolar wispy cytoplasmic processes (Diff-Quik)

69

malignancy.45,46 While uncommon, these neoplasms exhibit a wide variety of microscopic patterns including squamous cell carcinoma, epidermoid nonkeratinizing carcinoma, epithelioma-like carcinoma, sarcomatoid carcinoma, clear cell carcinoma, basaloid carcinoma, mucoepidermoid carcinoma, small cell carcinoma, and undifferentiated (anaplastic) carcinoma.45,46 Figures 4.13 and 4.14 give examples of smear material obtained from thymic carcinomas.

Germ Cell Neoplasms Seminoma (Germinoma) Mediastinal seminomas almost always arise within the thymus and appear cytologically and histologically identical to those occurring within the testicle. Histologically, these neoplasms are composed of compact nests of large polygonal cells with large nuclei and prominent nucleoli. A prominent component of lymphocytes is frequently present as is a granulomatous reaction. Seminomas are immunoreactive for placental alkaline phosphatase, OCT4, CD117, and SALL4. Aspirates from seminomas are usually cellular and composed of dispersed neoplastic cells in a tigroid background (Figs 4.15 to 4.18).47-49 The nuclei are fragile with chromatin smearing and smudging. Intact nuclei are ovoid and contain prominent nucleoli (Figs 4.17 and 4.18). The chromatin is coarse and often clumped with areas of clearing best seen in H&E or Papanicolaoustained material. Occasional mitotic figures are present in some smears. Characteristically, a population of mature small lymphocytes is scattered among the neoplastic cells, and epithelioid histiocytes including granuloma are often found.

Fig. 4.13: Thymic carcinomas yield on aspiration groups of anaplastic cells with large hyperchromatic irregular nuclei (Diff-Quik)

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Fig. 4.14: Cells obtained from thymic carcinomas can have a polygonal or spindle shape and contain pleomorphic nuclei (Hematoxylin and Eosin)

Fig. 4.15: Seminomas are characterized by a “tigroid” background (Diff-Quik)

Diagnostic Criteria

Diagnostic Pitfalls

• • •

Cellular smears Single dispersed cells without significant clustering Fragile cytoplasm with most cells being represented by naked nuclei Tigroid background Nuclear smudging Nuclei are large with a vesicular chromatin and distinct nucleoli Intact cytoplasm is pale and often vacuolated Lymphocytes present in background Granulomatous response variably present.

Seminomas must be separated from lymphomas and thymomas due to the heavy lymphocytic infiltrate found in some seminomas. Careful search for the characteristic large nuclei with stripped cytoplasm is necessary to establish the diagnosis of seminoma when a lymphoid component is prominent. The presence of a tigroid background and lack of lymphoglandular bodies aid in the separation of seminoma from lymphoma.

Fig. 4.16: The individual cells of seminomas have fragile cytoplasm which is often stripped from the associated nuclei. The nuclei are large with prominent nucleoli (Diff-Quik)

Fig. 4.17: The nuclei of seminomas are large with prominent nucleoli and clumped chromatin (Papanicolaou)

• • • • • •

Embryonal Carcinoma Embryonal carcinomas are poorly differentiated malignancies that are often highly necrotic. The cells comprising

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Fig. 4.18: The nuclei of seminomas often demonstrate chromatin clumping and clearing. Nucleoli are often huge (Hematoxylin and Eosin)

Fig. 4.19: Cytologically, embryonal carcinomas are characterized by large primitive cells forming large groups. The individual cells have retained cytoplasm surrounding large pleomorphic nuclei (Diff-Quik)

these tumors lie both individually and in clusters. Nuclear atypia is marked. Characteristically, these lesions lack the lymphoid component seen in seminomas and have a greater tendency toward cell clustering than is characteristic of seminomas. Cytomorphologically, these malignancies are characterized by cells with retained cytoplasm surrounding large pleomorphic nuclei (Figs 4.19 and 4.20). The nuclear chromatin is coarse and irregular. Nucleoli are large. The cytoplasm is usually retained and is pale or may be vacuolated (Fig. 4.21). The background is bloody or may contain necrotic debris. It lacks the tigroid appearance characteristic of seminomas.

Yolk Sac Tumor Yolk sac tumors may occur in either a pure form or admixed with other germ cell elements.50 Mediastinal yolk sac tumors, like their testicular counterparts, can present with a number of morphologic appearances including microcystic, endodermal sinus, myxomatous, papillary, endometrioid and hepatoid.51,52 Cytologically, yolk sac tumors present as poorly cohesive epithelial-like cells displaying moderate pleomorphism (Fig. 4.22). The nuclei are round and often surrounded by vacuolated cytoplasm (Fig. 4.23). Rarely, a fibrillary background can be present.52,53 In general, the cytologic findings are nondescript other than being compatible with a poorly differentiated malignancy.

Fig. 4.20: The cell groups seen in embryonal carcinomas may contain gland-like structures (Diff-Quik)

Fig. 4.21: Cells of embryonal carcinomas usually retain their cytoplasm which appears pale or finely vacuolated (Diff-Quik)

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Fig. 4.22: Yolk sac tumors are characterized by poorly cohesive epithelial-like cells showing moderate nuclear pleomorphism (Diff-Quik)

Fig. 4.23: The nuclei of yolk sac tumors are round and surrounded by finely vacuolated cytoplasm (Diff-Quik)

Lymphomas of the Mediastinum

The vast majority of Hodgkin lymphomas involving the thymus and anterior mediastinum are of the nodular sclerosis type. Smear preparations obtained from these lymphomas are characterized by smears of high cellularity dominated by a mixed lymphoid infiltrate (Fig. 4.24).56-58 Admixed with the background population

of lymphocytes are a variable number of eosinophils, plasma cells and histiocytes. The characteristic cell is the Reed–Sternberg cell. The Reed–Sternberg cells are immunohistochemically reactive for panB markers: CD45 and EMA. Characteristically, the Reed–Sternberg cells are CD30 positive. Classic Reed–Sternberg cells have large lobulated nuclei that may appear symmetric or have complex multilobed or multinucleated forms (Figs 4.25 and 4.26). The chromatin pattern is coarse and irregularly distributed. Nucleoli are often huge, frequently the size of a small mature lymphocyte (Fig. 4.27). The cytoplasm is often abundant but may be stripped from the nucleus. The classic Reed–Sternberg cell has a symmetric owl’s

Fig. 4.24: Aspirates from Hodgkin’s lymphoma are cellular. The cell population is polymorphous with a prominence of small lymphocytes (Diff-Quik)

Fig. 4.25: The characteristic Reed-Sternberg cell has a large lobulated nucleus with a prominent nucleolus (Hematoxylin and Eosin)

The majority of mediastinal lymphomas fall into one of three major subtypes including nodular sclerosis form of Hodgkin lymphoma, mediastinal large B-cell lymphoma, and T-lymphoblastic lymphoma.54,55

Hodgkin Lymphoma

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Fig. 4.26: Reed-Sternberg cells are characterized by two or more lobes and nucleoli the size of small lymphocyte nuclei (Hematoxylin and Eosin)

Fig. 4.27: Nucleoli of Reed-Sternberg cells are frequently the size of mature lymphocyte nuclei (Hematoxylin and Eosin)

eye appearance, or other forms of Reed–Sternberg cells exist including lacunar cells with irregular multilobulated nuclei and often inconspicuous nucleoli.

hypercellular but when sclerosis is present, the cell yield may be low. The smears are composed of noncohesive/ discohesive individual cells generally three times the size of a mature lymphocyte (Fig. 4.28). The amount of cytoplasm is variable often scant in amount (Fig. 4.29). The nuclei are round or may have a convoluted nuclear shape. The chromatin is vesicular in H&E and Papanicolaoustained preparations, and nucleoli are often prominent. The background contains large numbers of lymphoglandular bodies. Flow cytometry and immunohistochemical studies reveal these neoplasms to be monoclonal B-cell lymphomas.

Mediastinal Large B-cell Lymphoma Mediastinal large B-cell lymphoma occurs almost exclusively in the anterior mediastinum often in association with the thymus.56,59 This lymphoma occurs twice as frequently in women as in men and may lead to superior vena cava obstruction or symptoms secondary to a mediastinal mass. The tumors are frequently bulky. Cytomorphologically, these neoplasms are large cell non-Hodgkin lymphomas. The smears are usually

Fig. 4.28: Smears of large B-cell lymphomas are characterized by a monomorphous population of lymphoid cells usually three times the size of mature lymphocytes (Diff-Quik)

Fig. 4.29: The large B-cells have small amounts of cytoplasm surrounding the large nucleus (Diff-Quik)

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Fig. 4.30: Smears of lymphoblastic lymphomas contain a population of noncohesive lymphoid cells demonstrating a high nuclear/ cytoplasmic ratio (Diff-Quik)

Fig. 4.31: Granulomatous lymphadenopathy is cytologically characterized by scattered aggregates of epithelioid histiocytes. These cells have moderate to abundant cytoplasm and oval to elongated nuclei (Diff-Quik)

Diagnostic Criteria

frequently used to document the presence of granuloma in patients suspected as having sarcoid. The involved lymph nodes contain many noncaseating granulomas. Cytologically, these granulomas are characterized by scattered aggregates of epithelioid histiocytes (Fig. 4.31). These histiocytes have abundant pale cytoplasm and oval or spindle-shaped nuclei. The cytoplasm often has a syncytial appearance.

• • • • •

Noncohesive large cells approximately three times the size of a normal lymphocyte Cells have variable amounts of cytoplasm Nuclei are round or convoluted Chromatin has a vesicular appearance with prominent nucleoli (best seen on Papanicolaou or H&E staining) Background contains numerous lymphoglandular bodies.

Lymphoblastic Lymphoma Aspirated smears from lymphoblastic lymphomas are characterized by a monomorphic population of small- to medium-sized lymphocytes.56,60 The neoplastic cells have very high nuclear:cytoplasmic ratios (Fig. 4.30). Tingible body macrophages are frequent in the background. The neoplastic cells have a finely granular nuclear chromatin and small nucleoli. Nuclear membrane irregularities are variably present. Most examples demonstrate a high mitotic count. Because nuclear membrane features are important in the recognition of this form of lymphoma, Papanicolaou or H&E stains are of diagnostic importance. The monotony of cell type and the high mitotic index are important for the diagnosis of these neoplasms. Similarly, flow cytometry is important in the recognition of these lymphomas.

Granulomatous Lymphadenopathy Mediastinal lymph nodes are often affected by sarcoid, tuberculosis and other granulomatous diseases. FNA is



REFERENCES

1. Rosai J. Rosai and Ackerman’s surgical pathology. Edinburgh: Mosby/Elsevier; 2004. p. 437. 2. Blegvad S, Lippert H, Simper LB, et al. Mediastinal tumours. A report of 129 cases. Scand J Thorac Cardiovasc Surg. 1990;24(1):39-42. 3. Cohen AJ, Thompson L, Edwards FH, et al. Primary cysts and tumors of the mediastinum. Ann Thorac Surg. 1991;51(3):378-84. 4. Oldham HN Jr, Sabiston DC Jr. Primary tumors and cysts of the mediastinum. Monogr Surg Sci. 1967;4(4):243-79. 5. Shimasato Y, Mukai K. Tumors of the mediastinum. In: Rosai J, Rosai J (Ed). Atlas of tumor pathology, 3rd series, Fascicle 21. Washington DC: Armed Forces Institute of Pathology; 1997. 6. Harewood GC, Pascual J, Raimondo M, et al. Economic analysis of combined endoscopic and endobronchial ultrasound in the evaluation of patients with suspected non-small cell lung cancer. Lung Cancer. 2010;67(3): 366-71. 7. Lin LF, Huang PT, Tsai MH, et al. Role of endoscopic ultrasound-guided fine-needle aspiration in lung and mediastinal lesions. J Chin Med Assoc. 2010; 73(10): 523-9.

Mediastinum 8. Micames CG, McCrory DC, Pavey DA, et al. Endoscopic ultrasound-guided fine-needle aspiration for non-small cell lung cancer staging: a systematic review and metaanalysis. Chest. 2007;131(2):539-48. 9. Catalano MF, Nayar R, Gress F, et al. EUS-guided fine needle aspiration in mediastinal lymphadenopathy of unknown etiology. Gastrointest Endosc. 2002;55(7):863-9. 10. Zeppa P, Barra E, Napolitano V, et al. Impact of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in lymph nodal and mediastinal lesions: a multicenter experience. Diagn Cytopathol. 2011;39(10):723-9. 11. Devereaux BM, Leblanc JK, Yousif E, et al. Clinical utility of EUS-guided fine-needle aspiration of mediastinal masses in the absence of known pulmonary malignancy. Gastrointest Endosc. 2002;56(3):397-401. 12. Savoy AD, Ravenel JG, Hoffman BJ, et al. Endoscopic ultrasound for thoracic malignancy: a review. Curr Probl Diagn Radiol. 2005;34(3):106-15. 13. Adler OB, Rosenberger A, Peleg H. Fine-needle aspiration biopsy of mediastinal masses: evaluation of 136 experiences. AJR Am J Roentgenol. 1983;140(5):893-6. 14. Adler O, Rosenberger A. Invasive radiology in the diagnosis of mediastinal masses. Use of fine needle for aspiration biopsy. Radiologe. 1979;19(5):169-72. 15. Weisbrod GL. Percutaneous fine-needle aspiration biopsy of the mediastinum. Clin Chest Med. 1987;8(1):27-41. 16. Powers CN, Silverman JF, Geisinger KR, et al. Fine-needle aspiration biopsy of the mediastinum. A multi-institutional analysis. Am J Clin Pathol. 1996;105(2):168-73. 17. Singh HK, Silverman JF, Powers CN, et al. Diagnostic pitfalls in fine-needle aspiration biopsy of the mediastinum. Diagn Cytopathol. 1997;17(2):121-6. 18. Geisinger KR. Differential diagnostic considerations and potential pitfalls in fine-needle aspiration biopsies of the mediastinum. Diagn Cytopathol. 1995;13(5):436-42. 19. Levine GD, Rosai J. Thymic hyperplasia and neoplasia: a review of current concepts. Hum Pathol. 1978;9(5):495-515. 20. Marino M, Müller-Hermelink HK. Thymoma and thymic carcinoma. Relation of thymoma epithelial cells to the cortical and medullary differentiation of thymus. Virchows Arch A Pathol Anat Histopathol. 1985;407(2):119-49. 21. Suster S, Moran CA. Thymoma classification: current status and future trends. Am J Clin Pathol. 2006;125(4):542-54. 22. Rosai J, Sobin LH. Histological typing of tumors of the thymus. WHO international histological classification of tumors. Berlin: Springer Verlag; 1999. 23. Tao LC, Pearson FG, Cooper JD, et al. Cytopathology of thymoma. Acta Cytol. 1984;28(2):165-70. 24. Tao LC. Lung, pleura and mediastinum. In: Kline TS, (Ed). Guides to Clinical Aspiration Biopsy. New York: IgakuSkoin; 1988. 25. Wakely PE Jr. Fine needle aspiration in the diagnosis of thymic epithelial neoplasms. Hematol Oncol Clin North Am. 2008;22(3):433-42. 26. Chhieng DC, Rose D, Ludwig ME, et al. Cytology of thymomas: emphasis on morphology and correlation with histologic subtypes. Cancer. 2000;90(1):24-32.

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27. Ali SZ, Erozan YS. Thymoma. Cytopathologic features and differential diagnosis on fine needle aspiration. Acta Cytol. 1998;42(4):845-54. 28. Shin HJ, Katz RL. Thymic neoplasia as represented by fine needle aspiration biopsy of anterior mediastinal masses. A practical approach to the differential diagnosis. Acta Cytol. 1998;42(4):855-64. 29. Dahlgren S, Sandstedt B, Sundström C. Fine needle aspiration cytology of thymic tumors. Acta Cytol. 1983;27(1): 1-6. 30. Suster S, Rosai J. Multilocular thymic cyst: an acquired reactive process. Study of 18 cases. Am J Surg Pathol. 1991;15(4):388-98. 31. Siegal GP, Dehner LP, Rosai J. Histiocytosis X (Langerhans’ cell granulomatosis) of the thymus. A clinicopathologic study of four childhood cases. Am J Surg Pathol. 1985; 9(2):117-24. 32. Akhtar M, Ali MA, Bakry M, et al. Fine-needle aspiration biopsy of Langerhans histiocytosis (histiocytosis-X). Diagn Cytopathol. 1993;9(5):527-33. 33. Layfield LJ, Bhuta S. Fine-needle aspiration cytology of histiocytosis X: a case report. Diagn Cytopathol. 1988;4(2): 140-3. 34. Katlic MR, Wang CA, Grillo HC. Substernal goiter. Ann Thorac Surg. 1985;39(4):391-9. 35. Wick MR. Mediastinal cysts and intrathoracic thyroid tumors. Semin Diagn Pathol. 1990;7(4):285-94. 36. Moran CA, Suster S. Primary parathyroid tumors of the mediastinum: a clinicopathologic and immunohistochemical study of 17 cases. Am J Clin Pathol. 2005;124(5): 749-54. 37. Moran CA, Suster S. Neuroendocrine carcinomas (carcinoid tumor) of the thymus. A clinicopathologic analysis of 80 cases. Am J Clin Pathol. 2000;114(1):100-10. 38. Reed JC, Hallet KK, Feigin DS. Neural tumors of the thorax: subject review from the AFIP. Radiology. 1978;126(1): 9-17. 39. Simpson I, Campbell PE. Mediastinal masses in childhood: a review from a paediatric pathologist’s point of view. Prog Pediatr Surg. 1991;27:92-126. 40. Miller TR, Bottles K, Abele JS, et al. Neuroblastoma diagnosed by fine needle aspiration biopsy. Acta Cytol. 1985;29(3):461-8. 41. Silverman JF, Dabbs DJ, Ganick DJ, et al. Fine needle aspiration cytology of neuroblastoma, including peripheral neuroectodermal tumor, with immunocytochemical and ultrastructural confirmation. Acta Cytol. 1988;32(3): 367-76. 42. Palombini L, Vetrani A, Vecchione R, et al. The cytology of ganglioneuroma of fine needle aspiration smear. Acta Cytol. 1982;26(2):259-60. 43. Dahl I, Hagmar B, Idvall I. Benign solitary neurilemoma (Schwannoma). A correlative cytological and histological study of 28 cases. Acta Pathol Microbiol Immunol Scand A. 1984;92(2):91-101. 44. Mooney EE, Layfield LJ, Dodd LG. Fine-needle aspiration of neural lesions. Diagn Cytopathol. 1999;20(1):1-5.

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45. Ritter JH, Wick MR. Primary carcinomas of the thymus gland. Semin Diagn Pathol. 1999;16(1):18-31. 46. Suster S. Thymic carcinoma: update of current diagnostic criteria and histologic types. Semin Diagn Pathol. 2005;22(3):198-212. 47. Akhtar M, Ali MA, Huq M, et al. Fine-needle aspiration biopsy of seminoma and dysgerminoma: cytologic, histologic, and electron microscopic correlations. Diagn Cytopathol. 1990;6(2):99-105. 48. Caraway NP, Fanning CV, Amato RJ, et al. Fine-needle aspiration cytology of seminoma: a review of 16 cases. Diagn Cytopathol. 1995;12(4):327-33. 49. Fleury-Feith J, Bellot-Besnard J. Criteria for aspiration cytology for the diagnosis of seminoma. Diagn Cytopathol. 1989;5(4):392-5. 50. Moran CA, Suster S, Koss MN. Primary germ cell tumors of the mediastinum: III. Yolk sac tumor, embryonal carcinoma, choriocarcinoma, and combined nonteratomatous germ cell tumors of the mediastinum—a clinicopathologic and immunohistochemical study of 64 cases. Cancer. 1997;80(4):699-707. 51. Weidner N. Germ-cell tumors of the mediastinum. Semin Diagn Pathol. 1999;16(1):42-50. 52. Yang GC. Demonstration of fibrils in the hyaline globules of yolk sac tumor with parietal differentiation in fineneedle aspiration smears. Diagn Cytopathol. 1994;10(3): 216-20.

53. Akhtar M, Ali MA, Sackey K, et al. Fine-needle aspiration biopsy diagnosis of endodermal sinus tumor: histologic and ultrastructural correlations. Diagn Cytopathol. 1990; 6(3):184-92. 54. Yousem SA, Weiss LM, Warnke RA. Primary mediastinal non-Hodgkin’s lymphomas: a morphologic and immunologic study of 19 cases. Am J Clin Pathol. 1985;83(6): 676-80. 55. Strickler JG, Kurtin PJ. Mediastinal lymphoma. Semin Diagn Pathol. 1991;8(1):2-13. 56. Kardos TF, Vinson JH, Behm FG, et al. Hodgkin’s disease: diagnosis by fine-needle aspiration biopsy. Analysis of cytologic criteria from a selected series. Am J Clin Pathol. 1986;86(3):286-91. 57. Jogai S, Dey P, Al Jassar A, et al. Role of fine needle aspiration cytology in nodular sclerosis variant of Hodgkin’s lymphoma. Acta Cytol. 2006;50(5):507-12. 58. Jiménez-Heffernan JA, Vicandi B, López-Ferrer P, et al. Value of fine needle aspiration cytology in the initial diagnosis of Hodgkin’s disease. Analysis of 188 cases with an emphasis on diagnostic pitfalls. Acta Cytol. 2001;45(3):300-6. 59. Addis BJ, Isaacson PG. Large cell lymphoma of the mediastinum: a B-cell tumour of probable thymic origin. Histopathology. 1986;10(4):379-90. 60. Jacobs JC, Katz RL, Shabb N, et al. Fine needle aspiration of lymphoblastic lymphoma. A multiparameter diagnostic approach. Acta Cytol. 1992;36(6):887-94.

Chapter

5

Lung and Pleura

Lester J Layield



CLINICAL CONSIDERATIONS AND TECHNICAL ASPECTS

The position fine needle aspiration (FNA) holds in the workup of pulmonary nodules has changed markedly over the last two decades. Before the advent of endobronchial ultrasound (EBUS)-guided fine needle aspiration, the finding of a new pulmonary nodule on imaging studies initiated a diagnostic workup involving radiologists, pulmonologists, thoracic surgeons, and pathologists. Patients with centrally located lesions would initially be investigated by sputum cytology followed by bronchial brushings and washings. While these techniques are minimally invasive, the detection rate for carcinoma is variable. When five specimens are utilized, a detection rate as high as 90–95% is achievable.1,2 Sputum most reliably detects central tumors,2 but some authors have demonstrated nearly equal sensitivity for peripheral neoplasms.3,4 With the development of bronchoscopy, bronchial brushing and washing specimens were utilized for follow-up of negative sputum specimens in patients with clinically suspicious lesions and for investigation of peripheral lung masses seen on chest X-ray.5-6 Overall sensitivity of bronchial brushings is 70–90% when two or more specimens are obtained.7,8 In the majority of patients, these techniques were used for late stage, generally inoperable lung cancer. When the more limited and less invasive cytologic procedures were unsuccessful in establishing a diagnosis of carcinoma, endobronchial

and transbronchial biopsy procedures were utilized. When lung nodules were distinctly peripheral and less likely to be successfully diagnosed by sputum, bronchial brush and bronchial washing cytologies, percutaneous fluoroscopic, or computed tomography-directed FNA were utilized. While percutaneous or transthoracic FNA is highly accurate, local hemorrhage and risk of pneumothorax represent infrequent but realistic hazards. The FNA biopsy of pulmonary nodules has a good-to-excellent diagnostic accuracy with a sensitivity as high as 89% and specificity as high as 96% in some large studies.9 A number of authors have investigated the impact of needle size and the nature of biopsy technique. Al-Damegh demonstrated that the use of a 25-gauge needle for transthoracic FNA reduced the incidence of pneumothorax without significantly reducing diagnostic yield.10 Greif et al. demonstrated that percutaneous core needle biopsy did not demonstrate substantial advantage over FNA in the evaluation of peripheral malignant lung lesions.11 With increasing experience utilizing transthoracic FNA, increasingly small lesions could be successfully aspirated and diagnosed by FNA.12 This resulted in the FNA technique being utilized for the preoperative diagnosis of apparently resectable lung cancers. In an attempt to improve overall diagnostic accuracy as well as to reduce the incidence of pneumothorax, endoscopic ultrasound (EUS)-guided and EBUS-guided techniques were developed.13-19 These techniques are reliable for both early and advanced carcinoma of the lung.14,18

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Some authors have described difficulty in reliably separating the subtypes of non-small cell carcinoma,14 while others have demonstrated good ability in separating squamous cell carcinomas from adenocarcinomas.20,21 This is particularly true when cytologic material is available for immunocytochemical staining. Material obtained by FNA is also practical for EGFR and KRAS molecular testing.20 The development of EUS-guided FNA and EBUSguided FNA has improved the sensitivity and specificity for the diagnosis of non-small cell carcinoma.20,21 When cell block material is available for immunohistochemistry, the separation of non-small cell carcinoma into adenoand squamous subtypes is improved.20 In addition to the utility of EUS-FNA and EBUS-FNA for the primary diagnosis of lung carcinoma, both techniques have been shown to be highly successful in staging patients with known lung cancer by sampling of mediastinal lymph nodes.22-28 Immediate assessment of FNA derived material appears to reduce false-negative lung FNAs.29-30

Contraindications Relatively few contraindications exist for FNA of pulmonary nodules. Patients who do not have a cough reflex or who are unconscious should not undergo transthoracic FNA. Similarly, patients with known arterial venous malformations or aneurysms are poor candidates for FNA of pulmonary lesions. Patients with a bleeding diathesis, pulmonary hypertension, or on anticoagulant therapy are generally excluded from pulmonary FNA. Similarly, patients with significant emphysematous change and numerous pulmonary blebs are less than ideal candidates for transbronchial FNA.

Specimen Adequacy Currently, no widely accepted criteria exist for defining a pulmonary FNA as satisfactory. The presence of obscuring blood or marked artifactual change should result in an aspirate being labeled as nondiagnostic. Nonspecific findings such as ciliated respiratory epithelium, mesothelial cells, and foamy histiocytes do not exclude a specific lesion including carcinoma and should not be taken in and of themselves as excluding malignancy.36-38 Various lesions including round atelectasis, focal hemorrhage, and imaging artifacts may result in pulmonary nodules that undergo FNA.39,40 Aspiration of these will yield only respiratory epithelium, blood, or macrophages. Clinical and radiographic correlations are helpful in assessing the adequacy of an FNA specimen.38 

CYTOLOGIC FEATURES OF NORMAL LUNG

Respiratory (Bronchial Epithelium) Ciliated bronchial epithelium is a frequent component of FNA specimens, particularly when EBUS procedures are used. It may be found in transthoracic and EUS-obtained specimens when the needle transverses a medium-sized bronchus. Bronchial epithelium is characterized by sheets and small strips of palisading columnar cells with cilia and prominent end plates (Figs 5.1 and 5.2). The cilia are best seen in Papanicolaou-stained preparations but are also prominent in air-dried Diff-Quik smears, where they appear as pink to purple structures. The ciliated cells are most prominent in small strips or along the edges of larger sheets.

Ancillary Techniques The use of immediate assessment allows tissue to be triaged into appropriate specimen types and transport media for optimizing the use of ancillary techniques. Ancillary techniques have been shown to be of considerable utility in the FNA workup of intrathoracic lesions.31 When a lymphoid lesion is suspected, material can be placed in specific media for flow cytometry. Currently, cell block preparations appear optimal for classic histochemical stains, immunocytochemistry, and molecular analysis.30-35 Immunocytochemistry for p63 and thyroid transcription factor 1 (TTF-1) is especially helpful in separating squamous cell carcinoma from adenocarcinoma.

Fig. 5.1: Benign respiratory (bronchial) epithelial cells demonstrating a columnal shape with fine cilia and cilial endplates (Diff-Quik, 1000×)

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Fig. 5.2: Benign respiratory epithelial cells with a prominent cilial endplate (Papanicolaou, 1000×)

Fig. 5.3: Irregular sheet of pneumocytes characterized by cells with bland nuclei. The cells lack cilia (Diff-Quik, 600×)

Bronchiolar epithelium is a nonciliated epithelium most frequently seen as sheets (Fig. 5.3). These sheets display irregular edges, and the component cells may show variable degrees of nuclear crowding and nuclear overlapping. The nuclei are usually small with a low nuclear to cytoplasmic ratio. When reactive atypia is present, both the bronchial and bronchiolar epithelium may demonstrate variable degrees of nuclear enlargement, nuclear irregularity, and hyperchromasia. In Papanicolaou-stained smears, nuclear outlines may become slightly irregular, and intranuclear cytoplasmic pseudoinclusions can be seen in bronchiolar epithelium. Such changes suggest the diagnosis of welldifferentiated adenocarcinoma. The maintenance of cilia

and end plates in bronchial epithelium helps separate reactive atypia from carcinoma (Fig. 5.4). Mesothelial cells may be found as a contaminant of pulmonary aspirates, particularly when a transthoracic approach is used. Mesothelium is readily recognized due to its monolayer sheet-like character (Fig. 5.5). Sheets of mesothelial cells show significant separation between individual cells yielding a sponge-like appearance. This apparent cell separation corresponds to the “windows” seen in fluid cytology. The cell sheets show intercellular bridges in Papanicolaou stained preparations. A zone of reactive/inflammatory change is frequently found around primary and metastatic pulmonary neoplasms. If the FNA needle samples this area and fails

Fig. 5.4: Aggregate of reactive bronchial epithelial cells with retained cilia (Diff-Quik, 400×)

Fig. 5.5: Sheet of bland mesothelial cells with clear spaces between cells corresponding to the “windows” seen in fluid samples (Diff-Quik, 400×)

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Fig. 5.6: Pulmonary macrophages lying singly and characterized by abundant foamy or granular cytoplasm and eccentric nuclei (Diff-Quik, 400×)

Fig. 5.7: Material from a pulmonary abscess characterized by large numbers of neutrophils lying in a necrotic granular background (Diff-Quik, 600×)

to sample the neoplasm, the specimen is often dominated by macrophages. The macrophages are dispersed as individual cells with abundant foamy or pigmentladen cytoplasm. The nuclei are often eccentric and relatively large with prominent nucleoli (Fig. 5.6). Pulmonary macrophages may be binucleated or multinucleated or appear as foreign body-type giant cells.

including pneumocystis, cryptococcus, and nocardia (Figs 5.8 and 5.9)43,44. Neutrophils may be numerous even when the inciting organism is a fungus.



SPECIFIC AND NONSPECIFIC INFLAMMATORY CHANGES

Bronchopneumonia (Acute Inflammation) When clinically and radiographically apparent, pneumonic infiltrates rarely if ever undergo FNA. However, localized lung abscesses may be aspirated to rule out carcinoma. Material aspirated from pulmonary abscesses is characterized by numerous neutrophils, degenerating cells, and necrotic material (Fig. 5.7). Secondary populations of macrophages, lymphocytes, and plasma cells are commonly present. When immediate assessment discloses purulent material, aerobic and anaerobic cultures should be obtained. Unless bacteria, acid-fast bacilli (AFB), or fungal organisms are seen, the etiology of an abscess usually cannot be determined. Vegetable material or fragments of striated muscle suggest aspiration as a potential cause.41,42 While special stains for organisms are of low yield, Brown and Brenn, Ziehl–Neelsen, and Gomori methenamine silver staining (GMS) may be of value. Special stains can demonstrate a variety of infectious organisms

Cytologic Features of Pulmonary Abscess • • • •

Large numbers of neutrophils and nuclear dust Granular and necrotic debris Variable numbers of macrophages, lymphocytes, and plasma cells Bacteria or fungi may be seen on special stains.

Diagnostic Pitfalls Associated with Pulmonary Abscess Acute inflammation may be associated with degenerating and necrotic squamous cell carcinomas as well as

Fig. 5.8: Fungal abscess with spherules (coccidioides immitis) and acute inflammatory cells (Papanicolaou, 400×)

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Fig. 5.9: Fungal abscess with abundant necrotic debris and fungal forms (GMS, 400×)

Fig. 5.10: Reactive pneumocytes associated with granulomatous inflammation (Diff-Quik, 400×)

other malignancies.45 Careful study of smear preparations is necessary to identify rare malignant cells in abscess material. A more common diagnostic problem is the overinterpretation of reactive changes in pulmonary epithelium surrounding abscess cavities. Fungal abscesses (mycetomas) are particularly prone to producing a lining composed of atypical metaplastic squamous epithelium. Aspiration of this epithelium yields sheets and clusters of markedly atypical cells that may be incorrectly diagnosed as carcinoma.42,46 Similar reparative changes can be seen with instrumentation, radiation therapy, chemotherapy, and thermal injuries.47-52 These reactive atypias demonstrate cellular enlargement, nuclear enlargement, nuclear hyperchromasia, nuclear pleomorphism, nucleolar enlargement, and even increased mitotic activity. In contrast to carcinoma, these atypical cells occur predominately in tight two-dimensional clusters rather than being arranged both in clusters and as single cells that are characteristic of carcinoma.46,47,51,52 Also of differential diagnostic aid is the fact that atypical reactive cells are usually present in small numbers and many of the cells with atypical nuclear features will retain cilia and terminal bars.46 Finally, demonstrating a spectrum of cells running from clearly benign to markedly atypical helps separate reactive changes from true malignancies.52,53

• •

Cytologic Features Favoring Reactive Bronchial Epithelium over Malignancy •

Single cell population demonstrating a spectrum of changes from clearly benign to atypical (absence of two distinct cell populations)

• • • • •

Smooth and regular nuclear membranes Respiratory epithelial cells with preservation of terminal plates and cilia Single atypical cells rare or absent, most atypical cells lie in clusters Atypical cells contain degenerative smudging nuclei with little crisp nuclear detail Rare or absent mitotic figures Some cell clusters retain cell polarity Cytoplasm of atypical cells often has a spreading “reparative” appearance.

Granulomatous Inflammation (Sarcoid and Granuloma of Infectious Etiology) Pulmonary granulomatous inflammation may be of an infectious etiology, a response to foreign material, or secondary to sarcoid. Granulomas, secondary to tuberculosis, are usually caseating and aspirates of these granulomas yield abundant necrotic material that is distributed with epithelioid histiocytes characteristic of granuloma. Multinucleated giant cells of Langerhans type and lymphocytes are common. Reactive bronchial epithelial cells or pneumocytes are often seen (Fig. 5.10). The Ziehl–Neelsen stain for mycobacteria is positive in approximately 50% of cases with active infection. An important component of needle aspiration in the backdrop of suspected tuberculosis is the distinction of typical from atypical mycobacteria. Fungal organisms also result in granulomatous inflammation and require distinction from tuberculosis. Microscopic evaluation, special stains, and culture are all useful in the specific diagnosis of

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Fig. 5.11: Epithelioid histiocytes granulomas (Diff-Quik, 40×)

noncaseating

Fig. 5.12: Fragment of a granuloma composed of epithelioid histiocytes with curved or “footprint” shaped nuclei (Diff-Quik, 200×)

tuberculosis, atypical mycobacteria, and fungi.54,55 While AFB stains may be negative in the face of active AFB infection, they are immensely helpful when positive. Noncaseating granulomas characterize sarcoid.56,57 The granulomas appear as tight balls of epithelioid histiocytes (Fig. 5.11). The individual histiocytes have footprint-shaped nuclei and pale cytoplasm (Fig. 5.12). The cytoplasm is characterized by indistinct borders, giving a syncytial appearance to the noncaseating granuloma. The epithelioid histiocytes are predominately found in tightly formed granuloma but may appear singly or in small clusters (Fig. 5.12). Unfortunately, there are no specific morphologic features for definitively characterizing a noncaseating granuloma as sarcoid. While uncommon

today, chronic berylliosis is often associated with noncaseating granulomas. Granulomatous reactions can occur in other clinical settings including the reactive zone around a carcinoma (particularly squamous cell carcinoma) and in response to crystalline material resulting from the use of illicit or therapeutic drugs. Granulomas also characterize rheumatoid nodules in the lung.58 Fine needle aspiration may disclose other infectious organisms including pneumocystis. Aspirates yield aggregates of foamy debris and histiocytes (Fig. 5.13). Highpower examination reveals the aggregates to be composed of numerous organisms with pale cytoplasm and dotlike inclusions best seen with Papanicolaou staining (Fig. 5.14).

