Practical Diagnostic Approaches in Non-Gynaecologic Cytology [1st ed.] 9789811529603, 9789811529610

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Practical Diagnostic Approaches in Non-Gynaecologic Cytology Min En Nga

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Practical Diagnostic Approaches in Non-­Gynaecologic Cytology

Min En Nga

Practical Diagnostic Approaches in Non-Gynaecologic Cytology

Min En Nga Department of Pathology National University Hospital Singapore

ISBN 978-981-15-2960-3    ISBN 978-981-15-2961-0 (eBook) https://doi.org/10.1007/978-981-15-2961-0 © Springer Nature Singapore Pte Ltd. 2021 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

To my husband, Tony, for always inspiring me to be better. To my mentors Felicity Frost, Greg Sterrett and Priyanthi Kumarasinghe, not only for schooling me in cytology but also for showing me the meaning of service and humility. And to my parents, Been Hen and Cheng Choo, who made me want to teach.

Foreword

Although there are many comprehensive cytology texts, there are not many that give a broad approach to cytopathology with practical tips. Such teaching is inherently difficult and is built on a very deep understanding of the ‘art and science’ of cytopathology in the back drop of very sound clinical knowledge. This book fills that void. It covers aspects that are beyond standard interpretation, and yet essential in achieving the final goal of cytopathology practice which is patient care. Thyroid, lymph node, salivary gland, effusion cytology and cytopathology of endoscopic ultrasound-guided (EUS) aspirates are given special attention with the novel slant of pre- and post-analytical issues. The absolute value of cytopathology as one of the most cost-effective diagnostic techniques ever invented, both in the developed and developing world has been established beyond doubt for decades. Notwithstanding that, the honest acceptance of what you can not do is absolutely well illustrated in Chap. 1. The reader is introduced to a humble beginning of the journey of cytopathology with self-learning along the way. This is an essential part of mastering cytopathology along with the essential requirement of in-depth clinical knowledge. Chapter 2 covers an integral part of pathology practice— writing a meaningful, clinically useful cytopathology report. All the hard work would be lost with a confusing report as the final cytopathology report is often the only communication with the clinician. Chapter 3 deals comprehensively with the triple approach which comprises clinical, cytological evaluation and ancillary testing, and Chap. 4 describes Rapid On-site Evaluation (ROSE). The system-based chapters expand on the basic essentials covered in Chaps. 1–4. The many figures and charts provide details and clues to differential diagnosis and cytopathological features. Thyroid cytopathology is covered in Chap. 5 with emphasis on terminology and new and evolving concepts. A useful description of architectural patterns is beautifully presented with very clear illustrations. Similarly, the other systemic chapters are covered in a manner that can be readily understood even by the uninitiated. Chapter 8, on EUS-guided aspiration of the gastrointestinal tract and pancreas, provides excellent practical tips. The value and limitations of ROSE are discussed with honesty, with reference to the role of telecytology. In pancreatic cytology, the Papanicolaou Society of Cytopathology Guidelines for Reporting of Pancreaticobiliary Cytology appears to be the flavour in the text. This system has earned its deserved recognition with the useful categories especially the category of ‘Neoplasm’. Here again, approaches in the vii

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f­oundational chapters are elaborated on with a very comprehensive discussion on site-specific neoplasms, non-neoplastic lesions and potential diagnostic pitfalls. In the time that I have known Dr Nga, she has never hidden her keen interest in unravelling the mysteries of the cells. The intricate details have always made her curious. Over time, she has practised and taught cytopathology with the same undiluted passion. She has keenly embraced and refined all the practical tips gathered throughout her practice, and this text is a fine testimony to her journey. This book will no doubt fill a void that is needed in the overall understanding of cytopathology that is not only restricted to the interpretation of material. I congratulate Dr Nga for undertaking such a task. Priyanthi Kumarasinghe Consultant Pathologist, Department of Anatomical Pathology PathWest Laboratory Medicine, Queen Elizabeth II Medical Centre Nedlands, WA, Australia Clinical Professor, Discipline of Pathology and Laboratory Medicine University of Western Australia Perth, WA, Australia Director, Western Australia, International Academy of Pathologists, Australasian Division Perth, WA, Australia

Foreword

Preface

The practice of cytopathology is very much like detective work. It is akin to peering through a keyhole, gathering minute and sometimes peripheral clues, meticulously studying them, and, hopefully, solving the mystery that each case presents. This is a book of approaches. It is written with the busy practicing pathologist and cytotechnologist in mind. It is not a comprehensive tome that systematically highlights the diagnostic features of specific entities in every organ system—this has been amply and beautifully covered in many excellent texts today, many of which have been my own silent teachers past and present. Instead, it is a practical guide on how to best combine the knowledge of cytomorphology with clinical knowledge and an understanding of ancillary tests, in order to obtain the most accurate and clinically helpful diagnosis. In these pages, practice-driven approaches will be provided, focusing on the salient, most distinguishing and unique cytomorphologic features of common and challenging entities. In tandem with this, relevant clinical aspects will be highlighted, hand in hand with ancillary tests. The algorithmic approaches provided here are not comprehensive; indeed, they are only a start—a template—on which you can build your own improved and tailor-­ made versions. As you read, you are encouraged to actively think of each case in its full clinical context, incorporating relevant clinical findings into the cytologic evaluation where possible. This practical guide also touches on the optimal use of limited cytologic material for ancillary testing, and how to maximise the tissue for diagnostic use, prognostication and prediction of response to therapy. I hope that you will find this to be a helpful companion in your journey to uncover the mysteries that each and every cell holds. Min En Nga

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Acknowledgements

I would like to express my sincere thanks to many who have, in one way or another, helped to make this book a reality. For your generous and inspired suggestions and comments—Tony Lim, Siaw Ming Chai, Gwyneth Soon, Yingting Mok, Nicholas Tan, Li Yin Ooi, Hui Min Tan, Jaslyn Lee, Gideon Tan, Noel Chia and Fredrik Petersson. For so kindly and willingly hunting down suitable illustrative cases—Zhen Qin Tan and Gaik Lan Lim. For so patiently guiding me through the murky waters of formatting—Darren Chua. For your immense support, friendship and incisive opinions, and for being our steadfast and able cytology director, Shaun Goh. To my other colleagues in cytopathology who provide unstinting support in the practice and teaching of cytology, Ju Ee Seet and Qasim Ahmed. For always putting the patient first and for being the absolute backbone of our cytopathology service—my thanks to our wonderful cytotechnologists— Hua Cheng Kee, Xu Xia, Vanessa Soh, Gaik Lan Lim, Zhen Qin Tan, Irene Lee, Li Yin Lim, Jocelycn Chung and Bridget Wong. For being a joy to teach and learn with, all trainees past and present in the Department of Pathology, National University Hospital—you are the motivation for this book, and the future of cytopathology. To all colleagues in the Department of Pathology, National University Hospital and National University of Singapore, my sincere thanks for your support and for the generous sharing of cases and knowledge in this ever fascinating discipline of Pathology. And, finally, my sincere thanks to Barbro Nilsson, who was my first teacher of cytology and who taught me how to ‘talk’ to the cells!

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About the Book

This book is written in two main parts—Part I, the foundational chapters, and Part II, the system-based chapters. Part I (Chaps. 1–4) comprises the foundational elements of the practice of cytology, with a strong emphasis on practice-based aspects. It highlights active ways in which to maximise learning from each case and provides a general approach (the triple approach) to the diagnostic process. The three elements of this approach are clinical information, cytology and ancillary tests. These chapters aim to equip one with the mindset of how to formulate the most helpful diagnosis for optimal clinical management. It also provides practical tips on the writing of a cytology report such as how to craft a thorough, concise and clear report. The question of when and how to write comments in the diagnostic report is also discussed. In Part II (Chaps. 5–9), several specific body systems will be covered, applying the approaches and methods laid out in Part I. The chapters begin with laying out the spectrum of common and important conditions that may be encountered in that system, followed by a discussion on the limitations of cytology. Thereafter, a framework is provided in terms of the triple approach, with a particular focus on morphological aspects. Morphology is covered in a step-wise fashion, moving from low-power (cellularity, architecture) to highpower analysis (nuclear and cytoplasmic features) and, finally, background material. In some chapters, for example, endoscopic ultrasound-­guided (EUS) FNA, basic imaging correlates are also described. Thereafter, specific algorithmic approaches to challenging areas, as well as pitfalls and danger zones are discussed, with practical tips and clues to help avoid misinterpretation. The focus of this book is on problem-solving from the perspective of clinical and morphological clues, rather than a detailed description of the cytologic features of specific entities. For more comprehensive morphological descriptions, the reader is referred to many excellent texts such as The Art and Science of Cytopathology (Richard Dr May, Am Soc Clinical Pathology, Chicago, IL.), Orell and Sterrett’s Fine Needle Aspiration Cytology (Edited by Svante R Orell and Gregory F.  Sterrett Elsevier, 5th edition 2012), Cytology: Diagnostic Principles and Clinical Correlates (Edmund S. Cibas and Barbara S. Ducatman, Saunders, 4th edition, 2014). These texts form the basis of the appreciation of diagnostic cytologic features of a wide spectrum of diagnostic entities, as well as an understanding of sound laboratory practices, complementing the clinical approaches provided in this book. xiii

Contents

Part I Foundational Approaches in Cytopathology 1 Basic Principles in Cytopathology: Things I Wish I Knew at the Start������������������������������������������������������������������������������������������   3 1.1 Introduction������������������������������������������������������������������������������   3 1.2 Four Principles in Cytology������������������������������������������������������   4 1.2.1 Know What Cytology Can and Can’t Do ��������������������   4 1.2.2 Have a Morphological Approach����������������������������������   4 1.2.3 Know the Major Classification Systems ����������������������   5 1.2.4 Accept That There Will Always Be Cells You Can’t Figure Out����������������������������������������������������   5 1.3 The Triple Approach ����������������������������������������������������������������   5 1.3.1 Cytomorphologic Evaluation����������������������������������������   6 1.3.2 Clinical Information������������������������������������������������������   6 1.3.3 Ancillary Tests��������������������������������������������������������������   7 1.4 Four Tips for Improving Diagnostic Proficiency����������������������   7 1.4.1 Compare, Compare, Compare��������������������������������������   7 1.4.2 Seek Out the Equivocal Cells on Immunohistochemistry ������������������������������������������������   7 1.4.3 Create a Mental or Photographic Archive��������������������   7 1.4.4 Curiosity Is a Good Thing: Follow That Case Up! ������   8 References������������������������������������������������������������������������������������������   9 2 The Cytology Report: Practical Tips�����������������������������������������������  11 2.1 Introduction������������������������������������������������������������������������������  11 2.2 Three Important Questions ������������������������������������������������������  12 2.2.1 What Is the Reason for the Biopsy/Sample?����������������  12 2.2.2 How Can I Provide the Most Accurate Information in the Clearest Manner Possible?��������������  12 2.2.3 When Should I Include a Comment?����������������������������  13 2.3 The Cytology Report: What Goes In?��������������������������������������  14 2.4 Levels of Diagnostic Certainty ������������������������������������������������  14 2.4.1 Brief Scenario ��������������������������������������������������������������  15 2.5 Example Reports����������������������������������������������������������������������  15 2.5.1 Conveying Uncertainty as to Whether the Sample Is Representative����������������������������������������  15

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2.5.2 Conveying Correlation with Relevant Past Medical History����������������������������������������������������  16 2.5.3 Recommendations for Further Management����������������  16 3 The Triple Approach in Cytopathology: Its Practical Application��������������������������������������������������������������������  19 3.1 Introduction������������������������������������������������������������������������������  19 3.2 Elements of the Triple Approach����������������������������������������������  19 3.2.1 Clinical Information������������������������������������������������������  20 3.2.2 Cytology (Gross and Microscopic Evaluation)������������  21 3.2.3 Ancillary Tests��������������������������������������������������������������  26 3.3 Applying the Triple Approach��������������������������������������������������  29 3.3.1 Start with the Most Basic Clinical Information������������  29 3.3.2 Form a Quick Diagnostic Impression ��������������������������  29 3.3.3 Perform a Careful Check of the Relevant Clinical History If Necessary����������������������������������������  29 3.3.4 Carefully Screen All Slides������������������������������������������  29 3.3.5 If Additional Material Is Available, Decide on Whether Ancillary Tests Are Required��������  29 3.3.6 Consult a Colleague If Necessary ��������������������������������  29 3.3.7 Craft the Cytology Report��������������������������������������������  30 3.4 Outline of a Case����������������������������������������������������������������������  30 4 On-Site Evaluation and the Provisional Report������������������������������  33 4.1 Introduction������������������������������������������������������������������������������  33 4.2 Sample Adequacy and Triage����������������������������������������������������  33 4.2.1 Sample Adequacy����������������������������������������������������������  33 4.2.2 Specimen Triage ����������������������������������������������������������  35 4.3 Provisional Diagnosis ��������������������������������������������������������������  36 4.3.1 Information That a Provisional Report May Include������������������������������������������������������������������  36 4.4 Sample Case for Provisional Report ����������������������������������������  36 References������������������������������������������������������������������������������������������  38 Part II System-Specific Approaches in Cytopathology 5 Approach to Thyroid Cytology ��������������������������������������������������������  41 5.1 Introduction������������������������������������������������������������������������������  42 5.2 Main Diagnostic Entities and Classification Systems��������������  42 5.2.1 Main Diagnostic Entities����������������������������������������������  42 5.2.2 NIFTP: A Recently Defined Entity ������������������������������  43 5.2.3 Major Classification Systems in Thyroid Cytology������  43 5.3 The Bethesda System for Reporting Thyroid Cytology�����������  44 5.3.1 Non-diagnostic��������������������������������������������������������������  44 5.3.2 Benign ��������������������������������������������������������������������������  45 5.3.3 Atypia of Undetermined Significance (AUS)/Follicular Lesion of Undetermined Significance (FLUS) ����������������������������������������������������  45

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5.3.4 Follicular Neoplasm (FN)/Suspicious for Follicular Neoplasm (SFN) ������������������������������������  45 5.3.5 Suspicious for Malignancy (SM)����������������������������������  45 5.3.6 Malignant����������������������������������������������������������������������  46 5.4 What Cytology Can’t Diagnose in the Thyroid������������������������  46 5.5 Brief Technical Notes����������������������������������������������������������������  47 5.5.1 Needle Gauge����������������������������������������������������������������  47 5.5.2 Ultrasound Guidance����������������������������������������������������  48 5.5.3 Collecting Material for Ancillary Tests������������������������  48 5.6 Triple Approach������������������������������������������������������������������������  48 5.6.1 Clinical Information������������������������������������������������������  49 5.6.2 Cytology (Gross and Microscopic Evaluation)������������  51 5.6.3 Ancillary Tests��������������������������������������������������������������  65 5.7 Specific Challenges������������������������������������������������������������������  67 5.7.1 Cystic Lesions��������������������������������������������������������������  67 5.7.2 Hurthle Cell-Rich Lesions��������������������������������������������  68 5.7.3 Lymphoid-Rich Lesions (When to Diagnose Lymphoid Atypia?) ����������������������  69 5.7.4 The Atypical FNA: When to Call Something FLUS/AUS?������������������������������������������������  70 5.8 Pitfalls ��������������������������������������������������������������������������������������  73 5.8.1 False Negatives/Undercalls������������������������������������������  73 5.8.2 False Positives��������������������������������������������������������������  74 5.8.3 Mimics��������������������������������������������������������������������������  76 References������������������������������������������������������������������������������������������  78 6 Approach to Lymph Node Cytology������������������������������������������������  81 6.1 Introduction������������������������������������������������������������������������������  81 6.2 Main Diagnostic Entities and a Working Classification System����������������������������������������������������������������  82 6.2.1 Main Diagnostic Entities����������������������������������������������  82 6.2.2 Working Classification System ������������������������������������  83 6.3 Lymphomas: Role of Cytology������������������������������������������������  85 6.4 Brief Technical Notes����������������������������������������������������������������  86 6.4.1 Needle Gauge����������������������������������������������������������������  86 6.4.2 Personal Protective Equipment ������������������������������������  86 6.4.3 Collecting Material for Ancillary Tests������������������������  86 6.5 Triple Approach������������������������������������������������������������������������  87 6.5.1 Clinical Information������������������������������������������������������  87 6.5.2 Cytology (Gross and Microscopic Evaluation)������������  90 6.5.3 Ancillary Tests�������������������������������������������������������������� 100 6.6 A Sequential, Pattern-Based Diagnostic Approach������������������ 102 6.6.1 Is This a Lymphoid Population? ���������������������������������� 103 6.6.2 What Is the Main Architectural Pattern?���������������������� 104 6.6.3 In a Lymphoid Lesion, What Is the Main Cell Make-Up?�������������������������������������������������������������� 107 6.6.4 If This Is a Non-lymphoid Lesion, Could This Be a Non-nodal Lesion?�������������������������������������� 108

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6.6.5 Is There Any Relevant Clinical Finding that Ties in with the Lymphadenopathy?���������������������� 108 6.7 Pitfalls �������������������������������������������������������������������������������������� 108 6.7.1 False Positives�������������������������������������������������������������� 108 6.7.2 False Negatives ������������������������������������������������������������ 113 6.7.3 Mimics�������������������������������������������������������������������������� 116 References������������������������������������������������������������������������������������������ 119 7 Approach to Salivary Gland Cytology �������������������������������������������� 123 7.1 Introduction������������������������������������������������������������������������������ 124 7.2 Main Diagnostic Entities and the Milan System���������������������� 124 7.2.1 Non-diagnostic�������������������������������������������������������������� 124 7.2.2 Non-neoplastic�������������������������������������������������������������� 126 7.2.3 Atypia of Undetermined Significance (AUS) �������������� 126 7.2.4 Neoplasm���������������������������������������������������������������������� 126 7.2.5 Suspicious for Malignancy ������������������������������������������ 127 7.2.6 Malignant���������������������������������������������������������������������� 127 7.3 Salivary Gland FNA: What Cytology Can and Cannot Do�������������������������������������������������������������������������� 127 7.4 What Do Clinicians Need from Us? ���������������������������������������� 128 7.5 Triple Approach, with a Pattern-Based Morphologic Approach ������������������������������������������������������������ 128 7.5.1 Clinical Information������������������������������������������������������ 128 7.5.2 Alarming/Red Flag Clinical Findings�������������������������� 130 7.5.3 Cytology (Gross and Microscopic Evaluation)������������ 130 7.5.4 Ancillary Tests�������������������������������������������������������������� 137 7.6 Specific Challenging Areas ������������������������������������������������������ 139 7.6.1 Cystic Lesions�������������������������������������������������������������� 139 7.6.2 Lymphoid-Rich Lesions������������������������������������������������ 141 7.6.3 Basaloid Neoplasms������������������������������������������������������ 143 7.6.4 Oncocytic/Oncocytoid Lesions ������������������������������������ 144 7.6.5 Low-Grade Malignancies���������������������������������������������� 146 7.7 Pitfalls �������������������������������������������������������������������������������������� 151 7.7.1 False Positives and Negatives �������������������������������������� 151 7.7.2 Mimics�������������������������������������������������������������������������� 151 References������������������������������������������������������������������������������������������ 154 8 Approach to Endoscopic Ultrasound-Guided FNA of the Gastrointestinal Tract and Pancreas ������������������������������������ 157 8.1 Introduction������������������������������������������������������������������������������ 157 8.2 EUS FNA: What’s Good to Know�������������������������������������������� 158 8.2.1 Needle Route���������������������������������������������������������������� 159 8.2.2 ROSE���������������������������������������������������������������������������� 159 8.2.3 Specimen Triage for Ancillary Testing ������������������������ 160 8.3 GIT�������������������������������������������������������������������������������������������� 160 8.3.1 Triple Approach������������������������������������������������������������ 161 8.3.2 Specific Challenges������������������������������������������������������ 168 8.3.3 Pitfalls (False Positives, False Negatives, Mimics)������ 170

Contents

xix

8.4 Pancreas������������������������������������������������������������������������������������ 174 8.4.1 Spectrum of Diagnostic Entities and the Papanicolaou Society of Cytopathology Guidelines���������������������������������������� 175 8.4.2 What Pancreatic Cytology Can and Cannot Diagnose�������������������������������������������� 176 8.4.3 Technical Issues in Pancreatic EUS FNA �������������������� 177 8.4.4 A Systematic Approach: Key Questions���������������������� 179 8.4.5 Triple Approach������������������������������������������������������������ 180 8.4.6 Specific Challenges������������������������������������������������������ 190 8.4.7 Pitfalls (False Positives, False Negatives and Mimics)������������������������������������������������������������������ 197 References������������������������������������������������������������������������������������������ 201 9 Approach to Effusion Cytology�������������������������������������������������������� 206 9.1 Introduction������������������������������������������������������������������������������ 206 9.2 Spectrum of Conditions and Classification Systems���������������� 207 9.2.1 Cytologic Classification Systems���������������������������������� 208 9.3 Technical Notes������������������������������������������������������������������������ 208 9.3.1 Collection Method�������������������������������������������������������� 208 9.3.2 Fluid Volume���������������������������������������������������������������� 208 9.3.3 Optimising Cellular Yield �������������������������������������������� 210 9.3.4 Sample Triage for Ancillary Testing ���������������������������� 210 9.4 Triple Approach������������������������������������������������������������������������ 211 9.4.1 Clinical Information������������������������������������������������������ 211 9.4.2 Cytology (Gross and Microscopic Evaluation)������������ 213 9.4.3 Ancillary Tests�������������������������������������������������������������� 229 9.5 Pitfalls �������������������������������������������������������������������������������������� 234 9.5.1 False Negatives ������������������������������������������������������������ 234 9.5.2 False Positives�������������������������������������������������������������� 235 9.5.3 Mimics�������������������������������������������������������������������������� 239 References������������������������������������������������������������������������������������������ 241 Index���������������������������������������������������������������������������������������������������������� 245

About the Author

Min  En  Nga, FRCPath, FRCPA, FIAC  is an alumnus of the Faculty of Medicine at the National University of Singapore (NUS). She was awarded fellowships by the Royal College of Pathologists of both the United Kingdom and Australasia, as well as the International Academy of Cytology. She has been practicing anatomical pathology at the National University Hospital’s Department of Pathology since 2005 and has previously served as the Director cytology section and quality assurance program in cytology. She is also an Associate Professor at the Department of Pathology at the Yong Loo Lin School of Medicine at the National University of Singapore. She is currently a council member of the Singapore Society of Cytology and editor of its CytoPage newsletter. Dr Nga received the Singapore Society of Pathology’s Becton Dickinson Research Award in 2012, as well as numerous NUS teaching excellence awards, and was included in the NUS Teaching Excellence Honour Roll in 2016. In 2019, she was selected by NUS as the recipient of the university’s Outstanding Educator Award. She is currently the supervisor of postgraduate cytology training in NUH and a frequent invited speaker at cytopathology workshops, including the Australian Society of Cytology Annual Scientific Meetings; International Academy of Pathology Cytology Companion Meetings. She also addressed the Indian Academy of Cytologists in November 2017 as the IAC Oration Lecturer.

xxi

Abbreviations

ACC AcCC AdCC AITL ALCL ATC AUS BCA BCAC BCC BCN BFN BL BRMs CEPA CHL DDx DLBCL DQT EBUS EMC EMR EUS EUS FNA FA FDCs FISH FL FLUS FNA FVPTC GEC GIST GMS HCA HCC HCN

Acinic cell carcinoma Acinar cell carcinoma Adenoid cystic carcinoma Angioimmunoblastic T-cell lymphoma Anaplastic large cell lymphoma Anaplastic thyroid carcinoma Atypia of undetermined significance Basal cell adenoma Basal cell adenocarcinoma Basal cell carcinoma Basal cell neoplasm Benign follicular nodule Burkitt lymphoma Benign/reactive mesothelial cells Carcinoma ex pleomorphic adenoma Classical Hodgkin lymphoma Differential diagnosis Diffuse large B-cell lymphoma DeQuervain’s thyroiditis Endobronchial ultrasound Epithelial-myoepithelial carcinoma Electronic medical records Endoscopic ultrasound Endoscopic ultrasound-guided FNA Follicular adenoma Follicular dendritic cells Fluorescent in situ hybridisation Follicular lymphoma Follicular lesion of undetermined significance Fine needle aspiration Follicular variant papillary thyroid carcinoma Gene expression classifier Gastrointestinal stromal tumour Grocott methenamine silver Hurthle cell adenoma Hurthle cell carcinoma Hurthle cell neoplasm xxiii

xxiv

HL Hodgkin lymphoma HPF High-power field I-131 Radioactive iodine IHC Immunohistochemistry IMS Infectious mononucleosis syndrome IMT Inflammatory myofibroblastic tumour IPCHT Intraperitoneal chemotherapy IPMN Intraductal papillary mucinous neoplasm ITPN Intraductal tubulopapillary neoplasm KFD Kikuchi–Fujimoto disease LBCL Large B-cell lymphoma LBL Lymphoblastic lymphoma LELC Lymphoepithelioma-like carcinoma LGBs Lymphoglandular bodies LGFL Low-grade follicular lymphoma LGMEC Low-grade mucoepidermoid carcinoma LH aggregates Lymphohistiocytic aggregates LM Leiomyoma LMS Leiomyosarcoma LOH Loss of heterozygosity LBC Liquid-based cytology MAC Mycobacterium avium complex MALT lymphoma Mucosa-associated lymphoid tissue lymphoma MCN Mucinous cystic neoplasm ME Myoepithelioma MEC Mucoepidermoid carcinoma MECA Myoepithelial carcinoma MM Malignant mesothelioma MPD Main pancreatic duct MSRSGC Milan System for Reporting Salivary Gland Cytopathology MTC Medullary thyroid carcinoma MZL Marginal zone lymphoma ND Non-diagnostic NEC Neuroendocrine carcinoma NET Neuroendocrine tumour NF Nodular fasciitis NHL Non-Hodgkin lymphoma NIFTP Non-invasive follicular thyroid neoplasm with papillary-­ like nuclear features NLPHL Nodular lymphocyte predominant Hodgkin lymphoma NMC Neoplastic mucinous cyst NPC Nasopharyngeal carcinoma NSHL Nodular sclerosing Hodgkin lymphoma PA Pleomorphic adenoma PC Pseudocyst PDAC Pancreatic ductal adenocarcinoma PDTC Poorly differentiated thyroid carcinoma

Abbreviations

Abbreviations

xxv

PEL Primary effusion lymphoma PLAC Polymorphous adenocarcinoma PMX Pilomatrixoma PSGPBC Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology PTC Papillary thyroid carcinoma PTCL Peripheral T-cell lymphoma RA Rheumatoid arthritis RBCs Red blood cells RCC Renal cell carcinoma ROM Risk of malignancy ROSE Rapid on-site evaluation RS cells Reed–Sternberg cells SC Secretory carcinoma SCA Serous cystadenoma SCC Squamous cell carcinoma SDC Salivary duct carcinoma SFN Suspicious for follicular neoplasm SG Salivary gland SLE Systemic lupus erythematosus SM Suspicious for malignancy SmCC Small cell carcinoma SPN Solid pseudopapillary neoplasm SUMP Salivary gland neoplasm of Uncertain Malignant Potential TBMs Tingible body macrophages TBSRTC The Bethesda System for Reporting Thyroid Cytopathology TCRBCL T-cell-rich B-cell lymphoma US Ultrasound WT Warthin tumour ZN Ziehl–Neelsen

List of Charts

Chart 5.1 Thyroid—main diagnostic entities��������������������������������������������  42 Chart 5.2 Common architectural patterns with diagnostic correlates ����������������������������������������������������������������������������������  53 Chart 5.3 Approach to cystic thyroid nodules������������������������������������������  67 Chart 5.4 Approach to Hurthle cell-rich lesions ��������������������������������������  69 Chart 5.5 Approach to lymphoid-rich lesions������������������������������������������  70 Chart 5.6 Atypical lesions������������������������������������������������������������������������  71 Chart 6.1 Lymph nodes—main diagnostic entities����������������������������������  82 Chart 6.2 Lymph node FNA: pattern-based approach������������������������������  95 Chart 7.1 Salivary gland aspirates—main diagnostic entities������������������ 125 Chart 7.2 Salivary gland cytology—broad approach according to clinical/imaging characteristics�������������������������������������������� 131 Chart 7.3 Approach to cystic lesions with Milan categories�������������������� 140 Chart 7.4 Approach to lymphoid-rich lesions������������������������������������������ 141 Chart 7.5 Approach to basaloid neoplasms���������������������������������������������� 144 Chart 7.6 Approach to oncocytic lesions with Milan categories�������������� 145 Chart 8.1 Spectrum of common/important EUS FNA lesions������������������ 158 Chart 8.2 Common/important pancreatic lesions ������������������������������������ 175 Chart 8.3 Pancreas—key questions in the cytologic evaluation of solid vs. cystic lesions���������������������������������������������������������� 183 Chart 8.4 Triple approach to pancreatic cystic lesions ���������������������������� 192 Chart 9.1 Spectrum of conditions encountered in serous effusions���������� 207 Chart 9.2 Broad morphologic approach to effusion cytology������������������ 215 Chart 9.3 Approach to atypical epithelioid cells�������������������������������������� 216

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List of Tables

Table 2.1 Table 3.1 Table 5.1 Table 5.2 Table 5.3 Table 5.4 Table 6.1 Table 6.2 Table 6.3 Table 6.4 Table 6.5 Table 6.6 Table 7.1 Table 7.2 Table 7.3 Table 7.4 Table 7.5 Table 7.6 Table 8.1 Table 8.2 Table 8.3 Table 8.4 Table 8.5 Table 8.6 Table 8.7

Subjective Levels of Diagnostic Certainty ������������������������������ 15 Types of Material for Ancillary Testing ���������������������������������� 28 Major Classification Systems in Thyroid Cytology ���������������� 44 Entities That are Not Readily Diagnosable on Cytology �������� 47 Nuclear Features of Various Thyroid Entities�������������������������� 58 Thyroid—Helpful Immunohistochemical Panels�������������������� 65 Lymphoma Diagnosis on Cytology: What Cytology Can and Cannot Do���������������������������������������� 85 Metastases With Origin-specific Morphological Features������������������������������������������������������������������������������������ 91 Metastases Without Origin-specific Morphological Features������������������������������������������������������������������������������������ 93 Specific Morphologic Features of Common B-Cell Lymphomas������������������������������������������������������������������ 93 Specific Morphologic Features of Other Lymphomas ������������ 94 Ancillary Tests in Lymph Node FNA������������������������������������ 101 The Milan System for Reporting Salivary Gland Cytopathology with Risk of Malignancy (ROM) ������ 125 Benign and Malignant Neoplasms that are Readily Diagnosable on Cytology������������������������������������������ 128 Main Morphologic Cell Types in SG Neoplasms������������������ 133 Stromal Features of Basaloid Tumours���������������������������������� 134 IHC Profiles of Specific SG Neoplasms�������������������������������� 138 Common False-Negative and -Positive Diagnoses���������������� 152 Most Common Mesenchymal Lesions by Location�������������� 161 Salient Morphologic Features of Gut Wall or Extramural Lesions���������������������������������������������������� 166 Useful Ancillary Tests in Gut Wall Lesions �������������������������� 169 The Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology���������� 176 Cytologic Diagnoses of Pancreatic Lesions �������������������������� 177 Imaging Findings in Solid Pancreatic Lesions ���������������������� 182 Clinical, EUS and Cytological Findings and Ancillary Tests in Pancreatic Cystic Lesions������������������ 191

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xxx

Table 8.8 Ancillary Tests in PDAC vs. Benign Ductal Cells ���������������� 196 Table 9.1 Specific Gross Appearances of Effusion Fluid���������������������� 214 Table 9.2 Salient Morphologic Features—Mesothelial cells, Adenocarcinoma, Macrophages �������������������������������������������� 217 Table 9.3 Cells with a Signet Ring Appearance in Effusion Fluids ������������������������������������������������������������������ 223 Table 9.4 Ancillary Tests for Benign vs. Malignant Mesothelial Cells�������������������������������������������������������������������� 232

List of Tables

Part I Foundational Approaches in Cytopathology

1

Basic Principles in Cytopathology: Things I Wish I Knew at the Start

Contents 1.1

Introduction

 3

1.2 1.2.1  1.2.2  1.2.3  1.2.4 

Four Principles in Cytology  now What Cytology Can and Can’t Do K Have a Morphological Approach Know the Major Classification Systems Accept That There Will Always Be Cells You Can’t Figure Out

 4  4  4  5  5

1.3 1.3.1  1.3.2  1.3.3 

The Triple Approach Cytomorphologic Evaluation Clinical Information Ancillary Tests

 5  6  6  7

1.4 1.4.1  1.4.2  1.4.3  1.4.4 

Four Tips for Improving Diagnostic Proficiency  ompare, Compare, Compare C Seek Out the Equivocal Cells on Immunohistochemistry Create a Mental or Photographic Archive Curiosity Is a Good Thing: Follow That Case Up!

 7  7  7  7  8

References

1.1

Introduction

Welcome to the beginning. I know that it is tempting to jump right into the system-specific chapters, but I encourage you to first take a step back and consider cytology as a discipline in itself. In this chapter, you will find some truths and insights into cytology that will hopefully help you approach cases with a stronger sense of direction and understanding. At times, cytology may seem even more challenging than histology—because of the minute nature of the sample. And yet, therein lies the thrill and satisfaction of the prac-

 9

tice of cytology—from the fact that one is able to formulate a clinically helpful diagnosis from a tiny sample at the end of a needle. In the practice of cytology, it is prudent to obtain as much information from as many sources as possible, not unlike piecing together clues in a mystery, until one can finally see the big picture. This is the triple approach, combining cytomorphology with clinical findings and ancillary tests. Of these, cytomorphology forms the backbone of the diagnostic workup, with the other two corroborating elements helping to build the most accurate diagnosis possible. In my practice,

© Springer Nature Singapore Pte Ltd. 2021 M. E. Nga, Practical Diagnostic Approaches in Non-Gynaecologic Cytology, https://doi.org/10.1007/978-981-15-2961-0_1

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1  Basic Principles in Cytopathology: Things I Wish I Knew at the Start

4

I  ­ prefer to first obtain an unbiased cytologic impression before delving into clinical findings, and then, finally performing ancillary tests if necessary. Practicing cytology has caused me to better appreciate subtle morphologic features and fine-­ tune my selection of ancillary tests because of the limited tissue volume available. And it is a bonus that these skills are fully transferable to the practice of histopathology! Over the years, I have also come to appreciate some truths about cytology, which I will share in the following section. I have gleaned these principles from my teachers, from conversations with peers, seniors and juniors and, most importantly, in my interactions with the cells themselves. Like all of medicine, the practice of cytology is a process of continuous learning as we coax the answers out of the cells that we examine every day.

1.2

Four Principles in Cytology

Here are four principles that are worth bearing in mind in the practice of cytopathology. 1. 2. 3. 4.

Know what cytology can and can’t do Have a morphological approach Know the major classification systems Accept that there will always be cells you can’t figure out

1.2.1 K  now What Cytology Can and Can’t Do Cytology holds some fundamental differences from histology because of the inherent nature of cytology specimens. In aspiration cytology, the nature of sample acquisition precludes definitive evaluation of specific architectural features and hence may limit diagnoses in certain instances. For example, in follicular neoplasms of the thyroid, capsular and vascular invasion—both architectural features—are the key differences

between follicular carcinoma and follicular adenoma. Thus, cytology is inherently unable to reliably distinguish between benign and malignant follicular neoplasms. Recognising these intrinsic limitations of cytology is a step towards the safe practice of cytology. It is thus the pathologist’s responsibility to know what cytology can and can’t do—based on the familiarity of diagnostic criteria in specific clinical contexts. This limitation should be reflected in the cytology report as an inherent characteristic of the test, and, where relevant, recommendations can be made for further diagnostic workup. More detailed examples will be discussed in the system-specific chapters.

1.2.2 Have a Morphological Approach Don’t panic—cytology is just like histology— it is about understanding morphology. In any specific organ site, one can expect to encounter a spectrum of common morphologic patterns, which can be architectural (e.g. honeycomb sheet) or cytomorphological (e.g. clear cells). It is helpful to have a good knowledge of the most likely differential diagnoses of some of these commonly encountered patterns, as this is a natural starting point for the diagnostic workup. One such example is a thyroid aspirate in which oncocytic cells are prominent. Knowledge of the differential diagnoses (oncocytic change in a colloid nodule, autoimmune thyroiditis, Hurthle cell neoplasm) would trigger an active search for features of individual conditions (e.g. colloid, lymphocytes, etc.), thereby helping one to narrow down the differential diagnosis. Thus, having a morphological approach allows for a more systematic workup of common diagnostic dilemmas. The system-specific chapters will provide approaches to specific common morphological patterns and their range of differential diagnoses and distinguishing features.

1.3  The Triple Approach

5

1.2.3 K  now the Major Classification Systems

1.2.4 A  ccept That There Will Always Be Cells You Can’t Figure Out

Over the last decade, there has been international recognition of the need for a more universal language amongst cytopathologists, to facilitate clarity of communication between pathologists and multidisciplinary clinical teams. There has been increasing uptake of the use of international reporting systems worldwide for commonly encountered organ sites such as the thyroid and salivary glands. It is therefore important to know the major reporting/classification systems as these provide a scaffolding for systematic reporting and categorisation of cytology cases. Some internationally and nationally applied classification systems are listed here.

The challenge in cytology is that whilst there will be many cells that are readily recognisable, there will always be some inscrutable cells that are unwilling to give up their secrets. It is part of our journey as diagnosticians to interrogate these cells by constantly comparing, observing and questioning their nature. Our ultimate goal is to gradually reduce the proportion of these unknown cells as our experience grows. Over the years, I have found some concrete ways in which we can increase our repertoire of morphologic clues—a lot of these are based on deliberate observation and comparison. These are shared in the last section of this chapter. Of course, a little extra effort is required, but, with energetic and active inquiry, one can increase one’s confidence and diagnostic proficiency. It is likely that there will always remain some cells that defy classification; however, as we continue to seek out specific subtle differences and clues, we will find that these cells become progressively fewer over time, as our repertoire of discriminatory cytomorphologic clues increases. In the next section, we will have a look at the triple approach—the multi-pronged approach to cytodiagnosis.

1. Thyroid –– The Bethesda System for Reporting Thyroid Cytopathology (2009; 2018) [1, 2]. –– The British Thyroid Association/Royal College of Pathologists (United Kingdom) Guidelines for Reporting Thyroid Cytology [3]. –– The Royal College of Pathologists of Australasia Thyroid Structured Reporting Protocol 2014 [4]. 2. Salivary glands –– The Milan System for Reporting Salivary Gland Cytopathology [5]. 3. Pancreas –– The Papanicolaou Society of Cytology Guidelines for Standardised Reporting of Pancreatic Cytology [6]. 4. Urine cytology –– The Paris System for Reporting Urine Cytology [7]. Some of these are elaborated on in the chapters. Several other international classification systems are currently underway during the time of writing of this book.

1.3

The Triple Approach

The optimal diagnostic approach involves THREE components: 1. Cytomorphologic evaluation 2. Clinical information 3. Ancillary tests The rationale behind the triple approach is introduced here, and a more detailed description of its application is provided in Chap. 3 (The Triple Approach in Cytopathology—Its Practical

6

1  Basic Principles in Cytopathology: Things I Wish I Knew at the Start

Application). Whilst it may not be necessary to delve into the detailed medical records or perform ancillary tests for every case, it is certainly helpful in some instances. The triple approach is most helpful in cases with atypical or non-­ classical cytologic findings, or where certain specific information is required by clinicians, e.g. the source of metastatic carcinoma in a lymph node aspirate. My usual practice is to first do a quick evaluation of the smears with minimal clinical information (age, gender, location) in order to form an unbiased opinion. Thereafter, I seek out more clinical information where necessary.

1.3.1 Cytomorphologic Evaluation Cytomorphology is the language of the cells and the crucial starting point of our diagnostic workup. In cytopathology, the appreciation of architecture is limited and somewhat more challenging than in histopathology. For example, the relationships between cells and surrounding stroma or cells and basement membrane are not reliably discerned on cytology. Therefore, we need to be as observant as we can with the available morphologic clues in cytology smears and not waste any clues. When evaluating cytology cases, we sometimes dive straight into assessing nuclear and cytoplasmic features, bypassing other valuable clues along the way such as architectural formations. Here are three elements that are sometimes overlooked: • Gross appearance of aspirated material or fluid in exfoliative cytology specimens • Architectural arrangements of lesional cells • Background material or cells Gross appearance of aspirated material may be a key diagnostic feature in some scenarios, e.g. the macroscopic presence of colloid in thyroid aspirates (this may wash off and be lost during the staining process, leaving the gross description as the only documentation of its pres-

ence). The gross appearance of mucoid material aspirated from pancreatic cysts is also an important diagnostic clue, as will be highlighted in Chap. 8. When evaluating architectural formations, we begin to appreciate how cells relate to each other and the surrounding connective tissue. These formations and relationships reflect what we see on histology, although they may be more subtle on cytology. For example, in thyroid cytology, a flat honeycomb sheet corresponds to a large thyroid follicle that has been disrupted and spread open during the smearing process, thereby reflecting the likely macrofollicular nature of the lesion. Diagnostically useful information may also be gleaned from background material in smears, such as the subtle presence of a tigroid background (lacy-appearing background due to glycogen leached from tumour cells in neoplasms such as seminomas, Fig. 3.3 in Chap. 3). Thus, for each sample that we evaluate, there are many layers that are open to scrutiny and observation. When we consciously seek them out, we increase our repertoire of visual diagnostic clues and hence our confidence in approaching similar cases. A more detailed step-by-step approach to cytomorphologic evaluation is provided in Chap. 3.