Fig. 5.13: Aggregate of pneumocystis organisms demonstrating a foamy appearance (Papanicolaou, 600×)

Fig. 5.14: Pneumocystis organisms with clear cytoplasm and dot-like inclusions (Papanicolaou, 1000×)

forming

tight

Lung and Pleura



PULMONARY NEOPLASMS

Primary Pulmonary Carcinomas The classification and treatment options for carcinoma of the lung have changed substantially since the development of targeted therapies. The International Association for the Study of Lung Cancer, the American Thoracic Society, and the European Respiratory Society issued an International Multidisciplinary Classification for lung adenocarcinoma.59 This substantially changed the classification of pulmonary adenocarcinoma and made a number of recommendations for diagnosis and ancillary testing. The International Multidisciplinary Classification of carcinoma recommended discontinuing the use of the term bronchioloalveolar cell carcinoma (BAC). Depending on the size of the lesion, neoplasms with this pattern were reclassified as either adenocarcinoma in-situ or invasive carcinoma and could have mucinous or nonmucinous morphology. In-situ neoplasms are a localized lesion less than or equal to 3 cm in size demonstrating a growth pattern characterized by neoplastic cells extending along preexisting alveolar structures (lepidic growth). These lesions lack stromal, vascular, or pleural invasion. The vast majority are nonmucinous and consist of type II pneumocytes or Clara cells. Similar neoplasms less than or equal to 3 cm in size with a predominately lepidic pattern and a focus of invasion less than or equal to 5 mm in any one focus are now designated minimally invasive adenocarcinoma. In these carcinomas, the invasive component is defined as (1) histologic subtypes other than a lepidic pattern (acinar, papillary, micropapillae, or solid) or (2) tumor cells infiltrating a myofibroblastic stroma. Carcinomas, regardless of size, demonstrating invasion of the lymphatics, blood vessels, or pleura are excluded from the minimally invasive adenocarcinoma group as are carcinomas demonstrating necrosis. Adenocarcinomas with a predominately lepidic pattern of growth and more than 3 cm are considered adenocarcinomas with a dominant lepidic growth pattern. The majority of these are of nonmucinous type. The remaining accepted types of adenocarcinoma are adenocarcinoma, predominately invasive with some nonmucinous lepidic component as well as invasive mucinous adenocarcinoma (formerly mucinous bronchioloalveolar carcinoma). Using these definitions, between 70% and 90% of surgically resected lung carcinomas are invasive adenocarcinomas. Many of these demonstrate a complex mixture of histologic patterns and are further subclassified according to the predominate subtype present.60-65 Classification of small cell undifferentiated carcinoma,

83

large cell neuroendocrine carcinoma, squamous cell carcinoma, sarcomatoid carcinoma, adenosquamous carcinoma, and large cell carcinoma has not been changed. Immunocytochemistry, especially the application of p63, TTF-1, and napsin-A, is very helpful in the separation of adenocarcinoma from squamous cell carcinoma.66-67 The subdivisions of adenocarcinomas (gland forming, micropapillary, lepidic growth pattern, mucinous differentiation, “colloid”, signet ring, and clear cell) have cytologic correlates for all but the lepidic growth pattern and perhaps the clear cell type. The accurate separation of adenocarcinoma from squamous cell carcinoma is of immense therapeutic importance. Current standard of care recommends the submission of material from adenocarcinomas for EGFR and EML4-ALK mutational analysis.68-71 Mutational analysis for other genes is currently under review but is not currently standard of care.

Squamous Cell Carcinoma Squamous cell carcinoma of the lung is characterized as keratinizing and nonkeratinizing subtypes. Welldifferentiated keratinizing squamous cell carcinomas are easily recognizable in both air-dried and Papanicolaoustained preparations.72,73 These neoplasms are characterized by a combination of single cells and cell sheets (Figs 5.15 and 5.16). The neoplastic cells show extensive keratinization (Fig. 5.17). Anucleated keratinized cells are frequent. Necrosis may be prominent. The individual cells have a glassy pale to medium blue (robin’s egg blue) cytoplasm (Fig. 5.18). In Papanicolaou-stained preparations, these same cells show marked cytoplasmic orangeophilia. The presence of single keratinized cells

Fig. 5.15: Aspirate of a keratinizing squamous cell carcinoma characterized by sheets of neoplastic cells some of which demonstrate keratinization (Papanicolaou, 600×)

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Fig. 5.16: Keratinizing well differentiated squamous cell carcinoma characterized by numerous anucleatic keratotic cells and keratotic cells with large atypical nuclei (Diff-Quik, 600×)

Fig. 5.17: Keratotic cells of a squamous cell carcinoma characterized by cells with abundant waxy hard appearing cytoplasm and a triangular to rhomboid shape (Diff-Quik, 600×)

with irregular hyperchromatic nuclei is diagnostic of squamous cell carcinoma. Care must be taken to separate malignant keratotic cells from metaplastic squamous cells demonstrating reactive atypia. With decreasing levels of keratinization, the presence of a perinuclear halo within the cytoplasm is helpful as is peripheral condensation of the cytoplasm.72,73 Despite the frequently well-differentiated nature of keratinizing squamous cell carcinomas, these neoplasms are often characterized by marked pleomorphism with bizarre cells including spindle forms, tadpole-shaped cells, and rhomboid cells (Figs 5.17 and 5.18). Rarely, aspirated material will preserve whorled keratin pearls best recognized in Papanicolaou-stained preparations

(Fig. 5.17). The keratinization may spin around the nucleus in rings or Herxheimer spirals. The orangeophilia of keratinized cells has a glassy, waxy, or laminated appearance (Fig. 5.19). Characteristically, keratinized cells are often large and have a relatively low nuclear cytoplasmic ratio. The nuclei are irregular and often angular. Nuclear pyknosis is common. While nucleoli may be present, these structures are often obscured by the dense chromatin and are not visible in adenocarcinomas. Because some keratinizing squamous cell carcinomas are cystic, necrotic granular debris may dominate the smears. In these cases, acute inflammation may be extensive and obscure the tumor cells simulating a benign abscess. A granulomatous reaction with multinucleated giant cells may also be seen.

Fig. 5.18: Well differentiated keratinizing squamous cell carcinoma with polygonal, oval and rhomboid shaped cells (Diff-Quik, 600×)

Fig. 5.19: Well differentiated keratinizing squamous cell carcinoma characterized by atypical cells with marked orangophilia of cytoplasm (Papanicolaou, 600×)

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Fig. 5.20: Sheet of nonkeratinizing cells obtained from a moderately differentiated squamous cell carcinoma (Hematoxylin and Eosin, 600×)

Fig. 5.21: Sheet of crowded epithelial cells with moderate amounts of cytoplasm surrounding large atypical nuclei. Keratinization of cytoplasm is not present (Diff-Quik, 400×)

Nonkeratinizing squamous cell carcinomas are more cohesive with fewer single cells (Figs 5.20 and 5.21). The nuclei of these carcinomas are often spindle shaped or elongated. The chromatin is dense and irregularly distributed. Nucleoli are of variable size and number (Fig. 5.22). In most cases, the cytoplasm of nonkeratinizing squamous cell carcinomas is denser than that seen in adenocarcinomas, and the cell membranes are better defined. The cell groups comprising nonkeratinizing squamous cell carcinomas tend to be irregular cohesive fragments that lack a flat sheet-like appearance. Despite the absence of true orangeophilia in nonkeratinizing squamous cell carcinomas, the cytoplasm usually has a dense appearance with distinct cell borders.

Nonkeratinizing forms lack keratin pearls and have few bizarre-shaped malignant cells. Moreover, a greater proportion of the cells lie in sheets and clusters than is characteristic of keratinizing squamous cell carcinomas. The cells are more uniform and the nuclear cytoplasmic ratio is higher than that seen in the keratinizing form of squamous cell carcinoma. Nonkeratinizing squamous cell carcinoma cells have centrally located nuclei with irregular nuclear membranes. The chromatin may be coarse and hyperchromatic, but it is more open than that seen in keratinizing squamous cell carcinomas.

Fig. 5.22: Poorly differentiated squamous cell carcinoma. Some cells contain prominent nucleoli. Cytoplasm appears abundant in many cells. Nuclei are irregular and hyperchromatic (Diff-Quik, 400×)

Diagnostic Pitfalls •

Necrosis in other forms of carcinoma may simulate keratinization • Inflammation and necrosis may be present in abscesses and mycetomas simulating necrotic squamous cell carcinomas • Squamous metaplasia with reactive atypia may simulate squamous cell carcinoma • Mucoepidermoid carcinomas and rare basaloid carcinomas may simulate nonkeratinizing squamous cell carcinomas. The presence of extensive necrosis seen in both abscess cavities and some other forms of carcinoma may simulate keratinization.74 Because many well-differentiated squamous carcinomas show extensive necrosis, its presence may result in the misidentification of metaplastic squamous epithelium associated with an abscess or poorly differentiated adenocarcinoma with extensive necrosis as a squamous cell carcinoma. True keratinization shows strong orangeophilia on Papanicolaou staining and a

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Adenocarcinoma

characteristic “robin’s egg” blue in air-dried preparations. Poorly differentiated squamous cell carcinomas can be confused with large cell anaplastic carcinoma and poorly differentiated adenocarcinoma. In the case of poorly differentiated adenocarcinoma, immunohistochemical staining for p63 and TTF-1 is helpful in the separation of these two malignancies.66,67 Squamous cell carcinomas are p63 positive but TTF-1 negative. Adenocarcinomas show TTF-1 positivity and p63 nonreactivity. In some poorly differentiated squamous cell carcinomas, single cell necrosis with cell clusters as well as palisading along the cluster edge may simulate the gland lumens or papillary fragments of adenocarcinoma. An additional issue with squamous cell carcinoma is the presence of a prominent population of multinucleated foreign body-type giant cells. These cells are formed in response to keratotic debris and do not indicate an infectious granulomatous process. Careful attention to the presence of keratotic debris and rare malignant cells is helpful in recognizing the squamous carcinomatous nature of the process. In some well-differentiated squamous cell carcinomas, only keratotic debris is aspirated. In the absence of malignant cells, a definitive diagnosis of squamous cell carcinoma cannot be made. Spindle cell forms of squamous cell carcinoma exist and may be mistaken for sarcomas. Most spindle cell squamous cell carcinomas appear cytologically as a mixture of spindle cells and classic nonkeratinizing squamous cell carcinoma. The cells occur either in small loose groups or as single cells. Marked pleomorphism is characteristic.

The cytomorphology of adenocarcinoma not otherwise specified is characterized by cohesive groups of cells that show differentiation toward acinar structures or papillary fragments.75-77 Mucin may be abundant in the background. The cells can form flat sheets as well as cell balls and papillations (Figs 5.23 to 5.25). The individual cells are of medium-to-large size with abundant cytoplasm (Fig. 5.26). Many cell groups show abortive acinar or glandular structures (Figs 5.27 and 5.28). The nuclei are round to oval and usually lie eccentrically (Fig. 5.29). Characteristically, a large solitary nucleolus is present. Extensive background mucin may be seen, resulting in the so-called “colloid” appearance. Additionally, signet ring cell forms and intracytoplasm mucin vacuoles (Fig. 5.29) may be seen along with cells showing a columnar differentiation. Adenocarcinoma in situ, microinvasive carcinoma, and adenocarcinoma with a lepidic growth pattern may not have a specific appearance. In some cases, lesions traditionally designated BAC have cytologic features favoring that diagnosis. These features include a population of atypical bronchioloalveolar cells with bland nuclear features. The nuclei show varying degrees of nuclear clearing, chromatin margination, nuclear grooves, or intranuclear cytoplasmic pseudoinclusions.78-80 BACs are also characterized by relatively large sheets or papillae of bland appearing cells lying in a clean or mucinous background (Fig. 5.30). While the cells show minimal nuclear atypia, they lack cilia and terminal bars. Despite these features, there is considerable overlap between classic pulmonary

Fig. 5.23: Moderately differentiated adenocarcinoma characterized by a cell cluster with gland-like rings of cells (Hematoxylin and Eosin, 600×)

Fig. 5.24: Papillary group of atypical cells obtained from a moderately differentiated adenocarcinoma (Papanicolaou, 400×)

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Fig. 5.25: Atypical cells forming a papillary aggregate (Diff-Quik, 200×)

Fig. 5.26: The individual cells of a moderately differentiated adenocarcinoma have abundant pale to clear cytoplasm (Diff-Quik, 400×)

Fig. 5.27: Cell cluster from an adenocarcinoma with a round glandlike structure (Papanicolaou, 400×)

Fig. 5.28: Moderately differentiated adenocarcinoma characterized by an acinar group (Diff-Quik, 200×)

Fig. 5.29: Cells of an adenocarcinoma may contain intracytoplasmic mucin vacuoles (Diff-Quik, 200×)

Fig. 5.30: Aspirate material obtained from an adenocarcinoma of the type originally called BAC. Note the cell sheet appears as a monolayer composed of relatively bland cells with pale cytoplasm (Hematoxylin and Eosin, 400×)

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adenocarcinomas and BACs. Because of the marked morphologic overlap, formal separation of BAC from adenocarcinoma appears imprudent and unnecessary.81,82 The current recommendations do not recognize a cytologic counterpart to adenocarcinoma with lepidic growth pattern.59

Neuroendocrine Neoplasms of the Lung (Carcinoids, Atypical Carcinoids, and Small Cell Anaplastic Carcinomas) Small Cell Anaplastic Carcinoma Small cell anaplastic carcinoma of the lung represents approximately 20% of all lung cancers and is the most aggressive member of the family of neuroendocrine neoplasms of the lung. These carcinomas occur most frequently in males with a median age of approximately 60 years. Eighty-five percent of patients are smokers.83 These neoplasms characteristically arise within the central portions of the lung making bronchoscopic biopsy and FNA the preferred diagnostic modalities. The neoplasms are often extensively necrotic and are usually widely disseminated at time of recognition. The histomorphologic features have been well described by Azzopardi,84 but subclassification has changed over the decades. Subcategories have included small cell carcinoma, mixed small cell/large cell, and combined small cell carcinoma. The mixed small cell/large cell subcategory was poorly defined with poor interobserver agreement.85 Subsequently, this category was deleted and a combined

Fig. 5.31: Small cell carcinoma characterized by groups of cells with scant cytoplasm and hyperchromatic nuclei. Nuclear chromatin streaking is prominent (Diff-Quik, 200×)

small cell carcinoma substituted. Currently, assignment of a primary pulmonary carcinoma to the small cell category is based on nuclear features as determined by light microscopy.86 At the present moment, cytologic subclassification of small cell carcinomas appears inconsistent and probably therapeutically unnecessary. Material aspirated from small cell anaplastic carcinomas is usually highly cellular.87-90 The cells are often individually dispersed, but small clusters and tight groups are present in many smears (Fig. 5.31). The individual cells are markedly pleomorphic with scanty or absent cytoplasm (Fig. 5.32). Extensive nuclear molding is present and mitotic figures common. The nuclei are easily disrupted on smearing giving rise to streaks of nuclear material (Fig. 5.33).90 The chromatin pattern is coarsely granular, and nucleoli are usually inconspicuous. However, in some aspirates, small distinct nucleoli can be found. The cytoplasm is very scant or absent in small cell anaplastic carcinomas, but paranuclear blue inclusions are a common feature of small cell anaplastic carcinomas and aid in their separation from lymphoma.91 Necrosis is a common finding in the background of aspirates from small cell carcinomas.

Diagnostic Criteria—Small Cell Carcinoma of Lung • • •

Cellular smears containing dispersed individual cells, cell clusters, and small tight groups Small-to-medium-sized cells with scant or absent cytoplasm Prominent nuclear molding

Fig. 5.32: Smear of small cell carcinoma characterized by poorly cohesive round cells with very scant cytoplasm and hyperchromatic nuclei. Nucleoli are absent and some nuclear molding is seen (Diff-Quik, 600×)

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• • • • • •

Irregular nuclei with coarse granular chromatin Nucleoli are usually absent but in some smears may be small and distinct Tear drop cells, smeared cells, and streaks or cords of disrupted nuclear material present Paranuclear blue cytoplasmic inclusions Abundant necrosis frequent Background lacks lymphoglandular bodies.

Differential Diagnostic Issues • •

•

Subtypes of small cell carcinoma may be difficult to distinguish from some non-small cell carcinomas Neuroendocrine markers do not establish diagnosis of small cell carcinoma; neuroendocrine markers positive in carcinoids, atypical carcinoids, non-small cell neuroendocrine-positive neoplasms, and large cell neuroendocrine carcinoma Small cell carcinomas must be distinguished from lymphoma and some metastatic tumors including melanoma.

Typical Carcinoid Tumor The central-type typical carcinoid tumor is the most common form of carcinoid tumor. It presents as a slowly growing solitary mass within a major bronchus. It may lead to either bronchial obstruction or hemoptysis.92 Histopathologically, these tumors are made up of small uniform cells with central nuclei and scant or no mitotic activity. The cytoplasm is finely granular and the cells lie in nests, ribbons, festoons, or cords. The nuclei are small

Fig. 5.33: Nuclear chromatin crushing and streaking characteristic of small cell anaplastic carcinoma (Diff-Quik, 600×)

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and bland with a characteristic salt and pepper chromatin pattern. Most cases are endocrinologically clinically silent. Immunohistochemically, neoplasms may be reactive with antibodies directed against serotonin, neuron-specific enolase, chromogranin, synaptophysin, and Leu7.93 Cytologically, typical carcinoid tumors are characterized by cell clusters with a trabecular acinar or palisaded appearance lying in a background of dispersed cells (Fig. 5.34).87,94,95 The individual cells have a monotonous appearance, are relatively small in size with modest amounts of intact often granular cytoplasm (Fig. 5.34). The nuclei are round or oval and have a fine stippled (salt and pepper) granular chromatin pattern (Fig. 5.35). Nucleoli, when present, are small. Some cells have a plasmacytoid appearance with eccentric nuclei and a “clock face” chromatin pattern. The background may have plexiform groups of small vessels to which are adherent small groups and individual neoplastic cells. The background is free of necrotic debris and mitotic figures are not seen.

Differential Diagnostic Issues • •

•

Carcinoid tumors must be distinguished from small cell anaplastic carcinomas Small cell anaplastic carcinomas contain greater degrees of nuclear atypia and the presence of nuclear molding and nuclear membrane irregularities Pulmonary hamartomas may be composed of small bland looking cells closely resembling those of a carcinoid tumor. The presence of chondroid aids in their distinction

Fig. 5.34: Smear of carcinoid tumor characterized by cell clusters and individual cells with bland oval nuclei and moderate amounts of cytoplasm (Diff-Quik, 400×)

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Fig. 5.35: Carcinoid tumor cells with a round to oval shape and bland nuclei with a “salt and pepper” chromatin pattern (Diff-Quik, 1000×)

Fig. 5.36: Spindle cell carcinoid characterized by a sheet of spindleshaped cells with oval to spindle-shaped nuclei (Papanicolaou, 200×)

•

These neoplasms occasionally metastasize to hilar lymph nodes and bone. Fine needle aspirates obtained from spindle cell carcinoids reveal a population of short “spindle” cells running in bundles or lying singly in the background (Fig. 5.36).98,99 The nuclei are bacillary in shape and have the same chromatinic features as typical carcinoids (Fig. 5.37). The nucleoli are small but distinct on hematoxylin and eosin (H&E) and Papanicolaou-stained preparations (Fig. 5.38). Mitotic figures are not seen and the background is free of necrotic debris.

Some low-grade adenocarcinomas (formerly BAC) may closely resemble carcinoid tumors, but the cells of these carcinomas have a more columnar appearance and a larger cell size. BAC-like adenocarcinomas are also more cohesive forming sheets and large clusters

Spindle Cell Carcinoid These carcinoid tumors are characterized by a spindle cell morphology and peripheral location.96,97 The neoplastic cells have a definitive spindle shape and form bundles. Occasional cases show a mixture of the spindle cell component with groups of polygonal cells similar to those in typical carcinoids. Many spindle cell carcinoids are subpleural and less than 2 cm in diameter.97

Fig. 5.37: Spindle cell carcinoid composed of cells with wispy spindled cytoplasm and oval to bacillary shaped nuclei with a “salt and pepper” chromatin pattern (Hematoxylin and Eosin, 400×)

Differentiated Neuroendocrine Carcinoma Atypical carcinoid tumors: Atypical carcinoids exhibit the overall architectural and immunohistochemical features

Fig. 5.38: The nuclei of spindle cell carcinoids have a “salt and pepper” chromatin pattern with some nuclei having small nucleoli (Papanicolaou, 600×)

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of carcinoid tumors but demonstrate nuclear atypia, increased mitotic activity, and, at times, foci of necrosis.100,101 Atypical carcinoids show a proclivity for lymph node metastases (70%) and are definitively more aggressive than their typical counterpart. Five-year survival is approximately 56% and ten-year survival is 35%.102 Cytologically, atypical carcinoid tumors have clearly malignant nuclear features but have a number of characteristics in common with typical carcinoid tumors. Specifically, these neoplasms are composed of cohesive aggregates of cells as well as a prominent population of single dissociated cells. The individual cells have smallto-moderate amounts of cytoplasm (Fig. 5.39). The nuclei have a neuroendocrine appearance with a salt and pepper chromatin (Fig. 5.40).86,103,104 The presence of necrotic debris and mitotic figures favors the diagnosis of atypical carcinoid tumor. These lesions lack the nuclear molding, smeared nuclear material, and extensive background necrosis characteristic of small cell anaplastic carcinoma. Despite these differences, separation of atypical carcinoids from small cell anaplastic carcinoma may be extremely difficult.

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Additionally, architectural features such as the formation of cell ribbons, cords, and rosette-like structures offer clues to their neuroendocrine differentiation.105,106 Cytologic preparations obtained from large cell neuroendocrine carcinomas are characterized by sheets, clusters, and individual cells showing marked nuclear pleomorphism. The individual cells have abundant cytoplasm surrounding hyperchromatic nuclei with irregular nuclear membranes. The chromatin pattern is frequently granular with variably prominent nucleoli. Mitotic figures are present in most smears. Clear-cut cytologic features indicating neuroendocrine differentiation are not present, but cell block preparations submitted for immunocytochemistry will demonstrate neuroendocrine markers. Characteristically, these carcinomas show strong positive staining for synaptophysin, chromogranin, and neuron-specific enolase. Separation on purely morphologic grounds from undifferentiated large cell carcinoma appears impractical.

Undifferentiated Large Cell Carcinoma

These malignancies are composed of larger cells than those characteristic of atypical carcinoid tumor and small cell anaplastic carcinoma. They are highly mitotically active usually with more than 10 mitotic figures per 10 highpower fields, and necrosis is often extensive. Clues to their neuroendocrine nature are supplied by immunopositivity for neuron-specific enolase, serotonin, or chromogranin.

These large cell carcinomas are pleomorphic malignancies without definitive evidence of differentiation toward either a squamous or glandular morphology.107 The individual tumor cells are large with generally abundant cytoplasm. The nuclei are pleomorphic with prominent nucleoli. The chromatin pattern is variable but may show coarse granularity or chromatin clearing with margination. These neoplasms probably represent poorly differentiated forms of other more common carcinomas particularly adenocarcinoma.

Fig. 5.39: Atypical carcinoid tumors are composed atypical cells which form loose groups or lie individually (Hematoxylin and Eosin, 400×)

Fig. 5.40: The nuclei of atypical carcinoid tumors have a “salt and pepper” chromatin pattern but show greater degrees of atypia than are seen in typical carcinoid tumors (Hematoxylin and Eosin, 600×)

Large Cell Neuroendocrine Carcinoma

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Fig. 5.41: Undifferentiated large cell carcinomas are characterized by large cells lying in small groups and as single cells. The nuclei are hyperchromatic and irregular (Diff-Quik, 600×)

Fig. 5.42: Smear preparation of an adenoid cystic carcinoma demonstrating rings of cells distributed around basement membrane material (Hematoxylin and Eosin, 200×)

Cytologically, these neoplasms are characterized by large cells lying in groups and individually (Fig. 5.41). The cells demonstrate marked nuclear pleomorphism and moderate-to-abundant cytoplasm. The cytoplasm is frequently ill defined. The nuclei are markedly pleomorphic with irregular nuclear membranes, chromatin clearing, and margination along with prominent nucleoli. Occasional examples will contain multinucleated neoplastic giant cells. An unusual feature is the presence of neutrophils adherent to some of the malignant epithelial cells.

Low-Grade Carcinomas of Bronchial Gland Origin (Formerly Bronchial Adenomas)

Fig. 5.43: Adenoid cystic carcinoma characterized by sheets of relatively bland cells surrounding cords and cores of basement membrane material (Hematoxylin and Eosin, 200×)

Fig. 5.44: Mucoepidermoid carcinoma characterized by cells with mucin vacuoles and intermediate cells (Diff-Quik, 400×)

A number of low-grade carcinomas of a morphology similar to that seen in the salivary gland occur within the main bronchi. These neoplasms include adenoid cystic carcinoma (Figs 5.42 and 5.43), mucoepidermoid carcinoma (Figs 5.44 and 5.45), and bronchial benign mixed tumors (Figs 5.46). The cytologic appearance of these neoplasms is identical to their salivary gland counterparts.108,109

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Fig. 5.45: Sheets of cells composed of intermediate cells and vacuolated mucin producing cells characterize intermediate grade mucoepidermoid carcinomas (Diff-Quik, 400×)

Fig. 5.46: Benign mixed tumors are characterized by oval, plasmacytoid and spindle-shaped cells lying in a chondromyxoid background (Diff-Quik, 400×)

Other Primary Pulmonary Neoplasms Chondroid hamartomas occur predominately in adult males and present as solitary or rarely multiple pulmonary nodules.110 Most occur in the lung parenchyma immediately beneath the pleura and are usually asymptomatic. These lesions are sharply delineated and lobulated giving a characteristic radiographic appearance. Histologically, pulmonary hamartomas are composed of benign hyaline cartilage arranged in islands and may be associated with adipose tissue or smooth muscle. Characteristically, the clefts between cartilaginous lobules are lined by ciliated or nonciliated respiratory epithelium. Some pulmonary hamartomas are associated with a nonfamilial syndrome

(Carney triad) characterized by multiple pulmonary hamartomas, epithelioid gastrointestinal stromal tumors, and extra-adrenal paragangliomas.111 The cytologic features of pulmonary hamartomas are well described and are characterized by fibromyxoid tissue, mucous, and occasionally mature cartilage (Fig. 5.47).112-114 Recognizable fragments of mature cartilage are present only in a minority of cases.112 Aspirates from pulmonary hamartomas are often of scant cellularity, and the background is usually clean without necrosis, blood, or inflammation.112,114 Frequently, cytologic material contains disorganized clumps of adipose tissue, smooth muscle, cartilage, and rarely bone.115 An epithelial element is generally present and composed of normal appearing bronchial cells forming sheets and clusters (Fig. 5.48).

Fig. 5.47: Plumonary hamartomas are characterized by chondromyxoid material in which are scattered bland pneumocytes and bronchial epithelial cells (Diff-Quik, 100×)

Fig. 5.48: The epithelial cells associated with pulmonary hamartomas are bland respiratory cells often with a columnar or cuboidal shape (Diff-Quik, 200×)

Chondroid Hamartoma (Pulmonary Hamartoma)

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Fig. 5.49: The background of pulmonary hamartoma is often rich in fibromyxoid or chondromyxoid material (Diff-Quik, 40×)

Fig. 5.50: Some aspirates of pulmonary hamartoma contain fragments of hyaline cartilage (Diff-Quik, 100×)

The fibromyxoid tissue characteristic of these lesions has a watery to mucinous appearance (Fig. 5.49). The fibromyxoid aggregates frequently have fibrillar edges and entrapped groups of bronchiolar epithelial cells. These latter epithelial cells have uniform round nuclei and a fine chromatin. Nucleoli are inconspicuous. Bland spindle cells are present within the fibromyxoid stroma. In less than 50% of cases, fragments of mature cartilage are seen (Fig. 5.50). Pulmonary hamartomas are a significant source of false-positive diagnoses.114 In a series by Hughes et al.,114 22% of cases were associated with a false-positive diagnosis of malignancy. The most common false-positive diagnoses were carcinoid tumor, adenocarcinoma, and small cell carcinoma. The dominance of an epithelial component (respiratory epithelium and bronchiolar epithelium) appears to result in a significant number of false-positive diagnoses of carcinoid tumor and adenocarcinoma.114

Sclerosing Hemangioma

Key Diagnostic Features—Pulmonary Hamartoma • • • •

Smears may be scanty but contain aggregates of fibromyxoid tissue Bronchiolar and respiratory epithelium often trapped within a fibromyxoid stroma Benign cartilage present in less than 50% of cases Fibromyxoid stroma has a feathery edge and contains bland spindle cells.

Sclerosing hemangioma is an uncommon neoplasm occurring predominately in adult women, where it presents as an asymptomatic small solitary nodule.116 Histomorphologically, these lesions present as a compact growth of polygonal cells arranged in solid or papillary patterns. Sclerotic areas are common. The individual cells have abundant eosinophilic cytoplasm. Areas of old hemorrhage including hemosiderin deposition are common. Cytologically, smears from these lesions are characterized by large tissue aggregates containing sclerotic fibrovascular cores and papillary excrescences (Fig. 5.51).116-121 These tissue fragments are composed of epithelial-like cells surrounding blood vessels and stromal tissue.117,119 The cells covering the fibrovascular cores are loosely cohesive (Figs 5.52 and 5.53) and have moderate amounts of eosinophilic cytoplasm, demonstrating a cuboidal, oval, or short columnar shape (Fig. 5.52). The nuclei of these cells have a finely granular chromatin and small but distinct nucleoli (Fig. 5.53). Many of the cells contain hemosiderin granules, and in other areas, red blood cells lie in close association with the tissue fragments. The fibrovascular cores may contain small capillary segments or be composed predominantely of sclerotic stroma (Fig. 5.53).

Diagnostic Pitfalls

Diagnostic Features

•

•

•

Entrapped respiratory and bronchiolar epithelium may show reactive atypia, leading to the misdiagnosis of carcinoid tumor or low-grade adenocarcinoma Cartilage present in less than 50% of cases resulting in underdiagnosis of lesion.

•

Large tissue fragments composed of loosely cohesive epithelial-like cells surrounding central fibrovascular cores Epithelial-like cells may have a cuboidal, columnar, or polygonal shape

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Fig. 5.51: Cytologically, sclerosing hemangiomas are characterized by papillary structures with fibrovascular cores (Hematoxylin and Eosin, 100×)

Fig. 5.52: The cells lining the fibrovascular cores of sclerosing hemangiomas are cuboidal or short columnar and have bland nuclei (Hematoxylin and Eosin, 400×)

•

columnar or cuboidal cells of well-differentiated adenocarcinoma. Differential diagnostic features favoring sclerosing hemangioma include a prominence of hemosiderin granules and red blood cells in the background as wells as the presence of distinctive fibrovascular cores. While these neoplasms form distinctive papillary excrescences, duct-like structures, mucin production, and true nuclear anaplasia are not seen. These features aid in the separation of sclerosing hemangiomas from adenocarcinomas. The epithelial cells adherent to the fibrovascular stromal cores probably represent reactive alveolar or bronchiolar cells.

• •

Epithelial-like cells have bland round or ovoid nuclei with small distinct nucleoli Many cells contain hemosiderin granules Red blood cells often free in the background.

Diagnostic Pitfalls •

Sclerosing hemangiomas may be mistaken for lowgrade adenocarcinomas, particularly those formerly known as BAC. Sclerosing hemangiomas are often mistaken for either a papillary-type adenocarcinoma or the form of adenocarcinoma formally known as BAC. The neoplastic cells adherent to the fibrovascular cores may be mistaken for the

Lymphomas of Pulmonary Origin

Fig. 5.53: The lining cells of sclerosing hemangiomas are loosely cohesive and cuboidal or short columnar in shape (Hematoxylin and Eosin, 600×)

Lymphoproliferative processes may involve the lung either as a primary lesion or as a component of diffuse disease.122,123 Primary lymphomas of the lung can be divided into six diagnostically useful categories: (1) large cell lymphoma conventional type, (2) small lymphocytic lymphoma, (3) plasmacytoma, (4) Hodgkin disease, (5) leukemias, and (6) lymphomatoid granulomatosis. Of these, large cell lymphoma of conventional type has the most characteristic cytologic findings, and along with low-grade lymphocytic lymphoma can be diagnosed from FNA specimens using cytomorphologic analysis and flow cytometry.124 Low-grade lymphocytic lymphomas are characterized by a monotonous population of small lymphoid cells and flow cytometrically proven monoclonality (Fig. 5.54). Large cell lymphomas arising within lung are usually of diffuse large B-cell type and are

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Fig. 5.54: Low-grade lymphocytic lymphomas are characterized by a noncohesive monotonous population of lymphoid cells. Lymphoglandular bodies are seen in the background (Diff-Quik, 400×)

Fig. 5.55: Large cell lymphomas are characterized by a monotonous population of large lymphoid cells. Lymphoglandular bodies are prominent in the background (Diff-Quik, 400×)

characterized by monomorphous proliferation, demonstrating monoclonality for B-cell markers (Fig. 5.55). Both large cell and small cell lymphomas characteristically have an abundance of lymphoglandular bodies.

Metastatic adenocarcinomas arising from the colon are characterized by abundant necrotic debris in which are admixed sheets, nests and clusters of large cuboidal to columnar cells with moderate amounts of cytoplasm (Fig. 5.58). The nuclei are large with coarse chromatin and often prominent nucleoli. Gland-like structures are frequent in which the neoplastic cells palisade around a central lumen. Renal cell carcinomas are characterized by nests and small sheets of cells with abundant pale, foamy, or even clear cytoplasm (Fig. 5.59). The nuclei are usually large often with chromatin clearing and macronucleoli

Other Rare Primary Neoplasms of the Lung Reports of the cytologic findings in a variety of rare primary pulmonary neoplasms have been made in the literature. These include pleuropulmonary blastoma,125 pulmonary carcinosarcoma,126 a variety of sarcomas,127,128 and inflammatory pseudotumor of the lung.129 

METASTATIC LESIONS TO THE LUNG

The lung represents a common site for deposition of metastatic disease and a number of malignancies metastasize to the lung with significant frequency. Most common among these are carcinomas of the breast, colon and kidney, and melanoma. Metastatic malignant melanoma can present with a wide range of cytomorphologies. Metastatic malignant melanoma can resemble a small lymphoid proliferation, myeloma, spindle cell sarcoma, or a pleomorphic malignancy without definitive direction of differentiation.130 Most commonly, malignant melanoma presents as a discohesive population of large pleomorphic cells (Fig. 5.56). These cells have abundant cytoplasm surrounding large nuclei with prominent nucleoli. Pigment is variably present and may be seen in both the neoplastic cells and associated histiocytes (Fig. 5.57).

Fig. 5.56: Aspirates of metastatic malignant melanomas contain a pleomorphic population of cells which range from lymphocyte-like cells to plasmacytoid cells to anaplastic giant cells (Diff-Quik, 400×)

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Fig. 5.57: Melanoma aspirates may contain pleomorphic giant cells and histiocytes with abundant intracytoplasmic pigment (Diff-Quik, 400×)

Fig. 5.58: Metastases from colon adenocarcinomas are characterized by groups of columnar or cuboidal cells which may form gland-like structures (Diff-Quik, 200×)

(Fig. 5.59). The presence of macronucleoli is quite characteristic of metastatic renal cell carcinoma.131,132 Metastatic adenocarcinoma from a breast primary may show a relatively wide range of morphologies. Most commonly, metastatic deposits of mammary carcinoma reveal a pattern of individually dispersed cells among small cell aggregates and cell balls. These adenocarcinoma cells are of small-to-medium size and have modestto-moderate amounts of cytoplasm (Fig. 5.60). While the nuclei are enlarged, they have a bland chromatin pattern.

Occasionally, glandular/ductal structures are seen within the cell groups (Fig. 5.61). When linear groups of small bland carcinoma cells are seen, a lobular phenotype should be suspected.

Fig. 5.59: Metastases from renal cell carcinomas often contain groups of polygonal cells with abundant granular, pale or clear cytoplasm (Diff-Quik, 600×)



PLEURAL LESIONS

Malignant Mesothelioma The diagnosis of malignant mesothelioma generally requires the combination of cytomorphologic findings

Fig. 5.60: Metastasis from mammary adenocarcinomas are characterized by clusters of small to medium sized round to oval cells. The nuclei may be hyperchromatic but often have a bland appearance (Diff-Quik, 200×)

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Fig. 5.61: Metastases from mammary carcinomas may contain cell groups with duct-like or ring-like structures (Diff-Quik, 400×)

Fig. 5.62: Malignant mesotheliomas are characterized by tight groups and sheet-like arrangements of round, or polygonal cells. Clear spaces may be present between the cells within the sheet (Diff-Quik, 400×)

along with radiographic and clinical features. In most cases, the neoplastic cells are clearly malignant, but separation of malignant mesothelioma from adenocarcinoma is diagnostically challenging. In addition to the epithelioid type of mesothelioma, mixed and sarcomatoid forms occur.133,134 A variety of other subtypes are recognized histologically including desmoplastic mesothelioma and mesothelioma with rhabdoid features, but these are rarely recognized cytologically.135,136 The majority of mesotheliomas examined cytologically are of the epithelioid type. Epithelioid malignant mesotheliomas are cytologically characterized by papillary tissue fragments with fibrovascular cores, ball-like clusters, or cell sheets (Fig. 5.62).137-143 The individual cells are round to polygonal with generally round hyperchromatic nuclei often containing prominent nucleoli (Fig. 5.63). The cells may have a cytoplasmic zonation phenomenon with an endoand ectoplasmic appearance. Single vacuolated cells may be seen. The sarcomatoid and mixed forms are characterized by single disassociated cells and clusters of spindle cells. These cells have elongated or ovoid spindle-shaped nuclei, coarse chromatin, and prominent nucleoli. Naked nuclei may be seen in the background. In the biphasic form, these spindle-shaped cells are mixed with epithelial type cells. Immunohistochemistry is of immense help in separating mesothelioma from adenocarcinoma. Cell block material is especially helpful in the immunocytochemical differential diagnosis of these malignancies. Mesotheliomas react positively with antibodies directed

against EMA, calretinin, cytokeratins 5,6 and vimentin. Additionally, the use of WT-1 and thrombomodulin is helpful in identifying malignant mesothelioma. Malignant mesothelioma cells are usually nonreactive for CEA, B72.3, TTF-1, and Ber-Ep4. The accurate identification of a population of atypical cells as malignant mesothelioma requires separation of this entity from reactive mesothelial cells. At times, reactive mesothelial cells may be impossible to separate from malignant mesothelioma. In general, reactive mesothelial cells form larger clusters and more monolayer sheets than cell populations characteristic of malignant mesothelioma.

Fig. 5.63: The cells of epithelioid malignant mesothelioma are round to polygonal with hyperchromatic nuclei. Nucleoli may be prominent (Diff-Quik, 1000×)

Lung and Pleura

Fig. 5.64: Cells aspirated from solitary fibrous tumors are spindle shaped with wispy cytoplasm and bland plump spindle shaped nuceli (Diff-Quick, 400×)

The presence of papillary fragments, ball-like clusters with nuclear crowding, and marked nuclear pleomorphism along with necrosis favors the diagnosis of mesothelioma.

Solitary Fibrous Tumor (Benign Localized Mesothelioma) Solitary fibrous tumors arise from the pleura and form a spindle cell proliferation frequently with little nuclear atypia. These lesions are usually asymptomatic but may present due to pain, cough, or dyspnea.144,145 Microscopically, these neoplasms are characterized by a patternless pattern composed of relatively bland spindle cells. The fibroblast-like cells frequently squeeze between abundant collagen bundles and may even have a keloidlike pattern. In other cases, these neoplasms are characterized by a hemangiopericytomatous pattern. Cytologically, aspirates of these lesions are generally of low cellularity and are composed of bland spindle cells (Fig. 5.64). These cells may have a bipolar appearance or may present as naked nuclei. The cytologic features of solitary fibrous tumor are further described in the Chapter 12 on musculoskeletal lesions.

Askin Tumor (PNET) These small round cell malignancies are discussed in Chapter 12, which describes musculoskeletal lesions. 

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Chapter

6

The Breast

Shahla Masood, Marilin Rosa



INTRODUCTION

Fine needle aspiration biopsy (FNAB) of the breast is a well-known technique that can be used as a diagnostic and therapeutic procedure.1-4 It can confirm the diagnosis of malignancy in clinically or radiologically suspicious lesions, evaluate for tumor recurrence, corroborate the impression of a benign process avoiding unnecessary surgery, as well as evacuate clinically symptomatic cystic lesions.4-9 FNAB, when used as part of the triple test (physical examination, radiologic findings, and cytologic evaluation), has been demonstrated to be an excellent tool in the diagnosis of palpable lesions.1,2,5,6 The sensitivity, specificity, and accuracy of FNAB for breast malignancy range from 82% to 98%, from 77% to 100%, and from 79% to 97%, respectively, in the literature. In our own series, FNAB of palpable breast lesions had a sensitivity of 92%, specificity of 100%, accuracy of 94%, a positive predictive value of 100%, and a negative predictive value of 79%.10 The advantages of FNAB over other diagnostic techniques include cost-effectiveness, minimal discomfort for the patient, rapid results with potential bedside diagnosis, very low incidence of complications, and comparable results to core needle biopsy (CNB). In addition, when compared with CNB in palpable breast lesions, FNAB by moving the needle in different directions permits a better and a more extensive sampling (Fig. 6.1).2-7 FNAB is especially useful, when clinical presentation such

as a locally advanced breast cancer, nipple retraction, or axillary node involvement are considered when establishing the diagnosis. The diagnostic value of FNAB in the clinical management of breast lesions depends on many factors; therefore, the use of this technique might not be possible in all centers. First of all, a multidisciplinary approach is necessary

Fig. 6.1: Fine needle aspiration biopsy helps moving the needle in different directions that permits a better and a more extensive sampling of the lesion

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to increase FNAB’s role and to reduce its limitations. A careful selection of cases is also necessary to avoid insufficient specimens and false-negative results. In addition, accessibility to high-quality radiologic services to complement clinical and cytologic findings, availability of trained cytopathologists to perform and interpret FNA results, and accessibility to a laboratory with experience performing prognostic/predictive markers evaluation on cytologic material are essential.10-13 These are possibly some of the reasons behind the nationwide steady decline in the number of breast FNAB. A major disadvantage of the procedure is the inability to distinguish between highgrade in situ versus poorly differentiated invasive carcinomas. However, that would be a critical distinction only if neoadjuvant chemotherapy is a consideration.7 There is no doubt that changes in patterns of clinical practice that encourage clinicians to perform ultrasound–guided CNB in their offices have contributed significantly to this decline. 

INFLAMMATORY AND REACTIVE LESIONS

Clinically, inflammatory breast changes can be seen in different entities ranging from benign processes, such as mastitis, to aggressive malignant tumors, such as inflammatory carcinoma.14 The differential diagnosis of inflammatory breast lesions differs by age group and physiologic status. In pregnant and lactating women, the most common causes are acute mastitis, abscess, and, less often, malignancy. In nonlactating women, inflammatory

Fig. 6.2: Traumatic fat necrosis: Aspirations of fat necrosis reveal vacuolated histiocytes with engulfed lipid droplets (Diff-Quik stain, 40×)

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breast cancer, plasma cell mastitis, lymphoma, and generalized dermatitis, among others, should be considered.14,15

Fat Necrosis Clinical Features Fat necrosis is a benign inflammatory condition that can mimic cancer clinically and radiologically. It can be caused by previous biopsy or surgery, trauma, infectious processes, and radiation.14,16,17 It commonly affects perimenopausal women18 and may present as a palpable mass or as a mammographically detected abnormality.14

Cytologic Features Aspirates obtained from cases of fat necrosis are frequently hypocellular with a predominance of vacuolated or hemosiderin-laden histiocytes and degenerated fat (Figs 6.2 and 6.3). Multinucleated giant cells, fibroblasts, chronic inflammatory cells, dirty background, and rarely, calcified particles are also present. Reactive epithelial cells are usually a minor component, but their presence may raise the suspicion of malignancy if not interpreted carefully.7,18

Ancillary Studies Immunohistochemistry and molecular diagnostics play a small role in the diagnosis of fat necrosis. The histiocytes can be highlighted with antibodies against CD68 and are nonreactive for cytokeratins.

Fig. 6.3: Fat necrosis: Histologically, fat necrosis is characterized by the presence of foamy, vacuolated, and multinucleated macrophages with engulfed fat (Hematoxylin and Eosin stain, 40×)

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Mastitis Clinical Features Mastitis is an inflammatory condition of the breast that can be acute or chronic and infectious or idiopathic. According to the cause, clinical and pathologic presentation, and affected age group, mastitis can be classified into acute/puerperal mastitis, granulomatous mastitis, granulomatous lobular mastitis (idiopathic), and plasma cell mastitis. Clinical information is crucial for the diagnosis of the different forms of mastitis.7,14 Acute/puerperal mastitis is relatively common and typically occurs 2–3 weeks after lactation has started.19,20 It is usually the result of infection of the mammary ductal system with Staphylococcus aureus and beta-hemolytic streptococci.14 Clinically, the patient presents with a painful erythematous mass in the breast with associated edema, fever, and leukocytosis. It tends to be a localized process that requires timely treatment with antibiotics to avoid abscess and fistula formation.20 Plasma cell mastitis is a severe form of mastitis characterized by a prominent infiltrate of plasma cells around the ducts. The lesion is strongly associated with pregnancy and generally occurs within a few years after lactation has ended.14,16,21 Nipple discharge, nipple retraction, presence of a breast mass, and enlarge axillary lymph nodes are part of the clinical presentation.16 Granulomatous mastitis can be caused by a wide number of infectious agents such as mycobacterium, fungal organisms, and parasitic infections, as well as noninfectious etiologies such as sarcoidosis and foreign body reaction. When no cause for the disease is found clinically or after negative microbiology studies and special stains, the term granulomatous lobular mastitis (or idiopathic granulomatous mastitis) is used. The lesion usually appears after, rather than during pregnancy or lactation and similar to other forms of mastitis, can mimic cancer clinically and radiologically.16,22,23

Cytologic Features Cytologically, aspirations of acute mastitis predominantly consist of abundant neutrophils, foamy macrophages, reactive ductal cells with enlarged nuclei and prominent nucleoli, and a background of cellular debris.7 Granulomatous mastitis, including idiopathic forms, is characterized by cellular smears composed of multinucleated epithelioid histiocytes, multiple granulomas, lymphocytes, plasma cells, ductal epithelial cells with reactive changes, and presence of reactive fibroblasts (Fig. 6.4).

Fig. 6.4: Granulomatous mastitis: Aspiration of granulomatous mastitis reveals multinucleated epithelioid histiocytes in a background of macrophages, inflammatory cells, and cellular debris (Papanicolaou stain, 20×)

The cytologic hallmark of plasma cell mastitis is the presence of a conspicuous population of plasma cells. Lymphocytes and neutrophils are also present, but in fewer number. However, the cytologic picture of plasma cell mastitis will depend on the stage of the disease, and mature phase cases will have a less prominent inflammatory component. The presence of reactive atypia of the ductal epithelium can be worrisome for malignancy, especially in the clinical setting of a suspicious mass. Histiocytes, sometimes with a xanthomatous appearance, may be also seen.16,24

Ancillary Studies In the appropriate clinical scenario, no special studies are required for the diagnosis of acute/puerperal mastitis. Because granulomatous lobular mastitis is a diagnosis of exclusion, special stains and cultures for fungi, bacteria, and acid-fast organisms are mandatory.7 Multinucleated histiocytes are positive for CD68 and negative for keratins.

Subareolar Abscess Clinical Features Subareolar abscesses tend to occur in nonlactating premenopausal women and comprise about 90% of nonpuerperal breast abscesses. These abscesses are located in the retro and periareolar areas and are the result of obstruction of the lactiferous ducts by squamous metaplasia of the ductal epithelium.16,25 Subareolar abscesses

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Fig. 6.5: Subareolar abscess: Fine needle aspiration biopsy of a subareolar abscess consists of numerous acute inflammatory and anucleated squamous cells. Location of the lesion is important in establishing the diagnosis, since an infected epidermal inclusion cyst will have identical cytomorphologic features (Diff-Quik stain, 10×)

Fig. 6.6: Fibrocystic changes: Fine needle aspiration biopsy of fibrocystic disease yields a combination of benign ductal cells and apocrine cells (Diff-Quik stain, 10×)

have a slow clinical course with tendency to recur and form fistulous tracts in the periareolar area. They can also occur in men.14

debris or fluid levels. Additionally, there are mixed solid and cystic lesions, and cystic lesions with a mural solid nodule.26 Simple cysts can be safely followed without the need of confirmatory biopsy. Occasionally, simple cysts are aspirated for symptomatic relief because they are tender and uncomfortable for the patient. However, complicated cysts, mixed solid and cystic masses, and cystic lesions with a mural nodule need biopsy to exclude the possibility of malignancy.27

Cytologic Features The cytologic features of subareolar abscess overlap with those of infected epidermal inclusion cyst. Therefore, clinical presentation and location of the lesion are necessary for the correct diagnosis.7 Aspirates of subareolar abscess yield thick material consisting of numerous acute inflammatory and anucleated squamous cells (Fig. 6.5). Reactive squamous cells, histiocytes, and foreign-body giant cells with engulfed keratin debris are also present.24

Ancillary Studies In general, special studies are not needed for the diagnosis. Microbiology cultures may be useful in cases of secondary infection by skin microorganisms. 

NONPROLIFERATIVE BREAST CHANGES

Benign Cystic Lesions Clinical Features Cysts are the most common lesions of the female breast and are almost always benign. They usually arise in the lobules (terminal duct lobular unit of the breast, TDLU) and occur more often in premenopausal patients.7 Radiologically, breast cysts are classified as complicated or noncomplicated based on the presence of septations, echoes, and

Cytologic Features The cytologic diagnosis starts by looking at the gross appearance of the aspirated fluid, which may range from clear to cloudy or bloody, according to the underlying pathology. Simple cysts tend to produce clear fluid. Papillary lesions tend to produce bloody aspirations. However, infectious cystic lesions, galactocele, and epidermal inclusion cysts generally produce cloudy, yellowish or ‘dirty’ appearing fluid.7,28 The most common cells found in benign cystic aspirations are apocrine cells, foam cells, macrophages, and ductal epithelial cells (Fig. 6.6). Apocrine cells can be seen singly or can be arranged in flat sheets. They have abundant granular cytoplasm and eccentric nuclei with prominent nucleoli (Fig. 6.7). Foam cells can be of ductal cell origin or foamy macrophages. Their cytoplasm is abundant and vacuolated rather than granular. Ductal cells are usually scant and have “benign” morphology, and myoepithelial cells are readily identified.6,7

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Fig. 6.7: Apocrine cyst: A cluster of apocrine cells from a cyst aspiration is seen as a flat sheet. Apocrine cells have abundant granular cytoplasm, and eccentric nuclei with prominent nucleoli. A few degenerated macrophages are also seen in the background (Papanicolaou stain, 40×)

Fig. 6.8: Epidermal inclusion cyst: Aspiration of epidermal inclusion cyst yields abundant anucleated squamous cells (Diff-Quik stain, 20×)

Epidermal inclusion cysts are characterized by the presence of numerous anucleated squamous cells (Fig. 6.8). If infected or ruptured, inflammatory cells, debris, and multinucleated histiocytes can be seen. Infected epidermal inclusion cysts and subareolar abscesses have similar cytologic features; therefore, clinical and radiologic correlation is important for the diagnosis. The presence of reactive squamous cells from the cyst lining can be worrisome for malignancy; however, “reactive atypia” is characterized by cohesive groups instead of dyshesive atypical cells. In the presence of significant acute inflammation, the diagnosis of malignancy should be made with caution.7

There is controversy regarding the risk of developing breast cancer in patients with FA. Early studies showed that there was an increased risk in patients with complex FAs, similar to that of patients with proliferative breast disease (PBD) without atypia.30 More recent reports did not find an association between FA and subsequent breast cancer development.31,32

Ancillary Studies Special studies are not necessary for the diagnosis. 

FIBROEPITHELIAL TUMORS

Fibroadenoma Clinical Features Fibroadenomas (FAs) are benign tumors more commonly found in adolescents and young women. They also occur in postmenopausal women and account for up to 20% of breast masses in the population of this age. FAs arise from the epithelium and stroma of the TDLU. Clinically, they present as a palpable, firm, painless, mobile mass. In 15% of the patients, FAs are multiple.16,29

Cytologic Features Fibroadenomas are biphasic tumors and classically the aspiration consists of a mixture of epithelial and stromal components in a background of myoepithelial cells (Fig. 6.9). However, FAs are diverse tumors that may contain other proliferative lesions such as sclerosing adenosis, hyperplasia, apocrine metaplasia, secretory changes, and even carcinoma. Careful attention to this variability will help in avoiding cytologic misinterpretation. Similarly, when the three main components of FA are not present in the aspiration, the cytologic diagnosis is not always straightforward (Fig. 6.10).10 The epithelial component in FA consists of monolayer sheets of ductal cells mixed with myoepithelial cells. These sheets are often described as “staghorn” or “antler-like” configuration. One important clue to the benign nature of these epithelial sheets is the cellular cohesiveness and maintenance of cell polarity. The stromal fragments are usually fibromyxoid with variable cellularity. Commonly, the background of the aspirate is composed of numerous naked/bipolar nuclei that represent single myoepithelial cells. Their presence is a reliable feature in favor of FA.7,33

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Fig. 6.9: Fibroadenoma: Aspirations of fibroadenomas yield a biphasic population of ductal groups and stromal fragments in various amounts. The epithelial component consists of monolayer cohesive sheets of ductal cells admixed with myoepithelial cells. These sheets are often described as “staghorn” or “antler-like” configuration (Papanicolaou stain, 10×)

Fig. 6.10: Fibroadenoma: In the absence of stromal fragments, and when only abundant ductal fragments are aspirated, the distinction with proliferative breast disease such as usual ductal hyperplasia and papillary lesions may be difficult (Diff-Quik stain, 10×)

Ancillary Studies

The treatment of PT is excision with wide margins in order to avoid recurrences that are very common in these tumors.35

Cytologically, the most challenging differential diagnosis of FA is benign phyllodes tumor (PT). Some authors have recommended the use of proliferative markers such as Ki-67 to evaluate stromal proliferation and aid in this differential.16 However, these studies are better performed in tissue biopsies or surgical resections and lack practical use in cytology. If the possibility of PT is entertained cytologically, surgical excisional biopsy is necessary.

Phyllodes Tumor Clinical Features Phyllodes tumors (PT) are uncommon biphasic breast tumors that account for approximately 1% of all breast neoplasms. PT may occur in a wide age range but tend to affect older patients, usually a decade or two later than the average presentation of FA.7,16,34 Based on the histologic characteristics of the tumors, PT are subclassified as benign, borderline (low-grade malignant), or high-grade malignant. The distinction among these three subgroups is important for management and is also predictive of the probable clinical course.16 The patient usually presents with a firm to hard discrete palpable tumor. Although there are no specific clinical features that reliably separate FA, benign PT, and malignant PT, the diagnosis of PT may be favored if the tumor is larger than 4 cm and there is a history of rapid growth.16

Cytologic Features Benign PT demonstrates significant cytologic overlap with FA. FNA smears tend to be cellular, characterized by a biphasic population of epithelial and stromal fragments. The epithelial fragments are indistinguishable from those of FA, which even in benign cases can show hyperplasia and cytologic atypia. The stroma may have myxoid, chondroid, or mucinous changes. Cellularity of the stromal fragments, stromal/epithelial ratio, pleomorphism of the stromal cells, presence of atypical single stromal cells in the background, will increase with the tumor grade (Fig. 6.11). In malignant cases, stromal overgrowth may be so remarkable that cytologic smears may not show any epithelial component. In these cases, the distinction between malignant PT and pure malignant mesenchymal tumors is difficult. In contrast with FA, which are characterized by numerous bare bipolar nuclei in the background, PT more commonly show dissociated spindle or plump cells.7,35-37

Ancillary Studies Ki-67 may be helpful in distinguishing benign from malignant PT in diagnostically difficult cases.16,38

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cytologic criteria, this index makes it possible to define the continuous spectrum of changes in breast lesions and separate hyperplasia from neoplasia. The index has been used as a morphologic risk factor in chemoprevention trials and has been validated by others.42-44

Adenosis Clinical Features

Fig. 6.11: Phyllodes tumor: Fine needle aspiration biopsy of a benign phyllodes tumor shows stromal hypercellularity (Diff-Quik stain, 20×)

Genetic studies suggest that benign and malignant PT have distinct molecular profiles such as loss of p16INK4A, gain of 1q, and loss of 13q. These findings are not useful in daily cytopathology sign out.39,40 

PROLIFERATIVE BREAST DISEASE

Adenosis is a lobulocentric proliferative lesion derived from the terminal ductal-lobular unit composed of epithelial and myoepithelial cells (except in microglandular adenosis where myoepithelial cells are absent).16 Adenosis can be mammographically detected as a tissue distortion or by the presence of microcalcifications and can be suspicious for malignancy. When it forms a mass clinically or radiologically, the term adenosis tumor or adenoma may be used. Epithelial and myoepithelial hyperplasia may be present and these lesions are commonly named florid adenosis. The most common form of adenosis found in clinical practice is sclerosing adenosis, which is characterized by variable atrophy by compression of epithelial cells and significant lobular fibrosis.16,45

Cytologic Features

Proliferative breast disease (PBD) is a group of noncancerous conditions that may increase the risk of developing breast cancer. The term “proliferative breast disease” has now replaced the well-known and popular term of fibrocystic changes. PBD is subdivided into atypical and nonatypical.10 The “Masood Cytology Index” is a semiquantitative score based on comparison of the cytomorphology of mammographically detected lesions and their corresponding excisional biopsies (Table 6.1).41 Using strict

Cellularity of the smear varies with the degree of proliferative changes present in the lesion. In cases of sclerosing adenosis, FNAB shows varying amounts of small cohesive epithelial groups with acinar or tubular arrangements. Occasionally, tubules have an angulated configuration with pointed ends that differ from those seen in tubular carcinoma (TC) by the absence of atypia and the presence of myoepithelial cells. Epithelial cells in adenosis have bland cytologic features with small, uniform nuclei without atypia (Figs 6.12 and 6.13).45

Table 6.1: Masood cytology index Cellular arrangement

Cellular pleomorphism

Myoepithelial cells

Anisonucleosis

Nucleoli

Chromatin clumping

Score

Monolayer

Absent

Many

Absent

Absent

Absent

1

Nuclear overlapping

Mild

Moderate

Mild

Micronuclei

Rare

2

Clustering

Moderate

Few

Moderate

Micro and/or rare macronucleoli

Occasional

3

Loss of cohesion

Conspicuous

Absent

Conspicuous

Predominantly macronucleoli

Frequent

4

Sum of score: 6–10 normal; 11–14 proliferative; 15–18 atypia; ≥ 19 suspicious for cancer. Source: Modified from Masood et al.41

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Fig. 6.12: Sclerosing adenosis: Fine needle aspiration biopsy of a case of sclerosing adenosis yields a paucicellular sample composed of small glands similar to those seen in tubular carcinoma. However, glands in sclerosing adenosis lack pointed ends and cytologic atypia (Papanicolaou stain, 10×)

Fig. 6.13: Sclerosing adenosis: Histologically, sclerosing adenosis represents an acinar proliferation with lobular architecture and sclerosed stroma. The glands are crowded, compressed, and may be cytologically atypical; however, the acini retain a myoepithelial cell layer (Hematoxylin and Eosin stain, 40×)

In cases of microglandular adenosis, the smears tend to be hypocellular, composed of a monotonous population of epithelial cells with clear cytoplasm, uniform nuclei with small nucleoli, and absent myoepithelial cells. The paucicellularity of the smears and the absence of cytologic atypia are clues for the correct diagnosis.7

Cytologic Features

Ancillary Studies The presence of myoepithelial cells around the tubules can be highlighted with myoepithelial cell markers such as calponin, p63, and smooth muscle myosinheavy chain, among others performed on cell block material.7

Fine needle aspiration smears of benign pregnancy/ lactational changes are moderately to highly cellular and composed of a biphasic cell pattern of epithelial and myoepithelial cells. Epithelial cells are enlarged but mainly cohesive, although single cells are also seen. However, pleomorphism is minimal and the nucleolus is single and regular. Cytoplasm is abundant and vacuolated (Fig. 6.14). The background is granular and sometimes inflammatory with numerous foamy macrophages.7,47,48

Pregnancy-induced Changes Clinical Features During pregnancy and lactation, glandular expansion occurs as a result of progressive lobular enlargement with accumulation of secretory material in the lobular epithelial cells and lumina, accompanied by a relative decrease in fibrofatty stroma. When these changes occur as a localized adenomatous lactational hyperplasia forming a discrete nodule, the term lactating adenoma is used.7,16 Pregnancy-associated changes can occur de novo or involve previous lesions such as tubular adenomas and FAs. Other pregnancy-associated breast lesions include galactocele and, less commonly, invasive carcinoma.46

Fig. 6.14: Lactational changes: A case of lactational changes displays cohesive ductal cells with abundant vacuolated cytoplasm and minimal nuclear enlargement. Ductal cells are seen admixed with myoepithelial cells (Diff-Quik stain, 40×).

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Fine needle aspiration biopsy of galactocele yields a milky/cloudy fluid. The smears are low to moderately cellular and composed of numerous foam cells. A helpful diagnostic clue is disappearance or decrease in size of the mass after aspiration.7 In malignant cases, there is increased smear cellularity and malignant cell groups are arranged in three-dimensional clusters. Numerous isolated pleomorphic tumor cells are present in a necrotic background. Malignant cells are enlarged, irregular and reveal hyperchromatic nuclei, single or multiple nucleoli, accompanied by the presence of abnormal mitosis.7,47

Ancillary Studies Special studies are not contributory for the diagnosis.

Usual Ductal Hyperplasia Clinical Features Usual ductal hyperplasia (UDH) encompasses a spectrum of changes ranging from minimal stratification of intraluminal cells to proliferations that fall just short of atypical ductal hyperplasia (ADH).49 Unless the patient has a family history of breast cancer, proliferative disease without atypical hyperplasia increases the risk of breast cancer only slightly. The relative risk in the Nashville series in women without a family history of breast cancer was 1.3 times the risk in women with nonproliferative lesions and increased to 2.4 times in women with a family history of breast cancer.50

Cytologic Features Aspirations in cases of UDH are characterized by monolayer sheets of tight epithelial cells with maintenance of cell polarity, regular cellular spacing, and lack of nuclear overlap. Nuclei display fine chromatin, regular nuclear outlines, and small inconspicuous nucleoli. Myoepithelial cells can be identified admixed with the ductal epithelium (Fig. 6.15). There is also a lack of architectural atypia such as formation of cribriform spaces or micropapillae.7,10,51

Ancillary Studies High molecular weight cytokeratin (CK5/6) has been used in the differential diagnosis between UDH (positive staining) and atypical proliferative lesions (usually negative staining) in histology.52 However, although it is possible to apply to cytologic samples, its use is limited in this context.

Fig. 6.15: Proliferative breast disease without atypia: Aspirations of usual ductal hyperplasia yield tight monolayer sheets of ductal epithelium. Myoepithelial cells are seen as ovoid or spindle hyperchromatic nuclei admixed with the ductal epithelium (Papanicolaou stain, 40×)

Atypical Ductal Hyperplasia Clinical Features The diagnosis of ADH in FNAB is morphologically challenging, since the separation from low-grade ductal carcinoma in situ (DCIS) is usually not possible. Additionally, a significant discordance has been reported in cases of ADH diagnosed by minimally invasive procedures (including FNAB and CNB), with a significant number of cases having a concomitant carcinoma or proving to be either in situ or invasive carcinoma. Therefore, it is of general consensus that the diagnosis of atypia on FNAB should be followed by excisional biopsy of the lesion.10,53 Atypical ductal hyperplasia is a nonobligate precursor lesion, and women with it have a relative risk of developing breast cancer estimated to be approximately five times than that of women with nonproliferative lesions.50

Cytologic Features Cellularity varies according to the underlying lesion. Aspirations are composed of sheets of tightly cohesive epithelial cells with a variable degree of nuclear overlap and irregular intercellular spacing that varies with the degree of atypia. Nuclei are characterized by coarser granular chromatin and prominent or multiple nucleoli. A small number of single cells may be seen in the background. Architectural atypia in the form of occasional cribriform spaces and micropapillae is seen (Fig. 6.16). Myoepithelial cells are present.7,10,28

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Fig. 6.16: Proliferative breast disease with atypia: A small fragment of ductal epithelium with architectural atypia characterized by cribriform space formation (Diff-Quik stain, 40×)

Fig. 6.17: Papilloma: The hallmark of benign papillary lesions is the presence of papillary fragments with vascular cores and the lack of cytologic atypia as seen in this aspiration. Note that ductal cells are slightly monotonous but cohesive and bland appearing (Papanicolaou stain, 40×)

Ancillary Studies

Papillomas are well-defined benign tumors of the epithelium of mammary ducts. They arise more often from lactiferous ducts of the central part of the breast, but can also occur peripherally in any quadrant.16 Histologically, papillomas are composed of two layers of cells, one ductal epithelial cell layer and one myoepithelial cell layer, supported by a fibrovascular core. Papillomas are most common in women over 50 years of age and can occur also in men. Patients present with nipple discharge, a palpable subareolar mass or less frequently, a palpable peripheral mass.56

Immunostain for CK5/6 is of limited contribution for the differentiation with nonatypical lesions. Special studies are not contributory for the differential diagnosis of ADH versus low-grade DCIS in cytologic samples. 

PAPILLARY NEOPLASMS

Papilloma Clinical Features Papillary lesions of the breast include the spectrum of benign intraductal papillomas with and without atypia to papillary carcinoma. The assessment and categorization of papillary lesions remain one of the most challenging areas of breast pathology with a low accuracy for a correct diagnosis on minimally invasive procedures, since either many papillary lesions are not diagnosed as papillary or many of the diagnosed papillary lesions turn out not to be papillary.7,54,55 Additionally, correlation of benign cytologic findings with radiologic information does not assure a benign diagnosis, since there are no radiographic findings that can predict pathologic upstaging. Therefore, many authors support diagnosing all nonatypical and nonmalignant appearing papillary lesions on cytology as “papillary lesion” without further classification, with the recommendation of excisional biopsy.7

Cytologic Features Aspirations are usually cellular and characterized by the polymorphism of participating cells, which include ductal cells, apocrine metaplastic, and foam cells. The background is usually proteinaceous or bloody but not necrotic. Cells are arranged in three-dimensional papillary clusters or cell balls. Cell balls are defined as cohesive epithelial cell collections of variable sizes without fibrovascular cores, and in benign lesions tend to have a scalloped border (Fig. 6.17). Single epithelial cells are also seen in the background. Myoepithelial cells are present, admixed with the ductal clusters and in the background.54,55

Ancillary Studies In benign lesions, the presence of myoepithelial cells can be highlighted using myoepithelial cell markers

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(calponin, p63, and smooth muscle myosin-heavy chain, among others). Supported by positive expression of myoepithelial cells, the initial suggested diagnosis of an intraductal papilloma is important for communication with the patients. However, since the reported incidence of finding a more advanced lesion (ADH, DCIS, and invasive carcinoma) on follow-up excisional biopsy after the diagnosis of an intraductal papilloma obtained by minimally invasive procedures is up to 25%, all cases reported as papillary lesions on cytology should be excised for histologic evaluation. Therefore, the use of these markers has limited value on cytology.56-58

Papillary Lesion with Atypia and Papillary Carcinoma Clinical Features Papillomas can display focal proliferation of mildly atypical, monotonous cell populations identical to ADH or low-grade DCIS. When ADH or DCIS is found populating a lesion otherwise recognized as an intraductal papilloma, the term “atypical papilloma” is usually used. Criteria for the differentiation of ADH from low-grade DCIS within a papilloma have been established for tissue biopsies. Page et al.59 used criteria based on the extent of the abnormal population. Accordingly, ADH is diagnosed if the extent of the atypical population within the papilloma is less than 3 mm, and DCIS is diagnosed if the lesion is more than 3 mm in size. Conversely, Tavassoli60 uses the term “atypical papilloma” when less than onethird of the papilloma shows atypical features, and the term “carcinoma arising in a papilloma” when more than one-third but less than 90% of the papilloma is involved. Applying these criteria to cytologic samples is not possible. Therefore, accurate classification of papillary lesions with atypia in cytology is extremely challenging.61 Intraductal papillary carcinomas account for 2% of all breast cancers.62 Most occur during the fifth and sixth decades of life. Clinical and radiologic features of the lesion are important in the diagnostic workup, since the distinction between solitary, central papillary carcinomas from the multifocal, peripheral type, is necessary for treatment purposes.61

Cytologic Features The hallmark of any papillary lesion is the present of papillary clusters with fibrovascular cores. However, in

Fig. 6.18: Papillary carcinoma: A malignant papillary cluster with a well-defined fibrovascular core and epithelial cells with moderateto-high degree of cytologic atypia. Cells are more dyshesive than in benign lesions and anisonucleosis is evident. A few single, pleomorphic cells are seen in the background (Diff-Quik stain, 20×)

papillomas the papillae tend to be broader and in large papillary fragments. In contrast, papillary fronds are thinner and less evident in papillary carcinomas.54,55 The degree of atypia and cellularity of the aspiration varies. In cases of atypical papillomas, cytologic features may be identical to those seen in benign papilloma, or in cases of PBD with atypia.7 Papillary carcinomas, however, are characterized by highly cellular aspirates composed of a monomorphic population of ductal cells. The degree of atypia also varies, and papillary carcinomas are divided into low- and high-grade based on cell pleomorphism. The presence of large atypical single cells and numerous single columnar cells points to a malignant diagnosis (Fig. 6.18). Myoepithelial cells are absent. In frankly malignant lesions, the background is usually bloody with hemosiderin-laden macrophages and necrotic debris. A word of caution when dealing with papillary lesions in cytology is to be conservative when evaluating cytologic atypia, since intraductal papillomas, especially those with infarction or degeneration, may show marked cytologic atypia.55,63,64

Ancillary Studies Special studies are not contributory, since all papillary lesions with atypia diagnosed on cytology should be excised with adequate margins.