1.3.2 Clinical Information In many institutions, clinical information is available through electronic or physical patient records and through direct communication with the referring clinician. Clinical findings are useful in two ways: they tell us the reason for the biopsy and also provide relevant information that can help direct our evaluation. As mentioned above, I prefer to perform a quick cytologic evaluation first before hunting down more detailed clinical information. This helps to avoid bias, in the sense of prematurely agreeing with the clinical impression. In real-life practice, there is often scant relevant clinical information accompanying biopsy samples. Therefore, a sound knowledge of

1.4  Four Tips for Improving Diagnostic Proficiency

c­ytomorphology can help to fill the ‘clinical black hole’—by prompting us to raise relevant differentials based on specific morphologic findings. In these instances, after some extra digging, we might be rewarded with clinical findings that corroborate our cytologic impression. Pathologist-run fine needle aspiration (FNA) clinics allow the pathologist to have a direct interface with the patient. Every opportunity should be taken to take a focused history and perform a directed physical examination. This helps us gather helpful clues in the diagnostic process. Also, don’t forget to ask the patient if he or she is on blood thinners or anticoagulants! FNAs can still be performed, but a longer duration of compression after the procedure, and perhaps ice packs may also be considered in such situations. It must be remembered, however, that, at the end of the day, an awareness of the clinical findings is never a substitute for a sound knowledge of cytomorphologic features.

1.3.3 Ancillary Tests The third facet of the triple approach is ancillary testing. Adding to the challenge of limited morphological clues, cytologic material is often limited in terms of volume. Thus, in cytology, we have the added responsibility of maximising diagnostic yield whilst using tissue economically. In order to achieve this, we must have a sound knowledge of the most discriminatory, sensitive, specific and accurate ancillary tests. This challenge compels us to constantly upgrade our knowledge and scour the current literature for the most diagnostically helpful markers available. For this, I have found that an extremely helpful resource is Immunoquery (http://www.immunoquery.com/)—this excellent resource provides optimal immunohistochemical panels for selected differential diagnoses, together with links to peer-reviewed references. In the next section, I will share some tips that can help improve one’s diagnostic proficiency.

7

1.4

Four Tips for Improving Diagnostic Proficiency

Here are some practices that I have found to be extremely useful in improving one’s confidence and diagnostic proficiency. They are means of ‘interrogating’ the cells and coaxing answers out of them, and I still use them in my daily practice.

1.4.1 Compare, Compare, Compare Compare the ‘mystery cells’ to cells that are readily characterisable. For example, in a bronchial brushing, benign respiratory epithelial cells may exhibit a broad spectrum of reactive nuclear features. When one encounters ‘mystery cells’, active comparison between these and definite benign ciliated columnar cells or definite malignant cells can be extremely helpful. This is a way to train one’s eye to appreciate subtle morphologic features, in addition to the more apparent ones such as terminal bars and cilia. This exercise thus allows one to observe the range of nuclear and cytoplasmic variability amongst benign or malignant cells and hence adjust one’s threshold for the diagnosis of malignancy.

1.4.2 S  eek Out the Equivocal Cells on Immunohistochemistry Actively seek out the ‘equivocal cells’ on immunohistochemical preparations on cell blocks— don’t just look for the obviously abnormal ones. Although this takes time, it is a worthwhile exercise, as it may provide an answer to the nature of these challenging cells that straddle the grey area, thereby allowing us to appreciate more subtle cytomorphologic features.

1.4.3 C  reate a Mental or Photographic Archive If one has the means for photography, it can be an enormously helpful tool to permanently record

1  Basic Principles in Cytopathology: Things I Wish I Knew at the Start

8

a

b

c

d

Fig. 1.1  Smartphone pictures compared to microscope-­ mounted camera. (a) Original magnification picture taken on phone (Google Pixel 3) using the 20× objective (peritoneal fluid sample); (b) Magnified and cropped picture taken by phone using the 40× objective (colloid nodule,

thyroid FNA); (c, d) Mesothelial cells in an ascitic fluid sample, taken by phone and Olympus DP22 microscope-­ attached camera, respectively, using 40× objective—the quality is comparable

cells that are of learning value. Modern smartphones readily lend themselves to photography directly off microscope objectives, either held freehand or using specific adapters. This exercise enables one to build up a visual archive of pictures with high learning value. Figure 1.1 shows an example of the photographic quality that is possible using a smartphone to capture photomicrographs.

esting case. Correlation with the final histology can be a hugely rewarding experience and one that is always richly educational and contributes to strengthening one’s appreciation of both cytoand histomorphology. If our initial impression was erroneous, it is a good exercise to review the cytology in tandem with the histology, to recognise where we went wrong. Cytology and histology are essentially the same—but just viewed through slightly different lenses, and the more we appreciate this close relationship, the more we are able to demystify the practice of cytology. I have found it helpful to keep a record of cases that are of educational value and try to actively trace the histologic results whenever possible. Longitudinal tracking is one of the most

1.4.4 C  uriosity Is a Good Thing: Follow That Case Up! One of the most exciting things about the practice of cytopathology is the anticipation that follows the cytologic diagnosis of a challenging or inter-

References

valuable means of learning. It enables us to recognise our errors and adjust our thresholds so that we do not repeat our mistakes. We can also be affirmed from the times when cytology provided an accurate diagnosis that enabled prompt definitive management. Our ultimate goal is to progressively become more proficient in appreciating increasingly subtle diagnostic features, so that the proportion of mystery cells will decrease over time.

Take Home Points

• Know the limitations of cytology—this promotes safe practice. • Have a sound morphologic approach, with a ready list of differentials. • Apply the triple approach: cytomorphology, clinical information, ancillary tests. • Decrease the proportion of ‘unknown’ cells over time through thoughtful observation, comparison and follow-up of cases.

9

References 1. Cibas ES, Ali SZ. NCI thyroid FNA state of the science conference. The Bethesda system for reporting thyroid cytopathology. Am J Clin Pathol. 2009;132(5):658–65. 2. Ali SZ, Cibas E, editors. The Bethesda system for reporting thyroid cytopathology: definitions, criteria, and explanatory notes [Internet]. 2nd ed. Springer; 2018 [cited 2018 Jun 18]. https://www.springer.com/ gp/book/9783319605692. 3. Royal College of Pathologists (United Kingdom). Guidance on the reporting of thyroid cytology specimens [Internet]. http://ukeps.com/docs/thyroidfna.pdf. 4. RCPA—Cancer protocols [Internet]. [cited 2017 Mar 8]. http://www.rcpa.edu.au/Library/PractisingPathology/Structured-Pathology-Reporting-of-Cancer/ Cancer-Protocols. 5. Rossi ED, Faquin WC, Baloch Z, Barkan GA, Foschini MP, Pusztaszeri M, et al. The Milan system for reporting salivary gland cytopathology: analysis and suggestions of initial survey. Cancer Cytopathol. 2017;125(10):757–66. 6. Pitman MB, Centeno BA, Ali SZ, Genevay M, Stelow E, Mino-Kenudson M, et al. Standardized terminology and nomenclature for pancreatobiliary cytology: the Papanicolaou Society of Cytopathology guidelines. Diagn Cytopathol. 2014;42(4):338–50. 7. Barkan GA, Wojcik EM, Nayar R, Quek ML, Rosenthal DL. The Paris system for reporting urinary cytology: the quest to develop a standardized terminology. Adv Anat Pathol. 2016;23(4):9.

2

The Cytology Report: Practical Tips

Contents 2.1

Introduction 

 11

2.2  hree Important Questions  T 2.2.1  What Is the Reason for the Biopsy/Sample?  2.2.2  How Can I Provide the Most Accurate Information in the Clearest Manner Possible?  2.2.3  When Should I Include a Comment?  2.3

The Cytology Report: What Goes In? 

2.4  evels of Diagnostic Certainty  L 2.4.1  Brief Scenario  2.5 2.5.1  2.5.2  2.5.3 

2.1

Example Reports   onveying Uncertainty as to Whether the Sample Is Representative  C Conveying Correlation with Relevant Past Medical History  Recommendations for Further Management 

Introduction

The diagnostic pathology report is a permanent record of the pathologist’s professional opinion and, as such, must stand up to scrutiny in terms of accuracy and clarity. It is worth bearing in mind that the pathology report is not only meant for the managing clinical team—it may also be read by the patient, as well as other pathologists, for example, during multidisciplinary meetings, second opinion consults or in conjunction with future related biopsies. The main goal of a pathology report is to bring the clinical team closer to the most appropriate next step in management for the patient.

 12  12  12  13  14  14  15  15  15  16  16

Crafting a well-written report is a skill that takes time to hone, and one’s reporting style may evolve over time. There is an infinite spectrum of individual writing styles, and the contents of this chapter are not meant to dictate a specific style, but rather, to suggest some elements that one may consider including in a report, particularly for more junior practitioners. At times, comments may be included at the end of the cytology report. The utility of comments will be discussed here, with some examples given. Another area that will be discussed is the variability between terms used to convey diagnostic uncertainty—this is often not

© Springer Nature Singapore Pte Ltd. 2021 M. E. Nga, Practical Diagnostic Approaches in Non-Gynaecologic Cytology, https://doi.org/10.1007/978-981-15-2961-0_2

11

2  The Cytology Report: Practical Tips

12

s­ pecifically covered during postgraduate training and can be extremely subjective. The purpose of this chapter is not to lay down a prescriptive formula for report writing but, rather, to provide a guide to some of the elements within a cytology report. I also hope that this chapter will prompt active consideration of some of the finer points in the art of writing a report, such as how to organise the information given, how to craft a cogent comment, and how to reflect uncertainty in a balanced and measured manner.

This also helps to focus our ancillary tests on what is clinically relevant, in order to save time, money and tissue. With experience, it will become more apparent that some cases require more robust clinicopathologic correlation, whilst others may not require any more information than what is on the requisition form. Electronic medical records, where available, are extremely useful, as is discussing cases with clinicians or procedurists— the latter has the added advantage of building rapport and optimising the multidisciplinary approach.

2.2

Practical Tips: • Look through a summary list of previous biopsies from the patient. At a glance, we may be able to see that the patient has had a previous malignancy or infection, which may be relevant to the current sample. • Remember to include pertinent negative findings, if you know clinicians are asking a particular question. This saves time for both clinicians and pathologists, in reducing unnecessary requests for clarification or slide review.

Three Important Questions

As we craft our report, we are, in effect, answering a few questions. In our time spent in training and service work, many of us have ‘internalised’ these questions so much, so that they are second nature and we answer them subconsciously. But it is worth consciously considering these questions individually at some point, because they aid us in the crafting of a succinct, clinically helpful and relevant cytology report. Three important questions: 1. What is the reason for the biopsy/sample? 2. How can I provide the most accurate information in the clearest manner possible? 3. When should I include a comment?

2.2.1 W  hat Is the Reason for the Biopsy/Sample? Essentially, this translates to knowing the clinical question at hand. This is the single most important way to streamline a pathology report—by directing us to focus on what is clinically relevant; and also prompting us to include pertinent negative findings in our report. For example, if there is clinical suspicion of a mycobacterial infection in a cervical lymph node aspirate, it then becomes helpful for the pathologist to look out for granulomas or necrosis and to mention them even if we don’t see them.

2.2.2 H  ow Can I Provide the Most Accurate Information in the Clearest Manner Possible? We will focus on the microscopic description in this section, including some words of caution and several practical tips. In writing a microscopic description, one approach is to describe the morphological parameters in an organised manner, from low to high power.

2.2.2.1 Describing Microscopic Features • Overall cellularity • Architecture of main cellular components • Cytomorphologic features (nuclei and cytoplasm) of main cellular components • Background material or cells • Pertinent negative findings • Results of ancillary tests if applicable

2.2 Three Important Questions

In addition to answering a specific clinical question, there are usually two implicit aims of a pathology report: • To convey if the sample is adequate for diagnosis/management • To convey your level of certainty for the given diagnosis If there is uncertainty, a comment may help to convey the message—this is discussed in the following section.

2.2.2.2 A Word of Caution Here are some areas in which reports may potentially be misleading to clinicians and even other pathologists, in particular, when conveying a negative result. Two specific scenarios are described below:

13

in the report that one has looked for worrying nuclear features and not found them. Therefore, it is not uncommon to come across statements such as ‘There are no nuclear features of papillary thyroid carcinoma’ within the microscopic description. Whilst this statement is certainly true, there is a small danger of the word ‘carcinoma’ leading to misinterpretation by a quick scanning reader. Understandably, it is only a small risk, but it could be altogether avoided with an alternative statement such as ‘no significant nuclear atypia is seen’ or ‘no nuclear enlargement, inclusions or grooves are seen’. Furthermore, including terms such as ‘carcinoma’ routinely in benign reports might also impact database keyword searches, the results of which may become overpopulated with ‘false positive’ cases.

‘No Malignant Cells Seen’ Practical Tips: Although this is not universally agreed upon, it • Be organised and systematic—move from has been my practice to avoid using this statemorphology to ancillary tests, rather than ment when the sample is non-diagnostic. That is, jumping back and forth within the text. I only include this line when the sample is repre- • Short sentences are easier to read and sentative and adequate for cytologic evaluation. understand. Rationale: Whilst it is literally true that a non-­ • Consider breaking sections up into paragraphs diagnostic smear showing only blood or cyst (e.g. separate paragraphs for cytomorphology, macrophages shows no malignant cells, the cliniancillary tests and pertinent negatives). cian/reader might misinterpret this statement as • Be careful of wording the non-diagnostic/ the pathologist calling the sample ‘non-­ unsatisfactory report—avoid writing ‘No malignant’ or benign. This is an incorrect malignant cells seen’ in such reports (see assumption because, in fact, the pathologist in above text). unable to evaluate for malignancy due to the inadequacy of the sample. For example, in the context of a salivary gland 2.2.3 When Should I Include a Comment? cyst comprising only macrophages, the true nature of the lesion cannot be ascertained, whether neoplastic, benign or malignant. Comments are usually placed after the “diagnoTo avoid potential misinterpretation by clini- sis” section of the pathology report. They are not cians, a possible alternative but accurate state- necessary for all reports—only when there is ment is: ‘No well-preserved epithelial cells are uncertainty or when you want to relay a specific identified’. message to the clinician or future reviewing pathologist. Be Careful When Using the Word Like the rest of the report, the ideal comment “Carcinoma” in a Benign Report should be clear, concise and unambiguous. Think of a case of a thyroid FNA showing feaIn actual practice, many pathologists know tures of a colloid nodule. It is important to convey that the length of the comment is inversely

2  The Cytology Report: Practical Tips

14

p­roportional to the level of confidence in the diagnosis! Below are some reasons for writing comments, as well as some practical tips that one may find helpful when crafting them. Why Write Comments? 1. To convey uncertainty regarding whether a sample is representative (e.g. normal salivary gland elements seen only—sialadenosis vs. non-representative sample?). 2. To convey uncertainty in terms of diagnosis. 3. To direct the reader to refer to relevant other histology, cytology or molecular reports (e.g. cross-referencing concurrent core biopsy imprint cytology and core biopsy histology reports). 4. To make suggestions or recommendations in terms of further investigation or management. 5. To convey that the pathologist has noted relevant previous history or clinical findings or reviewed previous biopsies. 6. To include additional pertinent information regarding the behaviour of a particular lesion, especially if it is rare or uncommon. 7. To document communication with the clinical team. Practical Tips: • When a previous or concurrent relevant biopsy is reviewed in conjunction with the current sample, this should be mentioned in the comment, and the findings briefly described, together with their significance, e.g. ‘The previous lung core biopsy was reviewed (specimen number, date), showing small cell carcinoma with similar morphologic features to the malignant cells in the current cervical lymph node FNA.  Thus the current findings are in keeping with metastatic small cell carcinoma’. • The strength of recommendations differs from case to case. At times, they can be clear-cut and unambiguous, without the need for caveats/qualifiers, e.g. salivary gland neoplasm that requires complete excision. • In indeterminate cases, the wording for recommendations should permit some wiggle

room to allow for clinical judgement, e.g. ‘… excision biopsy may be considered in the appropriate clinical setting’. Examples of comments are provided in C (Example Reports) below.

2.3

 he Cytology Report: What T Goes In?

Most laboratories will have a fixed general template for reporting histology and cytology. We are, however, free to add elements that may be clinically relevant and helpful. In general, a complete cytology report should include the following components: 1 . Patient identifiers and demographics. 2. Specimen source (e.g. right thyroid nodule). 3. Clinical findings (optional). This is an optional section which can be added if pertinent clinical findings are forthcoming, particularly if the FNA is performed by the pathologist himself/herself. 4. Gross description. • Number of passes (for FNAs). • Volume and description of fluid or aspirated material (colour, texture, smell, if any). • Number of air-dried and alcohol-fixed slides. • Whether cell block is performed. 5. Microscopic description (discussed in previous section). 6. Diagnosis (includes diagnostic category and specific diagnosis). 7. Comments (if necessary).

2.4

Levels of Diagnostic Certainty

Not all diagnostic reports are created equal. As mentioned above, there may be subtle gradations of diagnostic certainty, and here are some commonly used phrases that might help convey

2.5 Example Reports

15

Table 2.1  Subjective Levels of Diagnostic Certainty

Diagnostic terminology 1. Cannot exclude E.g. “Basaloid lesion, adnexal neoplasm such as pilomatrixoma not excluded” 2. Raise the possibility of E.g. “Basaloid lesion, raising the possibility of pilomatrixoma” 3. Suggestive of E.g. “Basaloid lesion, suggestive of pilomatrixoma” 4.  Consistent with E.g. “Basaloid lesion, consistent with pilomatrixoma” 5.  No qualifier E.g. ‘Pilomatrixoma’

Own subjective level of certainty 40–60%

Polled range for level of certainty (5 consultants) 30–80%

60–70%

30–85%

70–95%

50–90%

95–99%

80–99%

My informal discussions have yielded both differences in the ranking of certainty amongst the various terms in Table  2.1, as well as some degree of variation in the rough percentages of diagnostic certainty that is accorded to each phrase. To my knowledge, there is no single unifying template that is available, given the inherently subjective nature of pathology reports; however, it is helpful to be cognizant of variations in the perception of both pathologists and clinicians as we routinely employ these terms in our daily practice. Conversations with clinicians may yield surprising opinions regarding the terms, in contrast to our assumptions amongst the pathology community.

2.5 >99%

99–100%

that, although at one’s discretion, speaking to the clinician can also be extremely useful in such settings. Below is a scenario in which there is some degree of diagnostic uncertainty. Table  2.1 shows the various terms that are often seen in the diagnostic line of reports and their corresponding subjective, perceived levels of diagnostic certainty based on my own thresholds, and that of an informal poll of five consultant pathologists.

2.4.1 Brief Scenario 49-year-old female. 1.5  cm lateral neck nodule. Cytology shows crowded sheets of basaloid cells and keratinised cells—raising the differential diagnoses of pilomatrixoma vs. other lesions such as metastatic squamous cell carcinoma.

Example Reports

Here are some sample reports illustrating the elements and tips mentioned above.

2.5.1 Conveying Uncertainty as to Whether the Sample Is Representative 2.5.1.1 Brief Scenario 44-year-old male, right posterior triangle lymph node. Direct FNA is performed. Cytologic material shows only adipose tissue. Pathologist not present at FNA procedure. No clinical information available regarding clinical features. Possibilities include incidentally sampled subcutaneous fat in a non-diagnostic sample vs. a lipomatous lesion, depending on the clinical examination findings. Note: The comments in this report convey some degree of uncertainty in the sense that the findings may be non-representative or representative, depending on the clinical picture. A recommendation/suggestion is included in the comment.

2  The Cytology Report: Practical Tips

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• Example report: • Gross description: • 2 passes. 2 air-dried smears and 2 alcohol-­fixed smears (2 HC, 2 Pap). • Microscopy: • Relatively hypocellular smears show only tissue fragments composed of mature adipocytes and occasional small blood vessels. There is no significant cytologic atypia. • No lymphoid or epithelial cell population is seen. • Diagnosis: • Right posterior triangle lump, FNA: –– Adipose tissue only (see comments). • Comments: The findings raise the possibility of a lipoma, with a differential diagnosis of incidentally sampled subcutaneous adipose tissue. Correlation with clinical findings is recommended—if the clinical impression is that of a cervical lymph node, the sample is not representative and repeat sampling, preferably under ultrasound guidance, may be considered.

2.5.2 Conveying Correlation with Relevant Past Medical History 2.5.2.1 Brief Scenario 67-year-old male with a history of rectal carcinoma now presents with a left upper lobe lung mass. CT-guided FNA is performed. Cytologic material shows classical cytomorphological features of colorectal carcinoma. You want to communicate that the relevant history is noted and that it had directed the ancillary testing panel performed. • Example report: • Gross description: • 2 passes. 2 air-dried smears and 2 alcohol-­fixed smears (2 HC, 2 Pap). • Cell block.

• Microscopy: • Cellular smears show cohesive crowded sheets of malignant cells with elongated, large hyperchromatic nuclei and relatively scant pale cytoplasm. In some areas, acinar formations are discernible, and focal nuclear palisading is noted. • Dirty necrosis is seen in the background. • Some alveolar macrophages are also identified. • Cell block shows some glandular and cribriform structures lined by cytologically malignant columnar cells. The malignant cells are positive for CK20 and CDX2 and negative for CK7 and TTF1. • Diagnosis: • Upper lobe mass, left lung, CT guided FNA: –– Adenocarcinoma, consistent with metastatic colorectal carcinoma. • Comments: The history of rectal adenocarcinoma is noted, status post anterior resection.

2.5.3 Recommendations for Further Management 2.5.3.1 Brief Scenario 63-year-old female. 2.5  cm left cervical lymph node. Direct FNA. Cytologic material shows features suspicious for non-Hodgkin lymphoma. Recommendation for excision biopsy is made.

• Example report: • Gross description: • 1 pass. 1 air-dried smear and 1 alcohol-­ fixed smear (1 HC, 1 Pap). • Microscopy: • Highly cellular smears show numerous lymphocytes comprising predominantly large cells with irregular nuclei and coarse chromatin. Frequent mitotic fig-

2.5 Example Reports

ures are present, as well as some apoptotic bodies. • Diagnosis: • Left cervical lymph node, FNA: –– Highly atypical lymphoid population, consistent with lymphoma with large cell morphology. • Comments: Excision biopsy is recommended for histologic evaluation. The diagnosis was communicated to Dr. John Smith at 3.00 pm on 2.1.2019.

Note that this is a strong recommendation because the lymph node requires histologic evaluation in light of the worrying cytologic findings. Recommendations may vary between institutions and should be adjusted accordingly. Note also the documentation of communication with the clinician. This should include the message, recipient’s name and time and date of the communication.

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Take Home Points

• Know the reason for the cytology sample  →  answer that clinical question in your cytology report. • Don’t forget to record gross appearance of aspirated material, it may provide diagnostic clues (e.g. colloid, mucus). • Write systematically—cellularity, architecture, cytomorphology, background, important negatives, ancillary tests. • Comments can help convey or explain uncertainty or doubts, as well as recommendations. • Be judicious in making recommendations; always bearing in mind that the clinical managing team may have many considerations when planning the next step.

3

The Triple Approach in Cytopathology: Its Practical Application

Contents 3.1

Introduction 

 19

3.2 3.2.1  3.2.2  3.2.3 

Elements of the Triple Approach  Clinical Information  Cytology (Gross and Microscopic Evaluation)  Ancillary Tests 

 19  20  21  26

Applying the Triple Approach   tart with the Most Basic Clinical Information  S Form a Quick Diagnostic Impression  Perform a Careful Check of the Relevant Clinical History If Necessary  Carefully Screen All Slides  If Additional Material Is Available, Decide on Whether Ancillary Tests Are Required  3.3.6  Consult a Colleague If Necessary  3.3.7  Craft the Cytology Report 

3.3 3.3.1  3.3.2  3.3.3  3.3.4  3.3.5 

3.4

3.1

Outline of a Case 

Introduction

We have seen in Chap. 1 that diagnostic cytologic evaluation includes the triple approach, which is the triad of clinical findings, cytomorphology and ancillary tests, where necessary. These will be discussed in more detail here, with a checklist of parameters that can be systematically evaluated in each of the three facets of this approach. A detailed discussion on the various elements of cytomorphology will be included, highlighting important elements that are sometimes left out, such as gross evaluation of the aspirated material.

 29  29  29  29  29  29  29  30  30

An example case will also be outlined in this chapter to illustrate how this approach may be used in real practice. But first, let us focus on each element of the triple approach in greater detail.

3.2

Elements of the Triple Approach

1. Clinical information 2. Cytology (gross and microscopic evaluation) 3. Ancillary tests

© Springer Nature Singapore Pte Ltd. 2021 M. E. Nga, Practical Diagnostic Approaches in Non-Gynaecologic Cytology, https://doi.org/10.1007/978-981-15-2961-0_3

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3  The Triple Approach in Cytopathology: Its Practical Application

The rationale behind this approach has been discussed in Chap. 1. Here, I will provide a more in-depth description of the individual elements that make up each prong of the triple approach.

3.2.1 Clinical Information Clinical correlation is helpful in two ways: • It tells us the specific reason for the diagnostic procedure. • It helps to direct our evaluation, e.g. alerting us to perform certain ancillary tests or prompting us to carefully look for subtle pathology such as viral cytopathic changes. Knowing some basic clinical information can also be reassuring in that it can corroborate our initial cytologic impression. Briefly, there are two main types of clinical information: 1. The reason for the cytology sample 2. Relevant clinical findings/investigations (current and past)

3.2.1.1 The Reason for the Cytology Sample In most laboratory practices, pathology samples/ slides are accompanied by a requisition form. Ideally, this would contain some relevant clinical information pertaining to the reason for the biopsy. Knowing the specific clinical question at hand brings focus to the handling of each case. This information also guides us in terms of the relevant positive or negative findings to include in our report. For example, in a lymph node aspirate in which a mycobacterial infection is suspected, it would be prudent to mention if granulomatous inflammation and necrosis is seen. When relevant clinical information is lacking, a quick phone call to the referring clinician or a targeted search in the electronic medical records (EMR) can be very enlightening.

3.2.1.2 Relevant Clinical Findings/ Investigations (Current and Past) Knowing the relevant current and past history as well as the results of relevant investigations can make a huge difference to how a case is worked up, particularly in a case featuring malignancy. Here are some examples of common helpful points in the clinical history: • Current clinical findings: –– Clinical impression of the current condition leading to biopsy. –– Current clinical presentation—relevant symptoms and signs. –– Location, appearance, size of any localised lesions. –– Intraprocedural notes during biopsy (e.g. endoscopic ultrasound findings in endoscopic ultrasound-guided FNA of a pancreatic lesion). • Relevant investigations: –– Imaging results—e.g. radiologically suspicious vs. low-risk lesion on imaging, single vs. multiple lung masses. –– Blood investigations (e.g. tumour markers, serologic markers in autoimmune disease, endocrine related investigations). –– Relevant microbiologic investigations. –– Fluid sample analysis (e.g. cyst fluid in cystic pancreatic lesions; biochemistry of needle washings from suspected parathyroid lesion). –– Flow cytometry findings. • Previous/current relevant history –– Infection. –– Malignancy—ideally, in addition to the site of any known malignancy; the histologic type and grade are also crucial, as well as the stage. Also, it is not uncommon to encounter patients with metachronous malignancies. If a cancer survivor presents with a new mass, a combination of radiologic findings and ancillary tests on cytologic material can often make the distinction between a late metastasis and a new primary.

3.2  Elements of the Triple Approach

–– Immunocompromised state—there is an increased risk of opportunistic infections and malignancy. –– If FNA is performed by the pathologist, it is prudent to ask if the patient is on blood thinners or anticoagulants, so that appropriate measures may be taken, e.g. longer duration of compression, use of ice packs. • Family history –– This is relevant in certain non-neoplastic conditions, e.g. Graves’ disease, as well as familial cancer syndromes.

Practical Tips: • If the FNA is performed by the pathologist, one should not waste the opportunity to take a quick directed history and perform a targeted physical examination. This provides a valuable clinical context to the case. • When hunting down clinical information, focus the search on specific relevant areas which may differ from case to case. Often, imaging, tumour markers, previous relevant histology, microbiological investigations and intraprocedural findings are amongst the most helpful findings. • If there is no access to clinical information and the diagnosis raises clinical questions or requires strong clinical corroboration, it is a good practice to relay this in the cytology report in the comments section, as discussed in Chap. 2.

3.2.2 Cytology (Gross and Microscopic Evaluation) Morphologic evaluation does not only refer to microscopic morphology but also includes gross morphology of cytologic material. For example, the appearance of cyst or effusion fluid should also form part of the diagnostic picture. A systematic approach is best, focusing sequentially on different elements during the screening process from low- to high-power evaluation.

21

Here are some important features to evaluate systematically when screening smears, moving  generally from low- to high-power magnification. 1. Gross appearance 2. Microscopic features –– Cellularity –– Architectural arrangements –– Cytomorphological features –– Background material or cells

3.2.2.1 Gross Appearance Although sometimes neglected, the gross appearance of cytologic material can be very relevant in the diagnostic workup. Exfoliative fluid samples or aspirated material (e.g. fluid from a cystic lesion) should be described in terms of: –– –– –– –– ––

Volume Colour Opacity (e.g. turbid, clear/serous) Viscosity Smell (if relevant)

Here are just a few examples in which gross evaluation is crucial in cytologic evaluation: –– Pancreatic cystic lesions—thick viscous mucoid fluid is almost always diagnostic of a neoplastic mucinous cyst such as an intraductal papillary mucinous neoplasm or a mucinous cystic neoplasm. –– Thyroid nodule FNA—obvious colloid seen grossly during the smearing process is an important clue to the benign nature of a thyroid nodule; this colloid may sometimes get washed away during the staining process. –– Mucinous material in a peritoneal fluid sample may suggest the diagnosis of pseudomyxoma peritonei. In turn, this should prompt the search for mucinous epithelial cells and an intra-abdominal or pelvic tumour on radiology.

22

3  The Triple Approach in Cytopathology: Its Practical Application

3.2.2.2 Microscopic Features A systematic approach that moves from low power (cellularity, architectural features) to high power (nuclear and cytoplasmic features) and background findings will be presented. Cellularity This refers to nucleated cells seen within the smear and does not include red blood cells. Smears can generally be classed as markedly hypocellular (sparsely cellular), hypocellular, moderately cellular and cellular (Fig. 3.1). There are no universally accepted quantitative benchmarks for this; however, a quick visual guide is that if representative material is readily identifiable on low power (e.g. two times objective), it may be considered cellular. This semi-­quantitative guide is arbitrary and based mostly on one’s gen-

eral impression. Nevertheless, it is worth providing a quick descriptor of the cellularity of any given sample in one’s cytology report, as this may prove helpful on retrospective clinicopathologic review. Sometimes, smears can be cellular and yet non-diagnostic. For example, in an endoscopic ultrasound-guided FNA (EUS FNA) of a pancreatic mass, there may be numerous sheets of contaminant gastrointestinal epithelium or benign pancreatic parenchyma but no definite lesional tissue. An example of a hypercellular but non-­ diagnostic transrectal EUS FNA is shown in Fig. 3.1d in Chap. 3. Conversely, smears may be sparsely cellular and yet diagnostic. For example, smears from a colloid nodule in the thyroid gland can have little or even no well-preserved follicular cells and yet

a

b

c

d

Fig. 3.1  Cellularity. (a) Hypocellular smears in a thyroid FNA (HC, 60×); (b) Moderately cellular smears in a thyroid FNA (HC, 40×); (c) Cellular smear (HC, 40×); (d) Highly cellular but non-diagnostic transrectal endo-

scopic ultrasound-guided FNA sample showing only colonic epithelial cells (HC, 40×); inset shows a higher power view of a single colonic crypt (HC, 400×)

3.2  Elements of the Triple Approach

be reported as benign if abundant colloid is present. As a ‘soft’ clue, it may be prudent to know that certain tumours/conditions tend to lend themselves to greater cellularity, whilst some are known to be hypocellular. For example, in the thyroid gland, papillary carcinoma tends to feature highly cellular smears, whilst some cases of Hodgkin lymphoma, namely the nodular sclerosis subtype, might yield surprisingly hypocellular smears even from a markedly enlarged lymph node. Other sparsely cellular malignancies including cystic epithelial malignancies in which the lesional cells are few and far between. Architectural Arrangements An initial distinction between dispersed cells vs. cohesive sheets is very helpful. For example, hae-

23

matolymphoid cells are often discohesive, as are malignant melanoma cells; whilst epithelial cells tend to form cohesive sheets. This is a particularly useful approach in evaluating lymph node aspirates and will be described in detail in Chap. 6. When cells are more cohesive, the architectural arrangements formed may provide clues to the diagnosis. Some of the commoner architectural formations are described here and demonstrated in Fig. 3.2. • Acinar formations—cells arranged in a gland-­ like formation with polarised nuclei away from a central lumen, e.g. adenocarcinoma such as prostatic acinar adenocarcinoma. • Rosettes—small rounded groups of cells that form a spokewheel-like arrangement radiating from a central acellular focus. They may be

a

b

c

d

Fig. 3.2  Architectural arrangements. (a) Syncytial sheet of malignant cells in undifferentiated nasopharyngeal carcinoma (Pap, 300×); (b) Acinar architecture in prostatic carcinoma (Pap, 300×); (c) Trabecular architecture in

hepatocellular carcinoma, with endothelial wrapping at the periphery of the tissue fragment (Pap, 400×); (d) Single file arrangement in lobular breast carcinoma in fluid (HC, 200×)

24

•

•

•

•

•

•

•

3  The Triple Approach in Cytopathology: Its Practical Application

seen in neuroendocrine tumours and some small round blue cell tumours such as retinoblastoma. Syncytial sheets—cells arranged in cohesive sheets with nuclear overlapping and ill-defined cytoplasmic membranes, e.g. undifferentiated nasopharyngeal carcinoma (lymphoepithelioma-­ like carcinoma, Fig. 3.2). Trabeculae—elongated, cohesive nests of cells, e.g. follicular neoplasm of thyroid, hepatocellular carcinoma (Fig. 3.2). Strips of cells—rows of columnar cells arranged in a picket fence-like formation, may exhibit nuclear pseudostratification, e.g. papillary breast lesions, endocervical cells in cervical smears. True papillary structures—finger-like fibrovascular stromal cores, sometimes branching, covered by epithelial cells. The epithelium may be seen in direct contact with the stromal cores or adjacent to them if they are pulled off during the smearing process. Papillary structures can be seen in papillary thyroid carcinoma and papillary lesions of the breast. Nuclear moulding—cells arranged in tight clusters with nuclei being pressed up against one another, with minimal intervening cytoplasm, e.g. small cell carcinoma, ­ medulloblastoma. Single file arrangements—cells arranged in a single row, sometimes resembling a vertical column, e.g. small cell carcinoma, lobular breast carcinoma. Sheets of cells with peripheral nuclear palisading—cells arranged in cohesive clusters with the more peripherally placed nuclei arranged in parallel arrays, usually perpendicular to the edge of the sheet. This may be seen in basal cell neoplasms of the salivary gland or colorectal carcinoma.

Cytomorphological Features This refers to specific nuclear and cytoplasmic features of the cells, which can be systematically evaluated by looking at nuclei and cytoplasm.

• Nuclear features –– Size. –– Shape—e.g. spindle, round, oval, angulated, bizarre. –– Location in cell—central vs. eccentric. –– Nuclear membrane—smooth, irregular, presence of pseudoinclusions or grooves. –– Degree of pleomorphism. –– Appearance of nucleoli—small, prominent, single, multiple, central, peripheral, etc. –– Chromatin pattern—fine vs. coarse, granular, speckled, pale, powdery, marginated, hyperchromatic, etc. • Cytoplasm –– Amount—reflecting N/C ratios. –– Texture—bubbly, vacuolated, delicate, dense, granular, waxy, ill- or well-defined. –– Colour. –– Presence of blebs or inclusions. –– Presence of cytoplasmic material/pigment pertinent to cell differentiation, e.g. melanin (melanocytes), mucin (glandular cells), bile (hepatocytes), etc. • Other features –– Mitoses—relative frequency, whether abnormal. –– Apoptosis—condensed inky black pyknotic nuclei or karyorrhectic material. –– Psammoma bodies—these are reddish in alcohol-fixed smears and colourless and refractile in air-dried smears (Fig. 9.5, Chap. 9) and may be missed if not specifically sought. They may be seen in benign cells (e.g. reactive mesothelial proliferations) or malignant lesions (e.g. c­ arcinomas with papillary architecture such as papillary thyroid carcinoma and papillary serous carcinoma of the ovary). Background Material or Cells Many elements may be seen in the background of smears—both non-cellular and cellular. These can sometimes be very helpful in the gathering of clues to the final diagnosis. These elements may arise from the following sources:

3.2  Elements of the Triple Approach

• Extracellular material –– Artefacts—e.g. ultrasound gel, appearing as reddish purple, coarse particulate material that is fairly opaque and homogeneous, and may sometimes be mistaken for necrosis (Fig. 3.3). –– Secretions—e.g. mucin, colloid. –– Cyst material—granular proteinaceous material, cholesterol crystals (Fig. 3.3). –– Stromal material—this is particularly important in salivary gland tumours, as well as some soft tissue entities. Stroma may be described in terms of colour (e.g. magenta, metachromatic), texture

25

(e.g. fibrillary, myxoid) and in the way it interacts with cellular components. –– Crystals—these may be seen within colloid in the thyroid, in cyst fluid (e.g. cholesterol crystals), joint aspirates, urine, etc. • Material derived from cells –– Lymphoglandular bodies—these are extremely helpful in indicating the lymphoid nature of cells, but do not d­ ifferentiate between benign and malignant lymphoid lesions. –– Tigroid background—this is seen in association with neoplasms with abundant cytoplasmic glycogen such as germinoma (Fig. 3.3) and clear cell sarcoma of soft tissues.

a

b

c

d

Fig. 3.3  Background material. (a) Ultrasound gel (HC, 400×); (b) Necrosis, which may sometimes be seen in the form of ‘rolled up’ granular material (HC, 60×); (c) Lacy,

tigroid background due to the presence of glycogen in seminoma (HC, 400×); (d) Cyst contents with cholesterol crystals and macrophages (HC, 100×)

26

3  The Triple Approach in Cytopathology: Its Practical Application

–– Necrosis—certain smear patterns should prompt one to look for necrosis. In particular, this should be sought in aspirates from lymph nodes with granulomatous inflammation, neuroendocrine tumours and high-­ grade malignancies. • Inflammatory cells seen in the background –– Lymphocytes—these are important in the context of FNAs of the thyroid and salivary glands, e.g. when oncocytic cells are identified—the presence of lymphocytes may point to possible lymphocytic thyroiditis or a Warthin tumour. –– Macrophages—these may indicate that an aspirate is from a cystic lesion and also help to give an idea of adequacy in the context of exfoliative respiratory samples (e.g. sputum). –– Acute inflammatory cells. –– Eosinophils—these may be important to look for in specific clinical contexts, e.g. classical Hodgkin lymphoma, parasitic or other infections, allergic or other systemic conditions. –– Red blood cells—whilst often ubiquitous in FNAs, erythrocytes are worth mentioning in certain specific contexts. Examples include urine samples in patients with haematuria and in cerebrospinal fluid samples in which significant haemodilution might render the specimen inappropriate for cytologic evaluation. Even in FNAs, it can be helpful to distinguish between ‘fresh’ and ‘old’ blood—the latter usually signifies bleeding prior to aspiration, e.g. haemorrhage into a pre-existing benign cystic colloid nodule. Red blood cells (RBCs) in fresh blood usually are round and evenly stained, whilst RBCs from ‘old blood’ may be irregular and wrinkled and exhibit uneven staining.

3.2.3 Ancillary Tests Ancillary tests include those done not only in the pathology laboratory but also in the microbiology, biochemistry and diagnostic molecu-

lar laboratories. Molecular testing (e.g. FISH testing, KRAS mutational analysis) is feasible on cytologic material from smears and cell blocks. There are some practical considerations regarding ancillary testing. 1. When to collect material for ancillary testing? 2. What is the most suitable material to perform ancillary testing on? 3. What tests to order?

3.2.3.1 When to Collect Material for Ancillary Testing? Ancillary tests are often performed when the sample shows malignant cells, for instance, to define the type of malignancy or ascertain the primary source. When unsure, it is good practice to collect material for possible ancillary testing at the time of rapid on-site evaluation (ROSE). Whilst it may not be necessary to actually perform ancillary tests on every malignant case, it is prudent to collect material upfront so that the option is available. Here are some general scenarios in which collecting material for ancillary testing upfront is recommended. More specific instances will be detailed in later chapters. • Deep seated aspirations—e.g. Endobronchial ultrasound (EBUS)-guided FNAs of mediastinal or lung lesions, endoscopic ultrasound (EUS)-guided FNAs or image-guided percutaneous aspirations of intra-abdominal organs. A standard protocol to collect material for cell block for such cases is reasonable because of the procedural risks involved and also for logistical reasons of cost and scheduling. • Suspected malignancy—in these instances one should also bear in mind the potential need for ancillary tests for prognostication and prediction of response to specific agents. • Suspected infection—material can be collected fresh for microbiological investigations or special stains, or cell block for PCR. • Unusual or unexpected findings at ROSE, because there may be a need for diagnostic

3.2  Elements of the Triple Approach

workup using ancillary immunohistochemistry.

tests

27

such

as

However, there are some scenarios in which ancillary testing for malignant samples may not be necessary: • When a follow-up histologic biopsy is planned, e.g. in a lymph node FNA which is suspicious for lymphoma. Ancillary testing on the cytology sample should be minimised in order to avoid the duplication of tests. • When previous histology or cytology slides are available for direct comparison with recurrent or metastatic disease.

3.2.3.2 What Is the Most Suitable Material to Perform Ancillary Testing On? The optimal sample depends on the nature of the ancillary test that one plans to perform. Even though optimal sample collection should entail a dedicated pass for each type of ancillary test; when necessary, a single specimen may be divided into multiple parts. The needle can also be rinsed after every smear before being discarded, to increase sample yield. Table 3.1 provides an overview of how some FNA and fluid samples can be collected for various types of ancillary investigations. This is not a comprehensive list, and more details will be provided in the system-specific chapters. Note that more than one ancillary testing method may apply to the same suspected diagnoses, e.g. in lymph node aspirates in which non-Hodgkin lymphoma is suspected, both flow cytometry and cell block may be considered. The choice of ancillary test will ultimately depend on the pathologist as well as the laboratory practice protocol. The preferred tissue preparation type for performing immunohistochemistry is the cell block. The method of cell block preparation varies between laboratories, and the specific methodology is not within the scope of this text. In my institution, we use the plasma thrombin clotting method for centrifuged material from fluid samples; and, for FNAs, prefer to use clotted material

expelled from the needle, which can be directly fixed in formalin. When processed, these small tissue fragments may also provide some valuable information on tissue architecture, which is a bonus to cytologic interpretation. Whichever method is used, validation of immunohistochemistry should be performed and appropriate controls must be applied. For fluid samples such as effusions, cell blocks are superior to additional cytospin preparations for performing immunostains. The reasons for this are discussed in the effusions chapter (Chap. 9). In my practice, I have found that for FNAs, sterile saline is one of the most versatile collecting media because the cellular material can be triaged to microbiology, haematology (flow cytometry may be performed if the sample is processed without delay) and pathology (for cell block).

3.2.3.3 What Tests to Order? When deciding on which ancillary tests to perform, several pertinent questions may be asked: What Is the Most Discriminatory Panel I Can Apply? In cytology, the sample size is extremely limited and hence only the most discriminatory panel should be applied. As far as possible, tissue use should be maximised, particularly if the cytology sample is the only sample available, e.g. in lesions that are surgically inaccessible. • It is advisable to decide on what markers to order only after relevant clinical information is ascertained. For example, in a patient with a history of lung adenocarcinoma with a new supraclavicular lymph node, before performing TTF1 on the lymph node FNA cell block, ideally, one should ascertain if the original lung adenocarcinoma was TTF1-positive and not assume it to be the case. • In a patient with a known history of malignancy, knowledge of the source of the malignancy and the histologic type can help narrow down the panel of ancillary tests required. • In seeking out the most discriminatory immunohistochemical panel, an extremely helpful online

3  The Triple Approach in Cytopathology: Its Practical Application

28

Table 3.1  Types of Material for Ancillary Testing

Suspected diagnosis Neoplasms (including Hodgkin or non-­ Hodgkin lymphoma)

Non-Hodgkin lymphoma Infection

Material for ancillary testing Cell block

Needle rinse (cell suspension) Cell block

Freshly aspirated material

Unstained smears

Source specimen (initial collection method) • Fluid (e.g. pleural effusion); cyst material (e.g. pancreatic cyst) • FNA blood clots/ solid tissue pieces • FNA needle rinse in saline FNA needle rinse in RPMI/other solution • Fluid (e.g. pleural effusion); cyst material (e.g. pancreatic cyst) • FNA blood clots/ solid tissue pieces • FNA needle rinse in sterile saline • Aspirated purulent material • FNA needle rinse in sterile saline FNA unstained smears (alcohol-fixed) Aspirated pancreatic cyst fluid

Pancreatic cystic neoplasm

Pancreatic cyst fluid (neat) or supernatant after centrifugation for cell block

Chylothorax or pseudochylous effusion

Serous effusion fluid sample

Serous effusion fluid sample

Parathyroid lesions

Needle wash

FNA needle rinse in a fixed volume of saline

resource is Immunoquery (http://www.immunoquery.com/). This resource allows comparisons of immunoprofiles between different entities, complete with links to relevant references. I have found this to be an ­invaluable resource, for both clinical practice and self-learning. How is Cytology Different from Histology, in Terms of Interpreting Immunostains? When interpreting immunostains, it is important to ensure that one is evaluating the correct cell

Ancillary tests • Immunohistochemistry • Molecular testing (e.g. FISH, PCR-based testing)

Flow cytometry (performed in haematology laboratory) • Special stains for organisms • Molecular testing (e.g. PCR-based testing) performed in microbiology laboratory

Smears for special stains for organisms; culture and sensitivity; performed in microbiology laboratory Special stains, e.g. Ziehl Neelsen (ZN), Grocott methenamine silver (GMS) • Biochemical analysis, e.g. CEA, amylase levels (performed in biochemistry laboratory); • Molecular testing (e.g. KRAS mutation testing) • Biochemical analysis, e.g. chylomicrons, triglycerides (performed in biochemistry laboratory) Parathormone concentration analysis (biochemistry laboratory)

population. Here are some points to bear in mind that apply specifically to the evaluation of immunohistochemistry on cytologic samples. • Quantitative assessments should be made with caution. The cellular milieu of cell blocks differs from that of solid tissue biopsies in that lesional cells might be detached without surrounding stroma and may also be intimately admixed with non-lesional cells. For example, in a cell block from a pleural

3.3  Applying the Triple Approach

effusion, ­macrophages and mesothelial cells are often also present. Quantitative markers (e.g. hormone receptor markers in breast carcinoma) should be interpreted with this in mind. • Always consider the needle route. Sometimes, benign, non-lesional epithelial cells may be present in addition to lesional cells, and these should not be misinterpreted as positive staining in lesional cells. For example, in an EBUS-­ guided FNA of a mediastinal lymph node, benign TTF-1positive cells from the lung/airways may also be present in addition to malignant cells within the lymph node. The next section outlines the chronological steps that one may follow in applying the triple approach.

3.3

Applying the Triple Approach

Here is an outline of my approach to individual cases, and how elements of the triple approach can be applied at various points.

3.3.1 S  tart with the Most Basic Clinical Information –– Age, gender, source of sample. –– Perform a quick scan at low power, to get an idea of the type of pathology one is looking at. –– Looking at cases ‘blind’ also helps one to strengthen one’s appreciation of cytomorphology.

3.3.2 F  orm a Quick Diagnostic Impression This will guide the clinical questions to ask and the detailed cytomorphologic evaluation, as well as ancillary tests performed.

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3.3.3 P  erform a Careful Check of the Relevant Clinical History If Necessary –– This is not required for all cases, but may be helpful in certain situations, e.g. a pleural effusion showing an adenocarcinoma may prompt a quick check for any known malignancy as well as correlation with imaging to narrow down the possible primary sites. –– At times, the cytologic picture is at odds with the clinical picture—and these cases may require more digging—it has been my experience that the cells don’t lie!

3.3.4 Carefully Screen All Slides –– Evaluate the morphologic features on low and high power microscopy as highlighted above. –– Don’t forget to look for background cells and non-cellular material as well.

3.3.5 I f Additional Material Is Available, Decide on Whether Ancillary Tests Are Required –– Available material may be in the form of cell block for FNAs, needle or brushing rinse material, or additional fluid in exfoliative cytology samples. –– Order ancillary tests as required, based on both cytomorphologic features and relevant history/clinical findings. –– If a cell block sections do not initially yield material, my usual practice is to evaluate two to three more consecutive 4-μm-thick levels, because sometimes cutting slightly deeper into the block will reveal diagnostic material.

3.3.6 Consult a Colleague If Necessary –– If one is unsure of the diagnosis, it is prudent to consult an experienced colleague. We all encounter challenging cases from time to time, and one of the richest resources that one

3  The Triple Approach in Cytopathology: Its Practical Application

30

can tap on is the experience of one’s colleagues. I have found that consultations with colleagues can be of great benefit to the diagnostic workup as well as my own learning. –– Of course, at times, opinions may differ significantly! In these instances a literature search may prove illustrative, as well as a deeper dig into the clinical notes. Differing opinions may be discussed with the clinical team, and, if required, an additional, external expert opinion may be sought. –– A word of caution regarding case consults—if one would like to include the name of the consulted pathologist in the report, permission should be sought. This is important as an act of professional courtesy and also for medicolegal reasons, because the consulted pathologist may then prefer to view the whole case and read the full final cytology report.

3.3.7 Craft the Cytology Report Incorporate morphologic features (including gross features where relevant, e.g. cyst fluid appearance), ancillary test results and relevant clinical findings. Include comments where necessary. The next and final section shows the example of a case and the actual application of the triple approach.

3.4

Outline of a Case

We have outlined the importance of the individual elements of the triple approach. But how do we actually begin to work on a case? Here is a real-life case with a step-wise description of the diagnostic workup.

Case History

• 67-year-old man with a direct FNA of a left supraclavicular swelling. • 2 passes yielding 4 slides—2 alcoholfixed, 2 air-dried smears. • There was no fluid aspirated, and no specific gross morphologic features of note. A cell block is being processed.

1. Perform a quick review of the slides to form an initial impression. The smear findings are shown in Fig.  3.4. What are your findings? Findings: • Branching tissue fragments composed of polygonal cells with relatively rounded, uniform nuclei and moderately abundant pale cytoplasm. • Higher power view reveals the presence of many small cytoplasmic vacuoles. 2. Form a quick diagnostic impression Impression: Metastatic carcinoma with clear cell morphology. • Differential diagnosis for metastatic clear cell carcinoma. –– Renal cell carcinoma (commonest scenario and hence first differential). –– Other clear cell tumours in a male patient: Adrenocortical carcinoma. Hepatocellular carcinoma. 3. Perform a careful check of the relevant clinical history • Any history of malignancy? (in particular, renal cell carcinoma). –– Medical records reveal a history of renal cell carcinoma, resected 7  years ago in another institution. • Any relevant imaging? –– No recent imaging investigations performed. • Any available previous histology slides or reports? –– The previous histology report reveals a clear cell renal cell carcinoma, Furhmann grade 2–3. 4. A cell block is available. Formulate your plan for ancillary testing There are two options for further evaluation: • Perform relevant immunohistochemical stains to confirm the impression. Recommended stains: RCC, PAX8 (±CD10—this is less specific). These should be sufficiently confirmatory in the appropriate clinical and morphological context.

3.4  Outline of a Case

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a

b

c

d

Fig. 3.4  Case example part 1. Cytologic features. (a, b) Medium low power views showing architectural features (HC and Pap, 100×); (c, d) Higher power views showing cytomorphologic features (HC, 300× and Pap, 400×)

• Alternatively, review previous histology slides if available. If the morphologic findings are similar in both the previous and current sample, a diagnosis of metastatic renal cell carcinoma may be made in the appropriate clinical and radiologic setting, even without immunostains.

Figure 3.5 shows the cell block with relevant immunohistochemical stains. 5. Generate the cytology report, incorporating relevant comments if necessary Sample of final diagnostic line with relevant comments:

3  The Triple Approach in Cytopathology: Its Practical Application

32

a

b

c

d

Fig. 3.5  Case example part 2. Ancillary tests on cell block. (a) Cell block showing a sheet of polygonal cells with abundant clear cytoplasm (HE, 300×); (b) IHC with

CD10 (300×); (c) IHC with RCC showing mostly membranous staining (400×); (d) IHC with PAX8 showing classical nuclear staining (400×)

• Left supraclavicular swelling, FNA: –– Metastatic clear cell carcinoma, consistent with metastatic clear cell renal cell carcinoma • Comments: The history of previously resected clear cell renal cell carcinoma is noted. The cytomorphologic and immunophenotypic features are in keeping with metastatic renal cell carcinoma. Please correlate with relevant clinical and radiological findings.

• It is good practice to do an initial quick screen without first looking at the detailed history, to form an unbiased cytologic impression. • Microscopic evaluation of cytologic material should be systematic—cellularity; architectural arrangement of cells; specific cytomorphologic features and any helpful background findings. • Knowing the reason for the biopsy and clinical picture helps one to focus on what to look for and narrow down ancillary tests. • When applying and evaluating ancillary tests such as immunohistochemistry, remember that there may be a more mixed cellular composition in cell blocks.

Take Home Points

• The three facets of cytomorphology, clinical information and ancillary testing form the basis of the triple approach.

4

On-Site Evaluation and the Provisional Report

Contents 4.1

Introduction

 33

4.2  ample Adequacy and Triage S 4.2.1  Sample Adequacy 4.2.2  Specimen Triage

 33  33  35

4.3  rovisional Diagnosis P 4.3.1  Information That a Provisional Report May Include

 36  36

4.4

Sample Case for Provisional Report

References

4.1

Introduction

One of the enduring advantages of cytology is the ability to perform rapid on-site evaluation (ROSE). ROSE may be performed for FNAs and core biopsy imprints, the latter being an excellent means of evaluating the adequacy of sampling in core biopsy specimens. This discussion on ROSE applies to conventional smears rather than liquid-­based cytology, in which aspirated material is directly expelled into preservative solutions. In many laboratories, air-dried smears stained with Romanowsky stains are used for ROSE.  Other methods of staining may also be employed, such as toluidine blue, rapid haematoxylin and eosin or rapid Papanicolaou stain on alcohol-fixed smears. There are two main reasons for on-site evaluation, which may vary depending on institutional practice:

 36  38

1. Sample adequacy and triage 2. Provisional diagnosis Practical points that are relevant to evaluating adequacy, performing triage and crafting a provisional report are discussed here, with an example of a case for provisional reporting presented at the end of the chapter.

4.2

Sample Adequacy and Triage

ROSE may be performed by either the cytologist (cytotechnologist) or the pathologist.

4.2.1 Sample Adequacy A sound appreciation of normal histology and normal cytology is important for ROSE.  For deep-seated FNAs such as endobronchial (EBUS)

© Springer Nature Singapore Pte Ltd. 2021 M. E. Nga, Practical Diagnostic Approaches in Non-Gynaecologic Cytology, https://doi.org/10.1007/978-981-15-2961-0_4

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4  On-Site Evaluation and the Provisional Report

or endoscopic ultrasound (EUS)-guided FNAs, a be required for ancillary testing. Therefore, basic understanding of the anatomical relationan adequate sample would ideally include ships between organs, and hence, the needle sufficient material for both cytologic evaluroute is also very important. In these contexts, ation and ancillary testing (e.g. flow cytomone should remember that a highly cellular smear etry and/or cell block, the latter for does not equate to an adequate smear because the immunohistochemistry and molecular needle may sample normal structures (e.g. brontesting). chial wall, gut wall) before passing into the • Active Communication Between the lesional area. An example of a cellular yet non-­ Cytology and Clinical Teams Is Important diagnostic smear is shown in Chap. 3, Fig. 3.1d. –– In likely infectious lesions, additional fresh It is also important to appreciate that the critetissue should be obtained for microbiologic ria for adequacy may differ in different scenartesting, and this should be relayed to the ios, depending on the extent of ancillary testing procedurist, so that more passes can be that is required on the cytologic sample. In the made. most practical clinical context, an adequate sam–– In a lesion in which the smear yields maligple is one that is sufficient not only for cytologic nant cells, it is reasonable to ask the prievaluation but also for ancillary testing, which mary clinical team if a larger histologic may be for diagnostic, prognostic or predictive biopsy will be performed, in order to have purposes. a better idea of the adequacy requirements Below are some points that are helpful to bear for that cytology sample. in mind when performing ROSE for specimen If there remains some uncertainty, one adequacy. can convey to the procedurist that the sample is ‘adequate for cytologic evalPractical Tips in Evaluating Specimen uation only, but more passes are Adequacy required if ancillary testing is needed, e.g. for tumour suptyping or • The Context of the Sampling Procedure Is prognostication’. Important –– Generally, it is safest to assume that ancilKnowing the context of the procedure helps lary testing is required in a malignant lesion one to ascertain if ancillary tests are needed on and obtain material for cell block whenever this particular sample. Two examples are propossible. As mentioned earlier, the stanvided here. dard practice in my institution is to obtain –– Example 1: FNA of a superficially located material for cell blocks for all deep-seated lymph node that is clinically suspicious for lesions and preferably for superficial FNAs lymphoma. An adequate sample simply that yield malignant cells. requires sufficient lesional cells to confirm • Reporting Adequacy and Uncertainty the clinical impression, and ancillary testAdequacy may be reported in three ing may not be necessary on this cytologic categories: sample if excision biopsy is to follow. In 1. Adequate this instance, cytology serves more as a tri 2. Inadequate age than as a diagnostic test—this choice 3. Descriptive when uncertain (this category depends on one’s institutional practice. should be minimised) –– Example 2: A retroperitoneal lymph node In most instances, the sample is either adethat is difficult to sample histologically. If quate or not. However, there are some ROSE reveals atypical lymphocytes, a furinstances in which there may be uncertainty. ther histologic biopsy may not be forthIn these instances, close communication with coming; hence, more cytologic tissue will the procedurist is particularly important. Here

4.2  Sample Adequacy and Triage

35

too much material is initially expelled onto are some scenarios in which it may be chalthe slide, excess material might be re-­ lenging to ascertain sample adequacy: aspirated back into the needle and saved for –– When imaging does not reveal a definite discytospin preparations or cell block. crete mass lesion, e.g. a bulky head of pan–– For air-dried smears, if there are visible creas, or a vague ill-defined mass. In these clots on the smear, it may be possible to instances, when the aspirate yields only norpick them up with a needle tip and save mal parenchyma, the on-site cytologist/ them for cell block. pathologist should communicate this clearly to the procedurist, and it is acceptable to write • Specimen Triage Should Be Individualised and Maximised a descriptive report such as ‘Benign pancre–– The choice of ancillary tests varies with the atic parenchyma; correlate with imaging’. site of the sample and the suspected Whether or not this is adequate sample ultidiagnosis. mately rests on close clinical and radiologic – – Material may be collected in many ways correlation. for ancillary testing. Some examples of –– In an endoscopic ultrasound-guided FNA of a specimen collection for triage have been cystic pancreatic lesion in which the needle shown in Table 3.1 in Chap. 3. traverses the gastric wall. In this context, gas–– It is prudent to be familiar with specific tric mucinous epithelium may be difficult to collecting protocols from various laboradistinguish from lesional tissue from a low-­ tory services that are available in one’s grade mucinous neoplasm. This difficulty clinical practice, in order to maximise should be related to the procedurist, who is specimen yield. usually able to rely on other parameters to – – Material that may be collected includes: assess adequacy, such as intraprocedural imaging findings and gross appearance of • Needle rinse in sterile saline (for microaspirated material. biological or biochemical tests or pro• When Adequacy Is Borderline cessed into cell block). When adequacy is borderline, it is prudent to • Clotted material expelled from the neeask for more passes where possible, to ensure dle, collected on a slide or specimen as far as possible that there truly is sufficient bottle (best for processing into cell material for diagnostic evaluation. block, for immunohistochemistry (IHC) and/or molecular testing). • Fluid that is directly aspirated, e.g. 4.2.2 Specimen Triage purulent material or cyst fluid (for biochemical analysis, microbiological tests Specimen triage consists of collecting and partior preparing additional cytospin tioning material for ancillary testing (e.g. cytolsmears). ogy, microbiology, biochemistry, haematology, • Unstained smears (for special stains, molecular testing). This has been previously e.g. for micro-organisms, or for fluoreshighlighted in Chap. 3. cent in-situ hybridisation [FISH]). Here are some practical points to note regard–– Fresh material is required for microbioling specimen triage: ogy, biochemical analysis and flow cytometry. Most other ancillary tests may be • Tissue Wastage Should Be Minimised performed on fixed material, e.g. IHC and –– For FNAs, the amount of material smeared molecular tests including PCR-based tests onto the slide should be small—a droplet and FISH.  Material from direct smears is of no more than 2–3 mm in diameter is sufalso suitable for molecular tests. ficient for preparing optimal smears. When

4  On-Site Evaluation and the Provisional Report

36

4.3

Provisional Diagnosis

In this context, the on-site evaluation is performed by a pathologist. The FNA may be performed by a clinician, pathologist or interventional radiologist. A provisional diagnosis is rendered upon quick evaluation of the air-dried smears, following which the clinician can decide on the next management step. Provisional reporting may be part of a one-stop diagnostic clinic, for example a breast clinic or pathologist-driven FNA clinic. A systematic classification system should be considered for provisional reports. Such systems have been proposed by Asharif M et  al. at the University of Minnesota [1] and Nayak A et al. in New York [2]. Both the authors reviewed the usefulness of ROSE in the context of evaluating mediastinal lymph nodes using EBUS-guided FNA and proposed a five-tier classification system. An example of such a five-tier system is: –– –– –– –– ––

Non-diagnostic Benign Atypical Suspicious for malignancy Malignant

During the time of evaluation for a provisional report, any uncertainty should be clearly conveyed to the clinician, particularly if this impacts the next step of management or investigation. For example, in a lymph node aspirate, there may be difficulty deciding if the malignant cells are those of a large or anaplastic cell lymphoma or a poorly differentiated carcinoma. In these instances, a differential diagnosis may be provided, and the findings clearly conveyed to the clinician in writing, and, preferably, also through direct communication. Often, the final cytology report will provide a definitive diagnosis, with the help of ancillary tests. It is far better for the diagnosis to be deferred to the final definitive diagnosis than for an erroneous diagnosis to be given in the provisional report, thereby resulting in inappropriate management or follow-up investigation.

4.3.1 Information That a Provisional Report May Include • Patient details, site and route of aspiration • Clinical findings (especially if the FNA is performed by the pathologist) • No. of passes • No. of slides evaluated (specify preparation type—usually air-dried smears) • Gross appearance of aspirated material (if applicable) • Summary of salient cytologic findings • Diagnostic category and specific diagnosis • Comment if necessary –– Recommending next step in investigation/ management –– Any other comments, e.g. reflecting uncertainty, differentials –– Documentation of communication with clinician –– Documentation of specimen triage if applicable (e.g. needle wash sent to microbiology laboratory) The next section will provide a walk-through of a case for provisional reporting, in which the FNA is performed by the pathologist.

4.4

 ample Case for Provisional S Report

Here is a sample case scenario in which a provisional report is requested by the clinician.

Case History

You are called to the pathologist FNA clinic to perform an FNA and provide a quick provisional diagnosis. The patient is a 45  -year-old man who presents with left cervical lymphadenopathy for 4  weeks. Examination reveals a slightly tender, 1.5 cm left-sided lymph node at level 3. A representative picture of the air-dried smear is shown in Fig. 4.1.

4.4  Sample Case for Provisional Report

37

Fig. 4.1  Sample case for provisional report. Air-dried smear of left cervical lymph node FNA (HC, 200×)

Q1: What is your impression? A: There is granulomatous inflammation on a background of acute inflammation. Necrosis is seen elsewhere in the smear. This is likely to be due to an infectious aetiology, with a mycobacterial infection (i.e. tuberculous lymphadenitis) being high up in the list of differentials. Q2: How would you proceed, after noting the findings from the first pass? A: After ensuring that the cytology team has donned appropriate personal protective equipment (e.g. N95 mask, gloves), I would perform one to two more passes to collect material for ancillary testing. The material would be triaged according to the steps below. It is preferable to perform dedicated passes for ancillary testing, rather than to only use needle washes from leftover material from passes. Aspirated material from second (±third) pass: – Needle rinse in sterile saline or neat purulent fluid (if present) sent to the microbiology lab without delay. – If there is sufficient additional material, a cell block may be considered for histochemical stains for micro-organisms and PCR. – If sufficient material, one unstained air-dried smear (for ZN stain). Q3: What would you write in your provisional report? A: Patient identifiers. Direct FNA of left cervical lymph node. Brief clinical findings: Patient noticed left cervical lymphadenopathy for 4 weeks, non-progressive. On examination, there is a mildly tender, mobile 1.5 cm left level 3 lymph node. Two passes performed. Less than 1 mL of yellowish purulent material aspirated. One air-dried smear evaluated. Smear shows well-formed epithelioid granulomas with necrosis and acute inflammation. No malignant cells seen. Diagnostic category: Benign. Provisional diagnosis: Necrotising granulomatous lymphadenitis; an infectious aetiology, in particular, a mycobacterial infection, should be excluded. Comment: Needle wash sent to microbiology laboratory; clinician Dr. J. Smith was informed of findings at (date and time).

4  On-Site Evaluation and the Provisional Report

38

Take Home Points

• ROSE serves the purposes of determining specimen adequacy and allowing for specimen triage. • In some instances, a provisional report is requested by clinicians. This should include a diagnostic category and the specific diagnosis as far as possible. • Close and active communication with the clinical team is essential in ROSE, particularly when there is uncertainty

References 1. Al-Qurayshi Z, Deniwar A, Thethi T, Mallik T, Srivastav S, Murad F, et al. Association of Malignancy Prevalence With Test Properties and Performance of the Gene Expression Classifier in Indeterminate

regarding specimen adequacy or diagnosis. • Initial sample preparation is important and should focus on maximising the sample yield. • Where possible, material should be collected for ancillary testing, particularly in malignant aspirates and aspirates obtained using more invasive methods such as endoscopic imageguided FNA.

Thyroid Nodules. JAMA Otolaryngol–Head Neck Surg. 2016. 2. Nayak A, Sugrue C, Koenig S, Wasserman PG, Hoda S, Morgenstern NJ. Endobronchial ultrasound-guided transbronchial needle aspirate (EBUS-TBNA): a proposal for on-site adequacy criteria. Diagn Cytopathol. 2012;40(2):128–37.

Part II System-Specific Approaches in Cytopathology

Introduction to Part II In Chaps. 5 to 9, practice-driven approaches specific to organ sites will be covered. Each chapter will be organised into sections which incorporate the triple approach, specific challenging areas and/or pitfalls. The discussions will be based on conventional cytology. Here is an outline of how each chapter is organised: • Most chapters will begin with an overview of main diagnostic entities within that organ or site, paying particular attention to the limitations of cytology in that specific context. • We then move on to the triple approach, which has been introduced in the previous chapters. In the cytomorphology section, a framework will be setup which includes definitions and descriptions of various morphologic features moving from lower to higher power—architectural features, cytomorphologic features (e.g. nuclear and cytoplasmic features) and background findings. • Thereafter, a system of algorithmic approaches will be provided, focusing on specific common and challenging areas. These charts are not meant to be comprehensive, but rather to be used as a basis for building up one’s approach in one’s own clinical setting. • Lastly, specific pitfalls will be discussed, highlighting some of the tips and clues that can help us navigate these pitfalls. It is suggested that the practical diagnostic approaches outlined here are best complemented by more comprehensive cytology reference texts that elaborate on detailed morphologic features. In this way, a balanced and working understanding of the diagnostic process can be appreciated from start to finish. Some examples of these texts are provided in the introductory pages of this book (About the Book).

5

Approach to Thyroid Cytology

Contents 5.1

Introduction

 42

5.2 5.2.1  5.2.2  5.2.3 

Main Diagnostic Entities and Classification Systems  ain Diagnostic Entities M NIFTP: A Recently Defined Entity Major Classification Systems in Thyroid Cytology

 42  42  43  43

The Bethesda System for Reporting Thyroid Cytology Non-diagnostic Benign Atypia of Undetermined Significance (AUS)/Follicular Lesion of Undetermined Significance (FLUS) 5.3.4  Follicular Neoplasm (FN)/Suspicious for Follicular Neoplasm (SFN) 5.3.5  Suspicious for Malignancy (SM) 5.3.6  Malignant

5.3 5.3.1  5.3.2  5.3.3 

 44  44  45  45  45  45  46

5.4

What Cytology Can’t Diagnose in the Thyroid

 46

5.5 5.5.1  5.5.2  5.5.3 

Brief Technical Notes Needle Gauge Ultrasound Guidance Collecting Material for Ancillary Tests

 47  47  48  48

5.6 5.6.1  5.6.2  5.6.3 

Triple Approach Clinical Information Cytology (Gross and Microscopic Evaluation) Ancillary Tests

 48  49  51  65

5.7 5.7.1  5.7.2  5.7.3  5.7.4 

Specific Challenges Cystic Lesions Hurthle Cell-Rich Lesions Lymphoid-Rich Lesions (When to Diagnose Lymphoid Atypia?) The Atypical FNA: When to Call Something FLUS/AUS?

 67  67  68  69  70

5.8 5.8.1  5.8.2  5.8.3 

Pitfalls False Negatives/Undercalls False Positives Mimics

 73  73  74  76

References

© Springer Nature Singapore Pte Ltd. 2021 M. E. Nga, Practical Diagnostic Approaches in Non-Gynaecologic Cytology, https://doi.org/10.1007/978-981-15-2961-0_5

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41

5  Approach to Thyroid Cytology

42

5.1

Introduction

5.2

Thyroid FNAs are one of the most frequently encountered cytological specimens. The FNA is a highly accurate diagnostic test for benign and malignant nodules of the thyroid. This chapter aims to provide one with the baseline knowledge of what can and can’t be diagnosed on thyroid cytology and to equip one with a framework of how to approach certain challenging aspects of thyroid cytology. First, we will walk through the main classification systems that are currently in use, followed by a look at specific aspects of the triple approach. Within the triple approach, special emphasis will be placed on cytologic evaluation, moving from low to high power features. Subsequently, challenging areas will be discussed with algorithmic approaches. We will also establish a threshold of what reasonably constitutes an ‘atypical’ nodule. Lastly, specific pitfalls will be highlighted, together with tips and clues that are helpful in avoiding misinterpretation.

 ain Diagnostic Entities M and Classification Systems

5.2.1 Main Diagnostic Entities A good place to start is by looking at the spectrum of main diagnostic entities in the thyroid gland that present as nodules or swellings (Chart 5.1). For rarer entities, e.g. spindle epithelial tumour with thymus-like differentiation (SETTLE), the reader is referred to other comprehensive cytology texts. It should be noted that whilst the commonest cause of a thyroid nodule, the colloid nodule, is readily diagnosed on FNA, there are some entities that cannot be definitively diagnosed on cytology. This may be due to specific architectural criteria for diagnosis that require histologic examination (e.g. capsular or vascular invasion in follicular adenoma vs carcinoma), or the need for ancillary tests (e.g. medullary thyroid carcinoma). These entities are noted in Chart 5.1 and Table 5.2 and will be highlighted in the section on what cytology can and can’t diagnose.

Thyroid nodules

Non-neoplastic

Neoplastic

Benign/Indolent neoplasms • Benign follicular nodule • Colloid nodule • Hyperplastic nodule • Benign follicular nodule (NOS) • Thyroiditis (thyroid may be asymmetrically enlarged) • Lymphocytic thyroiditis • Subacute granulomatous thyroiditis (DeQuervain thyroiditis) • Infectious thyroiditis • IgG4-related disease**

• Follicular adenoma** • Hurthle cell adenoma** • Non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP)** • Hyalinising trabecular tumour*

Malignant neoplasms • Follicular cell origin • Papillary carcinoma • Follicular carcinoma** • Poorly differentiated carcinoma** • Anaplastic carcinoma • Medullary carcinoma* • Lymphoma*

• Metastases*

Key: ** Cytology usually can’t definitively diagnose, histology required. *Challenging to definitively diagnose on cytology (may be possible with ancillary testing). NOS: Not otherwise specified NIFTP: Non-invasive follicular thyroid neoplasm with papillary-like nuclear features

Chart 5.1  Thyroid—main diagnostic entities

5.2  Main Diagnostic Entities and Classification Systems

The non-invasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP), a fairly recently described entity, is discussed here, focusing on its impact on thyroid cytology.

5.2.2 N  IFTP: A Recently Defined Entity The NIFTP came to prominence in 2016 when it appeared in the New York Times [1], after being featured in JAMA Oncology [2]. Essentially, NIFTP is the entity that used to be diagnosed as encapsulated follicular variant papillary thyroid carcinoma (FVPTC), without capsular or vascular invasion. In Nikiforov et al.’s seminal publication in JAMA Oncology, it was found that when strict diagnostic criteria were applied, the clinical outcome of NIFTP was indolent (no evidence of disease after a minimum follow-up of 10  years, n  =  109), compared to encapsulated FVPTC with capsular or vascular invasion, in which 12% of 101 patients suffered adverse events (e.g. recurrence, metastatic disease, nodal recurrence) [2]. Hence, they recommended that lobectomy was sufficient treatment for NIFTP. This work also led to the reclassification of NIFTP as a non-­ malignant entity, hence the removal of the term ‘carcinoma’. As some of the key diagnostic criteria for NIFTP—absence of capsular or vascular ­invasion—are histological, this entity is not diagnosable on cytology. NIFTP, along with FVPTC, may be suspected on cytology when there is microfollicular architecture together with some nuclear features that raise a concern for papillary thyroid carcinoma (PTC). These cases should not be classified as cytologically malignant, because this may result in overtreatment with near total thyroidectomy. In the next section, the Bethesda System for Reporting Thyroid Cytology will be discussed, and it should be noted that possible NIFTPs ­usually fall into the more indeterminate categories (III–V).

43

5.2.3 M  ajor Classification Systems in Thyroid Cytology There are several classification systems that are currently in use for reporting thyroid cytology, and these are presented in Table 5.1. There will of course be variations between different laboratories in terms of whether an in-house system is being used or one of the national/international systems presented here. Regardless of the system that is in use, it is important for pathologists and clinicians to speak the same language because this facilitates a logical management plan and also provides the basis for auditing diagnostic performance within and across institutions. Table 5.1 shows three systems that are in use in different parts of the world, with brief recommendations which have been provided by the authors of each system. This is not a comprehensive listing, and other systems such as the Papanicolaou system also exist [3]. One of the most widely used classification systems worldwide is The Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), which was originally presented in 2009 [8] and updated in 2018 [7, 9]. As with other systems that are currently in use, the original discussion included a multidisciplinary team of clinicians and radiologists in addition to pathologists, highlighting the importance of a shared understanding of the proposed diagnostic terminology. I will be using TBSRTC in this chapter, as it is familiar to many practicing pathologists, cytologists and clinicians globally. In the 2018 updated version, the diagnostic categories and morphologic criteria for each category are unchanged; however, there are a few minor points of difference which will be highlighted below. The main updates are the actual figures for the risk of malignancy (ROM) in each category, the impact of NIFTP on this classification system, and the additional recommendation of molecular testing in two of the indeterminate categories. These changes have been summarised in Table 5.1. A brief summary of each category and the main updates is presented next.

5  Approach to Thyroid Cytology

44 Table 5.1  Major Classification Systems in Thyroid Cytology BTA/RCPath Classification System (2016 revision) [4] Non-diagnostic—Thy 1/Thy 1c (“c” refers to cystic lesions)

Non-neoplastic—Thy 2/Thy 2c

Neoplasm possible—Thy 3 Two subcategories: 1. Thy 3a: samples that exhibit cytological/nuclear or architectural atypia and raise the possibility of neoplasia; or suboptimal samples with mild cytologic atypia Note: ‘In many cases, a repeat thyroid cytology sample is able to be placed into a more definitive category’ and 2. Thy 3f: samples suggesting follicular neoplasms Note: ‘… a repeat may help clarify the exact diagnostic category. Review of the cytology and/or MDT discussion locally or centrally may be of use to help in patient management’ (in most instances, the expected management for Thy 3f is lobectomy) Suspicious of malignancy— Thy 4

RCPA/ASC Australasian Classification System 2014, updated in 2019 [5] Non-diagnostic (includes cyst contents only without sufficient colloid or well-preserved follicular cells) Recommendation: Repeat FNA with ultrasound guidance Benign

Indeterminate/follicular lesion of undetermined significance Estimated risk of malignancy: Very low Recommendation: Repeat FNA after 3 months or shorter, depending on clinical circumstances. In cases with a concerning lymphoid population, repeat the FNA with material for flow cytometry Suggestive of a follicular neoplasm Estimated risk of malignancy: Low to moderate Recommendation: Refer to specialist surgeon

Suspicious of malignancy

Recommendation: Specialist referral Malignant—Thy 5

Malignant Recommendation: Specialist referral as appropriate

The Bethesda System for Reporting Thyroid Cytopathology 2018 [6, 7] Non-diagnostic or unsatisfactory Recommendation: Repeat FNA with ultrasound guidance Benign Recommendation: Clinical follow-up and correlate with sonographic findings Atypia of undetermined significance/follicular lesion of undetermined significance Risk of malignancy: –  10–30% pre-NIFTP –  6–18% post-NIFTP Recommendation: Repeat FNA, molecular testing or lobectomy (e.g. Afirma Gene expression classifier (GEC)—a benign result provides a high negative predictive value) Follicular neoplasm/suspicious for follicular neoplasm Risk of malignancy –  25–40% pre-NIFTP –  10–40% post-NIFTP Recommendation: Lobectomy or molecular testing (e.g. Afirma Gene expression classifier (GEC)—a benign result provides a high negative predictive value) Suspicious for malignancy Risk of malignancy –  50–75% pre-NIFTP –  45–60% post-NIFTP Recommendation: Near total thyroidectomy or lobectomy Malignant Recommendation: Near total thyroidectomy or lobectomy

BTA British Thyroid Association, RCPath Royal College of Pathologists, United Kingdom, RCPA Royal College of Pathologists of Australasia, ASC Australian Society of Cytology

5.3

 he Bethesda System T for Reporting Thyroid Cytology

‘­ Non-­ diagnostic; Insufficient material for cytologic evaluation’. –– Examples include quantitatively insufficient material (e.g. blood only, or cyst contents only 5.3.1 Non-diagnostic without follicular cells or colloid, fewer than 6 groups of 10 follicular cells) or poor quality of –– This term is used when there is insufficient wellmaterial (e.g. entrapment in blood clots, air-­ preserved material for cytologic evaluation. drying artefacts). –– The reason for the sample being non-­ –– Management: A suggestion in the 2018 diagnostic should always be stated, e.g. TBSRTC is to perform cell block in liquid-­

5.3  The Bethesda System for Reporting Thyroid Cytology

based cytology (LBC) preparations, which may sometimes yield diagnostic material. –– The suggested interval for repeat FNA, previously recommended to be ‘no sooner than 3 months’, has now been removed, due to insufficient evidence of overdiagnosis due to ­reactive or reparative changes in repeat FNAs performed within the 3-month period.

5.3.2 Benign –– As in the 2008 TBSRTC, this category includes benign follicular nodules (e.g. colloid nodules, hyperplastic nodules); DeQuervain (subacute granulomatous) thyroiditis and lymphocytic or other inflammatory thyroiditides. –– The presence of abundant colloid is sufficient for a diagnosis of a colloid nodule, even in the absence of 6 groups of at least 10 follicular cells each. –– Follow-up depends on imaging findings (as reflected in the American Thyroid Association Guidelines) [10], regarding interval of follow­up and need for repeat FNA.