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IN SITU LESIONS OF THE BREAST

Ductal Carcinoma In Situ Clinical Features Ductal carcinoma in situ (DCIS) comprises approximately 20% of histologically confirmed breast carcinomas in the United States. Approximately 80% of DCIS are diagnosed by mammography since very few cases present as a palpable mass.65 DCIS comprises a heterogeneous group of histopathologic lesions that have been classified into several subtypes based on the presence or absence of necrosis, the degree of nuclear atypia, and architectural pattern including micropapillary, papillary, solid, cribriform, and comedo.66

Cytologic Features

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Aspirations are composed of small-to-intermediate size monomorphic cells arranged singly and in clusters. The presence of numerous single cells is a useful feature that helps in the distinction of ADH versus low nuclear grade DCIS. The clusters are three-dimensional and may present in a variety of patterns including papillary, micropapillary, solid and cribriform (Fig. 6.19). Necrosis is usually absent. Scant myoepithelial cells may be present, but are not seen within the epithelial clusters.7,10,44,67,68 High nuclear grade DCIS is characterized by cellular smears composed of obviously malignant cells often in a background of necrosis (comedo DCIS). Neoplastic cells have irregular chromatin and nuclear outlines and display significant degrees of crowding and overlapping. Mitoses and apoptosis are frequent (Fig. 6.20).68,69

Cytologically, DCIS is classified into low nuclear grade and high nuclear grade and comedo versus noncomedo DCIS. Low nuclear grade DCIS is cytologically, and often histologically, difficult to differentiate from other proliferative breast lesions such as ADH. However, the distinction between high nuclear grade and comedo DCIS from invasive carcinoma is extremely challenging, and often not possible in cytology. These lesions constitute the “gray zone” in breast cytopathology. Smears in cases of low nuclear grade DCIS are usually hypercellular, according to the extent of the disease.

Ancillary Studies

Fig. 6.19: Ductal carcinoma in situ (DCIS): Fine needle aspiration biopsy of low nuclear grade DCIS yields a cellular aspirate composed of a monotonous population of ductal cells with minimal cytologic atypia. Occasional cribriform spaces are seen. The distinction with atypical ductal hyperplasia and well-differentiated invasive ductal carcinoma may be difficult in this case due to the dyshesive nature of the cells. These cases are sometimes classified as “borderline disease” on cytology (Diff-Quik stain, 20×)

Fig. 6.20: Ductal carcinoma in situ (DCIS): High nuclear grade DCIS is characterized by clusters of highly pleomorphic ductal cells in a background of necrosis. These cytologic features make highgrade DCIS indistinguishable from invasive ductal carcinoma in cytology. Cellular cohesiveness is a clue to the absence of invasion (Papanicolaou stain, 40×)

Special studies are not contributory since all atypical/ borderline lesions diagnosed on cytology require confirmatory core needle or excisional biopsy.

Lobular Neoplasia Clinical Features Lobular neoplasia is currently considered a nonobligated precursor for the development of carcinoma of ductal or lobular histology in either breast.70 This diagnostic term

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encompasses atypical lobular hyperplasia and lobular carcinoma in situ.71 Lobular neoplasia is multicentric in up to 85% of cases and bilateral in 30–67% of patients.

Cytologic Features Cytology smears of cases of pure lobular neoplasia (in the absence of associated proliferative lesions) are usually hypocellular.7,72 Neoplastic cells are small and monomorphic with a low degree of cytologic atypia and may be arranged singly or in loosely cohesive clusters. The presence of intracytoplasmic lumina has been regarded as one of the most helpful cytologic features.73

Ancillary Studies Since it may not be possible to separate lobular neoplasia from ADH and low nuclear grade solid DCIS on cytology, the use of E-cadherin, β-catenin, and p120 catenin is of diagnostic utility in this setting.74,75 

INVASIVE CARCINOMA

Breast cancer is the second leading cause of cancer death for women in the United States, after lung cancer. The National Cancer Institute estimates that a woman in the United States has a one in eight chance of developing invasive breast cancer during her lifetime. Breast cancer is more common in older women, with a median age of 61 years.76 Factors that increase a woman’s risk of breast cancer include older age, genetic factors, family history of breast or ovarian cancer, nulliparity, older than 30 years of age at first full-term pregnancy, alcohol consumption, use of combined postmenopausal hormone replacement therapy, postmenopausal obesity, and ionizing radiation. Only 5–10% of women with breast cancer have BRCA1 and BRCA2 gene mutations and 90–95% of breast cancer cases do not involve these inherited mutations.77 Estrogen receptor (ER)-positive cancers currently make up more than 80% of breast cancers diagnosed in women older than 45 years of age.78 The introduction of new technologies has made possible the subclassification of breast tumors into biologically different entities, since it has been noted that response to therapy and outcomes varies among tumors of apparently similar characteristics. Breast carcinoma can be categorized into five groups using gene expression profiles. These groups include normal breast like luminal A, luminal B, human epidermal growth factor receptor 2 (Her-2) positive, and basal phenotype.79-81 The majority of basal

phenotype breast cancers (BPBC) are triple negative (negative for ER, progesterone [PR], and Her-2). BPBC have demonstrated characteristic morphologic features, a significant decrease in survival, and poor prognosis. In addition, there is a strong association between this type of cancer and BRCA-1 mutations.79-87

Invasive Ductal Carcinoma, No Special Type Clinical Features Invasive ductal carcinoma (IDC) is the most common type of breast cancer, comprising up to 80% of cases. Although IDC is more common in older patients, older age is associated with more favorable prognostic tumor features.88 The IDC presents usually as a solid mass detected clinically or radiologically. Histologically, tumor grade ranges from well to poorly differentiated, and grading is attained using a combination of morphologic features that includes architecture, nuclear pleomorphism, and number of mitosis.16,70,89 Application of histologic grading to cytologic samples may be difficult mainly due to difficulties in detecting mitoses or tubules in cytology.90 Therefore, cytologically, ductal carcinoma is better graded as low-grade or high-grade based mostly on nuclear pleomorphism only.

Cytologic Features Aspirations are usually cellular. The degree of cellular pleomorphism varies with tumor grade and can be minimal in very well-differentiated tumors (Fig. 6.21). In moderately and poorly differentiated tumors, malignant cells are arranged in three-dimensional loose clusters, and numerous single malignant cells are usually evident (Fig. 6.22). The background may be clean, necrotic, inflammatory, or bloody. Significant anisonucleosis is present. Tumor cells have irregular nuclear contours, enlarged nuclei with significant hyperchromasia, coarse granular chromatin, and a prominent nucleolus (Fig. 6.23). Plasmacytoid cells, best appreciated in Diff-Quik stain, are common. There is an absence of myoepithelial cells.7,89,91

Ancillary Studies Immunohistochemical stains using E-cadherin, β-catenin, and p120 catenin can be used to separate ductal and lobular type in difficult cases.74,75 Additionally, complete tumor profiles, including ER, PR, and Her-2 can be performed in cases with available cells on the cell block or smears (Figs 6.24 and 6.25). Molecular studies for subclassification of breast invasive carcinomas into molecular subtypes can be performed on cytologic material.10,87,91-95

The Breast

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Fig. 6.21: Ductal carcinoma: Low-grade invasive ductal carcinoma may be difficult to distinguish from atypical ductal hyperplasia and low nuclear grade ductal carcinoma in situ (DCIS) in cytology as well as in histology. This figure depicts a case of low-grade invasive ductal carcinoma initially classified as ‘borderline breast disease’ mostly due to cohesiveness and uniformity of cells. However, when carefully assessed, the fragment displays lack of cell polarity, irregular nuclear outlines, slight overlapping, and anisonucleosis. These features go beyond what is expected in cases of DCIS or atypical ductal hyperplasia (Diff-Quik stain, 40×)

Fig. 6.22: Ductal carcinoma: Moderately differentiated invasive ductal carcinoma displays a more severe degree of cytologic atypia, nuclear overlap, pleomorphism, and anisonucleosis than low-grade ductal carcinomas. A well-formed glandular space is seen (Papanicolaou stain, 40×)

Invasive Lobular Carcinoma

of age.16 It generally presents as an ill-defined mass, but it can present only as a vague breast asymmetry, and can be radiologically occult. ILC differs from ductal carcinomas in the increased incidence of bilaterality, local recurrence after surgery, and its characteristic pattern of metastatic spread.7

Clinical Features Invasive lobular carcinoma (ILC) accounts for 5–10% of all invasive breast tumors.70 ILC can affect a broad age range with a median age at diagnosis between 45 and 56 years

Fig. 6.23: Ductal carcinoma: High-grade or poorly differentiated ductal carcinoma is characterized by obviously malignant cells with enlarged nuclei, significant hyperchromasia, and coarse granular chromatin. Malignant cells are dyshesive and single cells are common (Diff-Quik stain, 40×)

Fig. 6.24: Tumor markers: Estrogen receptor performed in cell block material is positive in a case of well-differentiated ductal carcinoma (40×)

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Fig. 6.25: Tumor markers: Cell block material of a case showing overexpression of human epidermal growth factor receptor 2 (Her-2) protein (3+) (40×)

Fig. 6.26: Lobular carcinoma: Neoplastic cells in lobular carcinomas are often single or arranged in loosely cohesive aggregates. Cells display a low-to-moderate degree of nuclear pleomorphism. Intracytoplasmic mucin vacuoles can be seen (Papanicolaou stain, 40×)

Cytologic Features

signet ring cells and intracytoplasmic vacuoles (targetoid mucin) are seen. The nucleus is slightly irregular, usually with a small nucleolus (Figs 6.26 to 6.28). Myoepithelial cells are absent.7

Smears of ILC are usually of low-to-moderate cellularity due to the associated stromal fibrosis.89 Neoplastic cells are often single or arranged in loosely cohesive aggregates. Occasionally, the characteristic Indian-file pattern can be observed in cytology.96 Tumor cells are of small-tomedium size and exhibit a low degree of cytologic atypia or pleomorphism, except in the pleomorphic variant that displays a significant degree of pleomorphism. Occasional

Fig. 6.27: Lobular carcinoma: A highly cellular aspiration of a case of lobular carcinoma reveals loosely cohesive aggregates, monomorphism, and occasional signet ring cells (Papanicolaou stain, 40×)

Ancillary Studies Immunohistochemical stains using E-cadherin, β-catenin, and p120 catenin can be used to separate ductal and lobule types in ambiguous cases.74,75

Fig. 6.28: Lobular carcinoma: Histologically, lobular carcinoma is characterized by low-grade dyshesive malignant cells (Hematoxylin and Eosin stain, 40×)

The Breast

Mucinous (Colloid) Carcinoma Clinical Features Pure mucinous cell carcinoma (MCC) accounts for approximately 2% of all breast carcinomas and is more common in older patients.16,70 MCC is a slow growing malignancy with a favorable prognosis. Clinically, it usually presents as a well-circumscribed mass and has a soft gelatinous consistency on sectioning. In addition to MCC, there is a variety of breast lesions that can yield abundant mucin on FNAB. The cytological differential diagnosis of MCC includes mucocele-like lesions, papillary carcinoma, FA, and ductal carcinoma with areas of mucinous differentiation.7,10,97-100

Cytologic Features Cellularity varies. The cytologic hallmark of MCC is the presence of abundant mucinous background. Malignant cells are seen singly and in tight clusters floating in a pool of mucin (Figs 6.29 and 6.30). Acinar and papillary formations and signet ring cells with intracytoplasmic mucin can be seen.101 Malignant cells display low-to-moderate cytologic atypia and a prominent nucleolus. Stromal fragments and branching capillaries are frequently seen. Psammoma bodies can be present.102 Myoepithelial cells are absent. This feature is useful in the differentiation with mucocele-like lesions in which myoepithelial cells are present.103,104 Additionally, MCC shows higher

Fig. 6.29: Mucinous carcinoma: Aspirations of mucinous carcinomas are characterized by abundant background mucin and floating clusters of low-grade ductal cells (Papanicolaou stain, 20×)

119

cellularity; abundant single, intact cells; three-dimensional cellular clusters in most cases; and variable nuclear atypia.7

Ancillary Studies Mucicarmine stain can be performed to highlight the mucinous background. Myoepithelial cell markers can be helpful in the differentiation with mucocele-like lesions.

Tubular Carcinoma Clinical Features Tubular carcinoma (TC) is an uncommon low-grade malignancy that accounts for less than 2% of all breast cancers.89 Although no general consensus exists, the majority of authors agree that at least 95% of the tumor should be of tubular morphology before this diagnosis is considered. Adherence to this strict histologic criterion is important for patient prognosis, since it is well-known that TC carries a favorable prognosis.105 Histologically, it is a well-differentiated tumor with a specific microscopic pattern of branched angular tubules embedded in a loose fibrous stroma. In cytology, the term “low-grade ductal carcinoma with tubular features” is preferred.7

Cytologic Features Cellularity varies, but smears are usually not very cellular due to stromal fibrosis.89 Aspirations are composed of ductal cells arranged as angulated tubular cellular

Fig. 6.30: Mucinous carcinoma: Histologically, similarly to cytology, abundant mucin is the hallmark of mucinous carcinomas. Malignant cells can be arranged in acinar, papillary, and cribriform formations (Hematoxylin and Eosin stain, 10×)

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clusters with peripheral perpendicular cellular arrangement (Fig. 6.31).106 Dispersed single cells may be present. The cells showed minimal atypia, fine chromatin, and inconspicuous nucleoli. Occasional cytoplasmic vacuoles are identified.107,108

Ancillary Studies Because the cytologic differential diagnosis of TC includes a variety of benign lesions such as FA, sclerosing adenosis, radial scar, and tubular adenoma, myoepithelial cell markers may be useful to confirm the diagnosis of malignancy.7 Immunohistochemical stains using E-cadherin, β-catenin, and p120 catenin can be used to separate TC from lobular carcinoma in difficult cases.74,75

Medullary Carcinoma Clinical Features Medullary carcinoma (MC) accounts for about 5–7% of all breast carcinomas. Patients with MC tend to be relatively young with a mean age at diagnosis ranging from 45 to 54 years of age. MC have several features in common with BRCA1-associated breast carcinomas, including relatively young age at diagnosis, poorly differentiated histology, lymphocytic infiltration, absence of hormone receptors expression, and frequent p53 alterations.16,109 MC belong to the ER(–), PR(–), Her-2 (–) group, harboring a basallike immunophenotype with a higher rate of cytokeratins

(CK 5/6) positivity than basal-like carcinomas.110 It is easily recognized as malignant on FNAB; however, these tumors are better classified as high-grade (or poorly differentiated) ductal carcinomas on cytology because histologic evaluation of the resected specimen is necessary to evaluate tumor borders.7

Cytologic Features Aspirations are usually very cellular and composed of predominantly single overtly malignant cells, naked atypical nuclei, or syncytial sheets of malignant cells.101 The cells display highly pleomorphic nuclei with prominent macronucleoli.89 Mitoses are evident. Numerous lymphocytes admixed with some plasma cells can be seen in the background. Hemorrhage and necrosis may be evident, especially in cystic tumors.111 Glandular formation and mucin production are not features of MC.7 The cytologic differential diagnosis of MC includes lymphoma, chronic mastitis, and intramammary lymph node.

Ancillary Studies When high-grade lymphoma is considered in the differential diagnosis, immunophenotyping by flow cytometry or immunostains for leukocyte common antigen (LCA) and cytokeratin are helpful.89

Metaplastic Carcinoma Clinical Features Metaplastic carcinoma (MtC) accounts for the less than 1% of all breast cancers. MtC constitute a heterogeneous group of tumors with an intimate admixture of adenocarcinoma with areas of spindle cell, mesenchymal, or squamous cell carcinoma.70 MtC are subclassified as pure epithelial (squamous cell, adenosquamous, mucoepidermoid, and carcinoma with spindle cell differentiation). A definitive pathologic diagnosis can only be made in the presence of both epithelial and mesenchymal components, or adenocarcinoma and squamous components. Therefore, it may not always be possible to make a specific diagnosis on cytology. Its clinical presentation does not differ from cases of IDC, of no special type.16 Median age at diagnosis is 55 years.70 MtC has an unfavorable prognosis, especially if the mesenchymal component predominates.7

Fig. 6.31: Tubular carcinoma: Aspirations of tubular carcinomas are composed of ductal groups with angulated “pointed” ends and peripheral perpendicular cellular arrangement. Nuclear atypia is minimal (Papanicolaou stain, 40×)

Cytologic Features Aspirations are usually cellular. Malignant cells can be isolated or in clusters.

The Breast

The background can be myxoid, necrotic, or inflammatory.89 The malignant epithelial component is usually high grade and reveals the same cytologic features of malignant cells seen in adenocarcinoma or squamous cell carcinoma. The mesenchymal cells are generally of spindle cell configuration, atypical and pleomorphic. Multinucleated cells, malignant osseous, or cartilaginous components can be seen. Abnormal mitotic figures are usually evident.112,113 If both components are not appropriately sampled during FNAB, the differential diagnosis with poorly differentiated ductal carcinoma, PT, and other spindle cell malignancies, including sarcomas, becomes difficult.7

Ancillary Studies A panel of immunostains including CAM 5.2, CK5/6, CK7, 34βE12 (K903), and the AE1/AE3 combination and p63, smooth muscle actin (SMA), and CD-10 may be used in the workup of MtC.16 The squamous areas are generally negative for ER and PR and positive for broad spectrum and high molecular weight cytokeratins (CK 5/6 and CK 34BE12). The spindle cell component may show positive reactivity for keratins, usually focally. Chondroid elements are frequently S-100 positive and may co-express cytokeratins, but are negative for actin.7 The routine use of immunostains is of limited value in the diagnosis of MtC on cytologic material.

Fig. 6.32: Granular cell tumor: Aspirations of granular cell tumors are large cells with abundant granular cytoplasm. The cytoplasm is fragile and easily disrupted during smearing resulting in abundant extracellular granular material (Papanicolaou stain, 40×)



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MESENCHYMAL TUMORS

Granular Cell Tumor Clinical Features Granular cell tumor (GCT) is an uncommon neoplasm that can occur in many sites throughout the body. About 5–8% of GCTs occur in the breast.114,115 GCT is considered to be of Schwann cell origin and is benign in the vast majority of cases. GCT is more common in middle-aged premenopausal patients, especially African-American women.116 It usually mimics malignancy at presentation since it usually presents clinically as a firm, painless mass, which may be fixed to the pectoral muscle or to the skin. Mammography often reveals a stellate lesion without calcifications.117

Cytologic Features Aspirates are moderately cellular consisting of single cells and relatively large cohesive groups of cells. Cells are characterized by ill-defined abundant granular cytoplasm, resulting in a syncytial appearance. Nuclei are bland, regular, and small. Nucleoli are inconspicuous. The cells are fragile, with stripped nuclei.118 The cytoplasm of single cells often appears to be disintegrating resulting in extracellular granular material (dirty background) (Figs 6.32 and 6.33). Mitosis and necrosis are not observed.119,120 Although malignant GCT is extremely rare,

Fig. 6.33: Granular cell tumor: Histologically, the tumor cells are round to polygonal with abundant eosinophilic granular cytoplasm, uniform nuclei with prominent nucleoli. The cells are arranged in nests, strands, cords, or sheets (Hematoxylin and Eosin stain, 40×)

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it should be considered if the tumor size exceeds 5 cm, and if necrosis, cellular pleomorphism, prominent nucleoli, and increased mitotic activity are present.7

Ancillary Studies The GCT cells are strongly immunoreactive with S-100 and also stain positively for CD68 and vimentin. They are negative for cytokeratin, epithelial membrane antigen (EMA), mucin, and estrogen and PR receptors. Cytoplasmic granularity can be highlighted using Periodic acid-Schiff (PAS) stain.7 The main differential cytologic diagnoses are histiocytic and apocrine lesions. Because of positivity with CD68, the distinction with histiocytes is sometimes challenging.120 Apocrine lesions are cytokeratin positive, thus can be easily differentiated if enough material is available to perform immunohistochemistry.7

Fibromatosis Clinical Features Fibromatosis (FM) is a locally aggressive fibroblastic proliferation that contains variable amounts of collagen. FM accounts for less than 0.2% of primary lesions in the breast. The affected age range is broad and patients range from 13 to 80 years at diagnosis. FM has been associated with trauma, breast implants, and Gardner syndrome.7,121 Fibromatosis is often clinically suggestive of a carcinoma. It presents as a palpable, painless, and firm mass.122 If present, dimpling of the overlying skin reinforces the clinical impression of carcinoma. Mammography often reveals a spiculated tumor. The frequency of local recurrence after local excision varies from 21% to 27%; however, FM lacks metastatic potential.123 Although FNAB may not render a definitive diagnosis of FM, it can confidently rule out malignancy and other more common lesions; therefore, it is useful in planning a surgical approach to the lesion.122

Cytologic Features Aspirates are moderately cellular and consist of a mixture of isolated spindle cells, stromal fragments, and inflammatory cells. The spindle cells have oval to elongated nuclei, fine chromatin, and small distinct nucleoli.121 The cytoplasm is pale with tapered ends and ill-defined borders. There is variation in size and shape, but marked nuclear pleomorphism is absent.122 Mitoses are absent. The stromal fragments are irregular in size and shape and composed of hyalinized collagen and scattered spindle cells.

The inflammatory cells consist of a heterogeneous population of small and large lymphocytes and a few plasma cells. The background is composed of amorphous proteinaceous material. Groups of benign ductal epithelial cells can be present.124,125

Ancillary Studies The spindle cells are positive for vimentin and a small portion of SMA and desmin. They are negative for S-100, CD-34, and cytokeratin.125

Myofibroblastoma Clinical Features Myofibroblastoma (MFB) is a benign breast stromal tumor that occurs mainly in older men and postmenopausal women with a range in age at presentation from 25 to 87 years. MFB generally presents as a well-circumscribed, rubbery, nonencapsulated, round to oval mass. Histologically, MFB is composed of bland-looking spindle cells, closely packed in short, straight, randomly intersecting fascicles or clusters of cohesive cells, separated by thick, hyalinized collagen bundles.126 Myofibroblastoma exhibits a wide variety of cytomorphologic features and architectural patterns; therefore, the differential diagnosis can be quite extensive,126,127 including reactive processes and benign neoplasms such as nodular and proliferative fasciitis, FM, spindle cell lipoma, neurofibroma, neurilemmoma, and leiomyoma.128 Unusual morphologic variants of MFB such as MFB with atypical cells, epithelioid MFB, myxoid MFB with or without atypical cells, and deciduoid-like MFB can lead to a misdiagnosis of gynecomastia, PT, or even malignancy.126

Cytologic Features Aspirates are characterized by moderate cellularity consistent of spindle or round to polygonal cells, according to the histologic variant. Neoplastic cells are haphazardly arranged and may show mildly pleomorphic nuclei.123,126 Nuclei contain a single nucleolus and occasional nuclear grooves and pseudoinclusions. No mitoses, significant nuclear atypia, or macronucleoli are present. The background contains scarce collagenous stroma and fragments of myxoid material.129,130

Ancillary Studies Most cases of MFB are positive for vimentin, desmin, CD34, ER, PR, and androgen receptors. Cytokeratin, EMA, S-100 protein, HMB-45, and c-Kit (CD117) are negative.126,131,132

The Breast

Sarcomas Clinical Features Sarcomas of the breast (SB) are extremely rare tumors that account for less than 1% of primary breast cancers.7,133,134 They usually present as a large, palpable mass. SB have a high risk of recurrence and are known to have a poor prognosis.7 SB should be differentiated from the two main entities in differential diagnosis: malignant PT and MtC.135 Cytologically, sarcomas are better classified as low grade and high grade. The most common sarcomas arising in the breast are fibrosarcoma, angiosarcoma, pleomorphic sarcoma, stromal sarcoma, liposarcoma, rhabdomyosarcoma, leiomyosarcoma, and myxofibrosarcoma.133,135,136 Angiosarcoma arising in the irradiated breast is being reported with increasing frequency.137 The interval between radiation and the diagnosis of angiosarcoma ranges from 3 to 12 years, with the majority occurring within 6 years post-treatment.16

Cytologic Features Cellularity and cytologic features vary with tumor type. Smears are usually composed of single spindle or round cells, or a dyshesive cell population. Tissue fragments with jagged, irregular borders and obvious running vessels can be seen. Cytoplasm is wispy and elongated. Nuclei are hyperchromatic with coarse granular chromatin and prominent nucleoli (Fig. 6.34). The presence of mitoses, pleomorphism, and necrosis correlates with the tumor

123

grade.89 Bizarre uninucleate/multinucleate giant cells can be seen in some tumors. The background can be bloody or necrotic.138

Ancillary Studies A panel of immunohistochemistry stains is necessary for diagnostic purposes. This panel should include two cytokeratins, vimentin, desmin, EMA, S-100, SMA, h-caldesmon, CD34, CD31, and others. Recently, molecular testing of sarcomas has become increasingly important, not only in the diagnostic approach of these lesions but also in regard to their prognosis and pathogenesis. A subset of sarcomas bears chromosomal abnormalities including reciprocal translocations, deletions, mutations, and amplifications. Besides their diagnostic value in sarcoma typing and subtyping, some of these abnormalities may also have an impact on treatment response and/or on prognosis.7 Conventional chromosomal analysis (karyotyping), fluorescence in situ hybridization (FISH), and reverse transcription chain reaction (RT-PCR) techniques can be performed on cytologic preparations such as cytospins, thin-layer slides, and smears.139 

LYMPHOPROLIFERATIVE DISORDERS

Lymphomas Clinical Features Breast lymphoma accounts for 1.7–2.2% of all extranodal lymphomas and 0.4–0.7% of all nonHodgkin lymphomas, with a roughly equal distribution between primary and secondary lesions. The majority are of B-cell type, especially diffuse large B-cell lymphoma, follicular lymphoma, Burkitt lymphoma, and lymphoma of mucosa-associated lymphoid tissue (Fig. 6.35). Lymphoma can mimic carcinoma clinically and also morphologically, and it can be especially difficult to differentiate from medullary breast carcinoma in small biopsies.

Cytologic Features

Fig. 6.34: Sarcoma: Aspirations of high-grade sarcomas of the breast yield malignant spindle-shaped cells characterized by a moderate amount of cytoplasm and large pleomorphic nucleus with coarse irregular chromatin (Diff-Quik stain, 63×)

Aspirations are usually highly cellular. The cytologic features are the same as those lymphomas arising elsewhere. The most common cytologic features characteristic of lymphomas are the dyscohesive cell pattern, the presence of lymphoglandular bodies, and the frequent crushing artifact. Morphology, cell size, and atypia will vary according to the type.

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Fig. 6.35: Lymphoma: Fine needle aspiration biopsy of large B-cell lymphoma involving the breast reveals dyscohesive large lymphoid cells with irregular nuclear contours, fine chromatin, and prominent nucleoli. Lymphoglandular bodies are seen in the background (Diff-Quik stain, 40×)

Fig. 6.36: Gynecomastia: Aspirations of gynecomastia are variably cellular and composed of a biphasic population of epithelial (left) and stromal fragments (right). The epithelial fragments are usually large, tightly cohesive, and usually display mild cytologic atypia (Papanicolaou stain, 10×)

Ancillary Studies

usually bilateral, but patients may present with asymmetrical or unilateral findings. Palpation usually demonstrates a palpable, tender, firm but mobile nodular tissue centrally under the nipple-areolar complex.144 The main use of FNAB in this setting is to rule out a malignant process, especially in those cases of unilateral gynecomastia.

Immunohistochemistry, flow cytometric analysis, and molecular studies are necessary for diagnosis and subclassification of lymphoproliferative disorders and can be performed on cytologic material. A basic panel of immunohistochemistry that includes a minimum of one B-cell and one T-cell marker can be performed on cell block material.140 If the suspicion of lymphoma arises after evaluation of the smears on-site, additional material should be submitted for flow cytometry studies. When compared with immunohistochemistry, flow cytometry allows for larger numbers of antigens to be tested and can improve the identification of dual antigen expression. Additionally, flow cytometry has the advantage of being rapid and highly sensitive.141,142 The diagnosis of lymphoma should not be made by cytology without the use of ancillary studies. 

LESIONS OF THE MALE BREAST

Gynecomastia Clinical Features Gynecomastia is a benign proliferation of the male breast glandular tissue usually caused by increased estrogen activity, decreased testosterone activity, or the use of numerous medications.143,144 It is the most common massforming lesion of the male breast.145 Gynecomastia is

Cytologic Features Smears are variably cellular composed of a biphasic population of epithelial and stromal fragments (Fig. 6.36). Epithelial fragments are usually large, tightly cohesive, often appearing as flat somewhat monolayered sheets. Occasional fingerlike projections reminiscent of FA are observed. Mild to marked epithelial atypia in the form of cellular crowding with nuclear overlap, nuclear hyperchromasia, high nuclear/cytoplasmic ratio, loss of polarity, and cellular dyshesion may be seen and can lead to a misdiagnosis of malignancy. Occasional naked bipolar to oval myoepithelial nuclei are present in the background.7,145-147

Ancillary Studies Noncontributory.

Breast Carcinoma Clinical Features Breast carcinoma among men is uncommon, accounting for not more than 1% of all breast carcinomas and for less than 0.1% of male cancer deaths.

The Breast

About 75% of patients present with a painless mass or nipple ulceration, retraction, or discharge. Approximately 85% of invasive breast carcinomas in males are of the ductal morphology.16 Cancer of the male breast is rare before the age of 30, but the risk increases with age; average diagnostic age is about 60 years.16,145 There is no proven link between gynecomastia and breast cancer. Metastatic tumors to the breast result in a large proportion of palpable masses in males. FNAB is very useful in separating primary tumors of the male breast from metastatic malignancy. This distinction would result in appropriate and timely patient management.7,148,149

Cytologic Features Aspirations of male breast carcinoma are similar to those seen in the female population. Smears are usually cellular. Malignant cells are characterized by high nuclear/ cytoplasmic ratio, and nuclear hyperchromasia with occasional prominent nucleoli. Occasional attempts at glandular formation are seen (Fig. 6.37).145,146

Ancillary Studies Metastatic tumors should be suspected when cytologic findings are not usual for a primary breast malignancy. The most common tumors that metastasize to the male breast are melanoma, lung adenocarcinoma, lymphoma, and prostate. A panel of immunohistochemistry stains that includes prostatic specific antigen (PSA), prostatic acid phosphatase (PAP), CK7, CK20, thyroid transcription factor 1 (TTF-1), S-100, ER, and PR is useful.7

Fig. 6.37: Male breast carcinoma: Aspiration of low-grade ductal carcinoma of the male breast showing malignant cells with moderate pleomorphism, overlapping and papillary configuration. Based on these cytologic features, metastasis from prostate should be excluded (Diff-Quik stain, 40×)

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Metastatic Tumors to the Breast Clinical Features Metastatic disease to the breast is uncommon and accounts for approximately 0.4–2% of all breast malignancies.150,151 In the majority of cases, a known history of disseminated malignancy is available at the time of the diagnosis. However, when the previous history is unknown, metastasis should be a consideration if the morphologic features of the tumor are unusual. Metastatic tumors to the breast often present as multiple masses.152 The distinction between primary and metastatic tumor to the breast is important, since the treatment and prognosis differ. The diagnosis of metastasis to the breast usually implies widely disseminated disease with a poor prognosis and short-term patient survival. Additionally, surgery and other treatment modalities could be avoided if the correct diagnosis is made initially.153,154 The most common tumors secondarily involving the breast are carcinomas (lung, ovarian serous carcinoma, squamous cell carcinoma of the cervix, endometrial adenocarcinoma, gastric carcinoma, and prostate carcinoma in males) followed by melanoma, sarcomas, and, less commonly, lymphomas (Fig. 6.38).150,154

Cytologic Features Aspirates are usually cellular and tumor classification is based on cell distribution pattern and individual cell morphology (Flow chart 6.1).

Fig. 6.38: Metastatic carcinoma: Metastatic small cell carcinoma from lung is characterized by cellular smears, cell dissociation, nuclear molding, coarse “salt and pepper” or dense chromatin, and inconspicuous nucleoli (Diff-Quik stain, 20×)

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Atlas of Fine Needle Aspiration Cytology Flow chart 6.1: The flow chart shows a practical morphologic approach to use in the differential diagnosis between metastatic and primary breast malignancies based on pattern recognition

Table 6.2: Immunohistochemical markers for most common tumors secondarily involving the breast and for breast primary malignancies Type of tumor

Immunohistochemical markers

Remarks

Lung

CK7 TTF-1 Napsin A

Up to 20% of lung adenocarcinomas can be negative for TTF-1 Squamous cell carcinomas will be negative for TTF-1 and CK7 in the majority of cases Lung carcinomas can be CK20 and ER positive

Gastrointestinal tract

Stomach: CK7, CK20 Colon: CK20, CDX2

Stomach: Difficult to distinguish from breast primary even with the use of immunohistochemistry, specially signet ring carcinoma CK20 is useful in this distinction since the presence of CK20 positivity in an adenocarcinoma is highly suggestive of nonbreast origin

Melanoma

S-100 Melan A HMB-45

Spindle cell variants can be confused with sarcoma or spindle cell carcinoma; therefore, S-100 should be used in poorly differentiated tumors Breast carcinomas can be S-100 positive More than one melanoma marker is necessary to establish the diagnosis Rarely, melanoma can be cytokeratin positive

Prostate

PSA PAP Contd...