5.3.3 Atypia of Undetermined Significance (AUS)/Follicular Lesion of Undetermined Significance (FLUS) –– The literature has shown that the true incidence of this diagnostic category exceeds the initial 2008 recommended incidence of up to 7%; hence, the recommended incidence has been revised upwards up to 10%. –– Post-Bethesda studies have shown that the presence of nuclear atypia is associated with a significantly higher ROM than architectural atypia alone because of the greater risk of PTC, hence its presence should be mentioned in the cytology report [11–14]. Nodules falling into this category are discussed in more detail later in Sect. 5.7 (Specific Challenges). –– Atypical lymphoid lesions are also included in this category, and these will be discussed later in Sect. 5.7 as well. –– ROM: The ROM is presented as both pre-­NIFTP and post-NIFTP figures, as seen in Table 5.1. It

45

is important to note the reduced incidence of malignant outcomes when NIFTPs are removed from the malignant outcome category (“postNIFTP”). This reduction would very much depend on the incidence of NIFTPs in individual laboratories—Bychkov et  al. showed that the reduction in ROM within Bethesda categories was lower in Asian countries with the exclusion of NIFTPs from the malignant outcome group [15]; hence, there is a need for individual laboratories to perform internal follow-up audits in this indeterminate category. –– Management: Repeat FNA, or molecular testing with a “rule out” test—i.e. the Afirma gene expression classifier (GEC). If the result is negative/benign, this accurately predicts a benign outcome [16].

5.3.4 F  ollicular Neoplasm (FN)/ Suspicious for Follicular Neoplasm (SFN) –– In addition to cases comprising microfollicular and trabecular formations with minimal colloid, cases in which FVPTC or NIFTP is suspected would often also fall into this category. –– ROM: Similar to the AUS/FLUS category, in the 2018 TBSRTC revision, the ROM in FN/ SFN is presented as both pre-NIFTP and post-­ NIFTP figures, shown in Table 5.1. In general, the ROM may fall between 10 and 40%. –– Management: Lobectomy or, alternatively, molecular testing with the Afirma GEC, as a ‘rule out’ test, due to its high negative predictive value within this category.

5.3.5 Suspicious for Malignancy (SM) –– Cases that may fall into this category include suspected FVPTC/NIFTP with well developed PTC-like nuclear features, suspected MTC without IHC confirmation and other suspected malignancies without sufficiently diagnostic cytologic features. –– ROM: Follow-up studies performed in the pre-NIFTP era yielded a high ROM of

5  Approach to Thyroid Cytology

46

50–75%; which, in Western studies, has fallen to 45–60% in the post-NIFTP era (Table 5.1). –– Management: Lobectomy or near total thyroidectomy.

5.3.6 Malignant –– Malignancies that can be diagnosed cytologically include PTC, medullary thyroid carcinoma (MTC) in the presence of confirmatory immunohistochemistry on cell block, anaplastic carcinoma, lymphoma (high grade lymphomas in particular), as well as metastases. Poorly differentiated thyroid carcinoma (PDTC) may be suggested but might exhibit considerable cytologic overlap with follicular neoplasms; this is discussed later in Sect. 5.8 (Pitfalls). –– TBSRTC 2018 recommends that strict architectural and cytomorphological diagnostic criteria be applied for a definitive cytologic diagnosis of papillary thyroid carcinoma. This is to avoid overdiagnosis and overtreatment in cases that turn out to be NIFTP. –– Management: Near-total thyroidectomy or lobectomy. For a more detailed reference to the updates in the 2018 TBSRTC, one is referred to the literature [7].

5.4

What Cytology Can’t Diagnose in the Thyroid

In reality, there are several entities that are not readily diagnosable on cytology. One reason is that the intrinsic nature of the FNA test precludes evaluation of architectural features such as capsular or vascular invasion, e.g. follicular/Hurthle cell adenoma and carcinoma. Another reason is that at times, ancillary testing is required to make a definitive diagnosis. For example, in MTC, cytology alone is able to raise a strong suspicion; however, immunohistochemistry with calcitonin is required to bridge the gap between a suspicious and an outright malignant diagnosis. Thus, the diagnostic certainty depends on whether a repre-

sentative cell block is available. Chart 5.1 shows these entities at a glance. Entities that are not readily diagnosed on cytology are summarised in Table 5.2, with their likely TBSRTC categories. Some of these may be diagnosed on cytology in the appropriate clinical setting, usually with the help of ancillary tests and clinical correlation, e.g. MTC, lymphoma. As a general rule, none of the follicular or Hurthle cell neoplasms (adenoma or carcinoma) can be diagnosed definitively on cytology, because the differentiation between adenoma and minimally invasive carcinoma hinges on the presence of capsular or vascular invasion. This requires histologic evaluation of the entire capsule of the nodule, because the invasion is microscopic and hence not appreciable grossly or on imaging. For this reason, intra-operative frozen section is also not helpful because the sampling of sections is effectively blind, due to the inability to visualise invasion with the naked eye. The management of cytologically diagnosed FNs/SFNs is conservative surgery with a possible view to completion thyroidectomy if malignancy is confirmed on histology. Alternatively, molecular testing using a test such as the Afirma gene expression classifier (GEC) may be helpful, as a ‘rule out’ test. A negative result has a reliably high negative predictive value, thereby allowing for surgery to be avoided or held off. At the current time, molecular testing for follicular patterned neoplasms has not reached a point where a reliable, highly sensitive and specific ‘rule in’ test is available, because there are still benign follicular nodules (adenomatoid nodules or follicular adenomas) that show overlapping molecular aberrations with follicular carcinoma. Hence, a positive test is generally not sufficiently specific for malignancy to warrant near-total thyroidectomy. In NIFTP, cytology has the same limitations as it does in follicular neoplasms—i.e. the absence of capsular or vascular invasion, a diagnostic criterion, cannot be established on cytology. In addition, several quantitative criteria are also applied to the diagnosis of NIFTP, in terms of a maximum allowable percentage of papillary architecture (less than 1%) and non-follicular architecture (less than 30%); these also generally require histologic evaluation [2].

5.5  Brief Technical Notes

47

FVPTC would have essentially identical cytologic features to NIFTP; hence, this is also a diagnosis that cannot be definitively made on cytology. These two entities most frequently fall into the AUS/FLUS, FN/SFN or SM categories in TBSRTC. One should take note that the nuclear features of PTC may not be fully present in some cases of FVPTC or NIFTP on cytology—one or two features may not be apparent, such as well-­ formed nuclear pseudoinclusions. Thus, knowing the inherent limitations of cytology is important as it informs one when to exercise appropriate caution in the cytologic diagnosis of thyroid nodules.

5.5

Brief Technical Notes

This section highlights some points to take note of, particularly if the FNA is being performed by the pathologist, or if rapid on-site evaluation (ROSE) is performed by the cytology team.

5.5.1 Needle Gauge The thyroid gland is a very vascular endocrine organ, thereby leading to potentially haemodiluted smears or blood clots that can obscure diagnostic material. It has been my experience that a 25G needle is well suited for thyroid aspirates. 23G needles are also commonly used and frequently yield satisfactory results and may better facilitate aspiration of cyst fluid. Negative pressure may be applied in the form of suction with a syringe; however, this often leads to more bloody aspirates. In most instances, I do not apply negative pressure in order to minimise haemodilution. However, in cystic nodules, aspiration of cyst contents is recommended, to dryness if possible. Complete collapse of the cyst suggests a benign nodule. If any solid areas remain, aspiration should be targeted at these areas, preferably under ultrasound (US) guidance.

Table 5.2  Entities That are Not Readily Diagnosable on Cytology

Nature of entity Nonneoplastic nodules Benign/ indolent neoplasms

Malignant neoplasms

Diagnostic entity Adenomatoid nodule with microfollicular architecture Follicular adenoma; Hurthle cell adenoma Hyalinising trabecular tumour

NIFTP FVPTC Follicular carcinoma Poorly differentiated thyroid carcinoma (PDTC) Medullary thyroid carcinoma Low-grade lymphoma (e.g. MALT lymphoma)

Most likely TBSRTC category FLUS

Other possible TBSRTC categories FN/SFN

FN/SFN

FLUS

Malignant (PTC) (diagnostic pitfall— overcall) FN/SFN FN/SFN FN/SFN FN/SFN

SM (PTC); FN/SFN; AUS/FLUS (Ki-67 immunohistochemistry on cell block may allow definitive diagnosis—see text)

SM AUS

SM SM – SM, malignant (ultrasound may show extrathyroidal extension, a helpful clue to the malignant nature) Malignant (can be definitively diagnosed with cell block and IHC—calcitonin, ±CEA) SM, malignant (flow cytometry and/or ancillary testing on cell block may enable definitive diagnosis)

5  Approach to Thyroid Cytology

48

5.5.2 Ultrasound Guidance

Ancillary tests fall into these main forms:

The American Thyroid Association recommends that thyroid FNAs be performed under US guidance [10]. I have observed that there is sometimes a tendency in US-guided FNAs for the needle to remain in the thyroid gland for a somewhat longer duration than with direct FNAs, hence leading to clot formation. This renders the smears more challenging to interpret because of artefactual alterations in architecture of cell groups entrapped in clots, as well as partial obscuration of nuclear detail by clumped red blood cells (RBCs). Feedback to clinicians and radiologists may help to ameliorate this issue. A duration of approximately 7–10 s should suffice for the FNA procedure. A good indication of when to withdraw the needle is when any amount of blood-stained material enters the needle hub. The other phenomenon sometimes encountered in US-guided FNAs is excessive US gel that is not wiped off from the skin prior to needle entry, thereby being seen in the smears as coarse reddish deposits (Fig. 3.3 in Chap. 3).

1. Cell block –– For immunohistochemistry (IHC) and molecular testing. –– Can be used for suspected MTC, metastases, lymphoma, parathyroid lesions, etc. 2. Flow cytometry –– Useful in suspected non-Hodgkin lymphoma, particularly low-grade lymphoma (e.g. MALT lymphoma). 3. Needle wash/fresh aspirated material –– Needle wash in an appropriate volume of saline may be sent for analysis for parathormone levels in a suspected parathyroid nodule. –– In likely infective lesions, purulent material or needle rinse in sterile saline may be sent for microbiological investigations. 4. Smears or needle wash in preservative solution –– These may be used for molecular testing, e.g. in indeterminate nodules or lymphoma.

5.5.3 Collecting Material for Ancillary Tests

Various discriminatory ancillary tests will be elaborated on in more detail under the Triple Approach.

Additional material may be collected for ancillary tests in the following scenarios:

5.6

• Suspected primary malignancy other than follicular or papillary thyroid carcinoma—MTC, lymphoma. • Other suspected neoplasms in which immunohistochemistry is helpful, e.g. hyalinising trabecular tumour. • Suspected secondary malignancy (metastases). • Suspected parathyroid lesion. • Suspected infectious aetiology. • Indeterminate nodules for which molecular testing is planned.

As mentioned in Chap. 3, the soundest approach to cytologic diagnosis incorporates three main elements. Of these, morphology forms the backbone of the diagnostic workup; however, knowledge of the clinical picture and access to ancillary tests can be invaluable in helping one to best answer the clinical question at hand. If clinical information is not forthcoming, important clinical considerations may be included in the ‘comments’ section of the cytology report. We will look at each aspect of the triple approach in turn.

Triple Approach

5.6  Triple Approach

Triple Approach 1. Clinical Information 2. Cytology (gross evaluation) 3. Ancillary tests

49

and

microscopic

5.6.1 Clinical Information If the FNA is performed by the pathologist, every opportunity should be taken to obtain a quick history and perform a focused physical examination. When clinical information is not available and a particular diagnosis requires clinical corroboration, e.g. autoimmune thyroiditis or subacute granulomatous thyroiditis, it may be helpful to write a comment on specific clinical features to note by the clinical team. We will briefly look at some pertinent points in the clinical history, physical examination and imaging here.

5.6.1.1 Clinical History Here are some pertinent points to take note of in the clinical history: (a) Duration of nodule/swelling • Longstanding nodules that are not progressively enlarging will mostly be relatively indolent, even if neoplastic. • A short history of a month or so should prompt one to think of conditions such as DeQuervain thyroiditis (DQT) or other inflammatory or infectious causes. In the consideration of DQT, one should ask for a recent history of viral illness (e.g. fever, malaise), and pain in the thyroid area. • The sudden appearance of a nodule may be sometimes seen when there is haemorrhage into an existing cyst, and attention to the nature of the fluid aspirated may help to confirm that impression.

(b) Progression of the nodule—new or enlarging nodules are of some clinical concern. • A progressively enlarging nodule, whether noted by the patient or on serial imaging, is a worrying feature for malignancy. • In anaplastic carcinoma, the clinical picture is that of an elderly patient with a history of a rapidly enlarging mass with accompanying progressively severe obstructive symptoms. • In a patient with longstanding Hashimoto thyroiditis, recent enlargement of the thyroid gland is a worrying feature that should alert one to the possibility of lymphoma arising in this background. (c) Obstructive symptoms such as hoarseness of voice, difficulty swallowing and breathing (stridor, dyspnoea/shortness of breath). • These indicate clinically significant disease that mechanically hinders important physiological activities, and surgery may be a consideration, even in cytologically benign goitres. Sometimes, an FNA is performed to exclude malignancy before surgery for a clinically benign but compressive goitre. • Obstructive symptoms may also be an indicator of malignancy, coupled with a progressively enlarging mass. (d) Symptoms of abnormal thyroid function • Most thyroid nodules that are referred for FNA are non-functioning nodules. If the clinical history suggests hyperthyroidism, the case is best discussed with the endocrinologist, regarding the optimal management of the thyroid nodule and the necessity of an FNA. (e) Past history • Radiation to the head and neck region might predispose one to thyroid carcinoma, whilst at the same time causing worrisome reactive nuclear atypia. • Previous treatment of the thyroid gland with radioiodine may also give rise to

50

large atypical hyperchromatic nuclei that are reactive in nature (see Sect. 5.8, Pitfalls). • History of thyroid disease (e.g. nodular goitre, Hashimoto thyroiditis) with a new nodule or mass is a worrying sign for possible malignancy. • History of treatment of thyroid disease is helpful, as certain medications, e.g. carbimazole can cause reactive cytologic atypia. (f) Family history • History of Graves’ disease, thyroid malignancy or endocrine disease (e.g. familial MTC, MEN 2 syndrome) may point to certain pathologies in the thyroid.

5.6.1.2 Physical Examination Physical examination should be focused and directed, depending on the clinical information provided. Here is a brief outline of some clinical signs that point to specific conditions: –– Hoarseness This, particularly if onset is recent, may be an ominous sign of invasion or compression on the recurrent laryngeal nerve, due to either malignancy or a sizeable goitre. –– Stridor This may rarely be noted in cases of significant compression of the airway, by either thyroid malignancy or a large goitre. –– Eye signs of Graves’ disease and hyperthyroidism (e.g. proptosis, lid lag, lid retraction) This may suggest Graves’ disease or a hyperfunctioning state, and prompt one to look up thyroid function test results, or look for other physical signs of thyrotoxicosis, e.g. tremor, tachycardia. Discrete nodules may sometimes occur in a background of Graves’ disease and may in fact be malignant, e.g. PTC. –– Characteristics of the nodule/mass: Even though thyroiditis (e.g. Hashimoto thyroiditis or DQT) usually results in a more diffuse swelling of the thyroid gland, sometimes the enlargement may be asymmetrical, giving rise to the clinical appear-

5  Approach to Thyroid Cytology

ance of a nodule. Also, hyperplastic nodules and neoplasms such as PTC may also occur in the setting of autoimmune (Hashimoto) thyroiditis. Ominous signs include: • Fixation to underlying structures—this suggests malignancy that extends beyond the thyroid gland. Rarely, benign conditions such as Riedel’s thyroiditis/IgG4-related disease or Hashimoto thyroiditis with extensive fibrosis may also cause this. • Non-movement with swallowing (this may indicate fixation to surrounding structures). Bear in mind that other non-thyroid swellings in the anterior neck location may also be a differential (e.g. lipoma). Tenderness (a tender nodule on palpation suggests a possibly inflammatory condition, in particular, DQT; with a differential diagnosis of infection). –– Other findings in the neck: Presence of enlarged cervical lymph nodes (this may be worrying in cases where there is a suspicion of PTC, MTC, PDTC or lymphoma).

5.6.1.3 Imaging Ultrasound imaging has become the standard of care in the diagnostic workup of discrete thyroid lesions. It is thus advantageous for the pathologist to know some of the basic sonographic features that suggest neoplasia or malignancy. It is also worth noting that in the 2018 updated TBSRTC, sonographic findings are used to guide the follow-up management of cytologically benign thyroid nodules. The 2015 ATA guidelines provide an outline of the features that are associated with a high suspicion of malignancy, which are listed here [10]: • Solid hypoechoic nodule or solid hypoechoic component of a partially cystic nodule with one or more of the following features: • Irregular margins (infiltrative, microlobulated). • Microcalcifications. • Taller than wide shape.

5.6  Triple Approach

• Rim calcifications with small extrusive soft tissue component. • Evidence of extra-thyroidal extension. Some of the more reassuring sonographic features are an isoechoic or hyperechoic nodule that is wider than tall and the presence of a halo around the periphery of the nodule. Summary of Alarming/Red Flag Clinical Findings that May Point to Malignancy The following are findings that are worrying for malignancy and that should prompt timely diagnosis and trigger other necessary investigations without delay.

• History of new or enlarging mass (especially on a background of Hashimoto thyroiditis). • Obstructive symptoms: Hoarseness (recent onset), stridor, dysphagia. • Fixation of the mass. • Cervical lymphadenopathy.

5.6.2 Cytology (Gross and Microscopic Evaluation) Evaluation of morphology starts with the gross appearance of aspirated material. Thereafter, we can apply a systematic approach to microscopic evaluation, progressively zooming in from low to high power, looking at both cells and background material. As outlined in Chap. 3, four main parameters should be examined on microscopy: cellularity, architectural arrangement, cytomorphology and background material and cells. Before we discuss microscopy, let us first have a look at the gross evaluation of aspirated material. This might not be readily appreciable in the setting of liquid-based processing methods; however, in conventional cytology, evaluation of gross appearance can yield some helpful diagnostic clues.

51

5.6.2.1 Gross Evaluation • Colloid –– The presence and volume of colloid is an important factor in the diagnostic evaluation of thyroid aspirates. In histology, benign nodules (e.g. dominant nodule in a nodular goitre) are often composed of large follicles which contain abundant colloid. This correlates with the relative abundance of colloid that is seen on cytology in benign nodules. –– Due to the physical properties of colloid, it tends to wash off during the staining process. Hence, it is important to always make an effort to identify colloid grossly and to document its presence at the time of smearing, e.g. during rapid on-site evaluation (ROSE). The gross description may be the only documentation of the presence of colloid, because of the tendency to slip off during staining. Sometimes, one may see empty spaces in which there are sparse erythrocytes (Fig.  5.1), suggesting that they were once occupied by colloid; however, it can be almost impossible to discern this in many instances. –– Colloid is recognised grossly as a shiny, glossy substance that is slightly thicker than water that appears similar to colourless nail varnish or dilute honey if admixed with blood. Figure  5.1 shows the gross appearance of a drop of colloid before it is smeared. The gross appreciation of colloid usually correlates with a benign nodule. Indeed, TBSRTC suggests that the presence of abundant colloid alone is sufficient for a benign diagnosis, even if the quantitative threshold for adequacy is not reached (i.e. at least 60 follicular cells). • Water-clear fluid from a parathyroid cyst –– Parathyroid cysts are rare. However, there is a very characteristic gross appearance of the aspirated material—that of thin, colourless, clear fluid that simply looks like water. Cytology may reveal sparse or no epithelial cells. If this appearance is noted grossly, it would be a good idea to send the aspirated fluid to the biochemistry laboratory for

5  Approach to Thyroid Cytology

52

a

b

c

d

Fig. 5.1  Colloid and its mimics. (a) Gross appearance of aspirated colloid, shiny liquid that resembles nail varnish (Photo: Dr Jaslyn Lee, Department of Pathology, National University Hospital, Singapore); (b) Low magnification view showing some colloid with intervening empty spaces where colloid has washed off (HC, 20×); (c) Plasma from

a lymph node aspirate mimicking thin colloid—a small epithelioid granuloma is present in the top right corner (HC, 40×); (d) Skeletal muscle bundles masquerading as colloid—note the parallel borders and the peripheral elongated nuclei (HC, 400×)

parathormone analysis, which is the most helpful diagnostic test in this context.

Hence, cellularity should not be used as a deciding factor between benign and neoplastic nodules. Architecture and cytomorphology are far more reliable parameters to determine the nature of the lesion. • TBSRTC mentions that for a diagnosis of FN/ SFN, the smears should be of at least moderate cellularity. Whilst this is true most of the time, there are instances in which follicular neoplasms can be fairly hypocellular, and the cytologic diagnosis can still be made if the architectural and cytomorphological features are suggestive. • Classical PTCs often produce extremely cellular smears, which on low power give the distinct impression of flat sheets of cells and sometimes folded tissue fragments, where cells outnumber colloid by far.

5.6.2.2 Microscopic Evaluation The four main parameters that will be discussed include: 1. 2. 3. 4.

Cellularity Architecture Cytomorphology Background

Cellularity • Traditional teaching dictates that benign nodules are usually hypocellular. In practice, I have found this to be true much of the time; however, it is also not uncommon to encounter benign follicular nodules with high cellularity.

5.6  Triple Approach

53 Common Architectural Patterns

Complex tissue fragments / Microbiopsies

Monolayered/flat sheets

Small crowded groups (Microfollicles, trabeculae)

Benign

Malignant

Benign

Malignant

Follicular patterned Neoplasm

Benign

Conglomerate of intact follicles +/blood vessels -BFN

True papillary fragments (with fibrovascular cores) -PTC

Honeycomb sheets -BFN

Syncytial sheets -PTC

Small round nuclei - FA, HCA - FC, HCC - PDTC (may show more intense crowding, mitoses, necrosis) Atypical nuclear features - FVPTC/NIFTP - MTC

Hashimoto thyroiditis

Key: BFN: Benign follicular nodule (e.g. colloid/hyperplastic nodule); FA: Follicular adenoma; FC: Follicular carcinoma; FVPTC: Follicular variant of papillary thyroid carcinoma; HCA: Hurthle cell adenoma; HCC: Hurthle cell carcinoma; MTC: Medullary thyroid carcinoma; NIFTP: Non-invasive follicular thyroid neoplasm with papillary-like nuclear features; PDTC: Poorly differentiated thyroid carcinoma; PTC: Papillary thyroid carcinoma

Chart 5.2  Common architectural patterns with diagnostic correlates

Architecture There are several commonly encountered architectural patterns in thyroid FNAs. Chart 5.2 summarises three of the more readily recognisable patterns and their associations with specific neoplastic and non-neoplastic entities. Some additional architectural patterns are described below. There are several usual architectural formations seen within some of the main diagnostic categories in TBSRTC: • Benign Flat honeycomb sheets with uniformly spaced nuclei and distinct cell borders (Fig. 5.2a, b) (as seen in a colloid nodule comprising ­macrofollicles); large tissue fragments comprising collections of intact follicles (Fig. 5.3d). • Malignant Syncytial sheets with overlapping nuclei (Fig.  5.2c, d), and true papillary tissue fragments with fibrovascular cores (Fig.  5.3c)— both seen in PTC. • Follicular neoplasm Microfollicular or trabecular arrangements, crowded clusters (Fig. 5.3a, b).

Here is a more detailed morphologic description of specific architectural patterns and their likely associated diagnoses: • Flat sheets –– Monolayered flat sheets can be seen in both benign nodules (colloid or hyperplastic nodules) and malignancies such as PTC. Attention needs to be paid to the specific arrangement of the cells within these sheets, which is quite distinct. –– Flat honeycomb sheets—This is one of the major benign architectural patterns in thyroid FNAs. In flat sheets that correspond to macrofollicles, the arrangement of the cells is more uniform and rigid, with nuclei appearing fairly equally spaced apart, often with distinct cell borders giving rise to the honeycomb arrangement (Fig.  5.2a, b). These are often referred to as ‘honeycomb sheets’. Their presence is reassuring and indicates that the nodule is likely to be a benign follicular nodule (BFN) . –– Syncytial sheets—Syncytial sheets are far more crowded and disorganised than flat honeycomb sheets. The nuclei often overlap one another, and the cytoplasmic

5  Approach to Thyroid Cytology

54

a

b

c

d

Fig. 5.2  Architectural arrangements I. Flat sheets. (a, b). Regimented, regular honeycomb arrangements from a macrofollicle in a colloid nodule (HC and Pap, 300×); (c,

d) Syncytial sheets with nuclear crowding and overlapping in PTC—a nuclear pseudoinclusion is seen in the air-­dried smear near the centre of the picture (HC and Pap, 400×)

b­ orders are usually indistinct, though not always (Fig.  5.2c, d). This is one of the commonest patterns seen in classical PTC. Another feature of the flat syncytial sheets in PTC is that they often have rather straight borders, sometimes with nuclei arranged in a peripheral row with a suggestion of nuclear polarity, similar to a row of columnar cells at the edge of the sheet (Fig. 5.2d). Compare and contrast the honeycomb and syncytial sheets demonstrated in Fig. 5.5. • Tissue fragments with intact follicles –– Intact follicular structures can sometimes be seen as isolated rounded groups of follicular cells with a very smooth outer border, with or without a central globule of colloid.

–– At other times, in benign follicular nodules, entire conglomerates of follicles are aspirated and remain intact in more complex tissue fragments or microbiopsies (Fig.  5.3d). Focusing up and down through such complex tissue fragments will reveal the individual follicular structures, which are often accompanied by small blood vessels running through the tissue fragments. On careful scrutiny, one can appreciate that these blood vessels are located between follicles rather than forming fibrovascular papillae within a true papillary structure. One should not thus mistake these f­ollicular conglomerates for true papillary structures. A direct comparison may be made in Fig. 5.3.

5.6  Triple Approach

55

a

b

c

d

Fig. 5.3  Architectural arrangements II.  Follicular and papillary structures. (a) Microfollicles in a follicular neoplasm (HC, 200×); (b) Trabecular arrangement in a follicular neoplasm (HC, 200×); (c) True papillary structures with fibrovascular cores lined by follicular cells in PTC

(HC, 40×); (d) Benign follicular nodule showing a tissue fragment composed of multiple intact follicles with some small blood vessels running through—this should not be mistaken for papillary structures (HC, 40×)

• Papillary tissue fragments –– These are tissue fragments in which there is a branching fibrovascular core that is covered by follicular cells (Fig. 5.3c). In a vast majority of cases, these indicate papillary architecture in classical PTC.  Rarely, true papillary structures may be encountered in benign lesions such as hyperplastic nodules or follicular neoplasms; however, the follicular cells will not display the nuclear features of PTC. Therefore, a definite diagnosis of PTC must also rely on the presence of corroborative nuclear features. –– Sometimes, the follicular cells in classical PTC separate from their fibrovascular cores during the smearing process and are seen in the form of finger- or cup-shaped sheets

with straight anatomical convex borders. These are not diagnostic of a true papillary architecture, but are suggestive of the presence of papillary structures, and should prompt a careful search for other features of PTC. –– Caution: The mere presence of blood vessels within a tissue fragment should not prompt one to diagnose PTC, as mentioned in the previous point. Figure 5.3 shows the differences between stromal vessels and true papillae. –– Papillary structures are further discussed in Sect. 5.8 (Pitfalls). • Microfollicles –– These are small rounded arrangements of follicular cells, arbitrarily defined as being

5  Approach to Thyroid Cytology

56

composed of less than 15 follicular cells [7], sometimes with a small central globule of colloid (Fig.  5.3). A predominance of microfollicles with scant accompanying colloid strongly suggests a FN. –– When microfollicles are admixed with a similar proportion of flat honeycomb sheets, a ‘FLUS’ diagnosis is warranted, because the findings are indeterminate between those of a BFN and an FN. Furthermore, rarely, microfollicular formations have been described in Hashimoto thyroiditis [17]. Cytologic criteria for the diagnosis of AUS/FLUS are further discussed in Sect. 5.7 (Specific Challenges). • Trabecular arrangements/clusters with nuclear crowding

–– These are more elongated groups of follicular cells with appreciable nuclear overlapping and crowding (Fig. 5.3). Predominance of this pattern also indicates a high likelihood of FN. Differential diagnoses include follicular adenoma, follicular carcinoma and sometimes even poorly differentiated thyroid carcinoma (PDTC, Fig. 5.4). –– Sometimes we encounter cell clusters with significant nuclear crowding where nuclei partially sit on top of each other or show some degree of overlap and crowding, without a definite microfollicular architecture. This is also an atypical architectural feature and, if predominant, should be categorized as FLUS or FN/SFN, unless other cytomorphologic features are present to suggest a specific entity such as MTC or

a

b

c

d

Fig. 5.4  Architectural arrangements III.  Less commonly encountered patterns. (a) Discohesive plump epithelioid cells in MTC; note the multinucleation and stippled chromatin (Pap, 400×); (b) A sheet of cells arranged in a whorled

pattern in PTC (HC, 200×); (c) An extremely crowded sheet of follicular cells in poorly differentiated thyroid carcinoma (PDTC) (HC, 200×); (d) A crowded sheet with a somewhat cribriform pattern in PDTC (Pap, 200×)

5.6  Triple Approach

metastatic carcinoma. Extremely crowded sheets such as those shown in Fig. 5.4c, d may be clues to the possible presence of a more sinister lesion such as PDTC, and prompt one to take a look at the clinical findings and imaging results. • Dissociated cells –– Follicular cells can be dispersed in several conditions ranging from colloid nodules to Hurthle cell neoplasms and MTC (Fig. 5.4); hence, this architectural feature alone does not reliably point to any specific diagnostic category. In this context, attention to the cytomorphologic features (nucleus and cytoplasm) are more illuminating. –– In aspirates composed almost exclusively of Hurthle cells, discohesion is one of several features that suggest a Hurthle cell neoplasm (HCN). Other features include the presence of traversing blood vessels running across tissue fragments and binucleation (Chart 5.4). –– Anaplastic carcinoma may also present with mainly dispersed, bizarrely ­pleomorphic cells which may sometimes be admixed with numerous neutrophils. –– Lymphocytes are also usually dispersed; however, these are usually readily distinguishable from thyroid follicular and other epithelial cells. Lymphoid-rich lesions will be discussed separately later in Sect. 5.7 (Specific Challenges). • Whorls –– This pattern is sometimes seen in PTC, in which the follicular cells appear to ‘whorl’ in a circular arrangement within tightly packed clusters (Fig.  5.4). It is less well documented than other architectural patterns in PTC (e.g. syncytial sheets, microfollicular arrangement in FVPTC). Cytomorphology This refers to nuclear and cytoplasmic features, as well as overall cellular morphologic features such as cell shape and size. On initial screening of the smears, one should first form an impression of the predominant

57

lesional cell appearance, e.g. small follicular cells or Hurthle cells. Here are some features that should be evaluated, divided broadly into cell shape/appearance, nucleus and cytoplasm: • Cell shape/appearance—Most follicular cells appear relatively cuboidal or polygonal in shape. One should note that if there is significant spindling of the cells, the possibility of MTC should be considered, and nuclear chromatin should be carefully evaluated. At times, cyst lining cells may also appear somewhat elongated, with cytoplasmic processes (Fig.  5.9a). Plasmacytoid cells, including binucleated cells, should also raise the possibility of MTC (Fig. 5.5). • Nucleus—Evaluate size (as compared to a red blood cell) and shape (round, oval, spindle, bizarre), location in cell (central vs. eccentric), chromatin pattern (pale, powdery vs. granular and euchromatic vs. coarsely granular/stippled), presence of nuclear grooves and pseudoinclusions, nucleolar prominence and location, presence and relative frequency of mitotic figures, pleomorphism. Table 5.3 describes the different appearances of nuclei of lesional cells that are likely to be encountered in thyroid aspirates, whilst some examples are shown in Fig. 5.5. –– Some benign conditions may harbour significant nuclear pleomorphism, e.g. Hashimoto thyroiditis or nodules in patients treated for Graves’ disease (Fig.  5.9), whilst neoplasms may conversely exhibit more monotonous nuclei, e.g. follicular neoplasm, Hurthle cell neoplasm). –– In hyperfunctioning follicular cells (e.g. Graves’ disease), the nuclei may appear mildly enlarged and exhibit pale chromatin. This is further discussed later in Sect. 5.8 (Pitfalls). –– Beware of mimics of nuclear pseudoinclusions such as red blood cells (RBCs) superimposed on follicular cell nuclei, artefactual holes or small cytoplasmic vacuoles or

5  Approach to Thyroid Cytology

58 Table 5.3  Nuclear Features of Various Thyroid Entities Diagnosis/Cell type Nuclear shape Round Follicular cells in BFN, FN Poorly differentiated thyroid carcinoma Papillary thyroid carcinoma

Usually round, occasionally irregular

Oval, some cells with irregular nuclear membranes. Longitudinal nuclear grooves and well-formed pseudoinclusions often present.

Nuclear size 1–1.5× red blood cell (RBC) diameter 1–1.5× RBC diameter, sometimes larger More than 1.5× RBC diameter

Hurthle (oncocytic) cells

Usually round

Variable in size, usually larger than RBC, can be several times larger

Medullary thyroid carcinoma

Round to ovoid eccentric nuclei (plasmacytoid or spindle-shaped cells). May be bi- or multinucleated. Bizarre nuclear shapes, including markedly irregular and spindle

Usually larger than RBCs and bystander follicular cell nuclei Very large nuclei (many times larger than RBCs)

Slightly irregular nuclei (not as round as thyroid follicular cells). Note: Naked nuclei from thyroid follicular cells are round and devoid of cytoplasm; whilst lymphocyte nuclei are usually slightly irregular and have a thin rim of cytoplasm (Fig. 5.8c).

Nuclei range from the size of RBCs to up to 4× the size, depending on the type of lymphocyte

Anaplastic carcinoma

Lymphocytes

Chromatin; nucleoli Fairly dark (euchromatic), finely granular chromatin (Fig. 5.5) Fairly dark, finely granular chromatin

Pale, powdery, small nucleoli close to nuclear membrane (Note: In toxic nodules or Graves’ disease, the nuclei may be enlarged and chromatin pale and open, reminiscent of PTC. However, nuclei are often round and cytoplasm is abundant and delicate.) Pale to euchromatic. A small nucleolus may be seen in benign nodules. A prominent macronucleolus may be seen in Hurthle cell neoplasms (HCNs).

Stippled, ‘salt and pepper’ chromatin. Nuclear inclusions may be present (Figs. 5.5c and 5.6d). Usual characteristics of malignancy—coarse chromatin, large nucleoli, may be hyperchromatic Usually fairly hyperchromatic

Nuclear pleomorphism Minimal

Usually fairly uniform nuclei

Mild to moderate

Benign Hurthle cell nodules in Hashimoto thyroiditis may exhibit significant nuclear pleomorphism and enlargement. Conversely, neoplasms may yield more uniform nuclei. Can exhibit marked variation in nuclear size

Highly pleomorphic, obviously malignant, not recognisable as thyroid in origin Reactive lymphoid population—mixed lymphoid population with predominantly small lymphocytes. Lymphoma—more likely to exhibit monotonous cell and nuclear size.

5.6  Triple Approach

59

a

b

c

d

Fig. 5.5  Distinctive nuclear features. (a) Benign follicular cells with small, round nuclei only slightly larger than RBCs, with characteristic finely granular chromatin (Pap, 400×); (b) PTC featuring larger, oval, overlapping nuclei with pale powdery chromatin and longitudinal grooves

(Pap, 400×); (c) MTC showing oval to spindle nuclei with stippled, salt and pepper chromatin (Pap, 400×); (d) FVPTC showing crowded arrangements of cells with oval, pale grooved nuclei similar to those of PTC (Pap, 600×)

bubbles that overlie the nucleus. True pseudoinclusions are usually round and are bordered by a sharp, well-defined nuclear membrane, and the contents are of similar appearance to the cytoplasm of the cell (Fig. 5.6). –– Nuclear pseudoinclusions are most commonly seen in PTC, but may also be present in hyalinising trabecular tumour (a neoplasm that is treated by lobectomy), MTC and metastatic carcinoma. Figure 5.6 shows some examples of real and ‘fake’ nuclear pseudoinclusions. Occasionally, pseudoinclusions have also been described in Hashimoto thyroiditis; however, I have not personally encountered true pseudoinclusions in this context [17].

• Cytoplasm—volume, colour, texture (dense, granular, delicate, vacuolated), cytoplasmic membranes (well- or ill-defined), shape. Here are some instances in which cytoplasmic findings are pertinent: –– Hurthle cells—Hurthle cells or oncocytes are recognised as such because of their relative abundance of cytoplasm, i.e. low N/C ratios. The cytoplasm is often relatively well-defined and has an opaque, granular and sometimes dense quality to it (Fig. 5.7). On alcohol-fixed, Papanicolaou-­ stained smears, the cytoplasm may appear variably blue, green or orange. Differential diagnoses of Hurthle cells or oncocytic appearing lesions range from benign to malignant entities, and a broad approach to

5  Approach to Thyroid Cytology

60

a

b

c

d

Fig. 5.6  Nuclear pseudoinclusions—real and ‘fake’. (a) ‘Fake’ inclusion (circled)—small, slightly irregular, and not surrounded by nuclear membrane, likely artefactual (HC, 400×); (b) PTC with a pseudoinclusion and other

classical nuclear features (Pap, 600×); (c) Well-formed pseudoinclusions in a hyalinising trabecular tumour (Pap, 600×); (d) MTC showing a pseudoinclusion in a large cell; note the binucleated cells in the background (Pap, 400×)

Hurthle cell-rich lesions is shown in Chart 5.4. Hurthle cell-rich lesions are discussed in Sect. 5.7 (Specific Challenges). –– Flame cells/cells with peripheral ‘fireflares’—Flame cells are recognisable in air-dried smears, as cells with abundant cytoplasm, with reddish bodies in their cytoplasm which appear darker at the edges (Fig. 5.7b). Sometimes these pinch off the cytoplasm and are seen adjacent to the cell membrane, hence the name ‘fireflares’. They are also known as ‘marginal vacuoles’. These bodies are thought to be dilated endoplasmic reticulum in metabolically active follicular cells [18]. Whilst they are most often seen in benign conditions (nodular goitre, toxic nodule, Hashimoto thyroiditis, etc.), they can also

be encountered in malignancies including follicular and papillary carcinoma. –– Paravacuolar granules—These are seen in air-dried smears as coarse dark blue granules in the cytoplasm within tiny vacuoles (Fig.  5.7e), and as brownish granules in Papanicolaou-stained smears and represent lysosomes containing haemosiderin or lipofuscin within dilated endoplasmic reticulum [19]. They are not specific for any particular condition and may be seen in non-neoplastic and, less frequently, neoplastic conditions. –– Vacuolated cytoplasm—The presence of numerous fine, tiny cytoplasmic vacuoles imparting a ‘bubbly’ appearance is very characteristic of cystic PTC (Fig.  5.7c). The lesional cells are often few in number

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61

a

b

c

d

e

f

Fig. 5.7  Cytoplasmic appearances. (a) Hurthle cells in Hashimoto thyroiditis with abundant granular to dense cytoplasm; note the lymphocytes in the background (HC, 400×); inset shows Hurthle cells from a Hurthle cell adenoma (Pap, 400×); (b) Follicular cells with peripheral ‘fireflares’ in a benign follicular nodule (HC, 400×); (c)

Cystic PTC showing cluster of cells with bubbly cytoplasm; (HC, 400×); (d) Macrophages from a thyroid aspirate to compare with (c) (HC, 400×); (e) Paravacuolar granules in follicular cells (HC, 400×); (f) Reddish cytoplasmic granules can rarely be seen in air-dried smears in MTC (HC, 600×)

and occur in small clusters with scalloped, flower petal-like borders, sometimes with a central psammoma body. If such cells are seen, one should search carefully for any

nuclear features of PTC as well as other cells with more classical features of PTC.  These vacuolated malignant cells should not be mistaken for cyst

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62

­ acrophages which may also be present in m the background—the latter are more discohesive and have smaller, blander nuclei. Figure  5.7 shows a direct comparison between cystic PTC cells and macrophages. –– Reddish cytoplasmic granules—These may rarely be seen in MTC (Fig. 5.7) and appear to be distinct in colour from the rest of the cytoplasm and somewhat coarser than the granular cytoplasm seen in Hurthle cells. They represent neurosecretory granules in the cytoplasm [20, 21]. –– Hyperfunctional cells—At times, in conditions such as partially treated Graves’ disease, the follicular cells have abundant cytoplasm and low N/C ratios. They are distinct from Hurthle cells, as the cytoplasm here is delicate, translucent and ill-defined. –– Reactive changes—In some nodules, particularly in those which have undergone cystic change, there is increased cytoplasm and sometimes elongated, tapering cytoplasmic processes, similar to those seen in cells with reactive changes in cervical smears (Fig. 5.9a).

as mentioned above, colloid may wash off during the staining process and leave behind telltale empty spaces (Fig. 5.1b)— these are recognisable due to the paucity of red blood cells, which are sometimes pushed to the periphery of the spaces. –– A word of caution: Beware of ‘fake’ colloid. • Skeletal muscle can sometimes closely mimic thick colloid (Fig. 5.1d). Clues to its nature are in its parallel cell borders, occasionally discernible peripheral nuclei and thin striations when viewed at high power. If a needle traverses skeletal muscle, this is also often rather painful, and the patient’s response may be a further corroborating clue (Fig. 5.1d). • Plasma might also mimic thin colloid, but this usually does not push apart the red blood cells, and is usually purplish in colour (whilst colloid may be bluish or purple) and is less likely to form cracks. In my own practice, the presence of homogeneous-­appearing purplish material between erythrocytes in air-­ dried smears is not sufficiently diagnostic of colloid, as I have frequently seen this in non-thyroidal aspirates (Fig. 5.1c). Background • Cyst contents Background elements include both cells and non-­ –– This appears as granular, pale material seen cellular material. It is a good habit to always in the background of smears. There are include evaluation of background material and often accompanying foamy cyst cells in the systematic evaluation of cytologic macrophages. smears, because this element may provide valu- • Amyloid able information that can help narrow down the –– This may be seen in the background in a differential diagnosis, or even pinpoint the proportion of MTC cases, usually fewer diagnosis. than half. It appears as extracellular clumps In the thyroid gland, these are some elements of amorphous, ‘fluffy’ to fibrillary material that should be actively sought: that is magenta-coloured in air-dried smears and may be blue-green in alcohol-­ • Colloid fixed smears (Fig. 5.8a, b). It may on occa–– This is visible in many forms and is well sion be challenging to distinguish from illustrated in most cytology texts. Thin colthick colloid. Amyloid may not declare loid can show a cracking appearance, thick itself upfront in smears, but should be colloid can appear as inspissated clumps; sought if the cytologic features suggest the and colloid seen in PTC may appear ‘gluey’ possibility of MTC. On the cell block, and extremely thick, being likened to Congo Red staining shows the classical chewing gum or bubble gum. In addition, apple green birefringence.