The Breast

127

Contd... Type of tumor

Immunohistochemical markers

Remarks

Squamous cell carcinoma

P63 CK5/6

Do not indicate tumor primary, only squamous differentiation Clinical history of a primary elsewhere is necessary for the diagnosis

Thyroid

TTF-1 Thyroglobulin Calcitonin for medullary carcinoma

Both markers should be performed to prove thyroid carcinoma since lung adenocarcinomas are also TTF-1 positive

Lymphoma

LCA (CD45)

Negative for cytokeratin

Neuroendocrine tumors

Chromogranin Synaptophysin CD56 NSE (low specificity)

Breast cancer may be focally positive for chromogranin

Sarcomas

Smooth muscle markers: h-caldesmon, actin Vascular markers: CD31, CD34, factor VIII Rhabdomyosarcoma: Desmin, myogenin

Since sarcomas are usually large tumors, a previous diagnosis is often available to the pathologist

Breast

ER, PR, GCDFP-15, mammaglobin, CK7

ER and PR are also positive in ovarian and endometrial carcinoma. Can be positive in lung, thyroid, and stomach carcinomas GCDFP-15 is a very specific but not a particularly sensitive marker for breast carcinoma Mammaglobin can be express by endometrial carcinoma. More sensitive but less specific than GCDFP-15

CK: Cytokeratin; TTF-1: Thyroid transcription factor 1; ER: Estrogen receptor; PSA: Prostatic specific antigen; PAP: Prostatic acid phosphatase; LCA: Leukocyte common antigen; NSE: Neuron-specific enolase; GCDFP-15: Gross cystic disease fluid protein-15

Table 6.3: CK7 and CK20 in the differential diagnosis Immunoprofile

Tumor

CK7 + CK20 +

Mucinous ovarian carcinoma Upper gastrointestinal tract Transitional cell carcinoma

CK7 + CK20 –

Breast Lung (usually) Endometrial adenocarcinoma Nonmucinous ovarian carcinoma

CK7 – CK20+

Colorectal adenocarcinoma

CK7 – CK20 –

Prostate

Ancillary Studies The use of immunohistochemistry is important to establish the diagnosis of metastatic disease (Tables 6.2 and 6.3).155 However, clinical history and thorough radiologic examination are the most helpful tools. A single positive immunostain should not be used as a confirmation or to render a definitive diagnosis.

For lymphoproliferative disorders, flow cytometry and molecular studies are necessary for diagnosis and subclassification. 

ACKNOWLEDGMENT

The authors would like to thank Dr. Amir Mohammadi for his useful comments and his help during the preparation of this chapter. 

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Chapter

7

Fine Needle Aspiration of Lymphoid Lesions

Hasan Ghaffar



INTRODUCTION

While there is a little doubt concerning the utility of fineneedle aspiration (FNA) for diagnosing metastatic solid tumors, considerable controversy exists surrounding its use in the workup of lymphoid proliferations.1 Despite the advantages of FNA, the National Cancer Comprehensive Network discourages reliance on FNA for the initial diagnosis of lymphoma except when a lymph node is not readily accessible for surgical excision.2,3 Much of the controversy stems from the inability to assess lymph node architecture in cytologic preparations and the challenge of procuring sufficient quantities of material to perform ancillary investigations. These hindrances can often be overcome with optimal cytologic preparations, rigorous sampling, and appropriate sample triaging. Abnormal lymphocyte morphology and evidence of monoclonality are essential requirements for lymphoma diagnosis, irrespective of the sampling modality. The former can be met in FNA samples if lymphocytes show significant cytomorphologic abnormalities, but detection of subtle cytologic abnormalities requires high-quality, well-stained samples. Architectural features play a more crucial role when cytomorphologic findings do not permit distinction of lymphoma from benign lymphocytes. While the term “monoclonality” implies the use of a molecular gold standard, the concept of monoclonality is less rigidly applied in practice and a variety of ancillary tools, both molecular based and other, are employed to infer that

a monoclonal population of lymphocytes exists.4,5 The need for ancillary studies highlights the importance of collecting sufficient numbers of cells by performing several needle passes and triaging samples appropriately. Although significant variation in the sensitivity, specificity, positive predictive value, and negative predictive value of FNA for diagnosing lymphoma is reported, it is clear that the best diagnostic yield is achieved when ancillary investigations are used in combination with morphology.6-8 The advantages of FNA are indisputable.9 FNA is safe and well tolerated by most patients. Complications such as bleeding resulting in clinically significant blood loss are uncommon. FNA is cost-effective and does not require an operating room setting or general anesthesia. Deep-seated lesions that cannot be easily excised may be amenable to FNA under ultrasound or computed tomography guidance.10 FNA is of particular benefit for patients who would otherwise be at increased risk of major complications if surgery was undertaken. Rapid diagnosis, sometimes within minutes, is possible, which is most beneficial for patients suffering from rapid clinical decline. FNA provides suitable samples for most types of ancillary studies used to diagnose and subclassify lymphomas. Although some limitations for lymphoma diagnosis exist, FNA remains the best initial diagnostic modality in certain clinical circumstances. Rapid on-site evaluation of FNA sampling is helpful for ensuring that lesions clinically suspicious for lymphoma are adequately sampled and that material is available for

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ancillary studies.10 Once aspiration of the targeted lesion is confirmed by identifying site-specific tissue and/or abnormal cells on direct smears, additional needle passes are triaged for appropriate ancillary studies. Morphologic clues are used to decide how subsequent needle passes should be processed. For example, if cells with cytologic characteristics of Hodgkin and Reed–Sternberg (RS) cells are identified in a background of mixed inflammatory cells, additional passes can be collected in fixative to make a cell block for immunocytochemical confirmation of classical Hodgkin lymphoma. Rapid on-site evaluation increases the likelihood of arriving at a conclusive and accurate interpretation and allows for cost-effective utilization of resources. Some lymphomas are more prone to false-negative FNA results than others. This is particularly true for Hodgkin lymphoma, where the neoplastic cells are frequently less abundant than the inflammatory cells that constitute the cellular environment and where fibrosis can interfere with obtaining a cellular sample.6 Partial involvement of a lymph node by lymphoma can also lead to falsenegative FNA findings. Negative FNA findings should therefore always be interpreted with caution and should prompt further clinical intervention in some instances. Additional sampling, either by repeat FNA, core biopsy or excision, should be pursued if the clinical pretest probability of lymphoma is high. 

TECHNICAL CONSIDERATIONS

Diff-Quik (DQ) and Papanicolaou (Pap) stains provide complimentary information with respect to the cytomorphologic assessment of lymphoid lesions. Many individuals prefer the DQ stain to the Pap stains for evaluating hematolymphoid cells because it is similar to the Wright–Giemsa stain used for peripheral blood and bone marrow aspirates. DQ stains are performed rapidly on air-dried direct smears or cytospins, while Pap stains require immediate fixation. Cytoplasmic characteristics such as the granules present in eosinophils and the cytoplasmic vacuolization evident in Burkitt lymphoma are readily recognized in DQ preparations but are not visualized in Pap-stained material. Background elements including lymphoglandular bodies are preserved in air-dried preparations but are often lost in fixed material. However, nuclear contours are more crisply defined using Pap stains. Cell size is important for grading non-Hodgkin lymphoma and can be evaluated using either type of stain. The routine use of both DQ and Pap stains is often helpful but individual preferences vary.

Fig. 7.1: Excessive crush artifact can limit interpretation in aspirates of hematolymphoid neoplasms as is illustrated in this FNA of a chest wall mass (Diff-Quik, high power)

High-quality preparations are crucial for accurately evaluating hematolymphoid processes. Crush artifact and stripped nuclei may significantly hinder interpretation, as lymphocytes are fragile (Fig. 7.1). This problem is exacerbated with forceful expulsion of aspirated material through the needle and excessive pressure used while spreading the sample between slides. Immediate fixation is essential for Pap staining as air-drying may cause cells to appear blown up. Excessive hemodilution can be problematic and may be unavoidable if the targeted lesion is highly vascularized or if bleeding occurs. In general, there is no substitute for adequate morphology as relying on ancillary studies alone can be misleading. 

ANCILLARY STUDIES

Immunophenotypic, cytogenetic, and molecular tools have become increasingly important adjuncts for diagnosing and subclassifying lymphomas.11 In addition, microbiology investigations are essential for the workup of some reactive lymphadenopathies such as those displaying granulomatous inflammation and/or necrosis. The results of these investigations should always be combined with cytomorphologic findings. In the final report, all immunophenotypic, cytogenetic, and molecular data are integrated with cytologic findings to produce a final diagnosis. Immunophenotyping is performed using immunocytochemistry or multiparameter flow cytometry. Charged cytospin slides, thin-layer slides, or paraffin-embedded cell block material can be used for immunocytochemistry.12 RPMI cell culture media is preferred for flow cytometry. Rigorous control of preanalytic variables is necessary

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in order to generate optimal flow cytometric data.13 Aspirates received in fixative solutions are rejected. Analysis is performed as soon as possible after collection as viability is time sensitive and nonviable cells may bind antibodies nonspecifically. Specimens requiring transport or short-term holding can be maintained at refrigerated temperature.14 The number of needle passes required for immunocytochemistry or flow cytometry depends on the yield of each individual pass, but a minimum of two passes with accompanying needle rinses is usually necessary for each test. Flow cytometry uses a laser source to determine the intrinsic properties of cells in fluid suspension using forward scatter, which is proportional to cell size and side scatter, which corresponds with the granularity of cells. Cells are incubated with groups of fluorochrome-labeled antibodies directed at cell surface markers. Permeabilizing agents can be used for intracellular antigens such as cytoplasmic light chains. Lasers excite the fluorochromes causing pulses of light to be emitted. Mirrors and filters separate light beams of different wavelengths, directing them to the appropriate detector. The light beams are converted into electrical signals, and a computer collates the events for display as dot plots or histograms. The selection of discrete populations of interest, or gating, defined on the basis of light scatter and/or fluorescence characteristics, allows for the characterization of individual populations. The sensitivity and specificity of flow cytometry for the detection of lymphoma increase with the number of antibodies and fluorochromes used. While the cellularity of FNA samples is not always sufficient for comprehensive analysis, a limited antibody panel that includes surface light chains and CD19 can be performed with less cellular samples to facilitate detection of a monotypic B-cell population. The kappa-to-lambda ratio in benign lymphoid tissue is typically 1.5, whereas ratios < 0.6 or above 6 are usually considered suspicious for lymphoma. Analysis of monotypic light chain expression in CD5+/CD19+ populations and CD10+/CD19+ populations further assists with diagnosis and aids with subclassification.15 T-cell lymphomas are more challenging to diagnose due to their variable patterns of antigen expression, but may demonstrate loss of pan-T-cell antigen expression, imbalanced CD4:CD8 ratios, and/or other immunophenotypic aberrancies. Flow cytometry offers some distinct advantages over immunocytochemistry. It can be performed more rapidly and allows multiple antigens to be evaluated simultaneously. It is superior for evaluating surface light chain expression, as detection of surface light chains by immunocytochemistry is often confounded by nonspecific

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background labeling. Conversely, flow cytometry is less effective than immunocytochemistry for characterizing large cell lymphomas and plasma cell neoplasms, in part due to loss of cells during processing.16 The sensitivity of flow cytometry is high, but admixed non-neoplastic cells if present in abundance may mask a clonal population. Taking into account these pros and cons, flow cytometry is the best test for distinguishing reactive lymphocytes from low-grade lymphoma; therefore, FNA samples containing monomorphous small lymphocytes or heterogeneous lymphoid populations should be routinely triaged for flow cytometric analysis. Immunocytochemistry allows for detailed correlation of morphology with protein expression. It is superior to flow cytometry for evaluating cytoplasmic light chains in plasma cells and for immunophenotyping large B-cell lymphomas (LBCLs). It is the method of choice for characterizing classical Hodgkin lymphoma for which flow cytometry is not helpful. However, validation of antibodies in cytologic samples is not easily performed and validation using formalin-fixed paraffin-embedded tissue does not account for variations in immunoreactivity, resulting from different sample preparation methods and fixatives. Consequently, careful attention should be paid to internal controls when interpreting immunocytochemical studies, and when possible confirmatory investigations such as fluorescence in situ hybridization (FISH) can be used. Aspirates consisting mainly of large lymphocytes, samples containing Hodgkin cells in a mixed inflammatory background, and plasma cell predominant infiltrates should be routinely triaged for immunocytochemistry. High-quality polymerase chain reaction (PCR) products are readily obtained from fresh or fixed FNA samples. PCR is performed to detect monoclonal immunoglobulin heavy chain (IgH) or T-cell receptor gene rearrangements in lymphoid proliferations17 and can be used to identify viruses associated with certain types of lymphoma such as human herpesvirus-8 in primary effusion lymphoma.18 In most lymphoid neoplasms, all cells derived from the malignant progenitor share the same antigen receptor gene configuration. In contrast, normal immune responses consist of polyclonal lymphocytes that express numerous different antigen receptors. Thus, PCR analyses of antigen receptor genes can aid in distinguishing polyclonal from monoclonal lymphoid proliferations. PCR analysis can be utilized as a backup tool when immunophenotyping fails to clearly demonstrate a monoclonal population in the context of suspected lymphoma. One notable pitfall is that the high sensitivity of PCR can sometimes lead to falsepositive results in limited samples due to the amplification

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of small clonal populations that are not clinically significant. In addition, lineage infidelity may be seen, particularly in precursor lymphoid neoplasms; therefore, PCR findings should not be used as the sole means of assigning lineage. PCR has evolved as an essential tool for the diagnosis of T-cell lymphomas. Chromosomal alterations play an important role in the characterization of malignant lymphoma. FISH uses fluorescent-labeled oligonucleotide probes that bind to DNA sequences. A variety of FISH probes are available to detect translocations and numeric abnormalities associated with specific lymphoma subtypes. Direct smears or cytospins are ideal for interphase FISH analysis as they provide a monolayer of cells with little nuclear overlapping.19 FISH has evolved as tool for the rapid detection of c-myc rearrangements that are crucial for the diagnostic evaluation of Burkitt lymphoma. 

DIAGNOSIS OF LYMPHOMA

Morphology forms the basis for the initial evaluation of lymphoid proliferations in cytologic samples and is used to guide subsequent workup. It is often stated that aspirates containing heterogeneous lymphocytes with scattered interspersed tingible body macrophages are likely reactive, that a predominance of monomorphous small lymphocytes is most closely aligned with low-grade lymphoma, and that populations dominated by large lymphocytes frequently represent high-grade lymphomas. These principles are useful for formulating a diagnostic approach, but many exceptions exist.20 There is considerable morphologic overlap between the cytomorphology of low-grade lymphomas and reactive lymphoid hyperplasia. Marginal zone lymphomas are characterized by

A

colonization of reactive lymphoid follicles by neoplastic small lymphocytes, and follicular lymphomas are comprised of small-cleaved centrocytes with variable numbers of admixed large centroblasts. As expected, the FNA cytology of both of these lymphoma types frequently demonstrates heterogenous lymphocyte populations. Likewise, reactive lymphoid proliferations may appear monomorphous and contain mainly small lymphocytes. A diagnosis of high-grade lymphoma may be confounded by a supervening reactive lymphoid infiltrate, and the proportion of large cells present in some forms of reactive lymphoid hyperplasia can be alarming. It is thus essential to consider a broad differential diagnosis when these patterns are encountered during the initial stages of working up the sample.

Reactive Lymphadenitis Reactive lymphadenitis can occur at any age and can involve any lymph node region, but cervical, axillary, and inguinal lymph node are most commonly involved.21 It is important to distinguish granulomatous lymphadenitis from other forms of reactive lymphadenitis.

Cytomorphology A range of lymphocyte morphologies is seen, including small lymphocytes, centroblasts, and immunoblasts. Tingible body macrophages, epithelioid histiocytes, plasma cells, neutrophils, and follicular dendritic cells may be present. Fragments of germinal centers containing conspicuous centroblasts, apoptotic cells, and mitotic figures may be identified. Clusters of histiocytes and multinucleated giant cells are characteristic of granulomatous inflammation (Figs 7.2A and B).

B

Figs 7.2A and B: (A) Granulomatous inflammation characterized by epithelioid histiocytes and multinucleated giant cells in a mediastinal lymph node aspirate from a patient with suspected sarcoidosis (Diff-Quik high power); (B) Granulomas are frequently well-represented in cell block preparations as is illustrated in this cell block from the sample shown in Figure 7.2A (Hematoxylin and Eosin, high power)

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Ancillary Studies T cells are often more abundant than B cells and flow cytometry reveals the B cells to be polytypic. If granulomatous inflammation or necrosis is present, cultures and special stains for infectious organisms should be performed.

Differential Diagnosis The differential diagnosis includes low-grade lymphomas that contain morphologically heterogeneous lymphocytes such as follicular lymphoma and marginal zone lymphoma. By flow cytometry, the B cells in low-grade lymphomas are usually monotypic, and in follicular lymphoma the monotypic population demonstrates CD10 coexpression. The differential diagnosis of granulomatous lymphadenitis includes classical Hodgkin lymphoma in which admixed eosinophils are often conspicuous and Hodgkin and RS cells are present.

Key Diagnostic Features • • •

Spectrum of small, medium, and large lymphocytes with bland cytomorphology Admixed tingible body macrophages Polytypic by flow cytometry

Hematolymphoid Neoplasms Hematolymphoid tumors are diagnosed according to the 2008 Word Health Organization (WHO) classification,22 which utilizes an integrated clinical, morphologic, immunophenotypic, cytogenetic, and molecular approach. The following discussion will emphasize tumors encountered commonly in FNA samples. Primary cutaneous lymphoproliferative disorders are not covered.

Plasmacytoma Plasmacytoma is a neoplasm derived from terminally differentiated B cells. Two types of plasmacytoma are recognized: solitary plasmacytoma of bone, a localized tumor of bone that is lytic on plain radiographs, and extraosseous (extramedullary) plasmacytoma, which arises in tissues other than bone. A monoclonal M-protein may be detected in the serum in a subset of patients, but this is generally of low quantity. Systemic bone marrow involvement is absent, and clinical manifestations of plasma cell myeloma are lacking. Plasmacytomas occur in middleaged patients with a higher prevalence in males and are often discovered incidentally. Vertebral body, rib, skull, pelvis, femur, clavicle, and scapula are common sites

Fig. 7.3: Plasmacytoma consisting of mature plasma cells with eccentrically placed nuclei, “clockface” chromatin, and perinuclear hofs (Papanicolaou, high power)

of involvement of solitary plasmacytoma of bone, and extraosseous plasmacytoma has a predilection for the upper aerodigestive tract. The majority of plasmacytomas respond well to radiotherapy.

Cytomorphology A pure population of plasma cells is present, characterized by eccentrically placed round nuclei with coarsely clumped (“clockface”) chromatin and perinuclear hofs (Fig. 7.3). Russell bodies, grapelike clusters of immunoglobulin-containing cytoplasmic inclusions, and various other types of inclusions may be seen. The plasma cells are usually mature and mitotic figures are not readily identified, but immature features including increased nuclear to cytoplasmic ratio, prominent nucleoli, and fine chromatin can be seen. Rarely amyloid may be detected in the background.

Ancillary Studies Plasmacytomas are positive for CD138, CD38, CD79a, and MUM-1, and show cytoplasmic light chain restriction. CD19 expressed in normal plasma cells is usually negative, and unlike normal plasma cells, aberrant expression of CD56, CD20, CD10, CD117, and cyclin D1 may be present. Cyclin D1 immunoreactivity correlates with t(11;14) and has been associated with lymphoplasmacytic morphology.

Differential Diagnosis Confirmation of cytoplasmic light chain restriction by immunocytochemistry permits distinction from

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non-neoplastic plasma cell proliferations. Exclusion of underlying multiple myeloma requires correlation with clinical findings, diagnostic imaging studies, and other laboratory investigations. Some carcinomas, such as medullary thyroid carcinoma, may demonstrate prominent plasmacytoid morphology. When suspected, cytokeratin immunocytochemistry should be used to supplement CD138 as this marker is widely expressed in epithelial tissues. A common diagnostic dilemma is distinguishing plasmacytoma from lymphoma with plasmacytic differentiation, particularly marginal zone lymphoma. Identification of a monotypic B-lymphocyte component favors a diagnosis of lymphoma over plasmacytoma.

Key Diagnostic Features • •

Pure population of mature plasma cells Positive for CD138, MUM-1, and cytoplasmic light chain restriction

Hodgkin Lymphoma Hodgkin lymphoma accounts for 30% of lymphomas and is divided into two clinically distinct types: classical and nodular lymphocyte predominant. Subclassification of classical Hodgkin lymphoma into nodular sclerosis, mixed cellularity, and lymphocyte-rich and lymphocyte-depleted subtypes requires assessment of tissue architecture and is not essential for treatment. The diagnosis of nodular lymphocyte predominant Hodgkin lymphoma often requires excisional biopsy; therefore, FNA is of limited utility for diagnosing this entity. Classical Hodgkin lymphoma has a bimodal age distribution: the first peak occurring at 15–35 years of age and a second peak occurring later in life. Cervical lymph nodes are most commonly involved, followed by mediastinal, axillary, and periaortic nodes. Extranodal involvement is rare except in immunosuppressed individuals. Most patients present with lymphadenopathy and a proportion exhibit B symptoms consisting of fever, night sweats, and weight loss. Treatment is based on staging using the modified Ann Arbor staging system. Chemotherapy and radiation have rendered the disease curable in the majority of patients.

Cytomorphology The sine qua non of classical Hodgkin lymphoma is the Hodgkin and RS cell, which is a large polylobated or multinucleated cell with at least two nucleoli in two separate lobes (Fig. 7.4).23 The nucleoli are eosinophilic and

Fig. 7.4: Classical Hodgkin lymphoma, with several multinucleated Hodgkin Reed-Sternberg cells in a mixed inflammatory background (Hematoxylin and Eosin, high power)

approach the size of a small lymphocyte. Mononuclear variants are designated Hodgkin cells. The background consists of mixed inflammatory cells, including varying proportions of histiocytes, eosinophils, plasma cells, and neutrophils. Granulomatous inflammation may be prominent.

Ancillary Studies The neoplastic cells are positive for CD15 and CD30, while negative for CD45, CD3, and CD20 by immunocytochemistry. Weak expression of PAX-5 is commonly seen. In 15–25% of cases, CD15 is negative. Epstein–Barr virus is demonstrable within the neoplastic population in approximately 25–50% of cases. Flow cytometry is not helpful for diagnosing Hodgkin lymphoma, and clonal gene rearrangements are not detected consistently unless specialized methods are used to separate the neoplastic cells from accompanying inflammatory cells.

Differential Diagnosis The inflammatory background may suggest a nonneoplastic process. A diligent search for RS cells and Hodgkin cells is necessary when a mixed inflammatory or granulomatous pattern is encountered. The differential diagnosis of classical Hodgkin lymphoma includes other types of lymphoma, mainly anaplastic large T-cell lymphoma (ALCL), nodular lymphocyte predominant Hodgkin lymphoma, and T-cell/histiocyte rich large

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B

Figs 7.5A and B: (A) Small lymphocytic lymphoma comprised of small lymphocytes with scant cytoplasm and clumped chromatin (WrightGiemsa, high power); (B) Small lymphocytic lymphoma, illustrating coarse “soccerball” chromatin pattern (Papanicolaou, high power)

B-cell lymphoma (TCRBCL).24 The large cells of ALCL display horseshoe-shaped nuclei and wreath-shaped nuclei. Nodular lymphocyte predominant Hodgkin lymphoma is defined by the presence of lymphocytic and histiocytic (popcorn) cells, which have nucleoli that are smaller than those of RS cells. The neoplastic cells of TCRBCL can resemble those of classical Hodgkin lymphoma but usually show more variation in size. There are immunophenotypic differences that help to separate these lymphomas from classical Hodgkin lymphoma and architecture may be informative when dealing with this differential diagnosis; therefore, excisional biopsy should be pursued if uncertainty exists. It is also important to be aware of non-neoplastic changes that can mimic Hodgkin lymphoma. Lymphadenitis induced by Epstein–Barr virus in infectious mononucleosis often contains Hodgkin-like cells. Follicular dendritic cells sometimes encountered in aspirates of non-neoplastic lymph nodes show some resemblance to the neoplastic cells of classical Hodgkin lymphoma. Diagnostic pitfalls can be avoided if careful attention is paid to the clinical history, immunophenotype, and cellular background.

Key Diagnostic Features •

• •

Hodgkin and RS cells in a background of mixed inflammatory cells including eosinophils and granuloma formation Large eosinophilic nucleoli Positive for CD15, CD30, and PAX-5 (weak); negative for CD3, CD20, and CD45

Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma Chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) is a mature B-cell neoplasm affecting older adults with a high prevalence in Western countries. Peripheral blood and bone marrow are commonly involved, and lymph nodes, liver, and spleen, are frequently infiltrated. Isolated lymph node involvement can be seen occasionally. The majority of patients are asymptomatic but some present with fatigue, lymphadenopathy, or splenomegaly. Cytopenias due to marrow infiltration, autoimmune hemolysis, or infection may occur. Progression manifests in the form of increased prolymphocytes, transformation to diffuse large B-cell lymphoma (DLBCL) (Richter syndrome), or transformation to classical Hodgkin lymphoma.

Cytomorphology There is a predominance of small, monomorphous lymphocytes with scant cytoplasm, coarse (“soccer ball”) chromatin, smooth nuclear borders, and inconspicuous nucleoli (Figs 7.5A and B). Scattered interspersed larger lymphoid cells, prolymphocytes, and paraimmunoblasts, are present. A marked increase in the proportion of large lymphocytes is indicative of transformation to large cell lymphoma.25

Ancillary Studies The neoplastic cells are surface light chain restricted and are positive for CD19, CD20, CD79a, CD5, and CD23 while negative for CD10, BCL-6, cyclin D1, and FMC-7.

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Expression of B-cell markers and surface immunoglobulins is often weak. A number of specific cytogenetic aberrations have been characterized in CLL/SLL and these have significant prognostic implications.

Differential Diagnosis The differential diagnosis is with other indolent B-cell lymphomas. In particular, it is important to distinguish CLL/SLL from mantle cell lymphoma, which is associated with a less favorable clinical outcome. Mantle cell lymphoma often displays more nuclear membrane irregularity and lacks admixed prolymphocytes and paraimmunoblasts. Expression of B-cell antigens and surface immunoglobulins is typically stronger in mantle cell lymphoma and CD23 is not expressed. Importantly, mantle cell lymphoma is characterized by t(11;14) that is absent in CLL/SLL.

Key Diagnostic Features • • • •

Small monomorphous lymphocytes with “soccer ball” chromatin Admixed prolymphocytes and paraimmunoblasts Positive for CD20, CD5, and CD23, and surface light chain restriction; negative for cyclin D1 Weak expression of pan-B-cell markers and surface light chain

Mantle Cell Lymphoma Mantle cell lymphoma is a low-grade B-cell lymphoma that comprises 40–60% has been associated with a worse prognosis. Cytogenetic analysis shows a t(11;14) involving cyclin D1 and IgH genes that can be demonstrated by FISH.

Differential Diagnosis CLL/SLL is the main differential diagnostic consideration.

Key Diagnostic Features • • • • •

Monotonous small-to-medium-sized lymphocytes Epithelioid histiocytes Positive for CD20, CD5, cyclin D1, and surface light chain restriction; negative for CD23 Cytogenetics: t(11;14) Bright expression of pan-B-cell markers and surface light chains

Marginal Zone Lymphoma Marginal zone lymphoma is a low-grade B-cell lymphoma with nodal, extranodal, and splenic presentations.26 Extranodal marginal zone lymphomas, also known as mucosa-associated lymphoid tissue (MALT) lymphomas, have a predilection for mucosal sites including the

Cytomorphology The lymphocytes are small to medium sized, strikingly monomorphous, and often display irregular nuclear membranes (Fig. 7.6A). Chromatin is somewhat dispersed and nucleoli are inconspicuous. Scattered epithelioid histiocytes are often present. The blastoid variant consists of larger cells resembling lymphoblasts or Burkitt lymphoma.

Fig. 7.6A: Mantle cell lymphoma, consisting of small lymphocytes showing striking monotony and slight nuclear membrane irregularity (Papanicolaou, high power)

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Fig. 7.6B: Mantle cell lymphoma. Gating of the lymphocytes was performed using CD45 versus side scatter (top) revealing a population of B-cells coexpressing CD5 (middle) and demonstrating surface light chain restriction (bottom). In addition, FMC-7 was positive and CD23 was negative (not shown)

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Fig. 7.6C: Mantle cell lymphoma. Immunocytochemical study for cyclin D1 (BCL-1) performed on a cell block from the case displayed in Figures 7.6A and B shows strong nuclear immunoreactivity (Cell block, high power)

Fig. 7.7: Marginal zone lymphoma. The neoplastic cells consist of monocytoid appearing small lymphocytes with clear cytoplasm (Diff-Quik, high power)

stomach where they most commonly arise in association with chronic Helicobacter pylori infection. Marginal zone lymphomas usually occur in adults but pediatric nodal marginal zone lymphomas are also described. The disease is indolent but incurable. Transformation to DLBCL may occur.

Differential Diagnosis

Cytomorphology A heterogeneous population of lymphocytes or a predominance of small lymphocytes may be seen. The neoplastic lymphocytes typically have moderate amounts of clear cytoplasm, coarse chromatin, inconspicuous nucleoli, and irregular nuclear membranes, imparting a monocytoid appearance (Fig. 7.7). Plasmacytic differentiation occurs more commonly in marginal zone lymphoma than in other types of low-grade lymphoma.

Ancillary Studies The neoplastic lymphocytes are positive for CD19, CD20, and CD79a, while negative for CD5, CD10, and BCL-6. CD43 and BCL-2 are frequently expressed. A monotypic B-cell population is seen by flow cytometry in most cases but may be difficult to detect in some instances due to the simultaneous presence of numerous reactive lymphocytes. In cases displaying plasmacytic differentiation, cytoplasmic light chain restriction can usually be demonstrated in the plasma cell component by immunocytochemistry. Cytogenetic abnormalities are observed in a subset of MALT lymphomas, including t(11;18) and t(14;18), trisomy 3, and trisomy 18.

The main differential diagnosis is with reactive lymphadenitis in which the B cells are polytypic. Marginal zone lymphomas showing extreme plasmacytic differentiation can be difficult to distinguish from plasmacytoma.

Key Diagnostic Features •

Monocytoid small lymphocytes with clear cytoplasm – May have a polymorphous appearance – Plasmacytic differentiation – Positive for CD20 and CD43 and light chain restriction; negative for CD5 and CD10.

Follicular Lymphoma Follicular lymphoma is a low-grade B-cell lymphoma of follicle center cell origin that occurs in adults. Sites of involvement include lymph nodes, spleen, bone marrow, peripheral blood, Waldeyer ring, and extranodal sites. Prognosis correlates with the extent of disease and the histologic grade. Transformation to DLBCL may occur.

Cytomorphology Follicular lymphomas consist of a mixture of centrocytes and centroblasts. Centrocytes are small lymphocytes with cleaved nuclear membranes occasionally imparting a bilobed appearance (“buttock cells”), inconspicuous nucleoli, and scant cytoplasm (Fig. 7.8). Centroblasts are large lymphocytes with vesicular chromatin and multiple peripherally located nucleoli. Grading is based

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Large B-cell Lymphoma

Fig. 7.8: Follicular lymphoma comprised on small monotonous lymphocytes, some displaying cleaved nuclei from a touch imprint of a bone biopsy (Wright Giemsa, high power)

LBCL is the most common type of non-Hodgkin lymphoma, arising either as de novo disease or from preexisting low-grade lymphoma that has undergone transformation. It may occur at any age and can involve a variety of sites, both nodal and extranodal. Most patients have no predisposing factors, but individuals who are immunosuppressed due to HIV infection, transplantation, or immunosuppressive medications are at increased risk, and there is a close association with Epstein–Barr virus infection in these patients.28 The morphologic spectrum of LBCL is broad, and several different subtypes have been defined. Patients typically present with a rapidly enlarging mass and may have B symptoms. The prognosis with treatment is variable, with complete remission achieved in approximately two-thirds of patients.

Cytomorphology on the proportion of centroblasts within neoplastic follicles. Some studies have shown that grading can be accurately reproduced in FNA samples, but this remains controversial.27

Ancillary Studies Follicle center cell markers CD10 and BCL-6 are typically expressed along with CD19, CD20, CD79a, BCL-2, and monotypic light chains, while CD5 is negative. Approximately 80% of cases show a t(14;18) involving IgH and BCL-2, which can be detected by FISH.

Differential Diagnosis The presence of monotypic immunoglobulin permits distinction of follicular lymphoma from reactive lymphoid hyperplasia, which may enter the cytomorphologic differential diagnosis due to the morphologic heterogeneity associated with both entities. Expression of follicle center cell markers assists in distinguishing follicular lymphoma from other low-grade lymphomas. A predominance of centroblasts rather than a mixed population of centrocytes and centroblasts suggests a diagnosis of LBCL.

Key Diagnostic Features • • •

Small cleaved centrocytes and variable numbers of centroblasts Positive for CD20, CD10, BCL-6, and BCL-2, and surface light chain restriction Cytogenetics: t(14;18) involving IgH and BCL-2

LBCL consists of large lymphocytes that frequently have centroblastic or immunoblastic morphology (Figs 7.9A and B). Nuclear contours are often irregular and mild to marked nuclear pleomorphism is seen. In some cases, extensive necrosis hinders cytologic interpretation.

Ancillary Studies CD45, CD19, CD20, and CD79a are positive. Other markers variably expressed include CD10, BCL-6, BCL-2, and MUM-1. The Ki-67 proliferation is usually >40%. Several cytogenetic abnormalities may be encountered, including t(14;18) and occasionally rearrangements involving c-myc.

Differential Diagnosis Distinguishing LBCL and other high-grade lymphomas from nonhematolymphoid malignancies such as small cell carcinoma may require immunocytochemistry for CD45, cytokeratins, and other markers. The cytomorphology of LBCL can overlap with Burkitt lymphoma, lymphoblastic lymphoma, and classical Hodgkin lymphoma. In general, a greater degree of nuclear size variation is seen in LBCL when compared with Burkitt lymphoma. Chromatin tends to be more evenly dispersed in lymphoblastic lymphoma than in LBCL. Distinguishing LBCL from reactive lymphoid hyperplasia with increased numbers of large lymphocytes is of critical importance. In the latter, the large lymphocytes generally lack overt cytologic atypia.

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B

A

Figs 7.9A and B: (A) Large B-cell lymphoma consisting of pleomorphic large lymphocytes with marked nuclear membrane irregularity and abnormal chromatin distribution (Papanicolaou, high power); (B) Large B-cell lymphoma comprised mainly of centroblasts in a cerebrospinal fluid (Papanicolaou, high power)

Key Diagnostic Features

Ancillary Studies

•

The B cells express monotypic light chains, CD19, CD20, CD79a, CD10, and BCL-6. BCL-2 is characteristically negative. The Ki-67 proliferation rate approaches 100%. Cytogenetics reveals t(8;14) involving c-myc and IgH or a variant translocation involving c-myc and either the kappa or lambda light chain locus.

•

Large lymphocytes (centroblastic, immunoblastic morphology common) with vesicular chromatin, nuclear pleomorphism often prominent, mitotic figures Positive for CD45, CD20; variably positive for BCL-2, BCL-6, CD10, and MUM-1; moderate-to-high Ki-67 proliferation rate

Burkitt Lymphoma Burkitt lymphoma is an aggressive B-cell lymphoma characterized by rapid growth and a translocation involving the myc oncogene. The endemic form is prevalent in equatorial Africa, affects the pediatric age group, and often involves the jaw. The sporadic form occurs in various parts of the world, has a broad age distribution, and frequently arises within the abdomen. Immunodeficiency-associated Burkitt lymphoma accounts for a significant proportion of lymphomas developing in patients with HIV. Presentation at nodal and extranodal sites is common, and a peripheral blood leukemic picture can be seen.