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63

a

b

c

d

Fig. 5.8  Background cells and material. (a, b) Amyloid in a case of MTC, appearing as vaguely fibrillary, clumpy extracellular material that can be easily mistaken for colloid (HC, 200× and Pap, 300×); (c) Bare round nuclei of

follicular cells (black arrowheads) and lymphocytes for comparison (yellow arrowhead) (HC, 600×); (d) Scattered lymphocytes and plasma cells in a case of Hashimoto thyroiditis (HC, 600×)

• Old blood (from previous haemorrhage) –– Grossly, old blood is more brownish than red. In old blood, the RBCs appear pale, wrinkled, shrunken and at times almost transparent, or they may show spotty staining in Papanicolaou-stained smears. In contrast, ‘fresh’ RBCs that are seen as a result of trauma induced by the FNA procedure appear round and show more even staining. Old blood is an indication of previous haemorrhage, e.g. into a cyst, and may explain alarming symptoms such as the abrupt onset of a thyroid lump. • Ultrasound gel material –– This has been previously described in Chap. 3 and may sometimes be mistaken for necrosis or inspissated colloid, but the reddish-purple colour and coarse

t­ exture are usually quite distinctive (Fig. 3.3). • Inflammatory cells –– Lymphocytes Lymphocytes may mimic naked follicular cell nuclei as they are similar in size, but with two main differences—lymphocyte nuclei are usually slightly irregular and not perfectly round like follicular cell nuclei, and there usually is a small thin rim of bluish cytoplasm around lymphocyte nuclei, as opposed to bare follicular cell nuclei (Fig. 5.8c). Once lymphocytes are identified, the next step is to decide if the lymphocytes are indeed from the tissue aspirated or if they are from the peripheral blood. This

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64

will be obvious when there are plenty of lymphocytes. However, at times, the cells are more sparse and this question arises. A rough method that I employ is to look at the numbers of lymphocytes in relation to the numbers of neutrophils. If the lymphocytes outnumber the neutrophils, then it is likely that they are from the lesional tissue, provided the patient does not have a peripheral lymphocytosis. Thereafter, their significance can be worked up following the steps outlined in Sect. 5.7 (Specific Challenges—Lymphoid-rich Lesions) and also presented in Chart 5.5. Lymphocytes may be seen in benign conditions such as lymphocytic thyroiditis (including Hashimoto ­ thyroiditis) and DQT. They may also be seen in lymphomas which may be primary or part of systemic disease, and even in PTC. It is not uncommon to find lymphocytes accompanying malignant cells in PTC, and histology of these tumours may show a lymphoid-rich stroma in close association with the papillary structures, for example, in Warthin-like PTC. –– Plasma cells The presence of occasional plasma cells is a helpful clue that suggests the possibility of autoimmune (lymphocytic) thyroiditis (Fig. 5.8c). Scattered plasma cells may be seen admixed with more abundant lymphocytes. The presence of numerous plasma cells and lymphocytes may raise a concern for IgG4-related disease, which is now believed to be the pathology seen in Riedel thyroiditis, although it has not be proven to be so in all cases [22, 23]. In these instances, clinicopathologic correlation must be performed, and cell block with IHC using IgG and IgG4 may be helpful if sufficient lesional cells are present. This is a diagnosis that is extremely challenging to make on cytology and is more suited to a histologic biopsy.

–– Macrophages These are frequently encountered in thyroid aspirates and, when in large numbers, indicate cystic change within the nodule. Sometimes, haemosiderophages are encountered—these contain coarsely granular haemosiderin which appears dark blue on Romanowsky stains and brown on the Papanicolaou stain. Haemosiderophages are indicative of previous haemorrhage and may be seen coupled with old blood. –– Multinucleated giant cells These likely represent multinucleated histiocytes and are not specific for any one condition. They may be seen in Hashimoto thyroiditis, palpation thyroiditis as well as subacute granulomatous thyroiditis/DQT. In the latter, the cells are often very large and contain a large number of nuclei, often in excess of 50. If this is present, one should also look for giant cells engulfing colloid, which is characteristic of DQT. This should also prompt one to look for poorly formed granulomas and to seek clinical correlation. PTC also frequently yields multinucleated giant cells (on histology, these are often seen within colloid in the tumour). • Skeletal muscle—This has been described above under ‘colloid’, and one should note its presence so as not to mistake it for thick colloid or amyloid (Fig. 5.1).

Summary of Salient Points Regarding Morphology

• It is important to document whether colloid is seen grossly, as it may be washed away during staining. • Neoplastic lesions can sometimes be hypocellular.

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• Recognise the main architectural patterns that fall into the benign, malignant and follicular neoplasm categories. • Significant nuclear crowding and trabecular arrangements are abnormal, raising a concern for a follicular-­ patterned neoplasm. • The cells in most benign follicular nodules and many follicular neoplasms have small nuclei (slightly larger than a RBC), whilst the nuclei of PTC are larger. • Significant nuclear pleomorphism may be seen in Hashimoto thyroiditis. • All smears should be systematically evaluated for cyto-architectural features, nuclear and cytoplasmic features and background cells/material.

5.6.3 Ancillary Tests Specimen collection for molecular testing was briefly touched on earlier, and molecular testing will not be elaborated upon in this text because its availability remains limited and this means of testing is currently still fairly costly.

Here, I will focus on three main types of ancillary tests: 1. Cell block 2. Flow cytometry 3. Needle washing/fresh aspirated material

5.6.3.1 Cell Block Cell blocks are useful when there are unusual findings in the smears, other than those of the usual colloid nodule and follicular cell-derived lesions and neoplasms. • Suspected MTC –– IHC bridges the gap between a ‘Suspicious for MTC’ and an outright ‘MTC’ diagnosis. The closest mimics of MTC are follicular or Hurthle cell neoplasm, PTC, hyalinising trabecular tumour and metastatic carcinoma. Table  5.4 shows useful IHC stains to distinguish between these entities. • Suspected parathyroid lesion –– Immunohistochemistry with PTH is extremely helpful in this context. Other helpful antibodies are included in Table 5.4. IHC can help with characterising the cells a parathyroid in origin; however, the distinction between hyperplasia, adenoma and

Table 5.4  Thyroid—Helpful Immunohistochemical Panels Suspected diagnosis MTC

Calcitonin CEA + ±

TTF1 +

Hurthle cell adenoma/ carcinoma PTC Hyalinising trabecular tumour Metastatic carcinoma

−

−

+

Thyroglobulin Remarks Can also correlate with − serum calcitonin levels + NA

− −

− −

+ +

+ +

−

± (depending on tumour)

± (positive in metastatic lung adenocarcinoma; negative in most others)

−

Parathyroid lesion

−

−

−

−

NA Ki-67 showing unique membranous reactivity [31] Site-specific antibodies: refer to Chap. 6 (approach to lymph node cytology) PTH, GATA3 positive. Can also perform needle wash for PTH assay in biochemistry lab

66

indeed even carcinoma is not definitively possible on cytologic grounds. • Suspected metastases to thyroid –– The thyroid is an occasional site for metastases, and commonest sources in clinical series include renal cell carcinoma, lung carcinoma, gastrointestinal malignancies and breast carcinoma [24]. If ROSE suggests metastasis, a cell block is invaluable in the diagnostic workup. –– Metastases may be identified clinically as PET-avid nodules in staging or follow-up investigations of patients with known malignancies, or rarely be the initial presentation in a case of an unknown extrathyroidal malignancy. –– The pathologist is often required to ascertain the origin of the malignancy—whether primary or metastatic, and, if metastatic, to ascertain the primary site where possible. Specific IHC panels are provided in Tables 6.2 and 6.3 in Chap. 6 (Approach to Lymph Node Cytology). –– Anaplastic thyroid carcinoma (ATC) or PDTC may sometimes be a differential diagnosis for metastases. PDTC is generally still positive for TTF1 and thyroglobulin; however, ATC might be negative for these two markers, whilst it is more likely to retain PAX8 reactivity [25, 26]. It should be remembered that PAX8 is also positive in Mullerian and renal tumours. • Suspected infectious aetiology –– We recommend that if infection is suspected, fresh needle rinse material in sterile saline, or neat fluid if available, be sent to the microbiology laboratory. However, cell block may be useful for the application of special stains for organisms or PCR-based testing for specific micro-organisms. • Suspected lymphoma –– Ancillary testing can be performed on cell block tissue, including IHC and clonality tests (e.g. IgH rearrangements). IHC can be very helpful in supporting a diagnosis of lymphoma and characterising the lym-

5  Approach to Thyroid Cytology

phoma. However, it should be noted that MALT lymphoma may not be readily diagnosed even on IHC on cell block material. A combination of flow cytometry and clonality testing may be more helpful. –– Needle washings may also be sent to the haematology lab for flow cytometry, particularly in suspected low-grade lymphomas (see below). –– A practical note is that if there is suspicion of lymphoma on the FNA and a larger tissue biopsy is forthcoming, ancillary testing on cytology should be limited accordingly, so as not to duplicate tests and incur unnecessary cost. • Indeterminate nodules in which PTC is not excluded –– In PTC, there have been studies showing limited usefulness of BRAF IHC in cell blocks in indeterminate nodules, due to both the low incidence of BRAF-mutated nodules in this setting and limited correlation with BRAF-mutation testing using molecular methods [27–30]. However, if the tissue is positive for the BRAF V600E mutation, the specificity for PTC is over 90%, provided BRAF IHC has been validated against BRAF mutation testing in one’s own laboratory setting.

5.6.3.2 Flow Cytometry Flow cytometry can be very useful when lymphoma is suspected, including MALT lymphoma or other low-grade non-Hodgkin lymphoma, which can be difficult to diagnose on cytology, even with the aid of IHC on cell block material. Dedicated passes should be made and the needle rinsed immediately in the preservative recommended by the haematology laboratory. In our institution, we use RPMI solution. At times, sterile saline is also appropriate, if the material is brought to the haematology laboratory for immediate processing. Lymphoid cells can be assessed for lineage markers as well as B-cell clonality.

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67

5.6.3.3 Needle Washing/Fresh Aspirated Material As mentioned above, if a parathyroid lesion is suspected, needle washings can be sent to the biochemistry laboratory for parathormone levels. The volume of saline used for the needle rinse is important, and this should be pre-established through communication with the biochemistry laboratory. (Alternatively, cell block with IHC may also be used to confirm the presence of parathyroid tissue.) Needle rinse in sterile saline or direct needle aspirates of purulent fluid may be sent to the microbiology laboratory if an infectious aetiology is suspected, for staining, culture and antibiotic sensitivity assays.

5.7

Specific Challenges

In my practice, I have found four specific morphologic scenarios to be particularly challenging in thyroid cytology. Here I will outline simplified approaches to each of these challenging scenarios. The approaches are written with the busy practicing pathologist/cytologist in mind and cover the major differentials within each area.

1. 2. 3. 4.

Cystic Lesions Hurthle cell-rich lesions Lymphoid-rich lesions The atypical FNA—when to call FLUS/ AUS?

5.7.1 Cystic Lesions In the literature, approximately 5–11% of cystic, non-diagnostic nodules have malignant outcomes [32]. In particular, it is not uncommon for PTC to have a cystic component; hence, all cystic thyroid nodules should be evaluated with care. Essentially, any lesion in the thyroid can undergo cystic change, whether neoplastic or not. Cystic lesions are challenging because they tend to yield low numbers of diagnostic cells, which often show degenerative changes. Aspiration is best performed under ultrasound guidance, with the aim of aspirating to dryness, with subsequent passes targeting any residual solid areas. Cysts that collapse fully after aspiration are likely to be benign; however, this information is often not available to the reporting pathologist. Chart 5.3 highlights my approach to cystic nodules. The first step is to recognise them as

Cystic nodules (Many cyst macrophages, background granular cyst material)

Abundant colloid?

Yes

No

Follicular/Other lesional cells* present?

Follicular/Other lesional cells* present?

No

Benign; Consistent with Colloid nodule

Yes

Yes

Evaluate based on: - Architecture - Cytomorphology

Key: * Rarely, other cysts such as parathyroid cysts or thymic cysts may occur. IHC may be most helpful in these contexts.

Chart 5.3  Approach to cystic thyroid nodules

No

Non-Diagnostic; Cyst contents only

68

cystic. If cyst fluid is present grossly, that is an obvious clue—the fluid is often thin and brownish. On microscopic examination, two main morphological findings point to the cystic nature of a nodule—the presence of appreciable numbers of foamy macrophages (i.e. obvious on 4X objective) and/or the presence of finely granular proteinaceous background material. Sometimes, cholesterol crystals may be present as well, also pointing to a likely cystic lesion (Fig. 3.3, Chap. 3). Following the recognition of a cystic nodule, one then applies the same approach as one would to a solid nodule—to look for follicular cells and colloid. These elements should be evaluated in the same way as they are in a solid nodule— architecture and cytomorphology of follicular cells, and amount and texture of colloid. If these diagnostic elements are absent or quantitatively insufficient, then the diagnosis would fall into the “Non-diagnostic” category. Usually, the epithelial cells seen in the context of cystic lesions are thyroid follicular cells, though, rarely, other cystic lesions may occur such as parathyroid cysts or thymic cysts. Cell blocks for IHC may prove helpful in these contexts, as well as needle washing for PTH in the context of a suspected parathyroid cyst. There are two additional points that should be noted: 1. Cystic degeneration may be associated with reactive cellular changes, either in the follicular cells within the cyst fluid, or the cyst lining cells. There may be significant nuclear enlargement, pallor and even nuclear grooves. A clue is the fact that these cells usually occur in loose groups with a streaming appearance, with the long axis of the cells pointing in the same direction, not unlike reactive endocervical cells in cervical smears (Fig.  5.9a). Also, the cells may contain increased amounts of cytoplasm with elongated, tapering cytoplasmic processes, which suggests their reactive nature. These reactive changes should not be mistaken for neoplastic nuclear features, in particular, for PTC.

5  Approach to Thyroid Cytology

Hence, recognising the cystic nature of the nodule is important, in order to avoid a potential overcall of atypia or malignancy. ­ The  ­presence of other benign elements and thin colloid are also helpful corroborative features of benignity. 2. Any of the thyroid follicular cell neoplasms can be cystic. One of the commonest tumours that tends to undergo cystic change is PTC, as mentioned above. The cells in cystic PTC have been described in Sect. 5.6.2 (Triple Approach—Cytology) and are shown in Fig. 5.7c. Because of the low N/C ratios and numerous cytoplasmic vacuolations, the malignant cells may be mistaken for macrophages (Fig.  5.7d). Features that favour PTC over macrophages are tight cohesion, the occasional presence of psammoma bodies within the cell clusters, the well-defined, punched out tiny cytoplasmic vacuoles and the presence of atypical nuclear features.

5.7.2 Hurthle Cell-Rich Lesions Hurthle/oncocytic cells are commonly encountered in thyroid aspirates and are most often seen in the setting of benign nodules, most commonly in colloid nodules/benign follicular nodules with oncocytic change. These have the other characteristics of colloid nodules—flat honeycomb sheets of bland follicular cells with or without abundant colloid. The main differentials of Hurthle cell-rich lesions include: • Non-neoplastic conditions –– Colloid/hyperplastic nodule with Hurthle cell change –– Hashimoto thyroiditis • Benign neoplasms –– Hurthle cell adenoma –– Hyalinising trabecular tumour • Malignant neoplasms –– Hurthle cell carcinoma –– PTC –– MTC

5.7  Specific Challenges

69 Hurthle Cell-rich Lesions

Non-Hurthle follicular cells also present

Pure Hurthle cell population

Are there lymphocytes?

Any nuclear features of PTC/ MTC?

Yes Atypical lymphoid population? (refer to Chart 5.5)

Yes • AUS; Atypical lymphoid cells • Suspicious for lymphoma

Yes

No Benign follicular nodule (likely Hurthle cell change/metaplasia)

No Benign: Lymphocytic thyroiditis (Raise the possibility of Hashimoto thyroiditis in comment).

• •

PTC MTC

No Morphologic features suggesting neoplasm - Dissociation - Binucleation - Transgressing vessels - Macronucleoli, cellular monotony

Yes FN, Hurthle cell type

No FLUS, Hurthle cell type Clinicopathologic correlation is helpful. TBSRTC suggests including a comment that these nodules are more likely to be benign in a background of: • Multinodular goitre • Hashimoto thyroiditis

Chart 5.4  Approach to Hurthle cell-rich lesions

An approach to Hurthle cell-rich lesions is outlined in Chart 5.4. In Hurthle cell-rich lesions, one should perform the following: • Clinicopathologic correlation (Hashimoto thyroiditis? multinodular goitre?) In these settings, TBSRTC suggests classifying pure Hurthle cell lesions as AUS/FLUS but including a comment to reflect the higher likelihood of a hyperplastic nodule in this setting, over a neoplasm [7]. • Search for non-oncocytic follicular cells, lymphocytes and colloid (these usually favour benign nodules—refer to Chart 5.4). • Search for architectural and cytologic features that favour HCNs, e.g. traversing blood vessels, cellular dissociation. • Search for nuclear features of PTC or MTC.

5.7.3 Lymphoid-Rich Lesions (When to Diagnose Lymphoid Atypia?) An approach to lymphoid-rich lesions is outlined in Chart 5.5.

The presence of significant numbers of lymphocytes can pose a diagnostic challenge in thyroid nodules. The most important considerations include lymphocytic thyroiditis (including Hashimoto thyroiditis) and lymphoma. In this setting, clinicopathologic correlation is particularly important, as many important clues can be gleaned from the clinical presentation and history. The commonest type of primary thyroid lymphoma is diffuse large B-cell lymphoma (DLBCL), followed by mucosa-associated lymphoid tissue (MALT) lymphoma [33, 34]. MALT lymphoma with transformation to DLBCL may also occur. MALT lymphoma is a particularly challenging diagnosis because there is significant morphologic overlap with Hashimoto thyroiditis— both conditions show a mixed lymphoid population admixed with Hurthle cells. In fact, it may not be possible to cytologically distinguish MALT lymphoma from florid lymphocytic thyroiditis. Thus, it is important to keep an eye out for the ‘red flags’ that should prompt more careful investigation, both clinically and on cytologic examination.

5  Approach to Thyroid Cytology

70 Lymphoid-rich Lesions Atypical clinical or cytologic features? • Elderly patient, new or enlarging nodule/swelling (+/- longstanding Hashimoto thyroiditis) • Monotonous lymphoid population • Predominance of medium-sized or large lymphocytes Yes • AUS; Atypical lymphoid cells or • Suspicious for lymphoma Recommend tissue biopsy or repeat FNA with flow cytometry and/or cell block

No Lymphocytes far outnumber follicular cells? Yes

No

Could this be a lymph node?

Yes - No follicular cells/colloid - Appropriate clinical/radiologic context Favour reactive lymph node Clinicopathologic correlation is helpful. Recommend follow-up and biopsy if persistent/enlarging.

No/Unlikely

Benign; Lymphocytic thyroiditis (raise possibility of Hashimoto thyroiditis as appropriate) Recommend follow-up and repeat FNA with flow cytometry/biopsy if swelling progresses

AUS; Atypical lymphoid cells Recommend tissue biopsy or repeat FNA with flow cytometry and/or cell block

Chart 5.5  Approach to lymphoid-rich lesions

These include: • Clinical findings: –– A history of longstanding Hashimoto thyroiditis with a recently enlarging goitre. –– Elderly patient with a significant lymphoid yield on FNA. –– Compressive symptoms and signs. • Cytologic findings: –– Marked predominance of lymphocytes over follicular cells (even with a mixed lymphoid population). –– Monotonous lymphoid cell population. –– Predominance of medium-sized to larger lymphocytes. When in doubt, a diagnosis of ‘Atypical lymphoid cells’ may be warranted, and ancillary investigations (e.g. flow cytometry) or a tissue biopsy suggested.

5.7.4 T  he Atypical FNA: When to Call Something FLUS/AUS? One of the most subjective elements of evaluating thyroid cytology is knowing when to call a nodule atypical, i.e. TBSRTC category III: FLUS/ AUS. The Bethesda authors have clearly recom-

mended that this category not be split into FLUS vs. AUS and prefer the term ‘AUS’ as it is less morphologically restrictive than ‘FLUS’. However, there are some who do split the category into ‘FLUS’ for lesions with some architectural atypia and ‘AUS’ for lesions with nuclear or lymphoid atypia, which is a neat divide and one that is understood by clinicians. I believe that this practice has some merits because there has been a clear demonstration in the literature that nuclear atypia carries a higher risk of malignant outcomes than architectural atypia alone [11–14]. Whichever terminology is used, the risk implications of the terms used must be understood by clinicians and, ideally, management guidelines based on follow-up studies within each institution. In a follow-up audit of 132 AUS/FLUS nodules that were surgically excised, we found that the rate of malignancy was 36.8% in the nuclear atypia group compared to 14.7% in the group with only architectural atypia (14.7%), p 4–5×

Type of lymphoma Classical Hodgkin lymphoma

Nuclear features Classical Reed– Sternberg cells (RS cells) are extremely large (visible on low-power microscopy) and contain at least two nuclei or nuclear lobes, each featuring a macronucleolus Sometimes, mononuclear RS cells are present, these are called Hodgkin cells

Other findings A mixed cell population is noted in the background, including small lymphocytes, plasma cells, eosinophils, and occasionally small epithelioid granulomas without necrosis. Note: NSHL may be associated with hypocellular smears

Differential diagnoses 1. Infectious mononucleosis (large, reactive immunoblasts) may resemble RS cells/Hodgkin cells. The distinction may be challenging and is aided by correlation with the clinical and serologic findings 2. Nodular lymphocyte predominant HL (popcorn cells have less prominent nucleoli than RS cells; however, cytologic distinction can be challenging). Histologic biopsies with IHC is recommended 3. T-cell-rich B-cell lymphoma. IHC is helpful in the distinction 4. Angioimmunoblastic T-cell lymphoma (large immunoblasts can resemble RS cells). Patients tend to present with generalised lymphadenopathy, unlike in HL

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95

Table 6.5 (continued) Cell size (relative to resting lymphocyte) Variable

>4–5×

Type of lymphoma Peripheral T-cell lymphoma

Anaplastic large cell lymphoma

Nuclear features Cell population may appear polymorphous with cells of different sizes, variable nuclear appearance and shape. Some nuclei may appear irregular, indented, grooved or lobulated Hallmark cells— very large cells approaching the size of RS cells, with horseshoe-, donut- or “embryoshaped” nuclei

Other findings Mixed cellular picture, which can mimic a reactive lymph node. Histiocytes, eosinophils and plasma cells may be seen

Differential diagnoses May potentially be mistaken for the mixed population of a reactive lymph node. Clinical clues: Patient often elderly and unwell (see Sect. 6.7)

The hallmark cells may at times resemble RS Reed–Sternberg cells (RS cells) cells, however, they are more numerous than RS cells. Necrosis and inflammatory cells, e.g. histiocytes, may be present in the background

1. Hodgkin lymphoma—though hallmark cells are usually more numerous than the usual sprinkling of Reed–Sternberg cells in HL 2. Carcinoma—due to the propensity of the large lymphoid cells to aggregate, and their large size that falls into the size range of epithelial cells

Lymphoid population or not?

Yes (Lymphoid)

Lymphoid: • Generally discohesive cells • Small cells with high N/C ratios • LGBs around lesional cells

Predominant architectural pattern?

Predominant architectural pattern?

Tissue fragments

Benign (lymphohistiocytic): • Reactive lymph node (LH aggregates) • Granulomatous lymphadenitis

Malignant: • FL • LBCL • ALCL • HL (Syncytial variant)

No (Non-lymphoid)

Dispersed cells

Mixed cell population: 1. Predominantly small lymphocytes: • Reactive lymph node • MZL 2. Predominantly Medium-sized or large lymphocytes: • FL • MZL 3. Scattered very large cells: • HL • TCRBCL

Monotonous cell population: • Lymphoma (Cell size helps categorise into specific types)

Key: ALCL: Anaplastic large cell lymphoma; FL: Follicular lymphoma; HL: Hodgkin lymphoma; LBCL: Large B-cell lymphoma; LGBs: Lymphoglandular bodies; MZL: Marginal zone lymphoma; TCRBCL: T cell-rich B-cell lymphoma

Chart 6.2  Lymph node FNA: pattern-based approach

Tissue fragments

Metastases: • Most metastatic carcinomas Others (Benign or malignant): • Non-nodal lesions e.g.pilomatrixoma, Warthin tumour, schwannoma • Granulation tissue

Dispersed cells

Benign: • Branchial cleft cyst/ Lymphoepithelial cyst Metastases: • Melanoma • Poorly differentiated/ Undifferentiated carcinoma • Neuroendocrine/ Small cell carcinoma • Gastric signet ring cell carcinoma • Lobular carcinoma of breast

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96

the specific morphological features within each broad pattern. 1.  Cohesive-Appearing Tissue Fragments

Definition: For the purposes of this text, tissue fragments are defined as cohesive-appearing sheets of cells that aggregate as a body on low power microscopy (using 2× or 4× objectives, Fig. 6.1). There may or may not be single dispersed cells in the background. Lymphohistiocytic (LH) aggregates representing germinal centre cells in a reactive lymph node are one of the commonest causes of tissue fragments in nodal aspirates (Fig.  6.2). These are described in detail in Sect. 6.6. Some lymphomas, e.g. follicular lymphoma, DLBCL and anaplastic large cell lymphoma, may also form

aggregates. Otherwise, lymphoid cells tend to be dispersed rather than to form cohesive-appearing sheets. When true cohesive, well-defined sheets of cells are seen, metastatic carcinoma should be high on the list. Figure 6.3 shows cohesive appearing lymphoid aggregates compared to true cohesive sheets in metastatic carcinoma (Fig. 6.5). 2.  Predominantly Dispersed Cells

Definition: For the purposes of this text, dispersed cells refer to cells that can be recognised as not adhering to adjacent cells. Hence, cells that may appear to be grouped together loosely are still considered dispersed if one is able to discern that the cell membrane of each cell is separate from that of the neighbouring cells (Fig. 6.1d).

a

b

c

d

Fig. 6.1  Adequacy and architecture in lymph node aspirates. (a) Hypocellular smears that are of borderline adequacy for evaluation of lymphoid pathology (HC, 40×); (b) Low-power view showing dispersed lymphocytes and some aggregates, likely representing germinal centres

(HC, 40×); (c) Mixed lymphoid population with predominantly small lymphocytes, classical picture of reactive lymph node (HC, 200×); (d) Aggregate of lymphocytes without true cellular cohesion; note the LGBs in the background (HC, 400×)

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a

b

c

d

Fig. 6.2  LH aggregates (germinal centre material). (a) Low-power view of LH aggregate, note the frayed edges (Pap, 40×); (b) High-power view LH aggregate, note variation in cell sizes (HC, 300×); (c) High-power view LH

aggregate, note variation in cell sizes. A tingible body macrophage (TBM) is present (Pap, 300×); (d) Follicular dendritic cell (black arrowhead) with TBMs from a germinal centre (Pap, 400×)

A general rule of thumb is that most lymphoid cells are discohesive, hence are disposed as dispersed cells in smears, whether benign or malignant. Metastatic malignancies tend to be more cohesive (especially in carcinomas). However, a few entities e.g. malignant melanoma may occur as more dispersed cells and hence cause some diagnostic confusion. Figures  6.6 and 6.7 show examples of lymphomas and non-lymphoid entities occurring as dispersed cells, respectively. The main differential diagnoses are discussed in Sect. 6.6. Table 6.2 summarises the salient morphologic features of certain metastatic malignancies that can be recognised on cytology, with both cohesive and dispersed cell arrangements. Following that, Table  6.3 lists metastatic malignancies in which the source may not be readily discerned from cytomorphologic features.

Cytomorphology Whilst cytomorphology is important, in a lymphoid cell population, the actual composition of the cell population is also very important, i.e. whether the population is monotonous or mixed (size-wise), and what the predominant cell size is. These distinctions are explained in more detail in Sect. 6.6. • Cell size –– Cell size may be determined by rough comparison with a small, resting lymphocyte (usually only marginally larger than a red blood cell, with condensed nuclear chromatin). –– Cell size is a very important clue in the distinction of lymphoid vs. non-lymphoid lesions. Lymphocytes are amongst the smallest nucleated cells in the body, and a

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a

b

c

d

Fig. 6.3  Tissue fragments—lymphoid vs. carcinoma. (a, b) Cohesive-appearing lymphoid aggregate in follicular lymphoma—note how the aggregated cells appear identical to the dispersed background lymphocytes (Pap, 300×;

HC, 400×); (c, d) True cohesive sheets in metastatic nonkeratinising squamous cell carcinoma (Pap, 300×; HC, 300×)

resting lymphocyte is only slightly larger than a red blood cell (approximately 6–9 μm in diameter). The smallest cell epithelial malignancies (e.g. small cell carcinoma) feature cells that are about 2–4 times the size of a resting lymphocyte, with other malignancies comprising even larger cells. –– Larger benign lymphocytes include centroblasts found in germinal centres (with round to oval nuclei and 2–3 peripherally arranged nucleoli) and immunoblasts (these usually contain a large single central nucleolus). These cells are usually 2–2.5 times the size of resting lymphocytes. Lymphomas may also arise from these cells. –– In the diagnosis of lymphoma, the cell size is a helpful clue to the type of lymphoma, particularly in B-cell lymphomas (Fig. 6.6).

–– Tables 6.4 and 6.5 highlight some of the commoner lymphomas with reference to the cell size. • Cell size variation (cell composition) –– This is also of paramount importance—a monotonous lymphoid cell population is worrying for non-Hodgkin lymphoma (NHL) whilst a mixed lymphoid cell population is more likely to be reactive in nature. Several caveats should be noted: • In a reactive lymphoid population, the predominant cell size is usually small (Fig.  6.1). A mixed but predominantly medium-sized lymphoid population may be a subtle sign of a low-grade lymphoma, e.g. marginal zone lymphoma (MZL). • MZL may be missed on cytology, closely resembling the mixed cell

6.5  Triple Approach

population of a reactive lymph node (Table  6.1). Figure  6.10 illustrates such a case. • LH aggregates from germinal centre material may comprise a seeming predominance of larger lymphocytes. These should not be mistaken for atypical lymphocytes. Helpful clues are the presence of tingible body macrophages (TBMs), follicular dendritic cells (FDCs) and the wide range of cell sizes seen, which is characteristic of germinal centre material (Fig. 6.2). –– A pattern of scattered singly occurring very large cells on a background of numerous smaller lymphocytes is suggestive of classical Hodgkin lymphoma (CHL), nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) and other rarer entities such as T-cell-rich B-cell lymphoma (TCRBCL) and anaplastic large cell lymphoma (ALCL). • Cell shape –– Lymphoid cells are usually of a fairly rounded appearance, whereas epithelial or mesenchymal cells may be polygonal/epithelioid, plasmacytoid, columnar, elongated, spindled or bizarrely shaped. –– Certain lymphoproliferative conditions may rarely appear as spindle cells, and these include follicular lymphoma and follicular dendritic cell sarcoma. –– Plasmacytoid cells may be seen in a range of conditions, in both nodal and non-nodal sites. Examples include plasma cells (benign and neoplastic—a pale staining perinuclear hoff is frequently present, Fig.  6.7), well-differentiated neuroendocrine tumours, certain carcinomas such as breast carcinoma, malignant melanoma (Fig.  6.7) and myoepithelial carcinoma (MECA). –– Larger, polygonal/plump cells may be seen in epithelioid granulomas—these comprise haphazardly arranged aggregates of large cells with elongated slipper-shaped nuclei and ill-defined cytoplasm (Fig. 6.12).

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–– Other cell shapes may be seen in metastatic malignancy, as shown in Table 6.2. • Cytoplasm –– Most lymphocytes, whether benign or malignant, contain little cytoplasm. Sometimes, the outer rim of cytoplasm appears bluish in Romanowsky stains; however, this feature may be seen in small cell carcinoma as well. –– Anaplastic large cell lymphomas and large B-cell lymphomas (LBCLs) may contain large amounts of cytoplasm to the extent that they can mimic carcinomas. Attention to other clues such as dispersed architecture and the presence of LGBs in the background will help one to classify these conditions correctly. –– Metastatic malignancies may also have specific cytoplasmic features, which are discussed in Table  6.2. One should also pay attention to intracytoplasmic substances within malignant cells that may provide a clue to the cell type, e.g. melanin pigment suggests a metastatic melanoma (Fig. 6.7) and intracytoplasmic bile pigment suggests hepatocellular carcinoma in the appropriate clinical contexts. • Nucleus –– In lymphoid lesions, particularly in lymphomas, the nuclear size, shape and chromatin pattern are extremely important (Fig.  6.6). Hence evaluation of both airdried and alcohol-fixed smears is essential. Tables 6.4 and 6.5 describe some of the more recognisable nuclear features of certain more commonly encountered lymphoproliferative diseases. –– In Sect. 6.6, the specific points of differences will be discussed between chromatin patterns in lymphoma vs. carcinoma. Background In lymph node aspirates, background material can provide very helpful clues to the possible nature of the pathology involving the lymph node, or even whether the aspirate is indeed from a lymph node or non-nodal structure.

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• Lymphoglandular bodies (LGBs) –– LGBs are extremely helpful clues that suggest the lymphoid nature of the aspirated cell. They are seen accompanying both benign and malignant lymphoid cell populations. –– LGBs can be appreciated on both alcoholfixed and air-dried preparations but are more readily appreciated on Romanowsky stains. They are of subcellular size and appear similar in colour and texture to the cytoplasm of lymphocytes—seen as small, rounded basophilic bodies of varying sizes that are seen in the immediate background of lymphocytes, possibly representing cytoplasmic fragments (Fig. 6.1). • Necrosis –– The presence of necrosis is always abnormal in nodal aspirates, with the two most common differentials being that of infection (e.g. necrotising granulomatous inflammation in mycobacterial or fungal infections, acute suppurative inflammation) and malignancy. –– Necrosis can be recognised as either amorphous, granular material that tends to form clumps or ‘rolls’ due to mechanical smearing (Fig. 3.3, Chap. 3); or as actual ‘cell carcasses’ with individual cellular outlines still visible, but with no appreciable internal cellular structures. –– Usually, the presence of other cellular elements is first noted (e.g. epithelioid granulomas or malignant cells), which then prompts one to look for necrosis. • Cyst material –– If the aspirate is confirmed to be from a lymph node, a cystic node is always abnormal and should raise the possibility of cystic metastasis. The location of the node is of paramount importance, for example, in a cervical lymph node, metastatic papillary thyroid carcinoma (PTC) and HPV-related oropharyngeal squamous cell carcinoma (SCC) would be high on the list of suspects, because the metastases often exhibit cystic change [7]. Specific cytomorphologic features can be sought, and the suspicion may be confirmed by IHC on cell

block (Tables 6.2 and 6.3). These cases will also benefit from clinicoradiologic correlation if available. –– In a cystic lesion in which the cytomorphologic findings are more bland, then nonnodal lesions should be considered, such as branchial cleft cysts or cystic salivary gland lesions that may be mistaken clinically for lymph nodes (e.g. Warthin tumour). In branchial cleft cysts, the findings usually include single occurring, bland superficial type squamous cells with small nuclei and very low N/C ratios. These will be further discussed in Chap. 7. • Micro-organisms –– These may be sought using special stains on smears or cell block, if the smears show infective features. –– In smears harbouring Mycobacterium avium complex (MAC) organisms, a particularly characteristic finding in Romanowsky-stained smears is the presence of ‘negative image’ thin, whitish rods seen within macrophages and also occurring loosely in the background [9, 10]. These negative images can be seen in Fig. 6.4a and b. Patients are often immunocompromised, and the cytologic picture may be that of granulomatous or suppurative inflammation. • Background material associated with certain malignancies may also be identified, such as extracellular mucin in certain adenocarcinomas and glycogen, imparting a tigroid, lacyappearing background due to the presence of glycogen in some tumours (e.g. germinomas, as shown in Fig. 3.3c, Chap. 3).

6.5.3 Ancillary Tests Material should be collected for ancillary tests if the following conditions are suspected: • • • •

Primary malignancy (lymphoma) Secondary malignancy (metastases) Suspected infectious aetiology Indeterminate FNA results

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Ancillary tests may be collected or processed in the following ways: 1. Cell block 2. Flow cytometry (haematology lab) 3. Needle rinse in sterile saline or neat fluid from sample (microbiology lab) 4. Needle wash for biochemical analysis (biochemistry lab) All samples should be collected and sent fresh, except in the case of the creation of a cell block, in which material may be allowed to clot and then can be placed into formalin. Table 6.6 summarises some the uses of ancillary tests in various suspected nodal pathologies. Here are some practical tips regarding the application of ancillary tests.

6.5.3.1 Suspected Lymphoma • When lymphoma is suspected, a practical and cost-effective approach is best. If a quick follow-up histologic sample is likely, ancillary testing should be minimised to avoid duplica-

tion of tests. However, if a histologic tissue sample is not forthcoming, then more extended ancillary testing panels are indicated, in order to allow for definitive management. Adequacy evaluation of FNA samples in this setting should also take into account that this may be the tissue sample for that particular patient. • Cell block or flow cytometry? This decision depends on the individual pathologist, the practice setting and type of lymphoma suspected. Here are two examples in which the type of lymphoma helps determine the usefulness of the test: –– If MZL is suspected, IHC on cell blocks may be of limited help, whilst flow cytometry may be more helpful. Even so, these cases may eventually require excision for demonstration of architecture. –– If Hodgkin lymphoma is suspected, flow cytometry is not helpful simply because of the relative paucity of neoplastic cells and the high possibility of a false-negative result. Cell blocks may be more informative if lesional cells are sufficiently sampled.

Table 6.6  Ancillary Tests in Lymph Node FNA Suspected diagnosis Metastases or lymphoma (including Hodgkin or non-Hodgkin lymphoma) Non-Hodgkin lymphoma

Infection

Material for ancillary testing Cell block

Needle rinse in preservative solution (e.g. RPMI solution), haematology lab Unprocessed material sent to microbiology lab Cell block

Unstained smears

Suspected metastases which produce assayable products (e.g. thyroid carcinoma)

Needle wash, to biochemistry lab

Source specimen (initial collection method) • FNA blood clots/ solid tissue pieces (preferably from dedicated pass.) • FNA needle rinse in saline FNA needle rinse in laboratory-specified solution

• Aspirated purulent material • FNA needle rinse in sterile saline • FNA needle rinse in sterile saline • FNA blood clots/solid tissue pieces FNA unstained smears (alcohol-fixed) FNA needle rinse in a fixed volume of saline

Ancillary tests • Immunohistochemistry • Molecular testing (e.g. FISH, PCR-based testing) Flow cytometry (usage of flow vs. cell block depends on individual pathologist and laboratory practice) Smears for special stains for organisms; culture and sensitivity • Special stains for organisms • Molecular testing (e.g. PCR-based testing) Special stains, e.g. Ziehl Neelsen (ZN), Grocott methenamine silver (GMS) Biochemical testing, e.g. thyroglobulin [8]

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a

b

c

d

Fig. 6.4  MAC organisms and granulation tissue. (a) Main picture and inset show negatively staining rods within the cytoplasm of multinucleated histiocytes (HC, 600×); (b) Same case showing negatively staining rods lying loosely in the background; inset shows ZN stain highlighting numerous intracytoplasmic acid-fast bacilli

(HC and ZN, 600×); (c) Low-power view of granulation tissue with a branching fine vascular network (HC, 40×); (d) High-power view of granulation tissue showing myofibroblasts and inflammatory cells loosely surrounding thin capillaries (HC, 200×)

6.5.3.2 Suspected Metastases Cell blocks are particularly useful in the setting of metastatic malignancy. Specific IHC panels that are helpful in ascertaining the source of malignancy are summarised in Tables 6.2 and 6.3.

• Cell blocks may provide some additional morphologic information in terms of architecture of tissue fragments, especially if they are formed from spontaneously clotted material. Sometimes, direct comparison of morphology on cell block with previous histology of a known malignancy can enable a definitive diagnosis, thereby obviating the need for IHC.

General Tips: • It is a good practice to provide a semi-quantitative description of the amount of lesional material present in the cell block (e.g. sparse or moderately abundant lesional cells), especially in the context of malignancy. This will be a helpful and readily accessible record and a rough gauge of how much archival material there is for further ancillary testing should the need arise, e.g. for the evaluation of predictive and prognostic markers.

6.6

 Sequential, Pattern-Based A Diagnostic Approach

The approach outlined here is summarised in Chart 6.2. In this section, a semi-sequential approach will be presented. Of course, in real life, the

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sequence is not truly linear because evaluation occurs simultaneously from several angles. However, this section provides a breakdown of the important questions to ask when evaluating lymph node aspirates. Here are some key questions that need to be addressed:

‘aggregates’ lack true cohesion—often the cellular outlines are distinct even in cells that are close to each other (Fig. 6.1d). –– In general, metastatic epithelial malignancies tend to exhibit true cohesion, forming syncytial tissue fragments with sharper, well-defined borders. –– Certain lymphomas may appear more aggregated; these are shown in Chart 6.2 and discussed in the next section on architectural patterns.

1. Is this a lymphoid population? 2. What is the main architectural pattern? 3. In a lymphoid lesion, what is the cell make-up? 4. If this is a non-lymphoid lesion, could this be a non-nodal lesion? 5. Is there any relevant clinical finding that ties in with the lymphadenopathy?

6.6.1 Is This a Lymphoid Population? One of the early questions one must answer when evaluating nodal aspirates is: Is this a lymphoid or non-lymphoid population? If this is a nonlymphoid cell population, the possibility includes metastatic disease, as well as aspirates from tissues other than a lymph node, such as an adnexal tumour. To answer this question, one can look at four helpful morphologic parameters: 1. 2. 3. 4.

Cell cohesion Cell size Lymphoglandular bodies Nuclear chromatin

6.6.1.1 Cell Cohesion –– Lymphocytes are seldom truly cohesive. At times, lymphoid tissue fragments may appear in lymphoid lesions, these often represent LH aggregates from germinal centres (Fig.  6.2). These are described below under ‘architectural patterns’ (Sect. 6.6.2). –– Aggregated lymphocytes can be recognised as lymphoid by their similarity to the more dispersed lymphocytes in the background. Focusing up and down shows that these tissue

Practical Tips: • Focus up and down at the periphery of the tissue fragments to appreciate the cytomorphologic features better. • Compare the cells within the aggregates to background lymphocytes to determine if the aggregates are composed of lymphoid cells or not—look at cell size and shape, nuclear appearance and N/C ratios.

6.6.1.2 Cell Size –– As mentioned in Sect. 6.5.2, lymphocytes are much smaller than epithelial cells, macrophages and mesenchymal cells. This is a very useful clue in determining if a cell population is lymphoid or not. A resting lymphocyte (slightly larger than an RBC, with condensed chromatin and sparse cytoplasm) is a useful ubiquitous cell for gauging of cell size. –– The cell size in small cell carcinoma is 2–4 times the size of a resting lymphocyte, in the range of the size of a large cell lymphoma. Other features that are most helpful are nuclear chromatin and the presence of LGBs; both are described below. –– Tables 6.4 and 6.5 provide a range of sizes for cells of different types of lymphoid malignancies. 6.6.1.3 Lymphoglandular Bodies LGBs have been described in Sect. 6.5. Here are practical tips in the evaluation of LGBs: • A well-prepared Romanowsky-stained smear is strongly recommended, in which LGBs are more readily appreciated.

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• They should be seen immediately around lesional cells, preferably where there are sparse bystander lymphocytes from the lymph node parenchyma.

6.6.1.4 Nuclear Chromatin –– The chromatin pattern in lymphocytes varies from fine in lymphoblastic cells to vesicular, coarse or condensed. In the small resting lymphocyte, the chromatin is relatively condensed. In lymphomas with smaller to medium-sized cells, chromatin is relatively dark or clumped, with the exception of lymphoblastic lymphoma (see Tables 6.4 and 6.5). –– In practice, the commonest lymphoma-carcinoma differential is large B-cell lymphoma (LBCL) with small cell carcinoma, and the chromatin pattern is the key discriminator. In LBCLs, the chromatin tends to be vesicular with some coarse clumps, and/or prominent nucleoli which may be central or peripheral. In contrast, in small cell carcinoma, the chromatin is relatively evenly granular without prominent nucleoli. Figure 6.12 compares and contrasts these two entities.

6.6.2 W  hat Is the Main Architectural Pattern? In this section, we will look at two main architectural patterns that are encountered in lymph node aspirates. The differential diagnoses can be seen at a glance in Chart 6.2. The two architectural patterns are: 1. Tissue fragments 2. Dispersed cells

6.6.2.1 Tissue Fragments These have been described in Sect. 6.5.2. Lymphohistiocytic aggregates, granulomas and metastatic carcinoma are some of the commonest entities that may be seen as tissue fragments. A description of the differential diagnoses is presented here, moving from lymphoid to non-lymphoid entities. (a) Lymphoid/histiocytic entities • Benign

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–– Lymphohistiocytic (LH) aggregates— These are the most frequently encountered tissue fragments seen in nodal aspirates, representing benign germinal centre material. On low power, they are recognised by their loose nature, which is due more to aggregation than true cellular cohesion. The edges of such tissue fragments are usually somewhat loose and frayed, without sharp borders as one would encounter in a sheet of epithelial cells. Other morphologic clues include the wide spectrum of cell types and sizes from smaller centrocytes to larger centroblasts (often with several marginated nucleoli); TBMs and occasional follicular dendritic cells (FDCs). FDCs are often binucleated with smooth outlined, oval nuclei, each containing a small, regular nucleolus, somewhat resembling ‘cartoon eyes’ [11]. Figure 6.2 shows some examples of LH aggregates in reactive lymph nodes. –– Granulomas—These are recognised by the fairly haphazard arrangement of large epithelioid cells with slippershaped nuclei and abundant, ill-defined cytoplasm (Fig. 6.12). • Larger granulomas, often associated with necrosis, are often seen in infectious conditions. • Smaller, well-formed granulomas without necrosis may raise the possibility of sarcoidosis after infection is excluded. • Very small granulomas comprising only a handful of epithelioid cells may also be seen in association with some malignancies including undifferentiated nasopharyngeal carcinoma, squamous cell carcinoma, Hodgkin lymphoma and germinoma. • Malignant –– Lymphoma—Whilst most lymphomas yield dispersed cells, occasional types may appear more cohesive. These include:

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• Follicular lymphoma (Fig. 6.3—note the similarity between aggregated and background dispersed cells). • Classical Hodgkin lymphoma, syncytial variant. • Anaplastic large cell lymphoma. • Occasionally, large B-cell lymphoma. ( b) Non-lymphoid entities • Benign –– Granulation tissue—This can be seen in the setting of acute suppurative lymphadenitis as branching fragments of thinwalled vessels, often appearing cellular due to closely intermingled mixed inflammatory cells and myofibroblasts (Fig. 6.4). They should not be mistaken for true papillary tissue fragments, which should be associated with epithelial cells. –– At times, an adnexal, soft tissue or

salivary gland lesion is mistaken for a lymph node, and these may give rise to some confusion when evaluating such aspirates. Examples include adnexal lesions such as pilomatrixoma (Fig.  6.9), or soft tissue lesions including lipoma or schwannoma. In order to avoid potential overdiagnosis as metastatic malignancy, one should always bear this possibility in mind. • Malignant –– Metastatic malignancy—Most metastases are epithelial in nature, and cells are usually arranged as cohesive sheets. Table 6.2 highlights certain architectural characteristics of some malignancies with cytologically distinct features—e.g. acinar formations, papillary structures. Figure 6.5 illustrates some examples.

a

b

e

c

d

Fig. 6.5  Metastases with characteristic cytologic features. (a) Discernible glandular openings and cells with elongated, palisaded nuclei in colorectal carcinoma (Pap, 300×); (b) Dirty necrosis in metastatic colorectal carci-

f noma (Pap, 100×); (c–f) Metastatic prostatic carcinoma, showing acinar formations in (c, d) (HC, 100×; Pap, 200×), (e) Cell block (HE, 40×) and (f) IHC on cell block (PSA, 200×)

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a

b

d

c

e

Fig. 6.6  Dispersed cell pattern in lymphoma—cell size and chromatin pattern. (a–c) These are taken at the same magnification to facilitate comparison of cell size. (a) Small lymphocytic lymphoma with ‘soccer ball’ chromatin (Pap, 400×); (b) Burkitt lymphoma—medium-sized

cells with relatively coarse chromatin (Pap, 400×); (c) LBCL—note the clumped chromatin (Pap, 400×); (d) T-cell lymphoma with very irregular nuclear outlines (Pap, 600×); (e) HL with readily identifiable RS cells at medium power magnification (HC, 200×)

6.6.2.2 Dispersed Cells Most lymphoid populations, whether reactive or malignant, will exhibit a more dispersed cell architecture; however, non-lymphoid lesions may also adopt this architectural pattern. Figures  6.6 and 6.7 show a range of lesions with dispersed cell patterns, while Tables 6.4 and 6.5 summarise morphological findings in the commoner lymphomas. Several common and important entities presenting mostly as dispersed cells will be described here, again, first looking at lymphoid and then non-lymphoid entities.

–– Benign or reactive lymph nodes often show numerous dispersed lymphocytes of varying sizes. Occasional LH aggregates may be admixed with the dispersed cells. Recognising the lymphoid population as benign relies on the presence of a mixed population with comprising predominantly small lymphocytes. • Malignant –– Malignant lymphomas also frequently present as dispersed, discohesive cells. Lymphomas tend to yield more monotonous populations, and these will be described in the next section on cell make-up in lymphoid lesions.

(a) Lymphoid entities • Benign

6.6  A Sequential, Pattern-Based Diagnostic Approach

(b) Non-lymphoid entities • Benign –– It is uncommon to encounter benign non-lymphoid lesions smearing as single dispersed cells; however, sometimes, branchial cleft or lymphoepithelial cysts may masquerade as nodal masses. On cytology, the picture is that of single bland squamous cells with low N/C ratios with or without accompanying lymphocytes. • Malignant –– Certain metastatic malignancies may exhibit a dispersed cell pattern. Some of these include: • Melanoma (Fig. 6.7). • Poorly differentiated/ Undifferentiated carcinoma (Fig. 6.7).

a

b

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• • • •

Small cell carcinoma . Neuroendocrine tumour. Gastric signet ring cell carcinoma. Lobular carcinoma of breast.

6.6.3 I n a Lymphoid Lesion, What Is the Main Cell Make-Up? Once one ascertains that one is dealing with a lymphoid cell population, it is important to answer these two questions: 1 . Is there a monotonous or mixed cell population? 2. What is the predominant cell size? Tables 6.4 and 6.5 summarise the salient morphological findings of some of the commoner

c

f

d

e

Fig. 6.7  Dispersed cells—plasmacytoid and non-lymphoid lesions. (a) Plasma cells with pale perinuclear hoff (HC, 400×); (b) Melanoma featuring dispersed plasmacytoid cells with melanin pigment (HC, 200×); (c) Medullary carcinoma showing dispersed plasmacytoid cells with

g stippled chromatin (Pap, 400×); (d, e) Metastatic undifferentiated NPC presenting as dispersed cells (HC, 200×; Pap 300×); (f, g) Corresponding post-nasal space biopsy (HE, 100×; IHC with MNF116 IHC, 200×)

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types of lymphomas. Below are the usual differentials based on cell composition and size.

6.6.3.1 Mixed Cell Population –– Benign/reactive pattern: Mixed population of lymphocytes of different sizes, with small lymphocytes predominating. –– Atypical pattern: Mixed population of lymphocytes with medium-sized or larger lymphocytes predominating. This raises a concern for lymphoma with mixed cell size (e.g. marginal zone lymphoma, follicular lymphoma). Note that LH aggregates in reactive lymph nodes may show a predominance of larger cells, e.g. centrocytes and macrophages; however, these should be readily recognised by the wide spectrum of cell sizes, the frequent presence of tingible body macrophages and follicular dendritic cells (Fig. 6.2). 6.6.3.2 Monotonous Lymphoid Population –– This is always worrying for lymphoma and should be flagged as at least atypical, if not suspicious or malignant. –– Again, the predominant cell size plays an important role. Table 6.4 provides a quick guide to three general size groupings in B-cell lymphomas—small, medium and large. In practice, it is useful to note the size of the abnormal lymphoid cell population in conjunction with the chromatin pattern, in order to ascertain if the cytomorphologic features are in keeping with a known lymphomatous entity. This can help increase one’s confidence in making the diagnosis of malignant lymphoma, particularly if there is no material for ancillary testing. Chart 6.2 summarises the evaluation of cell make-up in the broad diagnostic approach.

6.6.4 I f This Is a Non-lymphoid Lesion, Could This Be a  Non-nodal Lesion? –– As mentioned earlier, one should keep an open mind when evaluating lymph node aspi-

rates and always consider the possibility of a non-lymph node structure being aspirated. –– The main danger is in overcalling benign epithelial non-nodal lesions as metastases. Two particularly treacherous entities are the pilomatrixoma and Warthin tumour, in which metastatic carcinoma may be erroneously diagnosed. Some other examples of non-nodal lesions are provided in Charts 6.1 and 6.2. –– This pitfall is discussed in greater detail in Sect. 6.7.

6.6.5 I s There Any Relevant Clinical Finding that Ties in with the Lymphadenopathy? The importance of applying clinicopathologic correlation in the evaluation of nodal aspirates is undisputed, because nodal disease often reflects regional or systemic disease. Clinical information may not always be available; however, when it is, it can be enormously helpful on narrowing down differential diagnoses and enabling one to apply a more focused panel of ancillary tests.

6.7

Pitfalls

As in any other organ system, a basic knowledge of the ‘danger zones’ in lymph node cytology is helpful. We will look at three main areas of potential pitfalls: 1. False positives 2. False negatives 3. Mimics

6.7.1 False Positives False-positive diagnoses may occur in two forms: benign non-nodal lesions such as adnexal tumours, in which metastatic malignancy is erroneously diagnosed; and reactive lymph nodes in which an overcall of lymphoma may be made.

6.7 Pitfalls

6.7.1.1 Benign Non-nodal Lesions Diagnosed as Metastases As is reflected in Chart 6.1, sometimes, nonnodal lesions are clinically labelled as lymph nodes. These lesions may be benign or malignant, but, more importantly, they may give rise to false-positive diagnoses as metastatic malignancy. Some examples include adnexal skin lesions, lipomas, schwannomas or even salivary gland neoplasms. Sometimes, these may even be subcutaneous metastases that are not within lymph nodes, e.g. in renal cell carcinoma. To minimise the risk of overdiagnosis, it is a safe practice to always ensure that cytomorphologic features of malignancy are present before one makes a diagnosis of metastatic malignancy in a lymph node, rather than just the presence of epithelial cells alone. Two examples of potential false-positive diagnoses are described here. Pilomatrixoma Pilomatrixoma (PMX), also known as ‘pilomatricoma’ and ‘calcified epithelioma of Malherbe’ is a benign adnexal tumour that arises from the hair matrix, and frequently occurs in the head and neck region, which overlaps with the location of lymph nodes. Cytology reveals sheets of basaloid cells which may appear fairly crowded but relatively monotonous, with variably prominent nucleoli. Mitotic figures may be very numerous, contributing to the impression of malignancy. Dispersed squamoid appearing cells may also be seen in the in the Papanicolaou-stained smears, with pyknotic nuclei, or sometimes no discernible nuclei, and orangeophilic or yellowish cytoplasm, resembling individual keratinised cells encountered in SCC. PMX is most often mistaken for metastatic carcinoma, often squamous cell carcinoma, because of the presence of keratinised appearing cells, and the clinical impression of an enlarged lymph node. In a literature review of 16 cases, Wong et al. found that only 4 cases were correctly diagnosed on cytology, whilst the rest of the cases were diagnosed as carcinoma or suspicious for malignancy [12]. PMX has been erroneously diagnosed cytologically as squamous cell carcinoma,

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mucoepidermoid carcinoma and adenocarcinoma [12–14]. Tips and Clues: • Epidemiologically, PMX tends to occur in children and young adults; although they may present in later adulthood. Not infrequently, they occur in the head and neck region. Therefore, there should be an index of suspicion when a lump in the head or neck region in a child or young adult yields large sheets of basaloid cells and some individual keratinised cells. • Clinically, PMX is usually recognisable as a protruding skin lump and is somewhat more superficial than a lymph node. • There may be a gritty feeling during the FNA procedure, when the needle enters the lesion, due to the presence of calcifications. • Look for sheets of ‘ghost cells’—squamous cells in which the nuclei are no longer visible but appear as translucent ‘holes’ in the centre of the cell. These cells have fairly abundant cytoplasm. One may be able to see the transition between viable nucleated cells and ghost cells even on cytology—this is fairly diagnostic for PMX (Fig. 6.9). • PMXs often yield scattered macrophages including multinucleated histiocytes (Fig.  6.9), as well as sometimes calcified material in the background. Warthin Tumour Warthin tumour (WT) occurs most commonly in the parotid gland. Due to their location, WTs may give rise to swellings that may be clinically mistaken for cervical, pre-auricular or post-auricular lymph nodes. Furthermore, cystic degeneration is common, and squamous and even mucinous metaplasia may occur. Highly degenerate cells that slough into cystic spaces may appear orangeophilic and hence may closely resemble atypical keratinised squamous cells, thus mimicking SCC (Fig. 6.8). To make matters worse, the patients tend to have history of smoking as well. Tips and Clues: • One clue is that there is often relatively low cellularity, whilst metastatic carcinoma is

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•

•

•

•

often rather cellular. The caveat is that certain metastases, e.g. cystic metastases from HPVrelated oropharyngeal SCC may also be hypocellular. The atypical orangeophilic cells are fairly small—usually smaller than tadpole or fibre cells. In addition, the cytoplasm maintains some degree of translucency and is less dense and waxy as that of truly keratinised cells (Fig. 6.8). Have an index of suspicion: If the ‘lymph node’ location overlaps with that of a major salivary gland, the presence of individually occurring orangeophilic cells in a cystic background should always prompt the consideration of a WT. Therefore, one should actively search for flat sheets of oncocytic epithelium. Clinicopathologic correlation to ascertain if the needle aspirate is from a lymph node or salivary gland nodule may also be helpful. Have a healthy threshold for diagnosing metastatic SCC. A helpful feature is when there are cohesive sheets of malignant cells which can be evaluated for architecture. Malignant SCC will show crowded, overlapping nuclei and malignant nuclear features, as opposed to the flat sheets of bland oncocytes in WT (Fig. 6.8). When unsure, one may classify the case as ‘atypical’, provide the differential diagnoses and advise correlation with imaging as well as prior relevant history.

6.7.1.2 Benign/Reactive Lymphadenitis Mimicking Lymphoma Certain benign/reactive nodal conditions may mimic lymphoma due to the presence of increased numbers of larger lymphocytes. Here are a few examples. Infectious Mononucleosis and Similar Reactive Conditions Infectious mononucleosis syndrome (IMS), caused by the Epstein–Barr virus, usually affects teenagers or young adults. It is associated with systemic symptoms of fever and malaise, and often associated with pharyngitis and lymphadenopathy, which may be tender. Nodal aspirates exhibit a mixed lymphoid population which features a higher than usual

proportion of larger cells, namely immunoblasts, centroblasts and plasmacytoid cells. Both have been described earlier in Sect. 6.5.2. Immunoblasts often have a rim of bluish cytoplasm, and their nuclei contain a large nucleolus. They may be binucleated and hence closely resemble RS cells [15]. Aspirates may appear rather alarming due to the presence of large immunoblasts and centroblasts and may show features that are suspicious for lymphoma, in particular, LBCL or HL [16]. Tips and Clues: • In IMS, there is often an appreciable range of cells of different sizes—small, medium and large cells as opposed to two distinct populations of small and large cells in HL. Plasmacytoid cells also tend to be rather prominent in IMS. • In contrast to LBCL, in IMS, the predominant cell population is still that of the small lymphocytes. • If there is uncertainty, it is best to classify the case as ‘atypical’ and mention the differential diagnosis, recommending clinical and serologic correlation, close follow-up and biopsy if lymphadenopathy does not resolve. • The clinical scenario including the patient’s age and any regional or systemic signs may also be helpful in favouring reactive lymphadenopathy over that of lymphoma. • Certain other conditions may show a similar cytologic picture—these include other viral lymphadenitides (e.g. cytomegalovirus, Herpes simplex virus), drug-related lymphadenopathy (e.g. anti-convulsant therapy and drug hypersensitivity). Histiocytic Necrotising Lymphadenitis (Kikuchi-Fujimoto Disease and Similar Reactive Lymphadenitis) Kikuchi-Fujimoto disease (KFD) is a selflimiting lymphoid condition that affects young adults (particularly females) and sometimes children, of Asian ethnicity. Cytologic smears often show necrosis, karyorrhectic debris and varying numbers of ‘crescentic histiocytes’—

6.7 Pitfalls

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a

b

c

d

e

f

Fig. 6.8  Potential false positives: Warthin tumour vs. squamous cell carcinoma (SCC). (a–c) Warthin tumour. (a, b) Warthin tumour showing atypical squamoid cells on a cystic background (HC, 300×; Pap, 400×); (c) Histology showing Warthin tumour with inset showing atypical degenerate squamoid cells within cystic spaces (HE, 40×;

inset HE, 400×); (d–f) Metastatic SCC. (d) Dispersed keratinised ‘tadpole’ cells with waxy orangeophilic cytoplasm which is denser than the degenerative cytoplasm in the Warthin tumour cells (Pap, 200×); (e) Crowded sheets of malignant cells with overlapping nuclei (Pap, 300×); (f) Corresponding histology (HE, 300×)

histiocytes in which the nuclei are eccentric and crescent shaped, and the cytoplasm packed with karyorrhectic debris (Fig. 6.9). There is usually an absence of neutrophils or well-formed granu-

lomas. Many of the histiocytes and at least some of the crescentic histiocytes have been shown to be positive for myeloperoxidase (MPO) on IHC [17].

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The lymphoid population comprises a mixed cell population featuring small and some larger, activated lymphoid cells. The potential pitfall lies in the fact that in some areas, larger cells may predominate, as seen in

Fig.  6.9, and, when interpreted together with the presence of apoptotic cellular debris, may be mistaken for NHL [18]. These larger cells may be plasmacytoid monocytes or immunoblasts [19].

a

b

c

d

e

f

Fig. 6.9  Potential false positives: pilomatrixoma and Kikuchi-Fujimoto disease. (a–d) Pilomatrixoma. (a, b) Cohesive sheets of epithelial cells transitioning into characteristic ghost cells (Pap, 100× and 200×); (c) Multinucleated giant cells are a clue to the diagnosis (Pap, 200×); (d) Histology showing basaloid and ghost cells

g

(HE, 200×). (e–g) Kikuchi disease. (e) High-power view showing increased numbers of larger cells, with abundant karyorrhectic debris (HC, 300×); (f, g) Classical crescentic histiocytes containing karyorrhectic material (HC, 600×; Pap, 600×)

6.7 Pitfalls

Tips and Clues: • Caution should be exercised when a diagnosis of NHL is being considered in a young patient. • If karyorrhectic debris is seen, one should perform an active search for necrosis and crescentic histiocytes and also take note of the age, ethnicity and gender of the patient. • Other conditions may show similar cytologic features to KFD, such as autoimmune lymphadenopathy seen in systemic lupus erythematosus, which also tends to yield some plasma cells. It may hence be prudent to provide a morphologic diagnosis of ‘histiocytic necrotising lymphadenitis’, with the differential diagnosis of KFD and autoimmune lymphadenopathy or any other infectious or reactive lymphadenopathy that falls into the differential diagnosis.

6.7.2 False Negatives Table 6.1 lists some lymphomas that may be missed, i.e. mistaken for reactive lymph nodes on cytology. These are challenging because they are either comprised of mixed lymphoid cell population or have lesional cells that are relatively sparse and hence easy to miss. Here are the usual suspects: 1. Low-grade B-cell lymphomas 2. Hodgkin lymphoma 3. Peripheral T-cell lymphoma

6.7.2.1 Low-Grade B-Cell Lymphomas The two main entities here are marginal zone lymphoma (MZL) and low-grade follicular lymphoma (LGFL). Both these entities comprise a mixed lymphoid population, sometimes with an apparent predominance of small lymphocytes. Marginal Zone Lymphoma In my experience, the diagnosis of marginal zone lymphoma (MZL) presents one of the greatest challenges in cytology. This is because MZLs often feature a mixed lymphoid population and, not infrequently, germinal centre material including TBMs (Fig. 6.10). Thus, there is considerable

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morphologic overlap with benign/reactive lymph nodes. Unfortunately, cell blocks may also be of limited help because, unlike other low grade lymphomas, there is no clear IHC signature profile in MZL. Flow cytometry may prove more helpful in delineating a clonal B-cell population; however, it may not always be definitive [20]. In extranodal sites, the index of suspicion for mucosa-associated lymphoid tissue (MALT) lymphoma may be somewhat higher if lymphocytes far outnumber epithelial parenchymal cells, but this clue unfortunately does not apply to lymph node aspirates. Hence the cytologic diagnosis of nodal MZL remains extremely challenging. In a series of cytology samples of histologically confirmed MZLs (six extranodal and three nodal sites), Matsushima et al. showed that only three—all from extranodal sites—were called atypical or suspicious prospectively on cytology [21]. Amongst the three nodal lesions, all three were reported cytologically as reactive. In their review of the cytology, six of seven FNA cases showed a predominance of medium-sized rather than small lymphocytes, which was the clue to their lymphomatous nature. Crapanzano et al. looked at 13 FNAs of histologically confirmed nodal and extranodal MZLs, finding mixed lymphoid cell populations in all 13, but with a predominance of medium-sized cells [20]. On review, the authors identified germinal centre material (11 cases), plasma cells (7 cases) and tingible body macrophages (5 cases) readily, showing the overlap with reactive lymphoid populations. Tips and Clues: • In a mixed lymphoid cell population in which the predominant cell size is medium rather than small, there should be an index of suspicion of a low-grade lymphoma. Both authors in the above-mentioned case series concluded that the predominance of medium-sized lymphocytes rather than small lymphocytes was an atypical feature. Whilst helpful, this may be challenging to put into practice, due to preparation artefacts and some degree of subjectivity in the interpretation of these aspirates.

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a

b

c

d

Fig. 6.10  Potential false negatives: marginal zone lymphoma. (a, b) High-power view showing a mixed cell population comprising predominantly small lymphocytes—note the presence of a tingible body macrophage (black arrowhead) and follicular dendritic cell (white

arrowhead) in (b) (HC, 300×; Pap, 300×); (c) Histology of the excised node showing expanded pale marginal zones (HE, 40×); (d) High-power histology showing a variety of cell sizes in the expanded marginal zone, reflecting the mixed appearance on cytology (HE, 100×)

• For all lymph node FNAs which are reported as negative or reactive, one may elect to include a comment in the cytology report, e.g. ‘Further investigation (for example, histologic biopsy) may be considered if unexplained lymphadenopathy persists or progresses’. This is particularly prudent for patients who are middle-aged or older.

logic picture of low grade follicular lymphoma (LGFL) is that of a population of predominantly small lymphocytes and relatively few larger centroblasts. It is therefore not surprising that aspirates from LGFL may closely resemble reactive lymphoid hyperplasia.

Low-Grade Follicular Lymphoma FL usually occurs in middle-aged patients, who may sometimes present with widespread lymphadenopathy. It is graded on the basis of the numbers of centroblasts within the neoplastic follicles [3]. The WHO grading system has 3 grades, with grade 1 having the fewest number of centroblasts (0–5 per 40× high-power field). Thus, the cyto-

Tips and Clues: • In FL there is a tendency for neoplastic follicles to form small cohesive appearing lymphoid aggregates (Fig. 6.3). This recapitulates their follicular architecture on histology. • TBMs are usually not readily identified in LGFL (though they may be more readily seen in higher grade FLs, which would exhibit more obviously atypical cytologic features).

6.7 Pitfalls

• The predominant small cell population tends to appear cleaved [22]—with more grooved or deeply notched nuclei, compared to those of the small lymphocytes in a reactive lymph node. • In the context of an older patient with several sites of nodal enlargement, it is good practice to have a high index of suspicion and hence a low threshold for recommending histologic biopsy.

6.7.2.2 Hodgkin Lymphoma CHL is more common than nodular lymphocytepredominant Hodgkin lymphoma (NLPHL). The accuracy of cytologic diagnosis varies, with Chhieng et al. showing a cytologic pick-up rate of CHL (definite or suspicious diagnosis) in approximately 61% in 89 histologically confirmed cases [23]. In my own practice, I have found that the Reed–Sternberg (RS) cells usually do manifest themselves in the majority of cases, particularly if one has a high index of suspicion in the appropriate clinical context, e.g. young adult patient, large localised nodal mass. The RS cells of CHL are usually large enough to visualise on low power (using 4× and 10× objectives, Fig.  6.6). It is also not uncommon to find small epithelioid granulomas in the background, together with small lymphocytes, sometimes eosinophils and plasma cells. Differential diagnoses of CHL include TCRBCL, angioimmunoblastic T-cell lymphoma (AITL) and ALCL, which are less common than CHL. Immunohistochemistry is helpful in distinguishing between these entities; however, this may be deferred to the follow-up excision specimen, depending on the clinical context. NLPHL poses a greater diagnostic challenge because the abnormal LP cells (‘lymphocyte predominant’ cells, which were formerly called ‘L and H cells’) may not be as large or many as RS cells, and hence be more difficult to detect. At times, they may also be mistaken for immunoblasts and attributed to a reactive condition, e.g. viral lymphadenitis. In addition to scattered LP cells, there is a fairly monotonous population of small lymphocytes, sometimes with small groups of epithelioid histiocytes. The usual mixed cel-

115

lular picture including eosinophils and plasma cells that characterises CHL is not usually seen in NHPHL. In a series of six cases of histologically confirmed NLPHL, Subhawong AP et al. found that only two had been called atypical on preceding cytology, with the other four diagnosed as reactive lymph nodes [24]. Tips and Clues: • Careful screening is required in order not to miss RS cells or LH cells in CHL and NLPHD, respectively. In young patients with significantly enlarged lymph nodes, there should always be an index of suspicion. • In NLPHD, there may be a history of progressive transformation of germinal centres—this has been reported to occur synchronously or metachronously with NLPHD [25].

6.7.2.3 Peripheral T-Cell Lymphoma and Angioimmunoblastic T-Cell Lymphoma Peripheral T-cell lymphoma (PTCL) comprises a mixed bag of entities under the ‘T-cell lymphoma’ umbrella. Both PTCL and angioimmunoblastic T-cell lymphoma (AITL) may exhibit rather variable cytologic appearances. These entities may involve both nodal and extranodal sites. Patients are often clinically unwell, and disease may be widespread at the time of clinical presentation, with systemic B symptoms. Cytologically, these entities may be potential diagnostic pitfalls due to their polymorphous appearance. There may be cell sizes, imparting the appearance of a mixed lymphoid population. Plasma cells, smaller bystander lymphocytes and eosinophils may also be admixed with the neoplastic cell population. Figure 6.11 shows a case of AITL) that closely resembled a reactive lymph node, showing only subtle nuclear atypia such as mildly irregular nuclear membranes and slightly less condensed chromatin. At times, AITL) may even mimic HL, because scattered large Reed– Sternberg-like cells may also be identified in cytologic aspirates.

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a

b

c

d

Fig. 6.11  Potential false negatives: angioimmunoblastic T-cell lymphoma. (a, b) High-power view showing a mixed lymphoid population comprising predominantly small- to medium-sized lymphocytes and scattered plasma cells. On careful examination, the nuclear mem-

branes are slightly irregular, and chromatin is not as condensed as that of small resting lymphocytes (HX, 600×; Pap, 600×); (c, d) Histology showing effacement of nodal architecture with extension to extranodal adipose tissue and a mixed lymphoid population (HE, 20× and 100×)

Tips and Clues: • The clinical picture of a usually elderly, unwell patient is a clue to the presence of malignancy. There may be generalised symptoms such as fever, possible night sweats, and prominent lymphadenopathy. • The nuclei of neoplastic T cells are usually irregular with grooves and sometimes protrusions (Fig. 6.6). • The abnormal T cells may also contain more than the usual amount of cytoplasm that is associated with lymphocytes, and the cytoplasm often appears pale. • T-cell lymphomas tend to have mixed cell types in the background, including plasma cells, eosinophils and some histiocytes, imparting a rather polymorphous appearance—sometimes more so than in the usual reactive lymph node.

6.7.3 Mimics There are certain entities which may mimic each other, e.g. carcinoma vs. lymphoma. Mimics may fall into the same diagnostic category or, at times, different categories. Even if one malignancy is mistaken for another, errors in interpretation may still lead to clinically significant differences in management, e.g. a diagnosis of suspected lymphoma vs. one of metastatic carcinoma would prompt different follow-on investigations. Here are some examples of mimics encountered within lymph nodes.

6.7.3.1 Metastatic Small Cell/Poorly Differentiated Carcinoma vs. LBCLs Of the lymphomas, the most commonly encountered one that is likely to be misinterpreted as

6.7 Pitfalls

carcinoma is LBCL. Of the carcinomas, small cell carcinoma (SmCC) or other poorly differentiated carcinomas are the closest lymphoma mimics. In small cell carcinoma, the malignant cells are 2–4 times the size of resting lymphocytes, similar in size to LBCL. Both SmCC and LBCL may show apoptotic cells, frequent mitoses and necrosis. In addition, both may show nuclear streaking due to the fragile nature of the cells. A case of undifferentiated nasopharyngeal carcinoma is shown in Fig.  6.7, featuring dispersed cells that are difficult to differentiated from lymphomas with large cell morphology. Tips and Clues: • Nuclear chromatin pattern—The chromatin of SmCC is quite uniformly granular, whilst that of LBCL has a greater tendency to be clumped on a vesicular background, and nucleoli are more prominent (Fig. 6.12). It is also useful to apply the strict criteria for the exclusion of SmCC — prominent nucleoli are not a feature of SmCC — small nucleoli are allowed, but more prominent nucleoli are not. • Background LGBs should be fairly abundant in LBLC, and seen in close association with the lesional cells. In SmCC, they are usually not seen immediately around the tumour cells, though rare cases of SmCC with LGBs have been documented [26]. • In SmCC, one may sometimes encounter scattered very large single dispersed cells, which are several times larger than the surrounding malignant cells. I have found this to be a helpful clue in the diagnosis of SmCC vs. lymphoma. • Cell block with IHC will usually readily differentiated lymphoma from carcinoma.

6.7.3.2 Carcinoma vs. Anaplastic Large Cell Lymphoma ALCL has several morphological variants, of which classical ALCL is the commonest. Classical ALCL may mimic metastatic carcinoma, due to the large cell size and the tendency for the cells to form aggregates. The neoplastic cells (‘Hallmark cells’) of classical ALCL are typically large, approaching the size of RS cells in HL and hence epithelial cells. The nuclei tend to have a horse-

117

shoe or sometimes a ring shape and may also be ‘embryoid’ in appearance (resembling the shape of an embryo), multilobated or wreath-shaped [27]. Nucleoli are prominent, sometimes causing these cells to resemble RS cells. Cytoplasm can be fairly abundant, and these cells may tend to aggregate or occur singly. ALCLs may show a paucity of LGBs, thereby removing one helpful clue to the lymphoid nature of the lesional cells. Tips and Clues: • When in doubt, a cell block is handy, and one may apply a basic IHC panel of epithelial markers. • EMA should be avoided because it may be positive in ALCL. A useful panel would include CD30 and ALK (the latter may be negative in a proportion of ALCLs), as well as a panel of epithelial markers. • Most of the time, IHC is sufficient to distinguish between metastatic carcinoma and ALCL; however, if necessary, FISH on destained FNA smears or cell block material can be performed, for the characteristic t(2;5) (p23;q35): ALK and NPM translocation.

6.7.3.3 Small Cell Melanoma vs. Haematolymphoid Malignancy Melanoma is known, for good reason, as the great mimic. The cells may be disposed as dispersed large, plasmacytoid cells, cohesive sheets and also dispersed small cells which may closely resemble haematolymphoid cells. Table  6.2 briefly describes some of the morphologic features seen. In small cell melanomas, the cells are barely larger than resting lymphocytes, and show round to slightly irregular nuclei with vesicular chromatin and often have a small but prominent nucleolus. There is often a characteristic ‘nubbin’ of cytoplasm seen to one side of the nucleus. Figure 6.12 shows an example. Because of the small cell size and dispersed arrangement, these can easily be mistaken for haematolymphoid malignancies. Tips and Clues: • Melanoma cells in small cell melanoma often have a prominent nucleolus, which is uncommon in haematolymphoid neoplasms in that size range (small- to medium-sized cells).

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• The appearance of the little bulge of cytoplasm is very characteristic and is distinct from the cytoplasm in haematolymphoid neoplasms, which often surrounds the nucleus as a thin rim of bluish cytoplasm in Romanowsky stains. • These patients often have a history of melanoma—knowing this and the histologic type and appearance help one to avoid the pitfall of misinterpretation as lymphoma or leukaemia. • IHC on cell block will allow a definitive diagnosis.

6.7.3.4 Granulomas vs. Metastatic Nasopharyngeal Carcinoma Sometimes, seemingly unlikely mimics may surprise us. In my experience, there are times when the syncytial sheets in undifferentiated nasopharyngeal carcinoma (NPC) may bear a striking resemblance to epithelioid granulomas (Fig. 6.12e and f). Similarities: • Both granulomas and undifferentiated NPC are composed of cells with elongated nuclei occurring in syncytial sheets. • The cell sizes of both entities are similar.

a Fig. 6.12  Mimics. SmCC vs. DLBCL; small cell melanoma; granuloma vs. undifferentiated carcinoma. (a) Small cell carcinoma showing nuclear moulding and granular chromatin without coarse clumps or prominent nucleoli (Pap, 600×); (b) Diffuse large B-cell lymphoma with coarser chromatin and prominent nucleoli (Pap, 600×); (c) Small cell melanoma showing dispersed cells

• Lymph nodes harbouring metastatic NPC frequently also show small epithelioid granulomas that are separate from the sheets of malignant cells. It is important to distinguish between granulomas vs. tumour, so as not to assume all groups are granulomatous in nature and miss the metastatic malignancy. Tips and Clues: • The cellular sheets in NPC appear more tight, as the nuclei tend to show a greater degree of overlap because of cellular crowding and higher N/C ratios. • The nuclei in NPC tend to ‘stream’ in one direction, with their long axis parallel to the edge of the cell sheet, whilst the histiocytic nuclei in granulomas are more haphazardly arranged (Fig. 6.12). • On high-power magnification, the nuclei in NPC exhibit visible and usually prominent nucleoli, as opposed to the bland nuclei of epithelioid histiocytes. • IHC is very helpful: Cytokeratins, p63 and EBER-ISH are positive in undifferentiated NPC, whilst CD163 is positive in granulomas. These are mutually exclusive.

b with eccentric nuclei and a small ‘nubbin’ of cytoplasm (Pap, 600×); (d) Histology of small cell melanoma case, note the single melanophage (HE, 400×); (e) Granuloma showing a looser and more haphazard nuclear arrangement (Pap, 300×); (f) Metastatic undifferentiated nasopharyngeal carcinoma showing a crowded, syncytial sheet with nuclei ‘streaming’ in one direction (Pap, 300×)

References

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c

d

e

f

Fig 6.12 (continued)

Take Home Points

• Lymph nodes represent a microcosm of the body, being affected in particular by infections, systemic conditions and malignancies. • In the cytologic evaluation of lymph node aspirates, an early question is: Is this a lymphoid or non-lymphoid lesion? • In a lymphoid population, cell composition and cell size are very important parameters to evaluate. • Some lymphomas that may be missed on cytology include MAL, NLPHL and PTCL. • Certain metastatic malignancies feature morphologic clues that may alert one to the site of origin.