Cytomorphology The cells of Burkitt lymphoma are medium-sized monomorphous lymphocytes with round nuclei, coarse chromatin, and multiple small centrally located nucleoli (Figs 7.10A to D). On Romanowsky-stained smears, the cytoplasm is deeply basophilic and is filled with lipid vacuoles. Tingible body macrophages are scattered throughout and mitotic figures are encountered frequently.

Differential Diagnosis The differential diagnosis includes LBCL and lymphoblastic lymphoma. Lymphoblastic lymphoma can be distinguished by virtue of nuclear expression of terminal deoxynucleotidyl transferase (TdT). It is important to recognize that c-myc translocations are not specific for Burkitt lymphoma as other hematolymphoid neoplasms can be c-myc positive.

Key Diagnostic Features •

• •

•

Medium-sized monomorphous lymphocytes with deeply basophilic cytoplasm-containing tiny cytoplasmic vacuoles, mitotic figures Tingible body macrophages (“starry sky” pattern) Positive for CD20, CD10, BCL-6, and surface light chain restriction; negative for BCL-2; Ki-67 proliferation rate approaches 100% Cytogenetics: t(8;14) involving c-myc

Anaplastic Large Cell Lymphoma ALCL is a CD30-positive T-cell lymphoma that consists of pleomorphic cells. It most commonly occurs in the first

Fine Needle Aspiration of Lymphoid Lesions

A

B

C

D

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Figs 7.10A to D: (A) Burkitt lymphoma. Medium sized neoplastic lymphocytes containing small nucleoli. Note the presence of a mitotic figure (Papanicolaou, high power); (B) On Wright Giemsa stained preparations, the malignant lymphocytes display deeply basophilic cytoplasm and cytoplasmic vacuoles (Wright Giemsa, high power); (C) The Ki-67 proliferation rate approaches 100% (Cell block, high power); (D) FISH analysis using the c-myc breakapart probe reveals a c-myc rearrangement as indicated by splitting apart of the normal red-green fusion signal into one red (centromeric end of myc) and green (telomeric end of myc) signal (image provided by Dr CF Li, St. Michael’s Hospital, Toronto, Canada)

three decades and involves both lymph nodes and extranodal sites such as skin, bone, soft tissue, and lung. These tumors are subclassified on the basis of their expression or lack of expression of the ALK-1 protein that usually results from a translocation t(2;5) involving ALK and NPM genes. ALK-1-positive ALCL accounts for > 70% of cases of ALCL, tends to occur in younger patients, and has a more favorable prognosis than ALK-1 negative ALCL.

Cytomorphology The tumor cells are large and have pleomorphic nuclei, which may be horseshoe shaped (hallmark cells) or wreath shaped (Figs 7.11A and B). Multinucleation is common, and

cells resembling Hodgkin and RS cells may be present. The tumor cells often have vacuolated cytoplasm. Histiocytes and granulocytes may be present in abundance.

Ancillary Studies The tumor cells are CD30 positive and are variably positive for CD45. CD3 is negative in the majority of cases, but expression of other T-cell markers such as CD2 and CD4 is usually present. Epithelial membrane antigen, CD25, and cytotoxic markers (TIA-1 and granzyme) are frequently expressed. ALK-1 protein expression can be demonstrated with a high degree of sensitivity by immunocytochemistry. FISH probes are also available to detect translocations involving the ALK and NPM genes.

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A

B

Figs 7.11A and B: (A) Anaplastic large T-cell lymphoma: Pleomorphic tumor cells are present, some showing indented nuclei (hallmark cells; top) and multinucleated tumor cells (bottom) (Diff-Quik, high power). Images provided by Dr J Hunt, Baystate Medical Center, Massachusetts, USA

Differential Diagnosis

Cytomorphology

The prominent pleomorphism seen in ALCL in conjunction with other features, such as immunoreactivity for epithelial membrane antigen and lack of CD45 expression, can lead to an erroneous diagnosis of carcinoma. Although CD30 expression can be seen in some carcinomas such as embryonal carcinoma, unlike most carcinomas, cytokeratins are not expressed by ALCL. Classical Hodgkin lymphoma and TCRBCL also enter the differential diagnosis of ALCL.

The neoplastic cells are medium sized and typically have scant gray-blue cytoplasm, fine chromatin, convoluted nuclear membranes, and prominent nucleoli (Fig. 7.12). Tiny cytoplasmic vacuoles and scattered admixed tingible body macrophages may be present.

Key Diagnostic Features • •

•

Ancillary Studies Lymphoblastic lymphomas express the nuclear antigen TdT, and many are also positive for CD34, CD99, and CD1a. Cytoplasmic CD3 is often expressed in T-lymphoblastic lymphoma and is considered to be lineage specific.

“Hallmark cells”, wreath-shaped nuclei, multinucleated cells, marked pleomorphism Positive for CD30, epithelial membrane antigen, CD2, CD4, CD25, cytotoxic markers, ALK-1+/–, and CD45+/–; often negative for CD3 Cytogenetics: t(2;5) involving ALK-1 and NPM genes

Precursor Lymphoblastic Lymphoma Precursor lymphoblastic neoplasms most commonly involve the peripheral blood (precursor lymphoblastic leukemia), but occasionally presents with primary nodal involvement (precursor 3lymphoblastic lymphoma). T-lymphoblastic lymphoma accounts for 90% of the latter.29 T-lymphoblastic lymphoma commonly involves the mediastinum in adolescent males and is frequently accompanied by pleural effusions. If untreated, lymphoblastic lymphoma is highly aggressive, but many patients are cured with chemotherapy.

Fig. 7.12: Lymphoblastic lymphoma comprised of blasts with fine chromatin and basophilic cytoplasm containing tiny cytoplasmic vacuoles mimicking Burkitt lymphoma (Wright Giemsa, high power)

Fine Needle Aspiration of Lymphoid Lesions

A

147

B

Figs 7.13A and B: Primary effusion lymphoma showing large individually dispersed tumor cells with plasmablastic morphology (A—Diff-Quik, high power) that are immunoreactive for human herpesvirus-8 in a stippled nuclear pattern (B—Cell block immunocytochemistry, high power)

Other T-cell markers are variably positive and both CD4 and CD8 may be coexpressed. CD19, cytoplasmic CD79a, cytoplasmic CD22, and PAX-5 are frequently expressed in B-lymphoblastic lymphoma along with CD10, while CD20 is variably positive. Several cytogenetic abnormalities are described in B-lymphoblastic lymphoma, including t(9;22) and t(4;11) that are associated with less favorable outcomes. Clonality can usually be demonstrated by PCR analysis, but lineage infidelity is more common in precursor lymphoid neoplasms than in mature B-cell lymphomas.

Differential Diagnosis The main differential diagnosis is with mature B-cell neoplasms, especially Burkitt lymphoma. Morphologic distinction may be extremely challenging, and ancillary investigations including assessment for nuclear expression of TdT are essential.

Key Diagnostic Features • •

Medium-sized blasts with fine, homogeneous chromatin and scant blue cytoplasm Positive for Tdt, CD99, and CD1a

Other High-grade Hematolymphoid Neoplasms Several additional types of high-grade hematolymphoid neoplasms may be encountered in cytologic samples. B-cell lymphoma, unclassifiable, with features inter-mediate

between DLBCL and Burkitt lymphoma is an aggressive lymphoma that has overlapping morphologic, immunophenotypic, and cytogenetic characteristics.24 This diagnostic category is enriched with lymphomas that have genetic rearrangements involving both c-myc and BCL-2 or BCL-6, hence termed “dual translocation” or “2-hit” lymphomas, and are associated with an extremely poor prognosis. The differential diagnosis of high-grade hematolymphoid neoplasms showing plasmablastic features is complex and includes plasmablastic myeloma and high-grade lymphomas with preterminal B-cell differentiation. The later includes plasmablastic lymphoma, which typically arises in HIV-positive patients in the oral cavity and is linked with Epstein–Barr virus infection. Similarly, primary effusion lymphoma is an aggressive lymphoma with plasmablastic differentiation that presents in HIV-positive patients (Figs 7.13A and B), but unlike plasmablastic lymphoma, involves serous effusions without tumor masses. Primary effusion lymphoma is associated with human herpesvirus-8, which can be demonstrated by immunocytochemistry, as well as Epstein–Barr virus. Myeloid sarcoma is a tumor mass consisting of myeloblasts arising either de novo or in association with a myeloid neoplasm involving the bone marrow. These tumors display blastic morphology and show an immature myeloid phenotype with frequent expression of monocytic markers.

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REFERENCES

1. Khillan R, Sidhu G, Axiotis C, et al. Fine needle aspiration (FNA) cytology for diagnosis of cervical lymphadenopathy. Int J Hematol. 2012;95(3):282-4. 2. Hoppe RT, Advani RH, Ai WZ, et al. Hodgkin lymphoma. J Natl Compr Canc Netw. 2011;9(9):1020-58. 3. Zelenetz AD, Abramson JS, Advani RH, et al. NCCN clinical practice guidelines in oncology: non-Hodgkin’s lymphomas. J Natl Compr Canc Netw. 2010;8(3):288-334. 4. Jeffers MD, McCorriston J, Farquharson MA, et al. Analysis of clonality in cytologic material using the polymerase chain reaction (PCR). Cytopathology. 1997;8(2):114-21. 5. Jeffers MD, Milton J, Herriot R, et al. Fine needle aspiration cytology in the investigation on non-Hodgkin’s lymphoma. J Clin Pathol. 1998;51(3):189-96. 6. Chhieng DC, Cangiarella JF, Symmans WF, et al. Fineneedle aspiration cytology of Hodgkin disease: a study of 89 cases with emphasis on false-negative cases. Cancer. 2001;93(1):52-9. 7. Liu K, Stern RC, Rogers RT, et al. Diagnosis of hematopoietic processes by fine-needle aspiration in conjunction with flow cytometry: a review of 127 cases. Diagn Cytopathol. 2001;24(1):1-10. 8. Meda BA, Buss DH, Woodruff RD, et al. Diagnosis and subclassification of primary and recurrent lymphoma. The usefulness and limitations of combined fine-needle aspiration cytomorphology and flow cytometry. Am J Clin Pathol. 2000;113(5):688-99. 9. Cibas ES, Ducatman BS, (Eds). Cytology: Diagnostic principles and clinical correlates, 3rd edn. Philadelphia, Elsevier, 2009;537. 10. Nunez AL, Jhala NC, Carroll AJ, et al. Endoscopic ultrasound and endobronchial ultrasound-guided fine-needle aspiration of deep-seated lymphadenopathy: analysis of 1338 cases. Cytojournal. 2012;9:14-6413.95845. Epub 2012 May 5. 11. Jaffe ES. Hematopathology. 1st ed. Philadelphia, PA: Saunders/Elsevier; 2011. 12. Levitt S, Cheng L, DuPuis MH, et al. Fine needle aspiration diagnosis of malignant lymphoma with confirmation by immunoperoxidase staining. Acta Cytol. 1985; 29(5):895-902. 13. Calvo KR, McCoy CS, Stetler-Stevenson M. Flow cytometry immunophenotyping of hematolymphoid neoplasia. Methods Mol Biol. 2011;699:295-316. 14. Shetuni B, Lakey M, Kulesza P. Optimal specimen processing of fine needle aspirates of non-Hodgkin lymphoma. Diagn Cytopathol. 2012;40(11):984-6.

15. Skoog L, Tani E. Techniques. Monogr Clin Cytol. 2009; 18:5-10. 16. Savage EC, Vanderheyden AD, Bell AM, et al. Independent diagnostic accuracy of flow cytometry obtained from fineneedle aspirates: a 10-year experience with 451 cases. Am J Clin Pathol. 2011;135(2):304-9. 17. Zhang S, Abreo F, Lowery-Nordberg M, et al. The role of fluorescence in situ hybridization and polymerase chain reaction in the diagnosis and classification of lymphoproliferative disorders on fine-needle aspiration. Cancer Cytopathol. 2010;118(2):105-12. 18. Ochs RC, Bagg A. Molecular genetic characterization of lymphoma: application to cytology diagnosis. Diagn Cytopathol. 2012;40(6):542-55. 19. da Cunha Santos G, Ko HM, Geddie WR, et al. Targeted use of fluorescence in situ hybridization (FISH) in cytospin preparations: results of 298 fine needle aspirates of B-cell non-Hodgkin lymphoma. Cancer Cytopathol. 2010;118(5):250-58. 20. Dawsey SM, Korn EL, Layfield LJ. Morphometric analysis of the homogeneity of lymphoid cell populations in fine-needle aspiration cytology smears. Am J Clin Pathol. 1989;92(4):458-64. 21. Metzgeroth G, Schneider S, Walz C, et al. Fine needle aspiration and core needle biopsy in the diagnosis of lymphadenopathy of unknown aetiology. Ann Hematol. 2012;91(9):1477-84. 22. Swerdlow SH. International Agency for Research on Cancer, World Health Organization. WHO classification of tumours of haematopoietic and lymphoid tissues. 4th ed. Lyon, France: International Agency for Research on Cancer; 2008. 23. Skoog L, Tani E. Hodgkin lymphoma. Monogr Clin Cytol. 2009;18:49-52. 24. Das DK, Pathan SK, Mothaffer FJ, et al. T-cell-rich B-cell lymphoma (TCRBCL): limitations in fine-needle aspiration cytodiagnosis. Diagn Cytopathol. 2012;40(11):956-63. 25. Skoog L, Tani E. B cell neoplasms. Monogr Clin Cytol. 2009;18:19-37. 26. Skoog L, Tani E. Extranodal lymphomas. Monogr Clin Cytol 2009;18:60-3. 27. Brandao GD, Rose R, McKenzie S, et al. Grading follicular lymphomas in fine-needle aspiration biopsies: the role of ThinPrep slides and flow cytometry. Cancer. 2006;108(5):319-23. 28. Skoog L, Tani E. Immunodeficiency-associated lymphoproliferative disorders. Monogr Clin Cytol. 2009;18:53-5. 29. Skoog L, Tani E. T cell neoplasms. Monogr Clin Cytol. 2009;18:38-48.

Chapter

8

Liver

Harvey M Cramer



CLINICAL CONSIDERATIONS AND TECHNICAL ASPECTS

The liver is a structurally complex and metabolically active organ. It performs important functions in carbohydrate and porphyrin metabolism as well as important metabolic functions for lipids and amino acids. As such, the liver can display a wide range of changes related to storage and metabolic disorders both acquired and inherited.1 Moreover, the liver may be the primary target of a number of toxins including alcohol, acetaminophen, and halothane.2 Many of these toxins produce characteristic morphologic changes in the liver. A number of viruses predominantly or exclusively effect the liver, resulting in acute viral hepatitis that may ultimately lead to cirrhosis. The changes associated with these entities usually result in diffuse liver damage best investigated by core needle or open biopsy. Thus, fine needle aspiration (FNA) is not a preferred technique for the workup of metabolic and viral diseases within the liver.3,4 FNA is utilized for the investigation of localized processes that result in well-defined targets for image-guided FNA. A variety of imaging techniques are available for the workup of both localized and diffuse hepatic disease. Among those useful for localized disease are magnetic resonance imaging (MRI), ultrasound (US), computed tomography (CT), and nuclear scanning. CT and US are frequently used modalities for image-directed FNA. Focal abnormalities may be found in the liver incidentally,

secondary to workup of hepatic-related symptoms or as a part of a metastatic workup. When tissue diagnosis is necessary for selection of appropriate therapy, FNA represents an excellent minimally invasive technique for the diagnosis of these lesions. In the United States and Europe, the majority of focal abnormalities are due to metastatic neoplasms. However, in some African and Asian populations, hepatocellular carcinoma occurs with high frequency.5 FNA represents an excellent technique for the distinction of metastatic from primary malignancies. FNA is also helpful in the separation of hemangiomas, focal fatty change, focal nodular hyperplasia, and hepatic adenomas from malignancies. The precise role that FNA plays in the workup of suspected hepatocellular carcinoma has become controversial. Newer imaging techniques including Gd-EOBDTPA-MRI are being utilized in place of tissue diagnosis for the recognition of hepatocellular carcinoma in some regions of the world. Studies have shown that this technique has an accuracy of diagnosis comparable to that of a pathologist specializing in liver disease.6,7 Other authorities continue to recommend the use of FNA for the workup of hepatic nodules including those suspicious for hepatocellular carcinoma.7 Percutaneous (transabdominal) FNA performed under CT or US guidance is widely used and recognized as a safe, efficient, and minimally invasive procedure for the diagnosis of focal liver lesions. This technique is especially helpful in patients with advanced malignancies or those who are perceived to be poor operative

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candidates. The sensitivity and specificity of FNA for the detection of hepatocellular carcinoma are approximately 90% and 100%, respectively, when performed by trained radiologists and cytopathologists.8-10 More recently, endoscopic ultrasound (EUS)-guided FNA has become popular with improved sensitivity for small deeply situated lesions within the left lobe of the liver.11 Despite the high diagnostic accuracy of FNA, recent advances in clinical care and imaging techniques have resulted in controversy as when and where to use FNA. Contrast-enhanced US and dynamic MRI have a demonstrated accuracy, sensitivity, and specificity of 99.6%, 100%, and 98.9%, respectively.12 These accuracy statistics are similar to those achievable with FNA, and thus in many oncologists minds obviate the need for tissue diagnosis. These advances in imaging techniques have resulted in modifications of the European Association for the Study of Liver 2000 Conference and are also incorporated in the American Association for the Study of Liver Disease guidelines.13,14 Current guidelines suggest biopsy of nodules between 1 and 2 cm if no definitive diagnosis of hepatocellular carcinoma is reached on two sequential imaging modalities. A “wait and see” policy should be pursued with more frequent US surveillance for nodules under 1 cm.13,14 Suspected metastatic disease should undergo FNA for tissue confirmation.

Complications FNA of the liver is associated with few complications with only rare cases of severe or fatal hemorrhage being reported.15-17 Large studies have reported no mortality and only rare instances of significant hemorrhage.18 Extremely rare reports of bile peritonitis and needle tract seeding have been made, but these complications do not appear to be significant contraindications for hepatic FNA.19,20

Technical Aspects FNA is best performed under image guidance. Real-time US appears superior to CT imaging both for speed and cost. US is currently the most commonly used modality. More recently, EUS-guided FNA is gaining popularity over traditional percutaneous methods. A 22-or 23-gauge needle appears optimal for aspiration biopsies. Such needles can be obtained with a trocar that minimizes contamination along the biopsy tract. Local anesthesia is generally utilized and is injected into the skin, the parietal peritoneum, and at times along the planned biopsy pathway. Anesthetic should not be injected directly into the lesion of interest as this will be associated with a poor biopsy yield and perhaps distortion of the morphology of the material obtained. Small gauge core biopsies can also be utilized to maximize biopsy yield for cell block preparations (utilized for immunohistochemistry and molecular techniques). Rapid on-site evaluation (ROSE) can be of aid in assuring adequate material and for the triage of material to special studies. Most special studies including immunoperoxidase staining and molecular diagnostics can be performed on paraffin embedded cell block material.

Normal Hepatic Cytomorphology The normal liver aspirate will reveal a mixture of hepatocytes, Küpffer cells, and bile duct epithelium. Smears of morphologically normal liver are composed of large numbers of hepatocytes, predominantly forming sheets and clusters (Fig. 8.1). Single cells and nuclei devoid of cytoplasm are relatively infrequent. The sheets of

Contraindications Relatively few contraindications exist for hepatic FNA. A known bleeding diathesis is a contraindication, and prebiopsy prothrombin and platelet count should be obtained. Hemorrhage is a more significant risk when the lesion is immediately subcapsular and insufficient hepatic tissue is present to aid in compression of any potential hematoma. Hepatic hemangiomas do not appear to be a significant risk for hemorrhage as has been demonstrated in recent publications.21,22 Traditionally, hydatid cysts have been considered a contraindication for the use of FNA. However, recent series have not demonstrated a significant risk for allergic reactions following FNA of hydatid cysts.23,24

Fig. 8.1: Benign liver tissue characterized by hepatocytes forming tight clusters and sheets. Individual hepatocytes and “naked” nuclei are uncommon (Diff-Quik)

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Fig. 8.2: Benign hepatocytes with abundant cytoplasm and indistinct cell borders. Nuclei are bland-appearing and round to oval (Diff-Quik)

Fig. 8.3: Hepatocytes with abundant cytoplasm containing deep blue granules (Diff-Quik)

hepatocytes are composed of cohesive cells with dense cytoplasm and indistinct cell borders (Fig. 8.2). The nuclei are round or slightly ovoid and often contain a prominent nucleolus. The nuclei will vary mildly in size. The individual hepatocytes have abundant generally granular cytoplasm that will be vacuolated when a fatty change is present. Course intracytoplasmic pigment granules are common (Fig. 8.3) within hepatocytes and stain a green to black color in air-dried presentations, but brown with hematoxylin and eosin (H&E) and Papanicolaou staining. In most cases, this pigment represents lipofuscin. In rare cases, free bile pigment or intracytoplasmic bile pigment may be seen and has a more waxy appearance and forms larger aggregates. Bile duct epithelial cells are much less common than hepatocytes. These cells form small irregular monolayered sheets or occasionally tube-like structures. The individual cells are small with scant cytoplasm. The nuclei have a granular chromatin. Bile duct epithelial cells have inconspicuous nucleoli. Küpffer cells are rarely appreciated morphologically but when present are found within sheets of hepatocytes where they appear as small wedge-shaped cells composed predominantly of a nucleus. A variety of reactive processes can modify this basic smear pattern of benign hepatic tissue. Such changes usually result in decreased cohesion of hepatocytes with both degenerative and regenerative changes being present. Bile duct epithelial cells may become more prominent in cases of cirrhosis, and in cases of hepatitis

increased numbers of lymphocytes may be seen. These reactive changes may be associated with parenchymal disease and can be found in the reactive zone around both metastatic and primary hepatic neoplasms. These reactive changes include reduced hepatocyte cohesion so that a significant percentage of hepatocytes lie individually and may even be represented by “naked” nuclei. The nuclei may vary in size by fourfold or more. The associated cytoplasm often has a swollen edematous appearance and stains less uniformly than the normal hepatocyte. In many cells with reactive change, the periphery of the hepatocyte appears paler than the central zone. The cell border shows a greater distinctiveness. Fatty change is common and characterized by large numbers of intracytoplasmic vacuoles that can vary considerably in size. Multinucleated and binucleated hepatocytes occur with some regularity in reactive change. An important feature aiding in the distinction of reactive hepatocytes from those of hepatocellular carcinoma is that benign hepatocytes retain a relatively finely granular chromatin. The naked nuclei of hepatocytic origin seen in the background of reactive change do not show significant atypia. In some cases of reactive change, intranuclear cytoplasmic pseudoinclusions can be seen within hepatocytic nuclei. Nucleoli also become more prominent. Aspirates obtained from cirrhotic livers are associated with smears containing poorly cohesive hepatocytes showing degenerative and regenerative features. Significant variability in nuclear size and shape is common. Additionally, sheets and small clusters of bile duct cells are

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increased in number. Küpffer cells become more numerous and often lie along the edge of a loosely cohesive aggregate of hepatocytes. In cases of bile stasis, aggregates of a waxy blue-black (air-dried) or yellow-green (Papanicolaou and H&E staining) material are seen between hepatocytes or occasionally lying free in the background. Hemosiderin granules can be recognized in smears from examples of hemosiderosis. In this case, the pigment has a golden yellow or brown appearance and is present within both Küpffer cells and hepatocytes. Often hemosiderin is inseparable from lipofuscin in smeared preparations. Cell block preparations with appropriate staining for iron will separate the two pigments. 

LOCALIZED LESIONS AND LESIONS OF INFECTIOUS ETIOLOGY

Granulomatous Lesions Hepatic granulomas may be seen in association with a variety of infectious and noninfectious etiologies. Granuloma can be associated with cirrhosis, drug-induced hepatitis, and some viral diseases. Distinct epithelioid granulomas with or without Langerhans giant cells occur in tuberculosis, sarcoid, and many fungal infections. The cytomorphology of these lesions has been discussed in Chapter 5. Granulomata associated with tuberculosis and some fungal infections frequently show central necrosis, while those associated with sarcoid do not and are so-called noncaseating granuloma.

Abscess A number of infections may result in hepatic abscesses. When purulent material is obtained by FNA, specimens for cultures should be submitted to the microbiology laboratory. Routine smears should be prepared to search for neoplastic cells in the event that the abscess represents necrotic material from a malignancy. Colonic adenocarcinoma can frequently present as extensive necrosis in aspirated material. Abscesses secondary to pyogenic organisms are characterized by a marked neutrophilic infiltrate along with necrotic debris and cell fragments. Hepatocytes surrounding an abscess may demonstrate significant reactive change with degenerative cytoplasmic features (vacuoles) and large nuclei showing considerable anisonucleosis. The most commonly cultured bacteria from hepatic abscesses is Klebsiella.25

Hepatic invasion by amoeba is usually associated with abscess formation and abundant necrosis. The necrotic material characteristically contains few inflammatory cells, and trophozoites are not found within the central necrotic material. They may be recognizable in material obtained from the periphery of the abscess.25,26

Hepatic Cysts A variety of congenital and acquired cysts exist within the liver. Aspiration of most will yield a thin clear fluid with little or no cellular material. In some cases, congenital cysts of the liver contain a few monolayered sheets or strips of bland cuboidal epithelial cells. Scattered macrophages with cytoplasmic vacuolization will be seen. A second type of cyst occasionally seen within the liver is the ciliated hepatic foregut cyst. This cyst is characteristically located below the falciform ligament and may undergo FNA. Aspirated material is usually hypocellular and contains clusters of ciliated columnar cells.27,28 Fluid obtained from hydatid cysts is often thick and creamy in consistency.23,24 Light microscopic evaluation of smears will reveal fragments of laminated cyst wall and occasionally hooklets and scolices.23,29,30

Non-neoplastic Solid Lesions Focal fatty change of the liver can closely resemble a neoplasm on imaging and undergo FNA.31,32 Aspirated material produces cellular smears containing isolated hepatocytes or sheets of hepatocytes. The hepatocytes are characterized by an abundant cytoplasm containing large intracytoplasmic clear vacuoles that may displace the nucleus to the periphery of the cell.31,32 In some cases, these vacuoles flatten the nucleus against the cytoplasmic membrane, resulting in a signet ring cell appearance. In most cases, the nuclei are round and bland with a hyperchromatic nucleus. Nucleoli are present and the chromatin pattern is bland. Normal-appearing hepatocytes are interspersed between these vacuolated forms, and cells with an intermediate cytoplasmic appearance exist between the highly vacuolated and the nonvacuolated hepatocytes. PAS-staining of these cells reveals no evidence of glycogen. Rare examples of nodular extramedullary hematopoiesis occur within the liver. Aspiration of these nodules yields a population of hematopoietic cells, demonstrating trilineage differentiation (Fig. 8.4). Immature nucleated red blood cells as well as promyelocytes and megakaryocytes are found. The megakaryocytes are often binucleated and may resemble Reed–Sternberg cells or the cells of melanoma.33,34

Liver

Fig. 8.4: Extramedullary hematopoiesis is characterized by trilineage hematopoietic cells with fat in the background. The hematopoietic cells are of normal morphology (Diff-Quik)

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solid, and grayish-white. On cut section, a central white depressed area of fibrosis is seen from which broad bands of fibrosis radiate to the periphery of the lesion. This results in a stellate appearance. Histologically, all components of the normal liver lobule are present, including hepatocytes and bile ducts. Cytomorphologically, smears are cellular and composed predominantly of sheets and clusters of hepatocytes (Figs 8.5A and B). These hepatocytes have minimal nuclear enlargement or atypia. The chromatin pattern is bland, but nucleoli may be prominent as seen in normal liver tissue. Small sheets and aggregates of reactive biliary epithelium are present. Chronic inflammation and fibrous tissue may be present in smears and cell block preparations. Necrotic debris is not seen and bile aggregates are uncommon.38-40

Key Diagnostic Features

Focal Nodular Hyperplasia

•

Focal nodular hyperplasia occurs predominantly in women and is seen over a wide age range. The majority of cases occur in the third to fifth decades of life, but pediatric cases have been reported.35,36 In a majority of cases, the patient is asymptomatic and these lesions are unlikely to lead to significant hemorrhage. Focal nodular hyperplasia is usually solitary, but multicentricity has been reported in approximately one-fifth of adult cases. A tentative relationship with oral contraceptives has been proposed, but current data is less compelling for this relationship than it is for hepatocellular adenomas.37 The arteriographic appearance is characteristic with centrifugal filling pattern. A central scar is usually recognizable by either CT or US imaging. Grossly, examples of focal nodular hyperplasia are usually subcapsular,

•

A

B

• • • • •

Cellular smears composed predominantly of normalappearing hepatocytes Minimal nuclear enlargement within hepatocyte population Hepatocytes show a normal chromatin pattern with distinct nucleoli Nuclear atypia is absent Reactive biliary epithelium commonly present Necrosis and mitotic figures not seen Aggregates of bile may be seen but are uncommon

Ancillary Testing A variety of markers demonstrable by immunocytochemistry can aid in the separation of focal nodular hyperplasia from metastatic carcinoma. These include Hep Par-1, CD34, and CD10.

Figs 8.5A and B: (A) Aspirated material from examples of focal nodular hyperplasia is dominated by large groups of bland hepatocytes; (B) The individual hepatocytes have moderately abundant granular cytoplasm and round to oval nuclei with a bland chromatin pattern (Diff-Quik)

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Diagnostic Pitfalls Focal nodular hyperplasia should be distinguished from hepatic adenoma, hepatocellular carcinoma, and metastatic disease. The diagnosis of focal nodular hyperplasia by FNA requires close correlation with imaging findings and the clinical setting. Separation of focal nodular hyperplasia from hepatic adenoma is achieved by recognizing the presence of biliary epithelium within smears obtained from focal nodular hyperplasia. Bile duct epithelium is not seen in hepatic adenomas. Hemorrhage and necrosis appear to be more common in hepatic adenomas than in examples of focal nodular hyperplasia. While bile plugs (stasis) can be seen in examples of focal nodular hyperplasia, it is extremely uncommon in material aspirated from hepatic adenomas. Hepatocellular carcinomas are distinguished from examples of focal nodular hyperplasia by the lack of biliary epithelium in hepatocellular carcinomas. Moreover, degrees of nuclear atypia and hyperchromasia are greater in most hepatocellular carcinomas than are characteristic in examples of focal nodular hyperplasia. Hepatocellular carcinomas usually demonstrate a distinct trabecular pattern (Fig. 8.6). Binucleation and multinucleation are characteristic of hepatocellular carcinomas but are uncommon in examples of focal nodular hyperplasia. Nucleoli are more prominent, and

Fig. 8.6: Cell groups obtained from hepatocellular carcinomas have a distinctly trabecular appearance (Papanicolaou)

cytoplasm appears denser in hepatocellular carcinomas (Fig. 8.7). The presence of bile within hepatocytes favors the diagnosis of hepatocellular carcinoma over that of focal nodular hyperplasia.

Bile Duct Hamartoma Bile duct hamartomas are incidental findings discovered at the time of laparotomy or laparoscopy. They are asymptomatic and rarely come to attention preoperatively. Because of their size, they rarely, if ever, undergo FNA. The author is unaware of any descriptions of cytologic material from bile duct hamartomas obtained by FNA. Scrape preparations are of a low cellularity. The epithelial material obtained forms two-dimensional monolayer sheets of ductal epithelium. Most of the epithelium appears cuboidal rather than columnar.

Differential Diagnosis The cytologic differential diagnosis for bile duct hamartoma includes metastatic adenocarcinoma and cholangiocarcinoma. The gross appearance of the lesion is characteristic at the time of laparotomy or laparoscopy. These lesions appear as multiple tiny gray-white nodules on the capsular surface of the liver. Scrape cytology reveals a small number of bland epithelial cells. Material obtained from examples of cholangiocarcinoma and metastatic adenocarcinoma is composed of cells showing significantly greater degrees of nuclear atypia.

Fig. 8.7: Cells derived from examples of hepatocellular carcinoma characteristically have prominent nucleoli and often an optically dense cytoplasm (Papanicolaou)

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Hamartomas are composed of small bland ductal structures, while metastatic adenocarcinomas show gland or duct structures with greater degrees of architectural irregularity. 

PRIMARY HEPATIC NEOPLASMS

Hepatocellular Adenoma (Liver Adenoma) Hepatocellular adenomas are rare and are found almost exclusively in women. These neoplasms where there are believed to be related to the use of oral contraceptives.41,42 Other etiologic agents include anabolic steroid therapy. Hepatocellular adenomas are frequently asymptomatic, but they may present with severe often life-threatening peritoneal hemorrhage.43 While most women are in their third to fifth decades of life, occasional cases occur in children. While multicentric forms have been reported approximately 70% are solitary lesions.

Histopathologic Features Grossly, hepatocellular adenomas have a well-defined capsule and are grossly distinct from the surrounding liver. No scar is present and the neoplasm has a homogeneous appearance except where hemorrhage has occurred. Microscopic evaluation reveals a neoplasm composed of well-differentiated bland-appearing hepatocytes with abundant eosinophilic cytoplasm. Of diagnostic importance, no portal triads or central veins are present. Hepatocellular adenomas frequently show oncocytic change and may have pigmentation and Mallory alcoholic hyaline.

Cytomorphology Cytomorphologically, hepatocellular adenomas are characterized by a nearly uniform population of blandappearing hepatocytes (Fig. 8.8). These cells have a distinctly low nuclear cytoplasmic ratio with only mild nuclear enlargement. The nuclei have smooth nuclear membranes. Characteristically, hemorrhage and necrosis may be prominent, but bile duct epithelium is invariably absent. The cytoplasm of the hepatocytes may contain fat or glycogen. Rarely, granulomas are seen in the smeared specimen.

Key Diagnostic Features • •

Uniform population of bland hepatocytes Absence of bile duct epithelium

Fig. 8.8: Well-differentiated hepatocellular carcinoma with individual cells showing granular eosinophilic cytoplasm. Cells lie in sheets with a vague trabecular pattern (Hematoxylin and Eosin)

• • •

Hemorrhage and necrosis may be prominent Hepatocytic cytoplasm may have accumulations of fat or glycogen Granulomas may be present

Differential Diagnosis Hepatocellular adenomas must be distinguished from well-differentiated hepatocellular carcinomas and focal nodular hyperplasia. Distinction of hepatocellular adenomas from well-differentiated hepatocellular carcinomas is frequently cytologically impossible. Higher grade hepatocellular carcinomas show significant nuclear atypia with irregular nuclear membranes and often huge nucleoli. High-grade hepatocellular carcinomas show greater degrees of necrosis and mitotic activity. Separation of hepatocellular adenomas from examples of focal nodular hyperplasia may be extremely difficult. The separation depends on clinical imaging findings. The presence of biliary epithelium essentially excludes hepatocellular adenoma establishing the diagnosis of focal nodular hyperplasia when cytologic sampling has been adequate.