• The possibility of non-nodal lesions masquerading as lymph nodes should be considered, especially when the clinical and cytologic findings are at variance. • The type and extent of ancillary testing for lymphomas depends on individual institutional practice.

References 1. Nayak A, Sugrue C, Koenig S, Wasserman PG, Hoda S, Morgenstern NJ. Endobronchial ultrasound-guided transbronchial needle aspirate (EBUS-TBNA): a proposal for on-site adequacy criteria. Diagn Cytopathol. 2012;40(2):128–37. 2. Alsharif M, Andrade RS, Groth SS, Stelow EB, Pambuccian SE.  Endobronchial ultrasound–guided transbronchial fine-needle aspiration: the University

120 of Minnesota experience, with emphasis on usefulness, adequacy assessment, and diagnostic difficulties. Am J Clin Pathol. 2008;130(3):434–43. 3. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J (Eds). WHO Classification of Tumours of Haematopoeitic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017. 4. Ahuja AT, Ying M. Sonographic evaluation of cervical lymph nodes. Am J Roentgenol. 2005;184(5): 1691–9. 5. Boringi M, Bontha SC, Kaur M, Shireen A. Branchial cleft cyst—a case report with review of literature. J Orofac Sci. 2014;6(2):125. 6. Valentino M, Quiligotti C, Carone L. Branchial cleft cyst. J Ultrasound. 2013;16(1):17–20. 7. Goldenberg D, Sciubba J, Koch WM.  Cystic metastasis from head and neck squamous cell cancer: a distinct disease variant? Head Neck. 2006;28(7): 633–8. 8. Al-Hilli Z, Strajina V, McKenzie TJ, Thompson GB, Farley DR, Regina Castro M, et  al. Thyroglobulin measurement in fine-needle aspiration improves the diagnosis of cervical lymph node metastases in papillary thyroid carcinoma. Ann Surg Oncol. 2017;24(3):739–44. 9. Maygarden SJ, Flanders EL.  Mycobacteria can be seen as ‘negative images’ in cytology smears from patients with acquired immunodeficiency syndrome. Mod Pathol. 1989;2(3):239–43. 10. Ang GA, Janda WM, Novak RM, Gerardo L. Negative images of mycobacteria in aspiration biopsy smears from the lymph node of a patient with acquired immunodeficiency syndrome (AIDS): report of a case and a review of the literature. Diagn Cytopathol. 1993;9(3):325–8. 11. Mills SE, editor. Histology for pathologists: includes online access for fully searchable text and image bank. 4th ed. Philadelphia: Wolters Kluwer/Lippincott Williams & Wilkins Health; 2012. 1331 p. 12. Wong MP, Yuen ST, Collins RJ.  Fine-needle aspiration biopsy of pilomatrixoma: still a diagnostic trap for the unwary. Diagn Cytopathol. 1994;10(4):365–9; discussion 369–370. 13. Sharma D, Agarwal S, Jain LS, Kamal V. Pilomatrixoma masquerading as metastatic adenocarcinoma. A diagnostic pitfall on cytology. J Clin Diagn Res. 2014;8(10):FD13–4. 14. Sivakumar S.  Pilomatrixoma as a diagnostic pitfall in fine needle aspiration cytology: a case report. Acta Cytol. 2007;51(4):583–5.

6  Approach to Lymph Node Cytology 15. Tindle BH, Parker JW, Lukes RJ. ‘Reed-Sternberg cells’ in infectious mononucleosis? Am J Clin Pathol. 1972;58(6):607–17. 16. Stanley MW, Steeper TA, Horwitz CA, Burton LG, Strickler JG, Borken S.  Fine-needle aspiration of lymph nodes in patients with acute infectious mononucleosis. Diagn Cytopathol. 1990;6(5):323–9. 17. Pileri SA, Facchetti F, Ascani S, Sabattini E, Poggi S, Piccioli M, et  al. Myeloperoxidase expression by Histiocytes in Kikuchi’s and Kikuchi-like lymphadenopathy. Am J Pathol. 2001;159(3):915–24. 18. Viguer JM, Jiménez-Heffernan JA, Pérez P, López-Ferrer P, Gónzalez-Peramato P, Vicandi B.  Fine-needle aspiration cytology of Kikuchi’s lymphadenitis: a report of ten cases. Diagn Cytopathol. 2001;25(4):220–4. 19. Perry AM, Choi SM.  Kikuchi-Fujimoto disease: a review. Arch Pathol Lab Med. 2018;142(11):1341–6. 20. Crapanzano JP, Lin O. Cytologic findings of marginal zone lymphoma. Cancer. 2003;99(5):301–9. 21. Matsushima AY, Hamele-Bena D, Osborne BM. Fineneedle aspiration biopsy findings in marginal zone B cell lymphoma. Diagn Cytopathol. 1999;20(4):190–8. 22. Kishimoto K, Kitamura T, Fujita K, Tate G, Mitsuya T.  Cytologic differential diagnosis of follicular lymphoma grades 1 and 2 from reactive follicular hyperplasia: cytologic features of fine-needle aspiration smears with Pap stain and fluorescence in situ hybridization analysis to detect t(14;18)(q32;q21) chromosomal translocation. Diagn Cytopathol. 2006;34(1):11–7. 23. Chhieng DC, Cangiarella JF, Symmans WF, Cohen JM. Fine-needle aspiration cytology of Hodgkin disease: a study of 89 cases with emphasis on false-negative cases. Cancer. 2001;93(1):52–9. 24. Subhawong AP, Ali SZ, Tatsas AD.  Nodular lymphocyte-predominant Hodgkin lymphoma: cytopathologic correlates on fine-needle aspiration. Cancer Cytopathol. 2012;120(4):254–60. 25. Burns BF, Colby TV, Dorfman RF. Differential diagnostic features of nodular L & H Hodgkin’s disease, including progressive transformation of germinal centers. Am J Surg Pathol. 1984;8(4):253–61. 26. Murakami T, Kayano H, Itoh T, Shimizu Y, Ban S, Ogawa F, et al. Lymphoglandular bodies in malignant tumors: with special reference to histologic specimens. Ann Diagn Pathol. 2008;12(4):249–51. 27. Ng W-K, Ip P, Choy C, Collins RJ.  Cytologic and immunocytochemical findings of anaplastic large cell lymphoma: analysis of ten fine-needle aspiration specimens over a 9-year period. Cancer. 2003;99(1):33–43.

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Approach to Salivary Gland Cytology

Contents 7.1

Introduction

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7.2 7.2.1  7.2.2  7.2.3  7.2.4  7.2.5  7.2.6 

Main Diagnostic Entities and the Milan System Non-diagnostic Non-neoplastic Atypia of Undetermined Significance (AUS) Neoplasm Suspicious for Malignancy Malignant

 124  124  126  126  126  127  127

7.3

Salivary Gland FNA: What Cytology Can and Cannot Do

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7.4

What Do Clinicians Need from Us?

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7.5 7.5.1  7.5.2  7.5.3  7.5.4 

Triple Approach, with a Pattern-Based Morphologic Approach Clinical Information Alarming/Red Flag Clinical Findings Cytology (Gross and Microscopic Evaluation) Ancillary Tests

 128  128  130  130  137

7.6 7.6.1  7.6.2  7.6.3  7.6.4  7.6.5 

Specific Challenging Areas Cystic Lesions Lymphoid-Rich Lesions Basaloid Neoplasms Oncocytic/Oncocytoid Lesions Low-Grade Malignancies

 139  139  141  143  144  146

7.7  itfalls P 7.7.1  False Positives and Negatives 7.7.2  Mimics

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© Springer Nature Singapore Pte Ltd. 2021 M. E. Nga, Practical Diagnostic Approaches in Non-Gynaecologic Cytology, https://doi.org/10.1007/978-981-15-2961-0_7

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7  Approach to Salivary Gland Cytology

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7.1

Introduction

Salivary gland (SG) cytology is a particularly challenging but nevertheless rewarding area in cytology. Inherent difficulties may arise because there may be considerable cytomorphologic overlap between different entities. At times, non-­neoplastic and neoplastic entities may be difficult to tell apart, e.g. mucocoele with squamous metaplasia vs. low-grade mucoepidermoid carcinoma. In other instances, benign and malignant neoplasms may show very similar features (e.g., cellular pleomorphic adenoma vs. basal cell adenocarcinoma). To compound this issue, non-­SG structures such as lymph nodes may also masquerade clinically as SG lesions. At times, these difficulties may cause a degree of angst for the reporting pathologist. It is in these situations that attention to radiologic findings and close communication with the clinical team is most helpful, not only to increase one’s appreciation of clinicopathologic correlation, but also to build trust and rapport. Where present, uncertainty should be clearly reflected in the cytology report, along with suggestions for further investigation or specific correlation. In the following pages, you will find a practical approach to various challenging areas in ­salivary gland cytology, beginning with a broad scope of entities encountered, and an outline of what cytology can and cannot do.

7.2

 ain Diagnostic Entities M and the Milan System

The more common entities that one may encounter in SG aspirates are presented in Chart 7.1. This is not a comprehensive listing of all formal SG lesions or neoplasms—for the latter, the reader is referred to the most current WHO reference on head and neck tumours [1]. In 2015, the Milan System Taskforce, an international panel including anatomical pathologists, cytopathologists and clinicians, was convened to create a categorised system for reporting SG cytology, akin to

the Bethesda Reporting System for Thyroid Cytopathology. The result of this collaboration, the Milan System for Reporting Salivary Gland Cytopathology (MSRSGC), was published in 2018 [2, 3]. The MSRSGC aims to standardise cytology reporting into categories that reflect risk stratification and hence lead to meaningful management guidelines. Whilst many individual institutions are likely to already be using tried-and-tested diagnostic categories for SG cytology that predate the MSRSGC, this system is part of a worldwide move to standardise reporting terminology on a more global level. It is important to emphasise, however, that regardless of the system of terminology that is used, both clinicians and pathologists in one’s practice setting must be familiar with the terminology and management/risk stratification implications. Individual institution-­based audits are key to evaluating the performance of reporting systems and hence influence their optimal application. Since its publication, numerous authors have performed evaluations of the MSRSGC [4–12]. Table  7.1 summarises the MSRSGC as well as the category-specific risk of malignancy (ROM) presented in the Milan handbook [3]. The collated ROMs from one of the largest recent meta-­ analyses in the English language literature by Farahani et al. are also included [4]. A brief elaboration on each category as described in the in the Milan system is presented here.

7.2.1 Non-diagnostic This category is for aspirates in which material is quantitatively or qualitatively insufficient for a definitive cytologic diagnosis. Several points are worth noting: • All material should be processed for cytologic examination. • Aim for an incidence of less than 10%. • 60 cells is the arbitrary cut-off for cellular adequacy.

7.2 Main Diagnostic Entities and the Milan System

125 “Salivary Gland” Aspirates

Non-SG structures

SG lesions

Non-neoplastic entities

• •

• •

Sialadenosis Sialadenitis • Acute • Chronic (infectious, immune-related) Sialolithiasis Obstruction • Mucocoele*

Skin/Subcutaneous lesions

Neoplasms

Benign Neoplasms • Pleomorphic adenoma (PA) • Warthin tumour • Oncocytoma • Basal cell adenoma* • Myoepithelioma*

Malignant Neoplasms • Carcinoma ex-PA • Adenoid cystic carcinoma • Basal cell adenocarcinoma* • Mucoepidermoid carcinoma* • Acinic cell carcinoma • Secretory carcinoma* • Salivary duct carcinoma* • Epithelial-myoepithelial carcinoma* • Myoepithelial carcinoma* • Oncocytic carcinoma* • Polymorphous adenocarcinoma* • Lymphoepithelioma-like carcinoma • Lymphoma Metastases

Lymph node lesions

• Includes Benign and Malignant entities • • Adnexal tumours • Pilomatrixoma • • Cysts • Epidermal cyst • Branchial cyst • Soft tissue neoplasms • Lipoma • Nodular fasciitis • Schwannoma • Vascular neoplasms

Reactive/Inflamed lymph node Metastatic carcinoma Lymphoma

Key: “Salivary Gland”: FNAs labelled as salivary gland lesions *May not be definitively diagnosable on cytology

Chart 7.1  Salivary gland aspirates—main diagnostic entities Table 7.1  The Milan System for Reporting Salivary Gland Cytopathology with Risk of Malignancy (ROM) Milan System category (proposed incidence) I. Non-­diagnostic (up to 10%)

ROM as presented in the Milan book [3] 25%

III. Atypia of undetermined significance ( large bowel and oesophagus Oesophagus and Smooth muscle colorectum tumour (LM >>LMS) (Fig. 8.3) Schwannoma Stomach

Cellular Nests or sheets

Cellular Loosely aggregated or nests and dispersed cells

Necrosis; mitoses; Ki-67 nuclear labelling index increases with grade Challenging to ascertain on both cytology and histology. Necrosis, increased mitoses should be noted in the report

Plasmacytoid cells with moderately abundant, sometimes granular cytoplasm Plasmacytoid or plump spindle cells Delicate, relatively abundant cytoplasm, may have wispy cytoplasmic processes. Bare nuclei common Round to ovoid cells with relatively scant cytoplasm

Eccentric and round, with stippled, ‘salt and pepper’ chromatin

“Endocrine atypia” (anisonucleosis) Bi- or multinucleation frequent. Occasional nuclear inclusions. Chromatin stippled or dark and fairly coarse Central, uniform, round to oval nuclei with even chromatin; ± rare nuclear inclusions

Most behave in a benign fashion

Similar to spindle cell GIST

Ill-defined cytoplasm

Long, thin wavy to comma-shaped nuclei. Scattered large nuclei and occasional nuclear inclusions

Immunoprofile shows reactivity with smooth muscle markers (see Table 8.3)

May be negative for CD117, but usually positive for DOG1. DDx NET, carcinoma Less often FNAd because of more superficial location in deep mucosa to submucosa May be mistaken for NETs (carcinoids). Cytokeratin helpful in DDx with NET IHC shown in Table 8.3

Often noticeably less cellular than GISTs Location in GIT is very helpful Distinct from soft tissue schwannomas. (GIT schwannomas—more collagenous, with lymphoid cuff)

Closely resembles soft tissue schwannomas

1.  Location in GIT 2. Size 3.  Mitotic count per 5 mm2 Worrying for LMS: cellular, pleomorphic nuclei, mitoses, necrosis GIT schwannomas are usually benign

Moderately abundant, relatively well-defined and homogeneous

Relatively abundant homogeneous cytoplasm

Elongated, slender, usually somewhat shorter than GIST

Other remarks

Prognostic features

Round, relatively vesicular nuclei ± small nucleoli

Fibrillary, ill-defined. Bare nuclei may be present

Long, thin, wavy nuclei with even chromatin

Fairly cellular, compact fascicles ± nuclear palisading. Nuclei more crowded than LM Relatively low to moderate cellularity (well-spaced nuclei), fascicular arrangement Variable cellularity; fascicles or whorls; may have collagen bundles or myxoid stroma. Lymphoid cells are common

Relatively high cellularity; nests or sheets of polygonal cells Cellular Loosely aggregated nests, rosettes or dispersed cells

Cytoplasmic features

Nuclear features

Cellularity/architecture

Table 8.2  Salient Morphologic Features of Gut Wall or Extramural Lesions

166 8  Approach to Endoscopic Ultrasound-Guided FNA of the Gastrointestinal Tract and Pancreas

8.3 GIT

167

a

b

c

d

Fig. 8.3  GIST and leiomyoma. (a, b) GIST—cellular tissue fragment showing long, slender, relatively uniform nuclei (HC and Pap, 400×); (c, d) Leiomyoma—less cel-

a Fig. 8.4  Epithelioid GIST. (a) Relatively cohesive tissue fragment with some associated blood vessels (Pap, 40×); (b) Edge of tissue fragment showing plump polygonal cells with round nuclei (Pap, 300×); (c) Sheet of plump epithelioid cells with rounded nuclei and fairly abundant pale cytoplasm (HC, 300×); (d) Sheet of polygonal cells

lular appearing fascicles with somewhat shorter, uniform, spindle nuclei; note that the nuclei are more spaced apart than in the GIST (HC and Pap, 300×)

b with round to oval nuclei, small nucleoli and abundant delicate cytoplasm (HC, 600×); (e) Cells with round to oval nuclei and moderately abundant cytoplasm admixed with some lymphocytes (Pap, 400×); (f) Cell block with DOG1 IHC (Left: HE, 100× and Right: DOG1 IHC, 200×)

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c

d

e

f

Fig. 8.4 (continued)

ogy. Hence, ancillary tests are frequently performed on cell block material in order to ascertain the diagnosis. The expected findings are presented in Table 8.3.

for molecular testing, a record of diagnostic material should be preserved for future reference, e.g. in the form of ­photographs/digitised slides as digitally archived material.

2. Prognosis and Prediction of Response to Targeted Therapy –– IHC may provide prognostic information, e.g. Ki-67 nuclear labelling index in ascertaining the grade of NETs in the gut and pancreas. –– Molecular tests may provide helpful prognostic and predictive information, e.g. in GISTs, KIT mutation types and loci inform clinicians on the likely aggressiveness of the tumour as well as the likelihood of response to targeted therapy. One such example is the PDGFRA mutation, most frequently encountered in epithelioid GISTs, which has been found to confer resistance to imatinib [20, 21]. –– Molecular tests may be performed on smears if there is insufficient material in the cell block. If previously stained diagnostic smears are used

Table 8.3 highlights some of the useful IHC and molecular tests that may be applied to gut wall lesions.

8.3.2 Specific Challenges In this section, we will focus on specific challenges that stem from several practical questions that a pathologist may be faced with when encountering gut wall neoplasms. These are: 1. Spindle cell lesions: How far can I go without ancillary testing? 2. GIST: How much prognostic information can I provide? 3. NET: How accurately can I grade the tumour?

8.3 GIT

169

Table 8.3  Useful Ancillary Tests in Gut Wall Lesions Spindle cell lesions Neoplasm CK117 (c-KIT); DOG1 GIST (spindle + and epithelioid) (epithelioid GISTs may be CD117-­negative; but are usually DOG1-­positive) LM

Schwannoma

– (scattered CD117- or DOG1-­positive dendritic cells may be present) [18, 19] –

Epithelioid lesions Neoplasm DOG1; CD117

CD34

Desmin; SMA

S100

Molecular analysis/remarks

+ (small bowel GISTs are more likely to be negative)

–

–

–

+

–

KIT-activating mutations (particularly in exons 9 and 11, less often exons 13 and 17) and PDGFRA mutation Note—rare cytokeratin reactivity may sometimes be observed in GISTs H-caldesmon does not distinguish between smooth muscle tumours and GISTs, being positive in a majority of both tumour types

± (focal reactivity)

–

+

Differ from soft tissue schwannomas (GIT schwannomas lack NF2 gene mutation)

S100

Chromogranin; synaptophysin +

Cyto-­ keratin +

SMA, calponin –

Others

–

GATA3 is often positive

– +

SOX10 is positive H-caldesmon may be positive

NET

–

– (sustentacular pattern around cell nests) + – (sustentacular pattern around cell nests)

Paraganglioma

–

Granular cell tumour Glomus tumour

–

+

–

– (helpful in DDx from NET) –

–

–

–

–

8.3.2.1 Spindle Cell Lesions: How Far Can I Go Without Ancillary Testing? There is significant morphological overlap between various spindle cell lesions in the gut wall. Hence, whilst the cytologic features may favour a certain diagnosis, a definitive cytologic diagnosis can only be made with confirmatory ancillary tests on the cell block, usually in the form of IHC.  Without the benefit of ancillary testing, a morphologic diagnosis may be given and the main differentials highlighted, if possible, clearly stating the favoured diagnosis.

NA

As mentioned earlier, it is wise to collect material for cell block for all EUS FNA samples at the time of ROSE, in order to maximise diagnostic yield. However, sometimes, there may still be insufficient lesional material present for ancillary testing. If the initial HE level(s) in the cell block yields insufficient material for ancillary testing, it may be helpful to examine at least two to three further levels of the cell block, because lesional tissue may sometimes appear on further sections. Here is a sample cytology report without the benefit of an adequate cell block:

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• Microscopic description: Moderately cellular smears show tissue fragments composed of long fascicles of spindle cells with slender, long and wavy nuclei and relatively scant cytoplasm. The fascicles appear tight, with the nuclei closely opposed to each other. There is no significant nuclear pleomorphism; no necrosis is seen and mitoses are not readily identified. No tissue is available for ancillary testing. • Diagnosis: Gastric wall mass, EUS-­ guided FNA: –– Spindle cell neoplasm • Comments: The differential diagnoses include gastrointestinal stromal tumour (GIST) and a smooth muscle neoplasm such as a leiomyoma. In view of the location, the EUS findings and the cytomorphological features, GIST is favoured; however, a definitive cytologic diagnosis requires confirmatory immunohistochemical tests.

8.3.2.2 GIST: How Much Prognostic Information Can I Provide? As mentioned in Sect. 8.3.1.2, the risk stratification parameters of GISTs are well established— location in the GIT, size and mitotic count per 5 mm2. Two of these features should be clinically assessable—location within GIT and size. Mitotic count is usually definitively assessed on histology, as an area of 5  mm2 needs to be evaluated; and, usually several sample areas are evaluated. On cytology, this is difficult because of the scant amount of tissue, the presence of sometimes thick sheets of cells with overlapping layers, as well as the ‘dilution’ of lesional tissue with non-lesional material. Cell blocks are a closer match to histologic biopsies; however, some margin of error still exists. Ricci et  al. found suboptimal correlation between evaluating mitotic counts on FNA cell blocks as compared

to resected specimens, documenting that, even with cases in which cell blocks contained 5 mm2 of lesional tissue, the mitotic counts were significantly lower than those in the corresponding excised cases [22]. In some of the cases, the mitotic counts would have placed the patients into different risk stratification groups (5/5 mm2), with cytology usually underestimating the risk group. Therefore, unless there is a high mitotic count on cytology, formal evaluation of mitotic count for the purpose of risk stratification is best reserved for histologic resection specimens. Nevertheless, it is still helpful to perform evaluation of mitoses on the cell block, with a caveat or comment regarding the area examined, and a note that accurate evaluation is best performed on histology.

8.3.2.3 NET: How Accurately Can I Grade the Tumour? This question will be covered in more detail in the section on Pancreatic Cytology (Sect. 8.4.5.3). Essentially, the same principles apply to both GIT and pancreatic NET grading [23]. NETs of the GIT are usually more superficial within the gut wall, arising in the deep mucosa and sometimes extending into the submucosa. The more superficial lesions are usually accessible through luminal endoscopic biopsy; however, the deeper lesions may require EUS FNA to obtain lesional tissue. Once the diagnosis is made, it is helpful if the pathologist is able to ascertain the provisional grade of the tumour by evaluating the cell block and quantitating the Ki-67 labelling index. Studies comparing the degree of agreement between Ki67 labelling index on cell blocks and histologic samples are presented in the discussion of ancillary tests in the section on Pancreatic cytology.

8.3.3 P  itfalls (False Positives, False Negatives, Mimics) Some mimics exist, giving rise to diagnostic difficulties and potentially erroneous cytologic interpretation. These errors can be minimised if

8.3 GIT

one is aware these danger zones and the optimal IHC panels that can help solve these diagnostic dilemmas.

8.3.3.1 Epithelioid GIST The cytomorphologic features of epithelioid GISTs are summarised in Table 8.2. The cells are polygonal and may be arranged in cohesive sheets or nests, thus closely resembling epithelial cells. In particular, these lesions may be mistaken for NETs. Helpful features include the presence sometimes of an associated spindle cell component (GISTs may be mixed epithelioid and spindle cell), as well as the absence of the classical ‘salt and pepper’ stippled chromatin that characterises neuroendocrine tumours. Figure  8.4 shows the

171

morphologic features of epithelioid GIST, whilst Fig. 8.5 shows a NET. In the differentiation between epithelioid GIST, NET and another epithelial lesions, an IHC panel is suggested (Table  8.3) comprising DOG1 (positive in epithelioid GIST; CD117 can be done but may be negative); synaptophysin, chromogranin and CAM5.2 (these three are positive in NET but not in GIST). A small percentage of GISTs (up to 16%) may express cytokeratins (e.g. CAM5.2); however, the expression is usually focal and often shows a perinuclear dot-like pattern [24, 25].

8.3.3.2 Paraganglioma vs. NET Rarely, paragangliomas may be aspirated, either as retroperitoneal masses or masses adjacent to

a

b

c

d

Fig. 8.5  Neuroendocrine tumour. (a) Relatively loose aggregates of strikingly uniform cells with round nuclei and moderate amounts of cytoplasm (HC, 200×); (b) Rosette-like formations of uniform cells with round, eccentric nuclei. There is a close resemblance to acinar

formations (Pap, 200×); (c) Plasmacytoid appearance of individual cells is discernible (HC, 400×); (d) Alcohol-­ fixed smear showing cells with stippled chromatin, and a few cells with small but prominent nucleoli (Pap, 600×)

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the gut wall. The cytologic features show significant overlap with NET. Some subtle differences may be noted—paragangliomas are more likely to feature bare nuclei and may exhibit more marked ‘endocrine atypia’ and multinucleation. Also, the cytoplasm may sometimes appear more

wispy, with tapering cytoplasmic processes. The features of NET and paraganglioma can be compared and contrasted in Table 8.2 and illustrated in Figs. 8.5 and 8.6. A useful discriminatory IHC panel includes cytokeratins (e.g. CAM5.2 for NET) and GATA3

a

b

c

d

e

f

Fig. 8.6 Paraganglioma. (a) Low-power view showing fairly loose, trabecular-like arrangements composed of polygonal cells with fairly uniform nuclei (HC, 40×); (b, c) Air-dried smears showing variation in nuclear size, and some cells with delicate, wispy cytoplasm and others with vacuolated cytoplasm (HC, 200× and 400×); (d) Alcohol-­

fixed smear showing vague rosette like formations, comprising cells with round nuclei and abundant pale cytoplasm (Pap, 200×); (e) Nuclei show stippled ‘neuroendocrine-­ type’ chromatin (Pap, 400×); (f) Histologic section showing zellballen (well-formed nests) comprising cells with fairly abundant cytoplasm (HE, 200×)

8.3 GIT

(for paraganglioma) . Both tumours share reactivity with synaptophysin, chromogranin and S100, the latter showing a similar sustentacular cell staining pattern in both entities.

8.3.3.3 Other Less Commonly Encountered Lesions Inflammatory Myofibroblastic Tumour Inflammatory myofibroblastic tumours (IMTs) may be found in the abdominal cavity and may involve the GIT. A helpful clue is that they tend to occur in children and young adults, in contrast to GISTs and smooth muscle tumours. On cytology, cellularity is fairly modest with some plump to spindle cells with bipolar tapering cytoplasmic processes. The nuclei are oval and vesicular, and sometimes feature intranuclear inclusions. A mixed inflammatory cell population may be seen (lymphocytes, neutrophils, plasma cells and macrophages), which serves as a helpful clue to the diagnosis [26, 27]. Features that may portend a more aggressive behaviour include larger nuclei with prominent nucleoli and scattered mitotic figures. On IHC, the cells are variably positive for muscle markers such as SMA and desmin, and a proportion of cases may also show cytokeratin reactivity. ALK positivity is seen in approximately half of the cases. FISH for ALK gene rearrangement may be helpful and can be performed on cell block material. Differential diagnoses include other spindle cell tumours in the gut wall such as GISTs and smooth muscle tumours, as well as sarcomatoid carcinoma in more pleomorphic cases. Pancreatic Heterotopia Though uncommon, benign hyperplasias or heterotopias such as pancreatic heterotopia may be considered in the differential diagnosis when one encounters bland appearing epithelial cells in an aspirate from a mural mass lesion. Pancreatic heterotopia is a relatively uncommon, benign developmental mass-forming lesion that occurs in the wall of the gut, most frequently in the distal stomach, but also in the small bowel and colon.

173

The endoscopic view shows a mucosal-­ covered mass which may have an area of central umbilication through which secretions may be seen to exit. On EUS, the sonographic appearance may be hypoechoic or heterogeneous, and the margins may be somewhat irregular [12, 28]. Cytology reflects the histologic findings, usually showing varying proportions of pancreatic acinar tissue and sometimes ductal cells. Endocrine elements are generally not seen. The main differential diagnoses are that of needle placement into normal pancreatic parenchyma, well-differentiated adenocarcinoma (which would harbour more architectural and cytologic atypia) and NET. In the case of NET, there may be acinar-like clusters that are usually looser in formation, and the cells of NET appear more discohesive and are appreciably larger than pancreatic acinar cells (Fig.  8.8f) and exhibit stippled chromatin, which is not seen in acinar cells. Most cases of pancreatic heterotopia do not undergo malignant change. Brunner Gland Adenoma/Hyperplasia In rare instances, Brunner gland hyperplasia (sometimes termed ‘Brunner gland adenoma”), can form a sizeable duodenal submucosal mass that may cause obstructive symptoms or bleeding. Only very rare cytologic reports are present in the literature [29]. An example of a case in our files, diagnosed as ‘well-differentiated epithelial lesion’ is shown in Fig. 8.7. EUS may reveal a somewhat hypoechoic mass with internal cystic areas. Cytology aspirates show bland appearing epithelial cells with round, eccentric nuclei and abundant pale, delicate cytoplasm. Some of the cells may appear somewhat mucinous, with fairly abundant, pale flocculent cytoplasm. Some morphological clues that may be helpful include the presence of a stromal component, in the form of either smooth muscle bundles (bland spindle cells closely intermixed with the epithelial elements, Fig. 8.7a) or adipose tissue, as well as scattered background inflammatory cells [29]. The main differential diagnosis is well-differentiated adenocarcinoma. If, after

174

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a

b

c

d

Fig. 8.7  Brunner gland hyperplasia/adenoma. (a) Tissue fragment showing a spindle cell stromal component associated with bland epithelial cells at the superior aspect (HC, 40×); (b) Bland mucinous type epithelium (HC,

clinico-radiological-pathologic correlation is performed, one is still unable to make a definite diagnosis, then it is reasonable to provide a morphologic diagnosis such as ‘well-differentiated epithelial lesion’ and provide the differential diagnoses in the comments.

EUS FNA GIT: Take Home Points

• ROSE is especially important in EUS FNA, to assess sample adequacy and perform optimal specimen triage. • A cellular smear may be non-diagnostic because of potential contaminant cells. • Location in the GIT is important in the differential diagnosis of spindle cell lesions.

300×); (c) Sheets of bland epithelium with discernible acinar openings (Pap, 200×); (d) Histology showing lobules of bland mucous glands with some smooth muscle stroma (HE, 100×)

• Most gut wall lesions are mesenchymal lesions—chiefly GISTs and smooth muscle tumours. If epithelioid cells are seen in a mural lesion, the possibility of epithelioid GIST should always be considered. • Ancillary testing on cell block is extremely useful for definitive cytologic diagnosis; however, prognostication of GISTs is still suboptimal on cytologic samples.

8.4

Pancreas

The spectrum of conditions encountered in pancreatic EUS FNA ranges from non-neoplastic lesions to neoplasms which may be benign or

8.4 Pancreas

175

malignant. As in the gut wall, clinical and radiologic correlation plays a key role in the diagnostic workup, particularly for pancreatic cystic lesions. Thus it is good practice to have some basic knowledge of some expected findings in these other elements that can aid in diagnosis, beyond the scope of cytologic evaluation. Outline 1. Spectrum of diagnostic entities and the Papanicolaou Society of Cytopathology Guidelines 2. What pancreatic cytology can and cannot diagnose 3. Technical issues in pancreatic EUS FNA 4. A systematic approach: key questions 5. Triple approach 6. Specific challenges (false positives, false negatives and mimics)

8.4.1 Spectrum of Diagnostic Entities and the Papanicolaou Society of Cytopathology Guidelines Chart 8.2 summarises the spectrum of conditions seen in EUS FNA of the pancreas. In the context of pancreatic EUS FNAs, a natural

division lies between solid and cystic lesions, in terms of ­d ifferential diagnoses and diagnostic workup. The commonest solid lesions are pancreatic ductal adenocarcinoma (PDAC) and neuroendocrine tumour (NET)—both of which are relatively straightforward diagnoses. In contrast, in cystic lesions, the pathologist aims to ascertain the risk of malignancy of the lesion, and hence a large part of this lies in deciding if the lesion is a neoplastic mucinous cyst (NMC)—i.e. mucinous cystic neoplasm (MCN) or intraductal papillary mucinous neoplasm (IPMN). With this in mind, we can look at the specific clinical approaches. The Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology (PSGPBC) is a system of classification of pancreaticobiliary cytology, similar to the Bethesda and Milan Systems for thyroid and salivary gland cytology. It is part of a worldwide move to standardise terminology for the classification into specific diagnostic categories. In 2014, the guidelines were published in two major cytology journals, followed by the publication of the illustrated atlas in 2015 [30, 31]. Table  8.4 summarises the nomenclature for the six categories as well as the corresponding management guidelines.

EUS FNA of Pancreatic lesions

Solid lesions

Non-neoplastic: • Chronic pancreatitis • Autoimmune (IgG4) pancreatitis • Accessory spleen (tail of pancreas) • Hamartoma

Key: * Lymphoepithelial cysts are sometimes classified as neoplasms.

Neoplastic epithelial: • Ductal adenocarcinoma and variants (Colloid carcinoma, Undifferentiated carcinoma with osteoclast-like giant cells; Adenosquamous carcinoma) • Neuroendocrine tumour (NET), Grade 1-3 • Neuroendocrine carcinoma (large cell and small cell) • Acinar cell carcinoma • Solid pseudopapillary neoplasm (SPN; smaller lesions may be solid) • Pancreatoblastoma • Intraductal tubulopapillary neoplasm • Metastatic carcinoma Neoplastic non-epithelial: • Lymphoma • Lipoma • Solitary fibrous tumour • Schwannoma

Chart 8.2  Common/important pancreatic lesions

Cystic lesions

Non-neoplastic: • Pseudocyst • Lymphoepithelial cyst* • Ciliated foregut cyst • Cystic hamartoma

Mucinous neoplastic: • Intraductal papillary mucinous neoplasm • Mucinous cystic neoplasm Non-mucinous neoplastic: • SPN • Serous cystadenoma • NET with cystic change • Adenocarcinoma with cystic change • Acinar cell cystadenoma • Lymphangioma

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Table 8.4  The Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology [32] Diagnostic Category I. Non-­ diagnostic

II.  Negative for malignancy

III. Atypical

Summary of cytologic criteria/entities –  No lesional cells seen – Preparation artefact precludes cytologic evaluation –  Cystic lesions without mucin – Benign pancreatic parenchyma in the appropriate clinical setting – Cytologic features of a specific benign entity Diagnoses: –  Pancreatitis (acute/chronic/autoimmune) – Pseudocyst –  Lymphoepithelial cyst –  Accessory spleen – Cytologic or architectural atypia that is beyond what can be classified as reactive, but insufficient to call ‘suspicious for malignancy’

IV. Neoplastic (benign)

Neoplastic benign: –  Serous cystadenoma – Lymphangioma

   Neoplastic (other)

Neoplastic other: –  Neuroendocrine tumour (NET) –  Neoplastic mucinous cysts (NMCs): IPMN, MCN –  Solid pseudopapillary neoplasm (SPN)

V.  Suspicious for malignancy

Significant cytologic and architectural atypia, but qualitatively or quantitatively not reaching the threshold for a definitive diagnosis of malignancy

VI. Malignant

– Pancreatic ductal adenocarcinoma and variants – Poorly differentiated neuroendocrine carcinoma –  Acinar cell carcinoma – Lymphoma – Metastases

8.4.2 W  hat Pancreatic Cytology Can and Cannot Diagnose Just as this point applies to other organ sites, it is important to know the limitations of pancreatic cytology. Whilst Chart 8.2 summarises the common and important entities that one may encounter in pancreatic aspirates; not all are readily diagnosed on cytology. For a more comprehensive spectrum of mass forming conditions, please

Management ± risk of malignancy (ROM) – Close attention to clinical and EUS or other imaging findings—to proceed to surgery if indicated – Repeat FNA or tissue biopsy may be considered if required – Management is generally non-surgical unless symptomatic

–  ROM 25–100% (mean 58%) [32] – RFNA, biopsy, imaging and clinical follow-up –  Consider surgery in some cases Neoplastic benign: –  Consider surgery if clinically indicated

Neoplastic other: – NET—Resect, particularly for larger tumours – NMCs—Resect if specific criteria are met (clinical, imaging and cytologic criteria; see text) – SPN—Excision –  ROM 82–86% [32] – Ancillary testing, expert consultation, consider repeat sampling; multidisciplinary discussion – FISH results showing polysomy or demonstrable loss of SMAD4 protein expression render the case suitable for surgical referral [32] – Resect if operable and/or systemic therapy where appropriate

refer to texts of gastrointestinal and pancreatobiliary pathology [23, 32]. Table 8.5 summarises the readiness with which cytology is able to diagnose specific pancreatic lesions, based on personal experience. ‘Definitive diagnosis’ refers to the ready classification of the entity into a definitive diagnostic category, e.g. non-neoplastic, benign neoplasm or malignant. Of course, this is a subjective list and experience varies, and this depends on

8.4 Pancreas

177

Table 8.5  Cytologic Diagnoses of Pancreatic Lesions Definitive diagnosis usually possible (± cell block ancillary testing) Solid lesions (neoplastic) Ductal adenocarcinoma ✓ and variants NET ✓ Acinar cell carcinoma ✓ High-grade lymphoma ✓ Metastatic carcinoma Solid lesions (non-neoplastic) Chronic pancreatitis Autoimmune (IgG4-­ related) pancreatitis Cystic lesions (neoplastic) Neoplastic mucinous ✓ (Higher grade cysts) (MCN, IPMN) lesions more readily recognised)

Serous cystadenoma (SCA) SPN

Definitive diagnosis challenging without clinico-radiologic-­ pathologic correlation

Ancillary testing often not required IHC on cell block is very helpful

✓ ✓ ✓ (Histologic biopsy is preferable over FNA)

IHC may help (see Table 8.8) Cell block is essential for the evaluation of architectural features and ancillary testing

✓ (Low-grade neoplasms may be challenging to distinguish from contaminant gastric mucosa) ✓ (Smears usually of low cellularity)

Requires close correlation with clinical, EUS findings and cyst fluid analysis if available (see Chart 8.4, Sect. 8.4.6.1)

✓

Other neoplasms with ✓ cystic degeneration, e.g. adenocarcinoma, NET Cystic lesions (non-neoplastic) Pseudocyst (PC) ✓ (Correlation with clinical findings is important) Lymphoepithelial cyst ✓

Foregut cyst

Remarks

✓

EUS findings; IHC on cell block and fluid analysis is helpful Classical cytomorphology and IHC profile Cell block IHC is helpful

Cyst fluid analysis and correlation with radiologic findings is helpful Note: These are sometimes classified as neoplastic cysts; however, they are not included in the WHO classification of pancreatic neoplasms [23] Presence of ciliated epithelium is helpful, with radiologic correlation

8.4.3 T  echnical Issues in Pancreatic EUS FNA

8.4.3.1 ROSE In contrast to gut wall EUS FNAs where one tends to encounter mesenchymal lesions, one is more likely to come across epithelial lesions in pancreatic FNAs. Hence, the distinction between contaminant gut epithelium and lesional epithelial cells is particularly important. Some possible misinterpretations of normal epithelial tissues are:

The general technical points to consider when evaluating EUS FNAs have been discussed in Sect. 8.2. Here, specific points that are particularly relevant to pancreatic EUS FNAs will be highlighted.

• Gastric specialised epithelium –– Gastric parietal cells and chief cells (Fig. 8.1d,e) have been described in Sect. 8.2.1. To the unfamiliar evaluator, they

specific individual and institutional practice and the availability of clinical information and various ancillary tests. As a general rule, neoplastic solid lesions are more amenable to definitive cytologic diagnosis than are the other categories.

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8  Approach to Endoscopic Ultrasound-Guided FNA of the Gastrointestinal Tract and Pancreas

a

b

c

d

e

f

Fig. 8.8  Normal pancreas. (a) Low-power view showing lobules comprising acini (HC, 40×); (b) Lobule showing grape-like acini and a central duct (Pap, 20×); (c, d) Pancreatic acinar cells with round, eccentric nuclei and abundant flocculent to granular cytoplasm

(HC and Pap, 300×); (e) Pancreatic ductal cells showing a very uniform appearance and round to oval nuclei (Pap, 300×); (f) Comparison of smaller pancreatic acinar cells (centre) and larger cells from a NET, on the left and right (HC, 300×)

may appear rather abnormal and may be potentially mistaken for neoplasms, e.g. endocrine tumours. They are usually seen together with other gastric mucosal cells.