Hepatocellular Carcinoma While infrequent in the United States, hepatocellular carcinoma is very common in sub-Saharan Africa and East Asia. The majority of patients are over 50 years of age and are predominantly men.44 Hepatocellular carcinoma usually presents with abdominal pain, hepatic

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Fig. 8.9: Hepatocellular carcinoma with an insular to trabecular architectural pattern (Hematoxylin and Eosin)

Fig. 8.10: High-grade hepatocellular carcinoma with sheet-like growth pattern (Hematoxylin and Eosin)

enlargement, or ascites. Obstructive jaundice may also be a presenting manifestation. Serum alpha-fetoprotein (AFP) is often elevated especially in African and Asian patients. Etiologic agents include hepatitis viruses B and C, and infection by these viruses may represent the predominant etiologic agent for hepatocellular carcinoma. Other etiologies include cirrhosis (usually the macronodular type), adenomas-hyperplasia, thorium dioxide exposure, anabolic steroid usage, progestational agents, aflatoxins, schistosomiasis, and alpha-1 antitrypsin deficiency. Hepatocellular carcinoma shows one of three gross patterns presenting either as a single mass, multiple nodules, or as diffuse liver involvement. The carcinoma is usually unencapsulated grossly. Histopathologically, hepatocellular carcinomas usually demonstrate a

trabecular, solid, or tubular growth pattern (Figs 8.9 to 8.12).45-47 The tubular pattern may produce a papillary architecture and overlap the appearance of cholangiocarcinoma. The trabeculae of hepatocellular carcinomas are greater than two cells in thickness and are greater in thickness than benign hepatocellular plates. While the stroma surrounding groups of malignant hepatocytes is usually scant, a sclerosing form of hepatocellular carcinoma is known. The individual malignant hepatocytes vary considerably in morphology. Poorly differentiated hepatocellular carcinomas show marked nuclear pleomorphism with tumor giant cells. The nuclei and nucleoli are prominent and the cytoplasm is often scant. In better differentiated forms, the malignant cells closely resemble benign hepatocytes with abundant granular cytoplasm, relatively bland nuclei, and form a relatively well-preserved trabecular

Fig. 8.11: Hepatocellular carcinoma with trabecular growth pattern (Hematoxylin and Eosin)

Fig. 8.12: Well-differentiated hepatocellular carcinoma with atypical cells containing abundant granular cytoplasm (Hematoxylin and Eosin)

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Fig. 8.13: Hepatocellular carcinoma with eosinophilic intracytoplasmic inclusion bodies (Hematoxylin and Eosin)

Fig. 8.14: Material aspirated from hepatocellular carcinoma is composed of large trabecular groups lying in a background of individual atypical hepatocytes and many “naked” nuclei (Diff-Quik)

pattern.45,47 Some hepatocellular carcinomas may contain Mallory hyaline or pale bodies (Fig. 8.13). Additional microscopic subtypes occur including a clear cell form48 a fibrolamellar variant (in which tumor lobules of oncocytic cells are separated by wide fibrous bands)49,50 and a small cell variant.51 Recognition of these variants is important as they may show prognostic differences.45,48-51

as accurate as CT-guided FNA and may represent a superior approach.54,55 The simultaneous examination of smears and cell block appears to improve diagnostic accuracy.56 While generally regarded as safe, FNA of hepatomas has been occasionally reported to be associated with needle tract implantation57 and an increased incidence of reoccurrence of hepatocellular carcinoma following transplantation.57,58

Cytomorphology FNA has been demonstrated to be an accurate technique for the diagnosis of hepatocellular carcinoma.9,52,53 Overall sensitivity and specificity range from 78% to 95% and 97% to 100%, respectively.9,52,53 The use of US guidance improved sensitivity from 78% to 85% in one series.53 EUS-guided FNA has been shown to be at least

Cytomorphologic Features

Fig. 8.15: Large numbers of “naked” nuclei characterize fine-needle aspiration samples of hepatocellular carcinoma (Diff-Quik)

Fig. 8.16: Cells of hepatocellular carcinoma often show considerable variation in nuclear/cytoplasmic ratio and cytoplasmic volume (Papanicolaou)

Smears of aspirated material from hepatocellular carcinomas demonstrate moderate-to-high cellularity in a bloody background. The malignant hepatic component presents as stripped nuclei, individual intact hepatocytes, as well as variably sized cell groups (Figs 8.14 and 8.15).59-65

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Fig. 8.17: Moderately differentiated hepatocellular carcinomas demonstrate significant variability in nuclear size (Papanicolaou)

Fig. 8.18: Cluster of cells from an example of hepatocellular carcinoma, demonstrating marked variability in nuclear and cell size (Diff-Quik)

The morphology of the individual malignant hepatocytes depends on the degree of tumor differentiation.66,67 Cytoplasmic volume and nuclear/cytoplasmic ratio can vary considerably (Figs 8.16 to 8.18).64 Characteristically, the cells resemble hepatocytes with a polygonal shape, eosinophilic granular vacuolated cytoplasm, and a centrally placed nucleus (Fig. 8.19).68 The nuclei are characterized by a granular or coarse chromatin and a large central nucleolus. Intranuclear pseudoinclusions (cytoplasmic invaginations) are frequently seen (Fig. 8.20).67,68 Isolated tumor giant cells are a common characteristic feature of many hepatocellular carcinomas.68,69 A characteristic feature supporting the diagnosis of hepatocellular

carcinoma is a large number of naked nuclei in the smear background (Fig. 8.21).62 In the majority of smears, the cell groups have a trabecular appearance most prominent in better differentiated tumors.68 Some hepatocellular carcinomas demonstrate an acinar or tubular arrangement in the cell groups that may lead to confusion with metastatic adenocarcinoma or primary cholangiocarcinoma. More poorly differentiated hepatocellular carcinomas are characterized by smaller cell groups and an increasing number of individual free-lying cells. Kulesza et al.69 recommended a twotier grading system based on cohesiveness of cell fragments, numbers of individual neoplastic cells, number of atypical naked nuclei, as well as the prominence of the nucleoli.69

Fig. 8.19: The cells of hepatocellular carcinoma retain the appearance of hepatocytes but with alterations in nuclear/cytoplasmic ratio and nuclear appearance (Diff-Quik)

Fig. 8.20: Some cells derived from hepatocellular carcinoma demonstrate intranuclear cytoplasmic pseudoinclusions (Diff-Quik)

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Fig. 8.21: Hepatocellular carcinoma aspirates are characterized by large numbers of “naked” nuclei (Diff-Quik)

Fig. 8.22: Trabecular groups of hepatocellular carcinomas are wrapped by endothelial cells (Diff-Quik)

An important diagnostic clue for the recognition of hepatocellular carcinoma is the endothelial cell relationship with the malignant cell groups (Fig. 8.22). Characteristically, endothelial cells of the sinusoidal capillaries enclose or transverse hepatocyte trabeculae or tumor cell groups (Figs 8.23 and 8.24).63 However, as differentiation decreases, this feature may be lost. Occasional hepatocellular carcinomas contain large hyaline cytoplasmic inclusions best seen in air-dried preparations.70 Intracytoplasmic bile pigment and evidence of bile between cells substantiate the hepatocellular differentiation of the cellular component (Figs 8.25 and 8.26).

A number of cytomorphologically distinct subtypes of hepatocellular carcinoma exist. Aspirates obtained from fibrolamellar hepatocellular carcinomas are characterized by marked cellularity and a dispersed population of cells with a few loose clusters of large polygonal cells.71 These large polygonal cells often have bizarre shapes and abundant dense eosinophilic granular cytoplasm, giving an “oncocytic” appearance. The individual cells have large vesicular nuclei and may be binucleated. Characteristically, the cells have a very large single nucleolus.71-75 Of diagnostic importance, the cells showing hepatocytic differentiation are dispersed among bundles of spindle-shaped fibroblasts and fragments of lamellar collagen.71,75 Cytomorphologically, a lipid-rich variant exists characterized by scattered bland hepatocytes with abundant vacuolated cytoplasm. In Papanicolaou-stained preparations, these cells have a strikingly uniform bland appearance. The cytoplasm contains both micro- and macrovesicular steatotic changes.76 Separation of this variant of hepatocellular carcinoma from benign fatty liver is difficult, but benign fatty lesions are more often characterized by rigid cores of hepatocytes and larger tissue fragments, while well-differentiated hepatocellular carcinoma is characterized by loose cell aggregates and increased numbers of single cells.77 The majority of hepatocellular carcinomas are characterized in FNAs preparations by trabeculae three or more cells thick wrapped by a peripheral endothelium (see Fig. 8.22).78 An infrequent subtype of well-differentiated

Fig. 8.23: Endothelial cells surround and transverse hepatocyte trabeculae in hepatocellular carcinomas (Diff-Quik)

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Fig. 8.24: Large group of cells from a hepatocellular carcinoma showing the characteristic relationship between malignant hepatocytes and endothelial cells (Diff-Quik)

Fig. 8.25: Globules of bile are often seen in groups of malignant hepatocytes (Diff-Quik)

hepatocellular carcinoma is characterized by a microacinar and microtrabecular pattern (Figs 8.27 and 8.28).78 This variety of well-differentiated hepatocellular carcinoma contains numerous small hepatocytes with minimal nuclear atypia and reduced amounts of cytoplasm. The cells are grouped in micro-acini of five or more cells and microtrabeculae one or more cells in thickness. A peripheral endothelium is not apparent in these groups. Some transgressing capillaries are present.78 These hepatocellular carcinomas may be mistaken for metastatic neuroendocrine carcinoma.78

•

Diagnostic Criteria

• •

•

Moderate-to-high cellular smears with a bloody background

Fig. 8.26: Bile plugs are seen in aspirates from many hepatocellular carcinomas (Diff-Quik)

• • • • •

Neoplastic cells lie singly and in variably sized groups with a trabecular, acinar, or sheetlike arrangement Endothelial cells surround or transverse cell groups Many “naked” nuclei present in background Cells are polygonal with central nuclei and variable amounts of cytoplasm Most cells have abundant granular or vacuolated cytoplasm Occasional hepatocellular carcinomas are dominated by lipid-rich cells Nuclei are round with large central nucleoli Intracytoplasmic hyaline (eosinophilic) inclusions present

Fig. 8.27: Some hepatocellular carcinomas have sheets of cells containing a micro-acinar or microtrabecular pattern (Papanicolaou)

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Fig. 8.28: Some well-differentiated hepatocellular carcinomas are characterized by tight trabecular groups (Papanicolaou)

• • •

Intracytoplasmic bile or extracellular bile plugs may be seen Biliary epithelium absent In fibrolamellar variant, bundles of fibroblasts and aggregates of collagen are seen

Differential Diagnosis Hepatocellular carcinomas must be distinguished from liver cell adenomas, regenerative nodules, and metastatic carcinoma. Well-differentiated hepatocellular carcinomas may be impossible to separate from hepatocellular adenomas. Both neoplasms lack biliary epithelium. The presence of endothelial lined trabeculae and large numbers of naked nuclei in the background favor hepatocellular carcinoma over adenoma. Moreover, a significantly increased nuclear/cytoplasmic ratio favors hepatocellular carcinoma. With higher grade hepatocellular carcinomas become more easily recognized due to nuclear membrane irregularities, the presence of atypical mitotic figures, and macronucleoli. Separation of well-differentiated hepatocellular carcinomas from benign regenerative nodules is a significant diagnostic pitfall. Separation of these entities is optimized, when a dedicated radiologist-cytopathologist team is available and a combined cytohistologic approach with immunohistochemistry and clinical pathologic correlation is instituted.77 Smears obtained from cirrhotic patients are characterized by material lacking mitotic figures, transgressing epithelium, eccentric nuclei, and cells with scant cytoplasm.79 The presence of thick nuclear membranes, spindle cells, and abundant thick and monotonous

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cytoplasm favors cirrhosis. Large nucleoli, multiple nucleoli, increased nuclear/cytoplasmic ratio, and broad cores are rarely found in regenerative nodules and favor the diagnosis of hepatocellular carcinoma.79 Tissue fragments of rigid tissue cores also favor a benign nodule (see Fig. 8.1).77 The size of the dysplastic cells aids in the separation of hepatocellular carcinoma from the dysplasia seen in cirrhosis. Small cell dysplasia correlates with the presence of hepatocellular carcinoma, while large cell dysplasia is more frequently found in cirrhosis.80 A major problem in differential diagnosis for hepatocellular carcinoma is its distinction from metastatic carcinoma.81 Separation of metastatic adenocarcinoma from hepatocellular carcinoma is aided by the recognition of a trabecular pattern with endothelial cell wrapping characteristic of hepatocellular carcinoma (see Figs 8.22 and 8.28). Hepatocellular carcinomas frequently have a more prominent population of atypical naked nuclei than is common in metastatic adenocarcinomas (see Fig. 8.21). Moreover, bile and the presence of hyaline globules favor the diagnosis of hepatocellular carcinoma (see Fig. 8.25). Metastatic adenocarcinomas more commonly demonstrate an acinar or glandular pattern, and have vacuolated cytoplasm and greater degrees of background necrotic debris. Mucin is often demonstrable by special stains in adenocarcinomas but is absent in most hepatocellular carcinomas. Metastatic adenocarcinomas are often CEA positive, while hepatocellular carcinomas are AFP positive. Immunohistochemistry can be a great aid in the separation of hepatocellular carcinomas from metastatic adenocarcinomas and renal cell carcinomas. The combination of Hep Par 1 and MOC-31 helps establish a hepatocytic differentiation.82 Stepwise logistic regression analysis has shown that a panel of glypican-3, Hep Par 1, MOC-31, and CK7 is most helpful in distinguishing hepatocellular carcinoma from metastatic adenocarcinoma (Fig. 8.29).83,84 Cholangiocarcinoma may be difficult to distinguish from some hepatocellular carcinomas. Unlike hepatocellular carcinomas, cholangiocarcinomas are characterized by columnar cells that form ductal or acinar structures (Figs 8.30A and B). Bile plugs are common. The cytoplasm may take on a squamoid appearance or appear as signet ring cells with mucin filled vacuoles. Mixed hepatocellular carcinomas and cholangiocarcinomas occur and contain both hepatocellular and adenocarcinomatous elements. Other poorly differentiated malignancies must be considered when working up multiple or even large solitary hepatic nodules. Melanoma and renal cell carcinoma represent difficult differential diagnosis. Melanomas

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Hepatoblastomas occur almost exclusively in infants and are associated with a number of congenital abnormalities including hemihypertrophy, Wilms tumors, and familial colonic polyposis.85

Hepatoblastomas are solid, well circumscribed solitary masses. Microscopic evaluation demonstrates immature hepatocytic elements in the pure form, and in mixed tumors a combination of immature hepatocytic elements and a stromal component is present. The stromal component may be undifferentiated or osteocartilaginous. Pure epithelial hepatoblastomas can be divided into fetal and embryonal subtypes. The fetal subtype is composed of two cell thick irregular laminae resembling the fetal liver (Fig. 8.31). In the embryonal form, the hepatocytic elements are more immature and the pattern of growth is generally solid, but rosettes, papillary formations, and ribbons may be seen (Fig. 8.32).86,87 Rare cases of transitional hepatoblastomas/hepatocellular carcinomas have been reported.85 Fetal and embryonal hepatoblastomas can be distinguished cytomorphologically but mixed forms occur.88 Smears obtained from fetal type hepatoblastomas are highly cellular and composed of cells resembling fetal hepatocytes (Figs 8.33 to 8.36). These cells may form acini (Fig. 8.36) with or without a trabecular arrangement. The fetal hepatocytes have a low nuclear/cytoplasmic ratio, a polygonal shape, and round or oval nuclei (Fig. 8.35). Nuclear chromatin is finely granular. Nucleoli are inconspicuous. The cytoplasm is abundant and granular. As with hepatocellular carcinomas, numerous traversing capillaries are seen within and around the tumor cell tissue fragments.

A

B

Fig. 8.29: Hepatocellular carcinomas are strongly reactive with antisera directed against glypican-3 (immunochemistry)

frequently show greater degrees of nuclear atypia, larger numbers of intranuclear cytoplasmic pseudoinclusions, and may contain melanin pigment. Renal cell carcinomas can closely mimic hepatocellular carcinomas due to their oncocytic or clear cell appearance. Immunohistochemistry is of great aid in separating these lesions.

Hepatoblastoma

Figs 8.30A and B: Cholangiocarcinoma smears are characterized by sheets and clusters of cuboidal to columnar cells with significant nuclear atypia (A—Papanicolaou, B—Diff-Quik)

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Fig. 8.31: Fetal-type hepatoblastoma with nests of immature hematopoietic cells (Hematoxylin and Eosin)

Fig. 8.32: Hepatoblastoma composed predominantly of small immature cells resembling embryonal liver (Hematoxylin and Eosin)

The embryonal form has a small cell appearance (Fig. 8.37) with a lesser number of cells resembling a fetal epithelial component.88,89 Rosette structures can occur (Figs 8.38 and 8.39). With increasing immaturity of the embryonal type, smears of hepatoblastoma may resemble other poorly differentiated small round cell tumors of childhood.90 Aspirated material from mixed hepatoblastomas contains both the fetal epithelial elements and mesenchymal elements. These mesenchymal elements appear as stromal

fragments, extensive acellular stromal matrix (osteoid/ chondroid), or well-formed clusters of benign spindle cells (Fig. 8.40).88 Extramedullary hematopoietic elements may be found in all three forms of hematoblastoma (see Fig. 8.31). Rare mixed hepatocellular carcinoma and hepatoblastoma tumors have been reported. Smears from these neoplasms contain a distinct hepatocarcinomatous element and groups of smaller more primitive cells consistent with the embryonal subtype of hepatoblastoma.91

Fig. 8.33: Sheet of immature-appearing cells obtained by fine needle aspiration from a hepatoblastoma (Diff-Quik)

Fig. 8.34: Trabecular aggregate of immature-appearing hepatocytes obtained from a hepatoblastoma (Diff-Quik)

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Fig. 8.35: Immature hepatocytes characteristic of a fetal pattern hepatoblastoma (Diff-Quik)

Fig. 8.36: Cell groups from some fetal pattern hepatoblastomas may form acinar structures (Diff-Quik)

Diagnostic Features Epithelial Type, Fetal Subtype

Epithelial Hepatoblastoma, Embryonal Subtype

• •

•

• • • • •

Moderate-to-high cellular smears Individual cells with low nuclear/cytoplasmic ratio and polygonal shape Granular cytoplasm Round to oval nuclei with finely granular chromatin Inconspicuous nucleolus Encompassing sinusoidal endothelial cells and traversing capillaries Stromal cells show extensive cytoplasmic vacuolization

Fig. 8.37: The cells of an embryonal pattern hepatoblastoma have a small primitive appearance (Diff-Quik)

• • • • • •

Cellular smears composed of small oval immatureappearing cells High nuclear/cytoplasmic ratio Round or oval nuclei Scanty cytoplasm Coarse hyperchromatic nuclear chromatin with prominent nucleoli Frequent mitotic figures Lesser number of fetal pattern epithelial cells

Fig. 8.38: Embryonal pattern hepatoblastoma may contain rosettelike structures (Diff-Quik)

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Fig. 8.39: Rosette-like and acinar structure can be found in aspirates from hepatoblastoma (Papanicolaou)

Fig. 8.40: Aspirates of mixed pattern hepatoblastomas often contain prominent stromal components (Diff-Quik)

Mixed-type Hepatoblastoma

surrounding and intermixed with elongated branching bile duct structures. Cytologically, smears are of low-to-moderate cellularity and contain a mixture of individual epithelial cells, sheets of epithelial cells, and groups of spindle-shaped mesenchymal cells. Often fragments of a poorly vascularized myxoid/myxofibroid stroma are seen.93,94

• •

Epithelial cell component resembling fetal or embryonal elements Mesenchymal elements composed of stromal fragments, acellular eosinophilic matrix, and bundles or fascicles of spindle-shaped cells

Differential Diagnosis

Differential Diagnosis

Hepatoblastomas must be separated from hepatocellular carcinomas. Hepatocellular carcinomas show greater degrees of nuclear pleomorphism than seen in hepatoblastoma. Giant cells may be seen in hepatocellular carcinomas but are absent in hepatoblastomas. Hepatocellular carcinomas have greater numbers of atypical naked nuclei. Hepatoblastomas must be separated from other small round cell malignancies of childhood. Immunohistochemical findings are of great aid in this differential diagnosis. Please refer to Chapter 13 for further discussion of round cell malignancies of childhood.

Mesenchymal hamartomas have a distinct cytologic and histologic appearance separating them from metastatic disease, hepatocellular carcinomas, and hepatoblastomas. Age of presentation of mesenchymal hamartomas (infants) rules out the majority of myxoid sarcomas occurring in adults. The nonhepatocytic but epithelial appearance of the cells excludes hepatocellular carcinoma. The prominence of the mesenchymal component aids in exclusion of cholangiocarcinoma as does patient age.

Mesenchymal Hamartoma

Cholangiocarcinomas arise from intrahepatic bile ducts and may be associated with Thorotrast exposure,95 anabolic steroid use,96 and some congenital abnormalities of the bile duct.97 The majority of patients are over the age of 60 years and present with abdominal pain and weight loss. Grossly, these tumors are firm and white to whitetan with variable amounts of fibrous stroma. The majority arises close to the hepatic hilum.

Mesenchymal hamartomas are rare benign lesions occurring predominantly in infants.92 Grossly, they appear as solitary nearly spherical reddish nodules. While often asymptomatic, others present with abdominal swelling or as a palpable mass. Histopathologically, they are characterized by a well-vascularized connective tissue

Cholangiocarcinoma

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Microscopically, the carcinoma produces duct-like structures lined by cuboidal or columnar cells of varying degrees of differentiation (Fig. 8.41). These ductlike structures infiltrate and are surrounded by a fibrous response. Occasionally, a cribriform pattern of epithelium may be present. Cell morphology may vary considerably within a given epithelial structure. At the periphery of the neoplasm, the duct-like structures can spread between hepatocytes and along duct walls. Perineural invasion is common. Stains for mucin are generally positive. Some cases show extensive mucin production with either signet ring cells or broad expanses of mucin (mucinous cholangiocarcinoma).98 Modifications of this basic pattern can occur with a squamous, clear cell, or papillary component being present. Cholangiocarcinomas are consistently reactive with antibodies directed against keratin, EMA, and CEA. Characteristically, cholangiocarcinomas are reactive with antibodies directed against CK7 and CK19. The steaming pattern for CEA is not canalicular with polyclonal antisera, but is cytoplasmic and luminal.

Cytomorphology Smears derived from cholangiocarcinomas are characterized by smears of low-to-moderate cellularity containing individual and small clusters of columnar or cuboidal cells (Figs 8.30A and B and 8.42).99,100 These cells may form acinar structures or strips of epithelial cells. The cells may

Fig. 8.41: Cholangiocarcinoma with sclerotic background and tubular structures composed of cuboidal to low columnar cells (Hematoxylin and Eosin)

have a signet ring morphology or even a squamoid appearance. The cell strips demonstrate loss of nuclear polarity. Nuclear membranes are irregular, and nuclear chromatin is often coarse with variably sized nucleoli. Unlike hepatocellular carcinomas, cholangiocarcinomas invariably show ductular proliferation.99 Ten or more ductular clusters per smear are characteristic of cholangiocarcinoma and aid in discrimination of cholangiocarcinomas from metastatic adenocarcinomas.

Diagnostic Features • • • • • • •

Smears of low-to-moderate cellularity containing individual, strips, and duct-like cell aggregates Bile aggregates present Cells may have a signet ring cell or squamoid appearance Microacinar formation Irregular nuclear membranes Loss of nuclear polarity Sclerotic stromal fragments

Undifferentiated (Embryonal) Sarcoma Embryonal sarcomas are extremely rare malignancies occurring in children between 5 and 10 years of age.101 Most present with either an abdominal mass or pain. These malignancies present as large soft masses with both cystic and solid areas. Frequently a white or gelatinous mucoid appearance is present. Areas of necrosis and hemorrhage are common.

Fig. 8.42: Aspirates of cholangiocarcinoma contain cell groups and sheets composed of cuboidal or columnar cells (Diff-Quik)

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Embryonal sarcomas appear well circumscribed but infiltrate beyond the pseudocapsule into the liver. These neoplasms are a mixture of spindle-shaped and stellate cells in a myxoid stroma. Characteristically, the spindleshaped cells contain PAS-positive, diastase-resistant cytoplasmic globules. The nuclei are large and atypical and demonstrate marked hyperchromasia. Multinucleated tumor cells are common and the mitotic index is high. Interestingly, extramedullary hematopoiesis is common. Cytomorphologically, smears are characterized by oval to spindle-shaped cells in a myxoid background.102,103 The malignant cells lie both singly and in clusters. The cell population is a biomorphic and is composed of large cells with bizarre pleomorphic nuclei along with clusters of oval to spindle-shaped cells, demonstrating moderate nuclear atypia. Nucleoli are frequently prominent, and the chromatin is coarse in both cell types. Atypical mitotic figures, nuclear pyknosis, and debris are common.102,103

Hemangioma Hemangiomas are the most common benign neoplasm of the liver. They are generally found incidentally but may be associated with spontaneous hemorrhage or thrombocytopenic purpura. These neoplasms have both a characteristic imaging appearance and gross appearance that is spongy dark red. Microscopically, most hepatic hemangiomas are of cavernous type. Widely dilated nonanastamosing vascular spaces are lined by flat endothelial cells scattered among a fibrous tissue stroma. Cytomorphologically, smears are characterized

Fig. 8.43: Aspirates of hepatic hemangiomas have a background rich in red blood cells. Rare small groups of bland short spindle-shaped cells may be found and represent endothelial cells (Diff-Quik)

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by abundant blood in which there are scattered small numbers of short single spindle-shaped cells and rare cell clusters composed of similar cells (Fig. 8.43). The cell clusters are often tightly cohesive. Large fragments of fibrous tissue may be present (Fig. 8.44).

Angiosarcoma Angiosarcomas of the liver are high-grade lesions and may be related to Thorotrast usage. Cytologically, the tumor cells are large and often polygonal (Fig. 8.45), with large nuclei containing huge nucleoli. The cytoplasm is abundant with a micro/macrovesicular appearance.104-106 Some cells have very large intracytoplasmic vacuoles, giving them a signet ring appearance.106 In other cases, the cells are spindle shaped with large hyperchromatic nuclei and huge nucleoli (Fig. 8.46).

Angiomyolipoma Angiomyolipomas occasionally occur within the liver. Like their renal counterpart, they are composed of thickwalled blood vessels, smooth muscle, and adipose tissue. Some have a predominantly epithelioid appearance. The neoplasms are immunoreactive for actin, desmin, S100 protein, and HMB45. The latter immunoreactivity is diagnostically important. Cytomorphologically, these neoplasms are characterized by cellular smears lacking necrosis and inflammation. Small multilayered clusters of spindle and polygonal cells are found (Fig. 8.47). Many of the cells within the clusters

Fig. 8.44: Aspirates of hemangiomas may contain rare large stromal fragments (Diff-Quik)

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Fig. 8.45: Aspirates from angiosarcomas often contain clusters of atypical round to oval cells lining a stromal meshwork (Diff-Quik)

Fig. 8.46: Some angiosarcomas are characterized by large spindleshaped cells with hyperchromatic nuclei and huge nucleoli (Diff-Quik)

have an epithelioid appearance, while others are spindle shaped (Fig. 8.48). The nuclei appear benign or elongated and have no or indistinct nucleoli. Adipose tissue is relatively infrequent and is usually mixed with the spindle cell component (Fig. 8.49).

Metastatic carcinoma is the most common malignancy involving the liver. Carcinomas arising in the colon, lung, breast, pancreas, kidney, stomach, and gall bladder frequently metastasize to the liver. Direct extension from the gall bladder and extrahepatic bile ducts is

common. Malignant melanoma and many sarcomas of the soft tissue can also give rise to hepatic metastases. Separation of metastatic adenocarcinomas and renal cell carcinomas from primary hepatocellular carcinoma and cholangiocarcinoma is diagnostically challenging. Immunohistochemistry is of substantial help in differential diagnosis.82-84 A diagnostic panel glypican-3, Hep Par 1, MOC-31, and CK7 appears most helpful.83 In addition to immunohistochemical findings, cytomorphologic features on smear and cell block preparations are helpful. The presence of endothelial cells surrounding cell groups along with transgressing capillaries is strongly suggestive

Fig. 8.47: Fine-needle aspirations from angiomyolipomas contain thick aggregates of stroma and spindle cells with attached vessel fragments (Hematoxylin and Eosin)

Fig. 8.48: Angiomyolipomas produce smears with thick trabecular groups of spindle cells and small vessel fragments (Hematoxylin and Eosin)

Metastatic Disease to the Liver

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Fig. 8.49: While uncommon in aspirates, some smears will contain prominent collections of adipose tissue (Hematoxylin and Eosin)

Fig. 8.50: Aspirates of metastatic colonic adenocarcinomas are characterized by sheets of malignant epithelial cells in a necrotic background (Papanicolaou)

of hepatocellular carcinoma. A large number of atypical nuclei devoid of cytoplasm also favors hepatocellular carcinoma. The presence of mucin, signet ring-shaped cells, abundant necrosis, and columnar shaped cells favors metastatic carcinoma. Staining for S100 protein, melan-A, and HMB45 aids in the separation of melanoma from hepatocellular carcinoma and cholangiocarcinoma. Imaging studies are often of little help because hepatocellular carcinomas may present as multiple nodules similar to those found with widely metastatic disease. Cytomorphologically, colon carcinomas are characterized by clusters of atypical columnar cells often lying in a

necrotic background (Figs 8.50 and 8.51). The individual cells have large oval nuclei with prominent cytoplasm (Figs 8.52 and 8.53). Small gland-like or acinar spaces are present in some cell groups (Fig. 8.53). Smears of melanin can show several patterns including plasmacytoid (Fig. 8.54), anaplastic giant cell (Fig. 8.55), and spindle cell (Fig. 8.56) forms. Metastatic carcinoid tumors are characterized by individual and groups of relatively small bland cells (Fig. 8.57). The nuclei have a characteristic “salt and pepper” chromatin pattern. Immunocytochemical staining will reveal the neoplastic cells to be reactive for neuroendocrine markers (Fig. 8.58).

Fig. 8.51: Sheets of cell derived from colonic adenocarcinoma often have palisades of columnar cells along their edges (Papanicolaou)

Fig. 8.52: The individual cells aspirated from metastatic colonic adenocarcinomas have large nuclei with prominent nucleoli (Papanicolaou)

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Fig. 8.53: Clusters of malignant cells obtained from colonic carcinoma may contain small acinar structures (Diff-Quik)

Fig. 8.54: Aspirated cells from metastatic malignant melanoma may have a plasmacytoid appearance (Diff-Quik)

Fig. 8.55: Anaplastic giant cells characterize some aspirates from malignant melanoma (Diff-Quik)

Fig. 8.56: A subset of metastatic melanoma may have a spindle-cell morphology (Diff-Quik)

Fig. 8.57: Aspirates of metastatic carcinoid tumors contain groups of moderate size to small cells with nuclei showing “salt and pepper” chromatin pattern (Papanicolaou)

Fig. 8.58: Strong positive staining for synaptophysin is characteristic of metastatic carcinoid tumors (immunoperoxidase staining)

Liver



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1. Goodman ZD, Ishak KG. Medical diseases of the liver. In: Silverberg SG, DeLellis RA, Frable WJ, LiVolsi VA, Wick MR, editors. Silverberg’s principles and practice of surgical pathology and cytopathology. 4th ed. Philadelphia: Churchill Livingstone/Elsevier; 2006. P. 1503-8. 2. Geller SA, Petrovic LM. Biopsy interpretation of the liver. Philadelphia: Lippincott Williams and Wilkins; 2004. P. 50-170. 3. Geller SA, Petrovic LM. Biopsy interpretation of the liver. Philadelphia: Lippincott Williams and Wilkins; 2004. P. 111-121. 4. Goodman ZD, Ishak KG. Medical diseases of the liver. In: Silverberg SG, DeLellis RA, Frable WJ, LiVolsi VA, Wick MR, editors. Silverberg’s principles and practice of surgical pathology and cytopathology. 4th ed. Philadelphia: Churchill Livingstone/Elsevier; 2006. P. 1489-92. 5. Parkin DM, Muir CS, Whelan SL, et al. Cancer incidence in five continents. Vol VI. Oxford: Oxford University Press; 1992. P. 882-3. 6. Kudo M. Diagnostic imaging of hepatocellular carcinoma: recent progress. Oncology. 2011;81 Suppl 1:73-85. 7. Wee A. Fine needle aspiration biopsy of hepatocellular carcinoma and related hepatocellular nodular lesions in cirrhosis: controversies, challenges and expectations. Pathol Res Int. 2011;2011, article ID 587936, 17 pages, doi: 10.4061/2011/587936. 8. Hertz G, Reddy VB, Green L, et al. Fine-needle aspiration biopsy of the liver: a multicenter study of 602 radiologically guided FNA. Diagn Cytopathol. 2000;23(5):326-8. 9. Kuo FY, Chen WJ, Lu SN, et al. Fine needle aspiration cytodiagnosis of liver tumors. Acta Cytol. 2004;48(2):142-8. 10. Wang P, Meng ZQ, Chen Z, et al. Diagnostic value and complications of fine needle aspiration for primary liver cancer and its influence on the treatment outcome—a study based on 3011 patients in China. Eur J Surg Oncol. 2008;34(5):541-6. Epub 2007 Aug 30. 11. Maheshwari A, Kantsevoy S, Jagannath S, et al. Endoscopic ultrasound and fine-needle aspiration for the diagnosis of hepatocellular carcinoma. Clin Liver Dis. 2010;14(2):32532. doi: 10.1016/j.cld.2010.03.014. 12. Torzilli g, Minagawa M, Takayama T, et al. Accurate preoperative valuation of liver mass lesions without fine-needle biopsy. Hepatology. 1999;30(4):889-93. 13. Bruix J, Sherman M, Llovet JM, et al. Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL Conference. European Association for the Study of the Liver. J Hepatol. 2001;35(3):421-30. 14. Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology. 2005;42(5):1208-36. 15. Hertzanu Y, Peiser J, Zirkin H. Massive bleeding after fine needle aspiration of liver angiosarcoma. Gastrointest Radiol. 1990;15:43-6. 16. Riska H, Friman C. Letter: fatality after fine-needle aspiration biopsy of liver. Br Med J. 1975;1(5956): 517.

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17. Shariff S, Thomas JA, Kaliaperumal VG. An experience with ultrasonically guided liver aspirates from south India. Cytopathology. 1993;4(5):291-8. 18. Lundquist A. Liver biopsy with a needle of 0.7 MM outer diameter. Safety and quantitative yield. Acta Med Scand. 1970;188(6):471-4. 19. Onodera H, Oikawa M, Abe M, et al. Cutaneous seeding of hepatocellular carcinoma after fine-needle aspiration biopsy. J Ultrasound Med. 1987;6(5):273-5. 20. Malberger E, Edoute Y, Nagler A. Rare complications after transabdominal fine needle aspiration. Am J Gastroenterol. 1984;79(6):458-60. 21. Caturelli E, Rapaccini GL, Sabelli C, et al. Ultrasoundguided fine-needle aspiration biopsy in the diagnosis of hepatic hemangioma. Liver. 1986;6:326-30. 22. Nakaizumi A, Iishi H, Yamamoto R, et al. Diagnosis of hepatic cavernous hemangioma by fine needle aspiration biopsy under ultrasonic guidance. Gastrointest Radiol. 1990;15(1):39-42. 23. Das DK, Bhambani S, Pant CS. Ultrasound guided fineneedle aspiration cytology: diagnosis of hydatid disease of the abdomen and thorax. Diagn Cytopathol. 1995;12:173-6. 24. Pogacnik A, Pohar-Marinsek Z, Us-Krasovec M. Fine needle aspiration biopsy in the diagnosis of liver echinococcosis. Acta Cytol. 1990;34(5):765-6. 25. Wee A, Nilsson B, Yap I, et al. Aspiration cytology of liver abscesses, with an emphasis on diagnostic pitfalls. Acta Cytol. 1995;39:453-62. 26. Bhambhani S, Kashyap V. Amoebiasis: diagnosis by aspiration and exfoliative cytology. Cytopathology. 2001; 12(5):329-33. 27. Zaman SS, Langer JE, Gupta PK. Ciliated hepatic foregut cyst. Report of a case with findings on fine needle aspiration. Acta Cytol. 1995;39:781-4. 28. Hornstein A, Batts KP, Linz LJ, et al. Fine needle aspiration diagnosis of ciliated hepatic foregut cysts. A report of three cases. Act Cytol. 1996;40:576-80. 29. Parwani AV, Burroughs FH, Ali SZ. Echinococcal cyst of the liver. Diagn Cytopathol. 2004;31(2):111-2. 30. Firpi RJ, Lozada LR, Torres EA, et al. Fine-needle aspiration diagnosis of hydatid cyst. P R Health Sci J. 1999;18(2):129-31. 31. Zeppa P, Anniciello A, Vetrani A, et al. Fine needle aspiration biopsy of hepatic focal fatty change. A report of two cases. Acta Cytol. 2002;46(3):567-70. 32. Layfield LJ. Focal fatty change of the liver: cytologic findings in a radiographic mimic of metastases. Diagn Cytopathol. 1994;11(4):385-7; discussion 387-9. 33. Dardi LE, Marzano M, Froula E. Fine needle aspiration cytologic diagnosis of focal intrahepatic extramedullary hepatopoiesis. Acta Cytol. 1990;34:567-9. 34. Lemos LB, Baliga M, Benghuzzi HA, et al. Nodular hematopoiesis of the liver diagnosed by fine-needle aspiration cytology. Diagn Cytopathol. 1997;16:51-4. 35. Nguyen BN, Fléjou JF, Terris B, et al. Focal nodular hyperplasia of the liver: a comprehensive pathologic study of 305 lesions and recognition of new histologic forms. Am J Surg Pathol. 1999;23(12):1441-54.

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36. Stocker JT, Ishak KG. Focal nodular hyperplasia of the liver: a study of 21 pediatric cases. Cancer. 198115;48(2): 336-45. 37. Baum JK, Bookstein JJ, Holtz F, et al. Possible association between benign hepatomas and oral contraceptives. Lancet. 197327;2(7835):926-9. 38. Suen KC. Diagnosis of primary hepatic neoplasms by fineneedle aspiration cytology. Diagn Cytopathol. 1986;2(2): 99-109. 39. Ruschenburg I, Droese M. Fine needle aspiration cytology of focal nodular hyperplasia of the liver. Acta Cytol. 1989;33(6):857-60. 40. Kong CS, Appenzeller M, Ferrell LD. Utility of CD34 reactivity in evaluating focal nodular hepatocellular lesions sampled by fine needle aspiration biopsy. Acta Cytol. 2000;44(2):218-22. 41. Edmondson HA, Henderson B, Benton B. Liver-cell adenomas associated with use of oral contraceptives. N Engl J Med. 1976;294(9):470-2. 42. Shortell CK, Schwartz SI. Hepatic adenoma and focal nodular hyperplasia. Surg Gynecol Obstet. 1991;173(5):426-31. 43. Ameriks JA, Thompson NW, Frey CF, et al. Hepatic cell adenomas, spontaneous liver rupture, and oral contraceptives. Arch Surg. 1975;110(5):548-5. 44. Anthony PP. Hepatocellular carcinoma: an overview. Histopathology. 2001;39(2):109-18. 45. Chlebowski RT, Tong M, Weissman J, et al. Hepatocellular carcinoma. Diagnostic and prognostic features in North American patients. Cancer. 198415;53(12):2701-6. 46. Kondo Y. Histologic features of hepatocellular carcinoma and allied disorders. Pathol Annu. 1985;20 Pt 2:405-30. 47. Komatsu T, Kondo Y, Yamamoto Y, et al. Hepatocellular carcinoma presenting well differentiated, normotrabecular patterns in peripheral or metastatic loci. Analysis of 103 resected cases. Acta Pathol Jpn. 1990;40(12):887-93. 48. Wu PC, Lai CL, Lam KC, et al. Clear cell carcinoma of liver. An ultrastructural study. Cancer. 19831;52(3):504-7. 49. Berman MM, Libbey NP, Foster JH. Hepatocellular carcinoma. Polygonal cell type with fibrous stroma—an atypical variant with a favorable prognosis. Cancer. 1980; 46(6): 1448-55. 50. Goodman ZD, Ishak KG, Langloss JM, et al. Combined hepatocellular-cholangiocarcinoma. A histologic and immunohistochemical study. Cancer. 1985;55(1):124-35. 51. Zanconati F, Falconieri G, Lamovec J, et al. Small cell carcinoma of the liver: a hitherto unreported variant of hepatocellular carcinoma. Histopathology. 1996; 29(5):449-53. 52. Nazir RT, Sharif MA, Iqbal M, et al. Diagnostic accuracy of fine needle aspiration cytology in hepatic tumours. J Coll Physicians Surg Pak. 2010;20(6):373-6. 53. Crowe DR, Eloubeidi MA, Chhieng DC, et al. Fine-needle aspiration biopsy of hepatic lesions: computerized tomographic-guided versus endoscopic ultrasound-guided FNA. Cancer. 2006;108(3):180-5. 54. Prachayakul V, Aswakul P, Kachintorn U. EUS guided fine needle aspiration cytology of liver nodules suspicious for malignancy: yields, complications and impact on management. J Med Assoc Thai. 2012;95 Suppl 2:S56-60.

55. Khurana U, Handa U, Mohan H, et al. Evaluation of aspiration cytology of the liver space occupying lesions by simultaneous examination of smears and cell blocks. Diagn Cytopathol. 2009;37(8):557-63. 56. Liu YW, Chen CL, Chen YS, et al. Needle tract implantation of hepatocellular carcinoma after fine needle biopsy. Dig Dis Sci. 2007;52(1):228-31. 57. Saborido BP, Díaz JC, de Los Galanes SJ, et al. Does preoperative fine needle aspiration-biopsy produce tumor recurrence in patients following liver transplantation for hepatocellular carcinoma? Transplant Proc. 2005; 37(9):3874-7. 58. Pavan C, Parisi A, Girelli D. Recurrent needle-tract metastases of hepatocellular carcinoma following fine-needle aspiration. Intern Med J. 2007;37(2):134-6. 59. Bottles K, Cohen MB, Holly EM, et al. A step-wise logistic regression analysis of hepatocellular carcinoma. An aspiration biopsy study. Cancer. 1988;62:558-63. 60. Bottles K, Cohen MB. An approach to fine-needle aspiration biopsy diagnosis of hepatic masses. Diagn Cytopathol. 1991;7:204-10. 61. Pedio G, Landolt U, Zöbeli L, et al. Fine needle aspiration of the liver. Significance of hepatocytic naked nuclei in the diagnosis of hepatocellular carcinoma. Acta Cytol. 1988;32(4):437-42. 62. Pitman MB, Szyfelbein WM. Significance of endothelium in the fine-needle aspiration biopsy diagnosis of hepatocellular carcinoma. Diagn Cytopathol. 1995;12(3):208-14. 63. Sole M, Calvet X, Cuberes T, et al. Value and limitations of cytologic criteria for diagnosis of hepatocellular carcinoma by fine needle aspiration biopsy. Acta Cytol. 1993;37:309-16. 64. Wee A, Nilsson B, Chan-Wilde C, et al. Fine needle aspiration biopsy of hepatocellular carcinoma. Some unusual features. Acta Cytol. 1991;35:661-70. 65. Ali MA, Akhtar M, Mattingly RC. Morphologic spectrum of hepatocellular carcinoma in fine needle aspiration biopsies. Acta Cytol. 1986;30:294-302. 66. Pisharodi LR, Lavoie R, Bedrossian CWM. Differential diagnostic dilemmas in malignant fine needle aspiration of liver: a practical approach to final diagnosis. Diagn Cytopathol. 1995;12:364-71. 67. Soyuer I, Ekinci C, Kaya M, et al. Diagnosis of hepatocellular carcinoma by fine needle aspiration cytology. Cellular features. Acta Cytol. 2003;47(4):581-9. 68. Wee A, Nilsson B, Tan LKA, et al. Fine needle aspiration biopsy of hepatocellular carcinoma. Diagnostic dilemma at the ends of the spectrum. Acta Cytol. 1994;38:347-54. 69. Kulesza P, Torbenson M, Sheth S, et al. Cytopathologic grading of hepatocellular carcinoma on fine-needle aspiration. Cancer. 2004;102(4):247-58. 70. MacDonald K, Bedard YC. Cytologic, ultrastructural and immunologic features of intracytoplasmic hyaline bodies in fine needle aspirates of hepatocellular carcinoma. Acta Cytol. 1990;34(2):197-200. 71. Gulati G, Saran RK. Fine needle aspiration cytology of fibrolamellar hepatocellular carcinoma: recognizing the oncocytic hepatocyte. Indian J Pathol Microbiol. 2009; 52(2):288-9.

Liver 72. Montero A, Allende H, Tallada N, et al. Fine needle aspiration cytology of massive bilateral ovarian metastasis of fibrolamellar hepatocellular carcinoma. Acta Cytol. 2007;51(4):682-3. 73. Kaplan JS, Hoda RS. Clear cell variant of fibrolamellar hepatocellular carcinoma: diagnosis of recurrence by fineneedle aspiration. Diagn Cytopathol. 2007;35(7):459-62. 74. Crowe A, Knight CS, Jhala D, et al. Diagnosis of metastatic fibrolamellar hepatocellular carcinoma by endoscopic ultrasound-guided fine needle aspiration. Cytojournal. 2011;8:2. 75. Davenport RD. Cytologic diagnosis of fibrolamellar carcinoma of the liver by fine needle aspiration. Diagn Cytopathol. 1990;6:275-9. 76. Mitchell CM, Sturgis CD. Lipid-rich hepatocellular carcinoma in fine-needle aspiration biopsy. Diagn Cytopathol. 2009;37(1):36-7. 77. Yang GC, Yang GY, Tao LC. Distinguishing well-differentiated hepatocellular carcinoma from benign liver by the physical features of fine-needle aspirates. Mod Pathol. 2004;17(7):798-802. 78. Yang GC, Yang GY, Tao LC. Cytologic features and histologic correlations of microacinar and microtrabecular types of well-differentiated hepatocellular carcinoma in fineneedle aspiration biopsy. Cancer. 2004;102(1):27-33. 79. Geramizadeh B, Asadi N, Tabei SZ. Cytologic comparison between malignant and regenerative nodules in the background of cirrhosis. Hepat Mon. 2012;12(7): 448-52. 80. Hill KA, Nayar R, DeFrias DV. Cytohistologic correlation of cirrhosis and hepatocellular carcinoma. Pitfall in diagnosis? Acta Cytol. 2004;48(2):127-32. 81. Renshaw AA, Haja J, Wilbur DC, et al. Cytology Committee, College of American Pathologists. Fine-needle aspirates of adenocarcinoma/metastatic carcinoma that resemble hepatocellular carcinoma: correlating cytologic features and performance in the College of American Pathologists Nongynecologic Cytology Program. Arch Pathol Lab Med. 2005;129(10):1217-21. 82. Kakar S, Gown AM, Goodman ZD, et al. Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus metastatic neoplasms. Arch Pathol Lab Med. 2007;131(11):1648-54. 83. Saleh HA, Aulicino M, Zaidi SY, et al. Discriminating hepatocellular carcinoma from metastatic carcinoma on fine-needle aspiration biopsy of the liver: the utility of immunocytochemical panel. Diagn Cytopathol. 2009; 37(3):184-90. 84. Timek DT, Shi J, Liu H, et al. Arginase-1, HepPar-1, and Glypican-3 are the most effective panel of markers in distinguishing hepatocellular carcinoma from metastatic tumor on fine-needle aspiration specimens. Am J Clin Pathol. 2012;138(2):203-10. 85. Giardiello FM, Offerhaus GJ, Krush AJ, et al. Risk of hepatoblastoma in familial adenomatous polyposis. J Pediatr. 1991;119(5):766-8. 86. Schmidt D, Harms D, Lang W. Primary malignant hepatic tumours in childhood. Virchows Arch A Pathol Anat Histopathol. 1985;407(4):387-405. 87. Weinberg AG, Finegold MJ. Primary hepatic tumors of childhood. Hum Pathol. 1983;14(6):512-37.

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88. Barwad A, Gupta N, Gupta K, et al. Hepatoblastoma—an attempt of histological subtyping on fine-needle aspiration material. Diagn Cytopathol. 2013;41(2):95-101. 89. Parikh B, Jojo A, Shah B, et al. Fine needle aspiration cytology of hepatoblastoma: a study of 20 cases. Indian J Pathol Microbiol. 2005;48(3):331-6. 90. Philipose TR, Naik R, Rai S. Cytologic diagnosis of small cell anaplastic hepatoblastoma: a case report. Acta Cytol. 2006;50(2):205-7. 91. Canberk S, Uludokumaci A, Sonmez C, et al. Mixed hepatocellular carcinoma and hepatoblastoma: cytohistopathologic findings and differential diagnosis. Acta Cytol. 2013; 57(1):91-5. 92. Lack EE. Mesenchymal hamartoma of the liver. A clinical and pathologic study of nine cases. Am J Pediatr Hematol Oncol. 1986;8(2):91-8. 93. al-Rikabi AC, Buckai A, al-Sumayer S, et al. Fine needle aspiration cytology of mesenchymal hamartoma of the liver. A case report. Acta Cytol. 2000;44(3):449-53. 94. Jiménez-Heffernan JA, Vicandi B, López-Ferrer P, et al. Fine-needle aspiration cytology of mesenchymal hamartoma of the liver. Diagn Cytopathol. 2000;22(4):250-3. 95. Rubel LR, Ishak KG. Thorotrast-associated cholangiocarcinoma: an epidemiologic and clinicopathologic study. Cancer. 1982;50(7):1408-15. 96. Stromeyer FW, Smith DH, Ishak KG. Anabolic steroid therapy and intrahepatic cholangiocarcinoma. Cancer. 1979;43(2):440-3. 97. Gallagher PJ, Millis RR, Mitchinson MJ. Congenital dilatation of the intrahepatic bile ducts with cholangiocarcinoma. J Clin Pathol. 1972;25(9): 804–8. 98. Chou ST, Chan CW, Ng WL. Mucin histochemistry of human cholangiocarcinoma. J Pathol. 1976;118(3):165-70. 99. Sampatanukul P, Leong AS, Kosolbhand P, et al. Proliferating ductules are a diagnostic discriminator for intrahepatic cholangiocarcinoma in FNA biopsies. Diagn Cytopathol. 2000;22(6):359-63. 100. Fritscher-Ravens A, Broering DC, Sriram PV, et al. EUSguided fine-needle aspiration cytodiagnosis of hilar cholangiocarcinoma: a case series. Gastrointest Endosc. 2000; 52(4):534-40. 101. Stocker JT, Ishak KG. Undifferentiated (embryonal) sarcoma of the liver: report of 31 cases. Cancer. 1978;42(1): 336-48. 102. Kaur J, Dey P, Das A. Fine needle aspiration cytology of undifferentiated (embryonal) sarcoma of liver in an adult male. Diagn Cytopathol. 2010;38(7):547-8. 103. Gupta C, Iyer VK, Kaushal S, et al. Fine needle aspiration cytology of undifferentiated embryonal sarcoma of the liver. Cytopathology. 2010;21(6):414-6. 104. Wong JW, Bedard YC. Fine-needle aspiration biopsy of hepatic angiosarcoma: report of a case with immunocytochemical findings. Diagn Cytopathol. 1992;8(4):380-3. 105. Saleh HA, Tao LC. Hepatic angiosarcoma: aspiration biopsy cytology and immunocytochemical contribution. Diagn Cytopathol. 1998;18(3):208-11. 106. Cho NH, Lee KG, Jeong MG. Cytologic evaluation of primary malignant vascular tumors of the liver. One case each of angiosarcoma and epithelioid hemangioendothelioma. Acta Cytol. 1997;41(5):1468-76.

Chapter

9

Pancreas Cytology

Martha Bishop Pitman, Barbara Centeno



INTRODUCTION

Cytologic specimens are obtained from the pancreas by fine-needle aspiration biopsy (FNAB) of masses, by brushing the common bile duct or main pancreatic duct, or by aspirating contents of the main pancreatic duct. 

SPECIMEN PREPARATION AND TISSUE TRIAGE

The aspirated tissue from any lesion whether solid or cystic can be prepared in a number of different ways for cytologic analysis: direct smears, cytospins (Thermo-Shandon Instruments), liquid-based preparations—ThinPrep (Cytyc Corporation, Marlborough, MA) or SurePath Prep (TriPath, Inc., Burlington, NC), and cell blocks. Direct smears are recommended for tissue that is solid enough which it will make a typical, adequate smear. Fluid aspirated from a cyst is best maintained fresh and processed as a cytospin, so that all of the cellular and extracellular elements of the cyst fluid can be appreciated. Liquid-based preparations eliminate obscuring blood and inflammation and provide excellent cellular preservation, so this technique is recommended for brushing specimens that typically suffer from mechanical artifact caused during the direct smearing process. Having the cells in a liquid medium also makes the residual fluid readily available for ancillary testing such as fluorescent in situ hybridization (FISH). Liquid-based cytology (LBC) preparations are not recommended for cyst fluids, since fresh cyst fluid is required

for biochemical and molecular analysis. In addition, LBC preparation attenuates background mucin, making identification on routine stains challenging. Aspirates of solid masses rinsed in Roswell Park Memorial Institute Media (RPMI) can be sent for flow cytometry analysis, and needle rinsings enriched with dedicated FNA aspirates along with any small core biopsy placed in balanced saline, RPMI, or directly expressed into small bone marrow-sized formalin containing tubs provide tissue for cell block preparations. Cell block preparations provide tissue that may not only give a little architectural help in reaching a diagnosis but, most importantly, also provide tissue for ancillary testing. This is especially important for the solid cellular tumors (see Page 183) since this group of tumors often overlap in their cytologic features. Of the various techniques available, the colloidin bag technique provides a means of collecting all of the tissue through a filter mechanism that is embedded directly into paraffin.1

Rapid on-site Interpretation Rapid on-site interpretation (ROSE) is a time-consuming exercise that provides value when additional sampling of the lesion continues from a nondiagnostic immediate read of the sample. This is most valuable for solid mass lesions and complex cystic lesions with a solid component, but offers little to no benefit for FNA of a simple cyst where the aspirate drains only cyst fluid. For solid masses, ROSE may improve the diagnostic yield of endoscopic ultrasound (EUS)-guided FNA by ensuring that a diagnostic sample is obtained.2-6 For cysts, aspiration of the cyst contents

Pancreas Cytology Table 9.1: Pancreatic cyst fluid biochemical and molecular analysis results supportive of cyst type PCT

SCA

IPMN

MCN

CEA

↓

↓

↑

↑

Amylase

↑↑

↓

↑

↑↓

KRAS

–

–

+

+

GNAS

–

–

+

–

(PCT: Pseudocysts; SCA: Serous cystadenoma; IPMN: Intraductal papillary mucinous neoplasm; MCN: Mucinous cystic neoplasm)

only there is no demonstrable benefit of ROSE, since the immediate evaluation does not provide information that will influence the biopsy procedure. Appropriate triage of cyst fluid for biochemical analysis or molecular analysis is more important than ROSE.

Cyst Fluid Analysis: Biochemical and Molecular Testing The biochemical and molecular tests that can be performed on cyst fluids are dependent on the cyst volume (Table 9.1). Fresh unfixed sample is divided among cytology, biochemical, and molecular studies in order to answer two questions: (1). Is the cyst mucinous and (2). Is the cyst malignant?7 The first question can be answered from gross inspection of the fluid (thick, viscous, white fluid), from cytologic evidence of extracellular mucin, or from elevated carcinoembryonic antigen (CEA). The second question is answered from cytologic evaluation of the cells in the fluid. Evaluating the cyst fluid for CEA and amylase are the two most useful biochemical tests, and CEA is the most accurate test for the detection of a mucinous cyst.8-10 Although CEA elevation supports a mucinous cyst, the level of CEA does not correlate with malignancy. Rarely nonmucinous cysts and non-neoplastic mucinous cysts, such as gastrointestinal (GI) duplication cyst will demonstrate elevated CEA levels. KRAS molecular testing is helpful when CEA is low, and on the rare occasion that a non-neoplastic cyst demonstrates an elevated CEA.11,12 GNAS testing adds value by identifying an intraductal papillary mucinous neoplasm (IPMN).13 Neither KRAS nor GNAS, however, distinguishes benign from malignant cysts.

Accuracy and Limitations The accuracy of FNA in the diagnosis of pancreatic masses and cysts in particular requires a clear understanding of the classification and histologic interpretation of

175

pancreatic diseases14,15 as well as the potential pitfalls of the biopsy technique, for example, GI contamination. Understanding the diseases that affect the pancreas and the nomenclature used in histologic diagnosis helps to formulate an accurate and meaningful cytologic interpretation. A multimodal approach with coordination of clinical, radiologic, and ancillary laboratory tests with the cytologic diagnosis provides a clinically useful interpretation for patient management.16,17 The sensitivity of EUS-FNA of the pancreas averages 80% with specificity, ranging from 60% to 100%.18-34 Sensitivity may increase with increasing experience with the EUS-FNA technique.35,36 The specificity of EUS-FNA for solid pancreatic masses is >90%, but cystic lesions are more difficult to interpret and sensitivity and specificity are highly dependent on experience in interpretation.34,37,38 Although brush cytology has nearly 100% specificity, the sensitivity is approximately 50% due to false-negative sampling and interpretation.39 Definitive malignant interpretations are hindered by preparation artifact, leading to indeterminate interpretations that are directly related to a high threshold for malignancy, given the well-known atypia that generally occurs in inflamed, often stented ducts.40,41

Specimen Interpretation The interpretation of aspiration cytology smears is influenced by the technique used to obtain the specimen. Unlike percutaneous FNAB, EUS-guided biopsies puncture the gastric or duodenal wall thereby introducing epithelial and mucin contamination into the specimen. Identifying GI epithelial contamination is generally not an issue with poorly differentiated neoplasms where two populations of cells (one benign and one malignant) are readily recognized, but it can be a significant challenge with well-differentiated adenocarcinomas, pancreatic cysts in general, and pancreatic mucinous cysts in particular. The interpretation of bile duct brushing cytology requires a high threshold for a malignant interpretation, given the typical marked reactive atypia present in ducts that are inflamed, either due to inherent disease, instrumentation, or in-dwelling catheters. A cytologic diagnosis of a specific entity in general and of malignancy in particular requires both quantitative and qualitative cellular evidence of that disease entity. The initial approach to the evaluation of the cytologic specimen begins with a low power evaluation of the overall cellularity of the smears and background elements such as

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inflammation, mucin and necrosis. Low power also allows for a general evaluation of cellular composition, cell group architecture, and cellular cohesion. High-power evaluation of the individual cellular cytomorphologic features then follows where nuclear size, nuclear shape, chromatin pattern, nucleoli, and cytoplasmic features aid in determining the cell type. Ancillary studies may be useful; specific ancillary studies are covered with the discussion of each entity in this chapter. Combining all of these elements of the smear with the clinical and radiologic information establishes the cytologic diagnosis. Utilizing strict criteria for a cytologic diagnosis of malignancy maintains a low false-positive rate. Although sampling error is well recognized as the primary reason for false-negative FNABs, understanding and recognizing the cytologic features of the pancreatic lesions described below will help to reduce the contribution of underdiagnosis to the false-negative rate.

The Prebiopsy Differential Diagnosis The differential diagnosis of pancreatic masses is initially established prebiopsy from the radiographic appearance of the mass, and generally falls into two categories: solid versus cystic mass lesions. This information, combined with the location of the mass and the clinical information can assist in narrowing the differential diagnosis. Solid masses are much more common than cystic masses, and ductal adenocarcinoma is by far the most common neoplasm in the pancreas. Solid mass lesions in the differential diagnosis include chronic pancreatitis, neuroendocrine tumor, acinar cell carcinoma (ACC), pancreatoblastoma (PB), and metastatic neoplasms. The most common cyst in the pancreas is a pseudocyst. This non-neoplastic cyst needs to be distinguished from neoplastic mucinous cysts, serous cystic neoplasms, and solid neoplasms with cystic degeneration. Clinical presentation can raise concern for malignancy including obstructive jaundice in a patient with a cystic lesion in the pancreatic head42 and the sudden onset of diabetes. The appearance of the lesion radiographically can also help in establishing a level of suspicion for malignancy. Masses that invade or surround the major peripancreatic vessels, such as the superior mesenteric artery are almost always malignant. Cysts with an associated dilated main pancreatic duct, thick septa, or cyst wall, a solid mural nodule or cyst wall mass are imaging features that raise suspicion of malignancy.42,43 The radiographic appearance is also often crucial in providing an

accurate cytologic classification of neoplastic mucinous cysts, as connectivity of the cyst with the main pancreatic duct is frequently the only preoperative information that distinguishes a mucinous cystic neoplasm (MCN) from an IPMN. Details of the radiologic appearance are discussed in more detail with each disease entity below. The interpretation of pancreatic cytology specimens should not occur in a vacuum, and all clinical, radiologic, and ancillary tests need to be incorporated in order to provide the most complete and accurate cytologic diagnosis possible. 

NORMAL PANCREAS AND GASTROINTESTINAL CONTAMINANTS

It is very important to recognize the cytologic appearance of normal pancreatic tissue and GI contaminants to prevent inaccurate interpretations, especially falsepositive results. A significant pitfall in the interpretation of pancreatic cytology is the overinterpretation of normal pancreatic epithelium as neoplastic, and the interpretation of duodenal or gastric epithelium as lesional. The pancreas is a parenchymal-rich, stroma-poor organ composed of exocrine (acinar cells and ducts) and endocrine (Islets of Langerhans) cells. The vast majority of the pancreas is exocrine tissue, predominantly acinar epithelium. The FNA of normal acinar tissue can create a very cellular smear creating the appearance of a neoplasm. Benign acinar tissue is recognized as such by the cohesive, small grapelike clusters of cells singly and attached to loose connective tissue “vines” (Fig. 9.1). The round, regular nuclei are central to eccentric with uniform chromatin and conspicuous, often quite prominent nucleoli. The cytoplasm is typically abundant and granular staining blue-green with the Papanicolaou stain and purple with a Romanowsky stain, where granules can sometimes be very prominent (Fig. 9.2) or present as negative images of tiny vacuoles, a feature that helps to distinguish them from ductal cells (Fig. 9.3). The cytoplasmic granules may not be very visible in Papanicolaou-stained cells. The architectural arrangement of the cells is key to differentiating benign acinar cells from a neoplastic acinar cell proliferation,44 the latter generally forming irregular, large sheets often with single cells and stripped naked tumor nuclei (see acinar cell carcinoma). Normal pancreatic ducts appear as large, flat cohesive sheets of epithelium with round, uniform, evenly spaced nuclei yielding the classic glandular “honeycomb”

Pancreas Cytology

177

Fig. 9.1: Benign acinar tissue is recognized as such by the cohesive, small grapelike clusters of cells singly and attached to loose connective tissue “vines” (Papanicolaou, 10×)

Fig. 9.2: The cytoplasm of acinar cells is typically abundant and granular staining blue-green with the Papanicolaou stain and purple with a Romanowsky stain where granules can sometimes be very prominent (Diff-Quik, 60×)

appearance typical of ductal epithelium throughout the body (Fig. 9.4). The cells are all about the same size and shape and the nuclei are uniformly distributed in the sheet of cells in a geometric, lattice-like arrangement. The cytoplasm of normal ductal cells is not visibly mucinous. Ductal cells have round to oval nuclei, even chromatin and generally small, inconspicuous nucleoli. Benign islet cells are not often appreciated on aspirate smears of a non-neuroendocrine neoplasm. Cytologically, individual islet cells appear uniform and polygonal with

round nuclei, coarse clumped chromatin, small inconspicuous nucleoli, and pale amphophilic cytoplasm.

Fig. 9.3: Romanowsky stains create negative images of tiny vacuoles that represent the zymogen granules, a feature that helps to distinguish them (left) from ductal cells (right) (Diff-Quik, 40×)

Fig. 9.4: Normal pancreatic ducts appear as large, flat cohesive sheets of epithelium with round, uniform, evenly spaced nuclei yielding the classic glandular “honeycomb” appearance typical of ductal epithelium throughout the body (Papanicolaou, 40×)

Cytologic Features of Benign Pancreatic Tissue Normal Acinar Cells • • •

Cohesive, grapelike aggregates, singly, and attached to fibrovascular stroma Eccentrically placed, round nucleus with fine chromatin and small nucleolus Abundant granular cytoplasm.

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A

B

Figs 9.5A and B: Duodenal epithelial cells are nonmucinous cells with a brush border admixed with sporadically placed mucinous goblet cells that have the appearance of a fried egg on alcohol-fixed preparations. Intraepithelial lymphocytes (“sesame seeds”) are also sprinkled among the enterocytes (A) gastric epithelial cells typically present as smaller groups of glandular epithelium than duodenal epithelium in which surface foveolar cells display mucinous cytoplasm (B) (A. Diff-Quik, 20×, B. Papanicolaou, 40×)

Normal Ductal Cells •

Flat and cohesive sheets with even nuclear spacing within sheets • Round to oval nuclei with fine even chromatin • Inconspicuous nucleoli. With EUS-FNA, GI contamination from the stomach and duodenum must be recognized and distinguished from lesional tissue.45,46 GI epithelium from both the duodenum and the stomach typically presents as flat monolayered sheets of glandular epithelium with a uniform honeycomb pattern similar to normal pancreatic ducts. Duodenal epithelial cells are nonmucinous cells with a brush border admixed with sporadically placed mucinous goblet cells that have the appearance of a fried egg on alcohol-fixed preparations. Intraepithelial lymphocytes (“sesame seeds”) are also sprinkled among the enterocytes (Figs 9.5A and B). Gastric epithelial cells typically present as smaller groups of glandular epithelium than does duodenal epithelium. In gastric epithelium, surface foveolar cells display mucinous cytoplasm, often contained in the upper third of the cytoplasmic compartment (Fig. 9.6). Stripped naked nuclei with grooves may also be seen and should not be confused with neoplastic cells of a neuroendocrine tumor or solid pseudopapillary neoplasm (SPN).

Cytologic Features of GI Contaminants in EUS-FNA Duodenum • • • •

Flat and cohesive monolayered sheet with a honeycomb pattern Nonmucinous glandular cells with brush border Sporadically placed goblet cells with cytoplasmic mucin Lymphocytes (“sesame seeds”) in the epithelium.

Stomach • • •

Small sheets, strips, and occasionally single cells and gastric pits Visible cytoplasmic mucin in foveolar cells Grooved naked nuclei.



PANCREATITIS

Chronic Pancreatitis Clinical Features In the United States, chronic alcohol abuse accounts for roughly 70% of all cases of pancreatitis. Other causes of pancreatitis include duct obstruction from stones, abnormal anatomy, scarring, extrinsic compression, autoimmune disease (lymphoplasmacytic sclerosing pancreatitis), chronic malnutrition, and heredity.47-49 The condition is more common in men than women, generally in the

Pancreas Cytology

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Fig. 9.6: Active chronic pancreatitis demonstrates a background of acute inflammation admixed with necrotic debris composed of degenerating cells, foamy histiocytes, fat necrosis, and calcifications in variable amounts depending on the degree of tissue injury

Fig. 9.7: Autoimmune pancreatitis produces hypocellularity, cellular stromal fragments with crush artifact, and chronic inflammation with variable numbers of plasma cells in the background

40–60 years age range. Acute pancreatitis is the result of injury causing the release of pancreatic enzymes that essentially autodigest the pancreatic tissue and is almost always a clinical diagnosis where FNA is rarely performed. Active chronic pancreatitis or long-term chronic or autoimmune pancreatitis (AIP) with a radiologic appearance, demonstrating a mass lesion is what leads to an FNA in order to assess for carcinoma. Chronic pancreatitis results from progressive inflammation of the pancreas that destroys the exocrine component of the pancreas leading to a loss of acinar tissue, and sometimes endocrine tissue, with replacement fibrosis that is irreversible.47 This fibrosis is particularly prominent in AIP (also known as lymphoplasmacytic sclerosing pancreatitis). AIP is a duct-centric inflammatory process dominated by lymphocytes and plasma cells. Some forms of AIP are associated with a neutrophilic infiltrate that can dominate the inflammatory component, so the presence of neutrophils does not preclude the diagnosis of AIP.50 AIP is important to recognize at the time of FNA as AIP responds to corticosteroid therapy and a diagnosis may preclude unnecessary surgery.51

present. Focal involvement of the gland may occur, as well as dense expanses of fibrosis in more diffuse disease, both conditions create mass-like lesions that mimic pancreatic carcinoma.52,53 The classic feature of AIP on computed tomography (CT) is one of a diffusely enlarged, sausage-shaped pancreas with featureless borders surrounded by a low-density rim.54

Radiologic Features Most cases of chronic pancreatitis are associated with nonfocal lesions and irregular ductal dilatation that is often associated with stricture formation, obstruction and calcifications. Pseudocyst formation may also be

Cytopathologic Features Aspirate smears of pancreatitis are variable in the quality and quantity of tissue aspirated. Active chronic pancreatitis demonstrates a background of acute inflammation admixed with necrotic debris composed of degenerating cells, foamy histiocytes, fat necrosis, and calcifications in variable amounts, depending on the degree of tissue injury55 (Fig. 9.7).

Key Cytologic Features of Active Chronic Pancreatitis • • • • •

Acute inflammation Necrotic debris composed of degenerating cells Foamy histiocytes Fat necrosis Calcification. Clues to the diagnosis of AIP include hypocellularity, cellular stromal fragments with crush artifact, and chronic inflammation with variable numbers of plasma cells in the background but often none56,57 (Fig. 9.8). Cytologic “atypia” is common in chronic pancreatitis and AIP in

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Key Cytologic Features of Chronic and Autoimmune Pancreatitis • • • • • • • •

Background mixed inflammation and histiocytes Fat necrosis, calcific debris Cellular stromal fragments (AIP) Epithelial groups with only slightly crowded nuclei Absent or only rare isolated atypical cells Enlarged cells