• Pancreatic acinar cells –– These may occur in large intact lobules with stroma, as individual acinar arrangements or as dispersed cells (Fig. 8.8). –– They may be potentially mistaken for

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NETs. When compared directly, NET cells • If lymphoma is suspected, the aspirated mateare significantly larger than benign acinar rial may also be partitioned for flow cytomecells and exhibit classical stippled chromatry, but it would also be ideal to obtain a cell tin (Fig. 8.8f). block whenever possible. In some instances, a –– Benign acinar cells may also be potencell block may offer a greater benefit than flow tially mistaken for acinar cell carcinoma cytometry, because it may allow for a wider (AcCC) (Fig.  8.11)—the latter has a less range of ancillary and molecular tests and also rigid acinar architecture and higher N/C allows lesional tissue to be stored and ratios and does not yield intact lobules preserved. with stroma. • In cystic lesions, the cyst fluid should be parti• Gastric foveolar epithelium and mucin tioned for biochemical analysis (CEA, amy(Fig. 8.1) lase), molecular testing (e.g. KRAS and GNAS –– These may be potentially mistaken for low-­ mutational analysis) and cell block. All mategrade NMCs (IPMN, MCN) (Figs.  8.12, rial may be used, even the supernatant after 8.13, and 8.14). centrifugation of the cyst fluid for cell block. Chai et al. has shown that supernatant fluid is Practical Tips: suitable for both CEA analysis and molecular • Be familiar with the cytologic features of nortesting [33]. We have had similar results, showmal GI and pancreatic contaminants (Figs. 8.1, ing 100% concordance for KRAS mutational 8.2, and 8.8). Actively take note of these when analysis in paired samples of neat fluid and encountered in smears. supernatant (unpublished data). Supernatant • Know the needle route—this usually correcan thus yield diagnostically helpful results in lates with the location of the lesion in the panlow volume cystic lesions and may prove usecreas—transduodenal (pancreatic head and ful in a low volume sample. uncinate process lesions) vs. transgastric (pancreatic body and tail lesions). • Be aware of the EUS imaging appearances of 8.4.4 A Systematic Approach: Key Questions common and important entities. These are summarised in Tables 8.6 and 8.7. • Know the gross appearance of fluid aspirated In the evaluation of pancreatic lesions, a systemfrom cystic lesions (e.g. contaminant gastric atic approach is possible if one bears in mind some mucin is usually thin, compared to thick key questions early in the diagnostic process. viscous mucin from NMCs, Fig. 8.12).

8.4.3.2 Specimen Triage Because of the potential morbidity of pancreatic FNAs and the logistical and financial costs of the procedure, every effort should be made to ensure that the procedure yields sufficient tissue not only for cytologic evaluation, but for ancillary testing. Therefore, it is a good practice to perform cell blocks for all EUS FNAs whenever possible. • In certain entities, ancillary testing on cell block material is instrumental to the diagnosis, e.g. NET, autoimmune pancreatitis (AIP), solid pseudopapillary neoplasm (SPN), serous cystadenoma (SCA) and metastatic carcinoma.

8.4.4.1 Is the Lesion Solid or Cystic? The DDx and hence diagnostic workup is very different for solid vs. cystic lesions. The clinical questions to be addressed in solid and cystic lesions are summarised in Chart 8.3, and cystic lesions will be discussed in more detail in Sect. 8.4.6, as one of the challenging areas in pancreatic cytology. It has been my experience that the diagnostic pathway in solid lesions is somewhat more straightforward than that of cystic lesions, particularly in the context of a discrete pancreatic mass lesion. In this context, the two most common culprits are pancreatic ductal adenocarcinoma (PDAC) and NET.

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8.4.4.2 In Cystic Lesions: Is This a Mucinous or Non-mucinous Cystic Lesion? Cystic lesions include a plethora of non-­neoplastic and neoplastic lesions, the latter ranging from premalignant to malignant (Chart 8.2). In particular, the mucin producing cysts, which most often are neoplastic mucinous cysts (NMCs), are considered at least premalignant and require either close follow-up or resection, based on predetermined clinical, imaging and cytologic criteria. For these reasons, much of the evaluation hinges on whether the lesion is a mucinous cystic lesion, and hence an NMC. The two main types of NMCs are the intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN). Of the two, IPMNs are more common. The specific approach to cystic lesions is discussed later in Sect. 8.4.6.

8.4.5 Triple Approach The three elements of the triple approach are briefly discussed here. 1. Clinical information 2. Cytology 3. Ancillary testing

8.4.5.1 Clinical Information • Age and gender –– This is particularly relevant in cystic lesions of the pancreas (e.g. MCN and SPN almost always occur in females, with a different age distribution). In fact, sometimes these tumours are described as the ‘mother tumour’ (MCN—middle aged) and ‘daughter tumour’ (SPN—young women) tumours due to their distinct age groups. Of course, sometimes SPNs may remain undiscovered for years and present later in life. –– Table 8.7 shows the epidemiological features of pancreatic cystic lesions, together with other elements of the triple approach. • Symptoms –– The presence of obstructive jaundice in the setting of a pancreatic head lesion is an

ominous sign. In neoplastic mucinous cysts (NMCs), it is considered a high-risk feature which may be an indication for surgical resection of the lesion. –– Constitutional symptoms such as loss of weight and appetite are, as always, w ­ orrying because of the likelihood of malignancy. –– Endocrine tumours may be functional and give rise to symptoms attributable to their functional status, e.g. insulinoma. –– New onset of diabetes may also be a presenting feature of PDAC. –– Trousseau syndrome (migratory thrombophlebitis), or, simply a hypercoagulable state, may be present in pancreatic malignancy and rarely may be the first clinical manifestation. –– In the extremely rare AcCC, a minority of cases may present with panniculitis due to lipase hypersecretory syndrome. • Past history and family history –– A past history of malignancy is always relevant, because of the possibility of metastases to the pancreas or peripancreatic lymph nodes. –– MEN1 syndrome may be associated with pancreatic NET. –– Von Hippel Lindau (VHL) syndrome may be associated with pancreatic NET and SCA. –– Patients with a strong or multigenerational family history of pancreatic cancer may also be at increased risk of pancreatic cancer because of familial cancer syndromes (e.g. hereditary breast-ovarian cancer syndrome (HBOC), related to BRCA1 and BRCA2 gene mutations) as well as Lynch syndrome (associated with mutations in mismatch repair genes) [34]. • Physical examination –– Cachexia and jaundice are immediately noticeable on general inspection and are worrying signs for underlying malignancy. The presence of a palpable left ­supraclavicular lymph node is also a worrying sign of metastatic malignancy. –– Courvoisier’s law (Courvoisier’s sign) may be present on abdominal examination,

8.4 Pancreas

characterised by a palpable enlarged gallbladder in a jaundiced patient. This is an ominous finding which may point to pancreatic malignancy. • Blood investigations –– Tumour markers. Routine tumour markers performed in the context of suspected pancreatic malignancy include CA19-9 (reference range 33–37  U/mL) and CEA (reference range up to 2.5 mg/mL). CA19-­9 is more sensitive and specific, but may sometimes be elevated in non-malignant conditions such as cholangitis, cholelithiasis, biliary obstruction and pancreatitis, as well as other malignancies including colorectal, lung and ovarian carcinoma [35]. Elevated CA19-9 is a helpful supportive finding if cytologic features are suggestive of ductal adenocarcinoma, though its role in more recent years has evolved to also include monitoring for recurrent disease. –– Hormone assays on peripheral blood are helpful in the diagnosis of functioning NETs, in conjunction with imaging studies. • Imaging –– Location In some lesions, the location may be a classical feature that prompts consideration of that particular diagnosis. For example, lesions that are more likely to occur in the tail of the pancreas include SCA, MCN and accessory spleen. Table 8.7 summarises the most frequent locations of various cystic lesions within the pancreas. The specific location of the lesion in relation to the pancreas is an important piece of information, e.g. pancreatic mass vs. peripancreatic lymph node. –– Solid lesions The basic findings on EUS and CT/MRI are summarised in Table 8.6. Potential false-positive radiologic diagnoses include chronic pancreatitis and autoimmune pancreatitis (AIP), which may present with localised, mass-like lesions that may be almost indistinguishable from a neoplastic mass. This diffi-

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culty may be further compounded by the cytologic findings of reactive atypia. –– Cystic lesions The main question, as mentioned above, is whether one is dealing with an NMC (i.e. MCN or IPMN). The EUS features of NMCs and non-mucinous cystic lesions are summarised in Table 8.7. Distinction between MCN and IPMN depends on whether the lesion is connected to the pancreatic ductal ­system—IPMNs are connected, whilst MCNs are not. In NMCs, radiologic features also help determine the risk of malignancy—the lesional size, cyst wall thickness, presence of mural nodules and, in IPMNs, connection to the main pancreatic duct (MPD) or branch ducts. Main duct IPMNs carry a higher risk of malignancy. Worrying imaging features are summarised in Table 8.7.

8.4.5.2 Cytology (Gross and Microscopic Evaluation) Morphology includes gross and microscopic evaluation. Whenever assessable, the gross appearance of aspirated cystic material should be noted and documented. Gross Evaluation Gross evaluation is particularly important in the context of cystic lesions, in which the gross appearance of aspirated fluid is rather crucial. Table  8.7 summarises the gross appearance of pancreatic cyst fluid in some of the more common cystic lesions. Here are some parameters that should be evaluated when inspecting cyst contents: • Volume—This is highly variable. At one end of the spectrum, pseudocysts (PCs) often contain fairly large volumes of fluid, and at the opposite end, SCAs and sometimes NMCs may yield a very small volume of fluid. • Colour and clarity—Certain conditions produce clear, colourless fluid (e.g. NMCs) whilst pseudocysts tend to yield ‘dirtier’ appearing,

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Table 8.6  Imaging Findings in Solid Pancreatic Lesions Solid lesions Pancreatic ductal adenocarcinoma

Neuroendocrine tumour

Acinar cell carcinoma

EUS Usually hypoechoic, heterogeneous mass which may have cystic areas. Tumours larger than 3 cm may cause dilatation of pancreatic and/or common bile ducts There may be enlarged, hypoechoic, regional lymph nodes Rounded, well-defined, hypoechoic to nearly isoechoic homogeneous mass Aggressive tumours tend to exhibit more irregular margins Occasionally cystic (usually larger tumours) or mixed-solid cystic [12, 36] Rarely documented, isolated case reports describe a hypoechoic, homogenous mass [37, 38]

Lymphoma

Hypoechoic,, homogeneous mass, ± enlarged regional lymph nodes

Autoimmune pancreatitis

Two patterns: 1.  Diffuse—enlarged hypoechoic, pancreas 2.  Localised—hypoechoic mass (often in the pancreatic head), closely resembling a tumour [12]

Chronic pancreatitis

Abnormal parenchymal appearance— hyperechoic foci or strands (corresponding to areas of fibrosis) and hypoechoic, lobules and cysts; and main duct abnormalities—irregularity and dilatation. Stones appear as hyperechoic lesions with acoustic shadowing [12]

turbid brownish fluid. Lymphoepithelial cysts may produce pasty, yellow-greyish contents. • Viscosity—This is arguably one of the most important physical characteristics of fluid that is worth noting, particularly when an NMC is suspected. Contaminant mucin from the stomach is usually of limited volume and relatively thin, whilst lesional mucin from NMCs is usually of much higher viscosity. Some have suggested the use of the ‘string sign’ as a gauge of

CT or MRI CT scan: Hypodense mass MPD duct may show an abrupt cut-off and dilatation. Double duct sign—dilatation of both the bile and pancreatic ducts—may be seen in a head of pancreas tumour Contrast CT—well-­defined mass with enhancement during arterial phase

Usually large, often more than 10 cm. CT scan— circumscribed and enhancing mass. Cystic change and necrosis may be present (Note: Circumscription is generally not a feature of PDAC) CT: Usually homogeneous, poorly enhancing localised mass or diffuse lesion involving pancreas. The latter pattern may mimic pancreatitis Unlike PDAC, the MPD is usually not dilated, and, if the mass encases large vessels, it usually does not cause significant stenosis. Regional lymph nodes may also be enlarged [39] Diffuse or localised mass-like appearance. In the diffuse pattern, the pancreas may take on a ‘sausage’-like appearance. Usually peripancreatic fat planes are maintained. Contrast CT may show delayed or venous phase enhancement Unlike chronic pancreatitis, calcifications are usually not seen The MPD may show variable narrowing [40] Dilated and sometimes beaded or tortuous main pancreatic duct. Parenchymal or intraductal calcifications may be seen (favour pancreatitis over malignancy; unless there is dual pathology). Fluid levels or cyst formation (i.e. pseudocyst) may also be present [12, 41] Occasionally, involvement may be focal and appear as a localised pancreatic enlargement, mimicking malignancy

viscosity, defined variably as when the mucus ‘strings’ for a length of at least 3 mm or 1 cm when stretched between two surfaces [42, 43]. One author also additionally required a minimum duration of 1 s in which the mucin string had to hold its configuration [42]. When ­positive, the string sign has been shown to be a highly specific feature that favours NMCs, with a specificity of 95–100% for NMCs, using histologically confirmed cases as a gold

8.4 Pancreas

183 Pancreatic Lesions: Solid or Cystic?

Imaging Solid lesions

Cystic lesions

Benign or Malignant?

Mucinous or Non-mucinous cyst?

Clinical Imaging Tumour markers Cytology Benign: E.g. Chronic pancreatitis

Epidemiology EUS findings (Endoscopy, US) Cyst fluid analysis Cytology

Malignant: - Ductal adenocarcinoma - Neuroendocrine tumour (NET) - Others

Non-mucinous: - Neoplastic or not?

Non-neoplastic: - Pseudocyst - Lymphoepithelial cyst** - Others (chart 8.2)

Neoplastic: - Serous cystadenoma - Solid pseudopapillary neoplasm - Cystic NET - Others (Chart 8.2)

Mucinous: - MCN and IPMN*

Is there high grade cytologic atypia?

Key: *For practical diagnostic purposes, mucinous cysts in the pancreas are most likely to be neoplastic, though rarely, simple mucinous cysts may occur (refer to text). ** Lymphoepithelial cysts are sometimes classified as neoplasms.

Chart 8.3  Pancreas—key questions in the cytologic evaluation of solid vs. cystic lesions

standard [42, 43]. Figure  8.12a shows an example of the gross appearance of mucin from an IPMN. The viscosity of the mucus also translates to increased stickiness during the smearing process, and some resistance to the initial spreading of the material at the start of the smearing process. An experienced cytotechnologist will be able to appreciate this feature, which is a helpful clue to the thickness of the mucin. Microscopic Evaluation As with other chapters, microscopic features will be described systematically from low power (architectural formations) to high power (nuclear and cytoplasmic features) and finally background material and cells. Architecture

• Honeycomb sheets –– This regimented, highly regular arrangement of cells is the basic architectural unit for benign glandular cells from GIT contaminant (Figs.  8.1 and 8.2). In sheets of gastric foveolar epithelium, focusing

through various planes may highlight the sharply defined cell borders of the mucinous epithelial cells, reinforcing the polygonal shape of each cell within the honeycomb (Fig. 8.1c). –– Pancreatic ductal cells also appear as flat honeycomb sheets (Fig. 8.8e), these cells usually do not have a mucinous appearance and form smaller sheets than gastric foveolar epithelium, which may sometimes form large tissue fragments. • Drunken honeycomb sheets –– A more disorganised appearance, often referred to as a ‘drunken honeycomb’, may be seen in PDAC.  This is shown in Fig.  8.9. Focusing through the sheet reveals a disorderly arrangement of the cells, which are still arranged in a fairly flat sheet, but with some degree of nuclear overlapping and crowding, with uneven spacing between the nuclei, and hence loss of the regimented honeycomb arrangement. This atypical architectural feature is one of the features of neoplastic glandular cells.

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• Papillary structures –– True papillary structures require the presence of a fibrovascular core and associated overlying epithelium. SPN, although not forming true papillary structures, often shows tissue fragments with a thin, delicate stromal core, reminiscent of papillary structures (Fig. 8.10). These thin cores are curved and branching and are sometimes likened to the appearance of Mandarin characters. The epithelium that accompanies the stromal cores is often bland and uniform. –– IPMN and, less commonly, MCN may exhibit a papillary architecture on histology, but rarely yield true papillary structures on EUS FNA. The lesional cells are usually fairly few and are arranged as small sheets and clusters showing varying degrees of cytologic atypia. • Rosette/acinar like formations –– Normal pancreatic acini are not infrequently seen in pancreatic aspirates. These may occur within a large intact lobule, with the acinar structures appearing as a ‘bunch of grapes’ held together by stroma; or as individual scattered acini comprising bland acinar cells with low N/C ratios (Fig. 8.8). –– NETs may also feature vague rosette-like formations, sometimes forming an incomplete ring. The cells of NETs are often significantly larger than benign pancreatic acinar cells (Fig.  8.8f). NET cells also appear as dispersed single cells and have characteristic plasmacytoid appearance with stippled chromatin (Fig. 8.5). –– SPN may sometimes exhibit acinar-like formations (Fig.  8.10) but usually as a minor architectural component, with papillary-­ like structures (see above) and dispersed single cells forming the most part of the smears. –– Acinar cell carcinoma (AcCC) is an extremely rare tumour which may exhibit a variably prominent acinar architecture (Fig. 8.11). The malignant acinar cells are larger and exhibit more prominent nucleoli and higher N/C ratios than benign acinar cells. Also, the acinar pattern in AcCC is

more subtle and not as pronounced as that seen in benign acini. • Loose aggregates of dispersed cells and singly occurring cells –– In a lesion showing cohesive sheets of cells with high-grade cytologic atypia in keeping with at least high-grade intraepithelial neoplasia, the occurrence of single highly atypical cells is worrying for the presence of invasive malignancy. –– NETs often feature discohesive cells. They may be arranged in loose aggregates as well as single cells that are more spaced apart. One of the classical low power appearances of NETs is the presence of numerous uniform appearing cells which are loosely aggregated, without true cohesion (Fig. 8.5). –– SPN may also harbour numerous singly occurring cells with highly uniform, round to oval nuclei and fine chromatin (Fig. 8.10). –– AcCC may feature, in addition to many cohesive cell clusters, dispersed bare, round nuclei that are stripped of cytoplasm. These nuclei may exhibit variably prominent nucleoli. Some of these dispersed cells have a rim of delicate, ‘barely there’ cytoplasm, if one looks closely. This is in contrast to the cytoplasm of NET cells, which is usually somewhat denser and more well-defined. Cytomorphology

• Cell shape/type –– Plasmacytoid cells are seen in NETs, some metastatic tumours (e.g. melanoma, breast carcinoma). Actual plasma cells may be seen in AIP, and these are much smaller than the plasmacytoid cells of the above-­ mentioned neoplasms. –– Glandular cells from gut contaminant as well as low-grade glandular lesions, e.g. neoplastic mucinous cysts, may exhibit columnar shapes with well-defined cytoplasmic borders (Fig.  8.1c). Mucinous cytoplasm is usually recognisable in NMCs as pale, almost colourless, translucent and

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185

a

b

c

d

Fig. 8.9  Pancreatic ductal adenocarcinoma vs. chronic pancreatitis (CP). (a, b) ‘Drunken’ honeycomb appearance of malignant glandular cells showing uneven spacing and some nuclear overlapping and a moderate degree of nuclear pleomorphism (HC, 200× and Pap, 400×); (c)

Alcohol-fixed smear showing large malignant cells with cytoplasmic vacuoles, some cells have relatively low N/C ratios (Pap, 400×); (d) Reactive ductal cells in CP, showing some disruption of the honeycomb arrangement and mild variation in nuclear size (Pap, 400×)

sometimes fairly voluminous cytoplasm (Figs. 8.12, 8.13, and 8.14). –– Pancreatic acinar cells are usually polygonal with fairly abundant, delicately granular cytoplasm. • Nuclear appearance –– Nuclear pleomorphism and other features of malignancy are usually readily identified in PDAC.  The diagnostic threshold for PDAC is usually high in cytologic samples because the diagnosis usually signals a dismal prognosis, and hence, it is important to be certain before rendering the diagnosis. Potential false-positive diagnoses include chronic pancreatitis and AIP, which sometimes exhibit significant reactive nuclear atypia (see Sect. 8.4.6, Specific Challenges).

Figure 8.9 shows both PDAC and reactive nuclear atypia in chronic pancreatitis. Some texts suggest that a feature favouring adenocarcinoma over reactive atypia is the presence of a greater than fourfold variation in nuclear size within the same group; however, in my experience, this feature should not be used in isolation [44].These potential pitfalls are discussed further in Sect. 8.4.6. –– NETs, similar to those in other locations, also exhibit classical stippled, salt and pepper chromatin. In addition, small and fairly prominent nucleoli can be seen not infrequently (Fig. 8.5). –– AcCC usually has very prominent nucleoli and round nuclei with fairly smooth nuclear

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a

b

c

d

e

Fig. 8.10 Solid pseudopapillary neoplasm. (a, b) Thin delicate branching stromal cores with loosely attached, uniform lesional cells (HC, 40× and Pap, 40×); (c) Acinar-­like formations with many background single cells and naked

ovoid nuclei (HC, 200×); (d) Acinar-like arrangements composed of bland, uniform cells (Pap, 300×); (e) Cell block material stained with B-catenin, showing nuclear (and cytoplasmic) reactivity (B-catenin IHC, 100×)

membranes. The degree of nuclear pleomorphism varies depending on the grade of the tumour. Intranuclear inclusions may occasionally be seen. • Cytoplasm –– In some PDACs, the cytoplasm may be fairly abundant, leading to cells with relatively low N/C ratios (Fig. 8.9). The cytoplasm is pale and sometimes vacuolated. In the more well-differentiated tumours, the well-defined cell borders contribute to the appearance of the ‘drunken honeycomb’. For specific features of PDAC variants, the reader is directed to more detailed descriptive texts [23, 32]. –– In neoplastic mucinous cysts, particularly those comprising gastric foveolar-type

epithelium with low-grade dysplasia, the cytoplasm is pale mucinous and may be relatively voluminous (Figs. 8.12, 8.13, and 8.14). –– The cytoplasm of NETs is relatively homogeneous and, rarely, may contain reddish cytoplasmic granules. As mentioned above, a plasmacytoid appearance is characteristic of NET cells (Fig. 8.5). –– The extremely rare AcCC may exhibit delicate, flocculent, finely vacuolated to granular cytoplasm (Fig.  8.11). At times, the cells appear as naked, stripped nuclei. Background Cells/Material

• Inflammatory cells –– Acute and sometimes some chronic inflammatory cells may be seen in acute

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a

b

c

d

e

Fig. 8.11  Acinar cell carcinoma. (a) Low-power view showing discernible acinar arrangements of relatively uniform cells (HC, 100×); (b) Alcohol-fixed smear showing somewhat disorganised acinar arrangements of cells with round, eccentric nuclei (Pap, 300×); (c) Relatively round,

somewhat hyperchromatic nuclei. Prominent nucleoli are not a feature in this case (Pap, 600×); Inset shows well-­ formed acinar structure, note the relatively delicate cytoplasm (HC, 600×); (d, e) Acinar structures in cell block (HE, 100×; PAS, 200×)

pancreatitis. If accompanying granulation tissue is present, this is a helpful feature pointing to an inflammatory process. –– Chronic inflammatory cells may be seen in chronic pancreatitis, but may be relatively few in number. –– Lymphoplasmacytic cells, particularly plasma cells, may be prominent in AIP. In my experience, however, these are usually not readily found on cytological specimens. Cell blocks sampled by wider gauge needles may occasionally yield sufficient plasma cells to warrant IHC staining for IgG4 and IgG. • Calcifications –– These are helpful when present, to corroborate the clinical and radiologic impression

of chronic pancreatitis. In very challenging cases, however, PDAC may be accompanied by CP; hence, all cases should still warrant careful evaluation for malignant features. • Necrosis –– Fat necrosis may be seen in the context of pancreatitis. –– Coagulative necrosis and apoptotic tumour cells are seen in pancreatic neoplasms and malignancies. • Mucin –– Mucin may be seen as a component of GIT contaminant tissue from transgastric FNAs and in mucin-producing neoplasms. The cytologic distinction of thin mucin (gastric contaminant) from thick lesional mucin is

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b

d

c

e

Fig. 8.12  Neoplastic mucinous cyst—mucin and cytology. (a) Gross appearance of thick mucin in specimen bottle; bottom picture shows a positive ‘string sign’; (b) Low-power appearance showing colloid-like, thick mucoid material (HC, 20×); (c) Oncotic cells entrapped in

mucin in an otherwise acellular aspirate from a MCN (Pap, 400×); (d) Low-grade mucinous epithelium with preserved low N/C ratios and maintained nuclear polarity (HC, 300×); (e) Histology of an MCN with low-grade dysplasia. Note the ovarian-type stroma (HE, 200×)

thus important, and it is more readily performed on air-dried smears. When the mucin appears colloid-like in its staining characteristics, it is likely to represent thick lesional mucin from an NMC (Figs. 8.12 and 8.13). However, the appearance of thinner mucin does not exclude the possibility of an NMC. –– Oncotic cells—Xiao et  al. reported the presence of “oncotic cells”—degenerate singly occurring epithelial cells with eccentric, karyolytic nuclei and swollen, vacuolated cytoplasm, floating within pools of mucin—as a helpful feature that is present in mucin from NMCs but absent in GIT contaminant mucin [45]. I have seen these oncotic cells occasionally in NMCs, and an example is shown in Fig. 8.12c.

• Stroma –– The presence of fibrous stromal fragments in which cytologically malignant cells are embedded is a helpful clue in invasive carcinoma, reflecting the histologic appearance of stromal invasion. Such stromal fragments should also be sought in cell blocks. –– In AIP, cellular stromal fragments which contain chronic inflammatory cells are a useful morphologic feature (Fig.  8.15), though not always seen, particularly when a finer gauge needle is used. –– Chronic pancreatitis may also yield fragments of stroma within which are spindle cells; however, unlike AIP, the cellularity is lower, and chronic inflammatory cells do not predominate.

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a

b

c

d

Fig. 8.13  Intraductal papillary mucinous neoplasm. (a) EUS appearance of a multiloculated cyst (the connection to the MPD is not seen in this frame); (b) Air-dried smear showing abundant thick mucin (HC, 20×); (c) Alcohol-­

fixed smear showing mucinous columnar lesional cells with moderate nuclear atypia (Pap, 300×); (d) Resection specimen showing an intestinal type IPMN with low-­ grade (moderate) dysplasia (HE, 200×)

8.4.5.3 Ancillary Testing A spectrum of ancillary tests may be performed on cytologic material from pancreatic FNAs:

KRAS mutation analysis) and biochemical testing (e.g. CEA level) [33]. It is good practice to work out the specific protocol for sharing material between the cytology and other diagnostic laboratories, so that the specimen wastage is minimised. –– Table 8.7 shows some expected ancillary test results in various cystic lesions. • Cell block material –– IHC, FISH, molecular testing.

• Fresh aspirated material –– Microbiological tests (smears, culture, etc.). –– Flow cytometry for suspected lymphoma. • Cyst fluid –– Biochemical assays (either neat fluid or supernatant fluid after centrifugation— CEA, amylase). –– Molecular testing (KRAS, GNAS mutation testing and other molecular panels). –– Fluid analysis is an important part of the diagnostic workup. As mentioned above in Sect. 8.4.3.2, after centrifugation for cell block and/or smears, the supernatant fluid may be used for both molecular testing (e.g.

In solid lesions, several ancillary tests on cell block material may be helpful, particularly in certain scenarios: • NET—A standard panel of synaptophysin, chromogranin and Ki-67 is helpful, the latter for provisional grading. In the literature, the cyto-

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•

•

•

•

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histologic concordance of grading on cytology (cell block) using Ki-67 is relatively good and ranges from 73 to 85% [46–48]. Preoperative grading is helpful because it has a bearing on management. When quantitating Ki-67 nuclear labelling index, one should look for areas with the purest lesional cell population and be aware of potential contamination by other cell types. Quantifying mitotic figures may be attempted, but there may not be sufficient full high-power fields with lesional cells for a meaningful evaluation. CD56 is usually also positive and may be helpful if other neuroendocrine markers prove to be ‘uncooperative’. AcCC—IHC markers that are sensitive and specific for this rare diagnosis include trypsin, lipase, chymotrypsin and BCL10 [49]. A word of caution is that nuclear B-catenin staining may be present (as also seen in SPN). If these IHC markers are not available, histochemical staining with PAS and DPAS is also helpful, showing positively staining cytoplasmic zymogen granules (Fig. 8.11). Note that AcCC may at times show reactivity with neuroendocrine markers such as chromogranin A—in these instances, the staining is usually very focal and only seen in a minority of the lesional cells. It is also worth noting that mixed AcCC and neuroendocrine tumours do occur, and these are best evaluated on histological samples. Lymphoma—Lymphomas may be primary in the pancreas or secondarily involve it. Cell block material is suitable for IHC, FISH and molecular testing. Fresh needle-aspirated material may also be suspended in a liquid medium and submitted to the haematology lab for flow cytometry. Metastatic malignancy—Cell block is extremely helpful in this context, with specific IHC panels applied for suspected primary sites. Tables 6.2 and 6.3 in Chap. 6 shows some helpful IHC panels for specific malignancies. SPN—Smaller tumours may appear as solid masses on imaging. The unique IHC features of SPNs are covered under cystic lesions, in Table 8.7.

8.4.6 Specific Challenges There are several areas that are particularly challenging in EUS FNA cytology of the pancreas. We will focus on four main scenarios: 1. Cystic lesions 2. Chronic pancreatitis vs. PDAC 3. Autoimmune pancreatitis (IgG4-related disease) 4. Serous cystadenoma

8.4.6.1 Cystic Lesions Cystic lesions pose a diagnostic challenge for two main reasons: 1. Diagnostic cellular material is often sparse and may even be absent. 2. There is a considerable morphologic overlap between contaminant gastric mucosa and some low-grade neoplastic mucinous cysts (NMCs). The spectrum of conditions that may present radiologically as cystic pancreatic lesions is presented in Chart 8.2. Because of the paucity of diagnostic cellular material, the approach to cystic lesions is necessarily multipronged, ideally incorporating epidemiology, EUS findings, gross fluid characteristics and fluid analysis results (biochemical and molecular). Chart 8.4 shows how the triple approach may be applied to cystic lesions and Table 8.7 summarises the salient findings within each domain at a glance. When faced with a cystic lesion, the pathologist is required to address several important clinical questions (Chart 8.3): 1. Is this a Mucinous or Non-mucinous Cystic Lesion? Mucinous lesions, in most instances, refer to the two main types of neoplastic mucinous cysts: intraductal papillary mucinous neoplasm (IPMN) and mucinous cystic neoplasm (MCN). Of the two, IPMNs are more common. • Neoplastic mucinous cysts (NMCs) hold a significant risk of malignancy (synchronous or metachronous).

Mostly head, but can occur anywhere Main duct IPMN—on endoscopy, ampulla may appear patulous and extrude thick mucin Fluid collection in the lesser sac and sometimes within the pancreas

M > F 60–70 years

M > F Young to middleaged adults

F > M 60 years mean age

F >> M Adolescents and young adults

IPMN

PC

SCA

SPN

Anywhere in pancreas

Body and tail > head

Location in pancreas Body or tail (note—most aspirates are transgastric— potential pitfall)

Diagnosis Gender/age MCN F >> M 40–50 years

Usually unilocular, anechoic, thick-walled cyst with luminal debris but no mural nodules. Features of pancreatitis may also be present Circumscribed, multilocular microcystic mass with ‘honeycomb’ appearance; may have a central stellate scar ± calcifications. Uncommonly, SCAs are macrocystic and may closely resemble MCN on EUS Well-defined heterogeneous solid-cystic mass, ± peripheral calcifications. No septations

EUS findings Sharply demarcated cystic lesion ± thick septa. No communication with MPD. Worrying features: large size, irregular wall thickening or mural nodules Three types: Main duct, branch duct or mixed. Cystic lesion connected to MPD or side branches. Ductal system may be dilated. Worrying features: mural nodules or solid components

Blood-stained material

CEA level low (series of five cases, all less than 1 ng/mL) [62]

CEA 1000 U/L (unlikely if 90–100% for the diagnosis of neoplastic mucinous cysts) [55, 57], but sensitivity is limited (49–68%) [55, 58, 59] – GNAS mutation (sensitivity 47%, specificity 98.8% for detecting IPMN) [58]

Ancillary tests Cyst fluid: – KRAS mutation specific for neoplastic mucinous cyst but seen less frequently than in IPMN [55, 56]

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8  Approach to Endoscopic Ultrasound-Guided FNA of the Gastrointestinal Tract and Pancreas Pancreatic Cystic lesions: Triple Approach

Gross and Microscopic evaluation

Clinical Information

Epidemiology: • Age - Young female: SPN - Middle aged female: MCN • Gender - Male: MCN unlikely Background findings: • Pancreatitis (consider pseudocyst)

EUS findings: • Neoplastic mucinous cysts (NMCs): - Connection to pancreatic duct (IPMN) - Thickening/Solid areas - Patulous, oozing Ampulla (Main duct IPMN) - Needle route (gastric contaminant may resemble low grade NMC) •

Non-mucinous cysts: - SCA honeycomb appearance, central scar/calcifications - SPN solid-cystic appearance

Gross: • Thick viscous mucin (NMCs) • Turbid brown fluid (pseudocyst) • Serous fluid ( SCA) Microscopy: • Mucinous lesions - Mucinous cells (lesional or contaminant?) - Low or high grade atypia - Thick (colloid-like) mucin • Non-mucinous lesions - Characteristic cellular appearance - Cell block IHC (SCA, SPN, NET)

Fluid analysis

Biochemistry: • CEA >192 ng/ml* (favour NMC) • CEA < 5ng/ml (favour SCA) • Amylase 1000’s U/L (Pseudocyst) Molecular: • KRAS mutation (NMC) • GNAS mutations (IPMN)

Key: * Different CEA level thresholds may be applied in different laboratories, see Table 8.7. IPMN: Intraductal papillary mucinous neoplasm; MCN: Mucinous cystic neoplasm; NET: neuroendocrine tumour; NMC: Neoplastic mucinous cyst; SCA: Serous cystadenoma; SPN: Solid pseudopapillary neoplasm

Chart 8.4  Triple approach to pancreatic cystic lesions

• Uncommonly, simple mucinous cysts may occur (formerly known as ‘mucinous nonneoplastic cyst’). These contain serous or mucinous fluid and are lined by a single layer of bland gastric-type mucinous epithelium, without the ovarian type stroma that characterises MCNs. It is currently still not clear if these cysts are neoplastic or truly nonneoplastic; however, their behaviour has been generally shown to be clinically indolent [50]. Because of its relative rarity, this entity has been excluded from the main algorithm for cystic lesions (Chart 8.3). Hence, when a cyst that produces mucinous material is encountered in the pancreas, particularly if there is associated mucinous epithelium, it is best regarded as a neoplastic mucinous cyst. • It is generally difficult on cytology alone to distinguish between MCN and IPMN, without the help of clinical information (patient gender, EUS findings such as location and connection to the pancreatic ductal system). On cytology, both may yield honeycomb sheets with varying degrees of architectural and nuclear atypia, on a background of thick

mucin. Well-formed papillary clusters are more likely to be seen in IPMNs than MCNs [51]. In practice, a more clinically helpful approach for the pathologist is to ascertain if there is high-grade atypia. 2. If Mucinous: Is there High-Grade Cytologic Atypia? • The presence of high-grade cytologic atypia is an indication for surgical resection because of the concern for malignancy. • Features of low-grade atypia are: preserved honeycomb arrangement and some retained nuclear polarity, cells closely resemble gastric foveolar epithelium. • Features of high-grade atypia are: small, tight cell clusters, single atypical cells, high N/C ratios and irregular nuclear membranes. In the presence of high-grade cytologic atypia, it may not be possible to definitively determine if there is invasive malignancy or only intraepithelial disease. However, features that are highly worrying for invasive malignancy include the presence of single highly atypical cells, three-dimensional clusters, atypical mitoses and necrosis (Fig. 8.14).

8.4 Pancreas

If the Cystic Lesion Is Non-mucinous: Is It Neoplastic or Non-neoplastic? • Non-mucinous cysts are listed in Chart 8.3— these may be neoplastic or non-neoplastic. The pseudocyst (PC) is an important non-­ neoplastic differential diagnosis whilst the serous cystadenoma (SCA) and neoplasms with cystic degeneration (e.g. SPNs and NET) are considerations in the neoplastic category. It should be noted that smaller SPNs may be entirely solid, whilst larger tumours are more likely to undergo cystic change. • A word of caution: Fluid from lymphoepithelial cysts may yield high CEA and CA19-9 levels; the former may be in the range of 5000 to >30,000  ng/mL [52, 53]. Thus, cyst fluid analysis should not be used in isolation for

193

definitive diagnosis, without radiologic and cytologic correlation. Because of the importance of non-cytologic clues in the approach to cystic pancreatic lesions, it is helpful to be well acquainted with the epidemiological, EUS imaging features and molecular/biochemical profiles in various cystic pancreatic lesions. Chart 8.4 shows how various aspects of the triple approach can help in working out the nature of cystic lesions, while Table 8.7 links all the elements of the triple approach for quick reference. Management of NMCs International consensus guidelines have been written for the management of NMCs. For the

a

b

c

d

Fig. 8.14  IPMNs with low- and high-grade atypia. (a, b) An IPMN, gastric type, with low-grade dysplasia. Cytology shows mucinous lesional epithelial cells with moderate variation in nuclear size (HC, 200×; Pap, 400×); (c) Histology showing gastric type IPMN with low-grade

e

(moderate) dysplasia (HE, 200×); (d) A three-dimensional cell cluster with marked nuclear atypia in an IPMN (Pap, 600×). (e) High-grade atypia in an IPMN—single cells with high N/C ratios and a few apoptotic cells in the background (Pap, 300×)

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patient who is fit for surgery, management depends on the type of NMC, as well as some key radiologic and cytologic features:

marked nuclear atypia (Fig. 8.9d). Squamous metaplasia may be present. • The presence of islet cells—occurring singly or in aggregates. Islet cells are usually not discernible on pancreatic aspirates due to their paucity relative to other cell types. However, in CP, there is atrophy of the acinar cells, ­leading to a prominence of islet cells, which may be recognisable on cytologic aspirates. • Fragments of fat necrosis may be present.

1. MCN and main duct IPMN • Surgical resection is recommended, particularly for larger lesions because of the significant risk of malignancy. Small main duct IPMNs measuring less than 10  mm are a relative indication for surgery and may alternatively be watched [64–66]. 2. Branch duct IPMN In practice, however, the cytologic diagnosis • The risk of malignancy is not negligible may prove challenging for several reasons: but is less immediate, and these may be watched if there are no worrying or high-­ 1. The radiologic features may be those of a localrisk radiologic or cytologic features. ised mass-like lesion and can be virtually indistinguishable from PDAC. Table 8.6 describes Worrying or high-risk features: the imaging features of both entities. 2. The aspirates are relatively paucicellular. • International consensus criteria (revised 3. Reactive cytologic atypia may be present Fukuoka criteria) as well as European consenwithin the pancreatic ductal cells, mimicking sus criteria, largely based on radiologic appearneoplasia. Nuclear enlargement, prominent ance, have been established. These relate to the nucleoli and moderate variation in nuclear size of the lesion, the presence and size of size may be present. mural nodules, the appearance of the MPD, the 4. The hyperplastic endocrine elements may thickness of the cyst walls, as well as the presmimic a pancreatic NET, especially if radiolence of obstructive jaundice [64, 65]. ogy shows a mass lesion. False-positive diag• On cytology, the presence of high-grade cytonosis of NET in CP has been described [67]. logic atypia suggests in situ or possibly even 5. Helpful features of fibrotic stroma, calcificainvasive malignancy and is an indication for tions and inflammatory cells may not be pressurgery in the setting of an operable lesion. ent in a significant proportion of cases. 6. PDAC may sometimes coexist with CP.

8.4.6.2 Chronic Pancreatitis vs. PDAC The diagnosis of chronic pancreatitis (CP) is based on clinico-radiologic and pathologic correlation. The cytologic diagnosis may sometimes be challenging because of radiologic and cytologic overlaps with PDAC. Cytologic features of CP include:

• Generally limited cellularity, particularly later in the course of the disease. • Relatively few chronic inflammatory cells. • Some fibrous stromal tissue fragments, with or without calcifications. • Generally few acinar cells. • Ductal cells showing reactive changes, with some disruption of the honeycomb architecture and mild nuclear enlargement but without

For these reasons, the FNA may be categorised as non-diagnostic, atypical or even neoplastic or suspicious for neoplasia. Siddiqui et  al. reviewed 367 cases of pancreatic FNA with cyto-­ histological correlation and found the false-­ positive rate to be 1.1% (cases diagnosed as malignant), in which all four false-positive cases were instances of CP [67]. The main differential diagnoses are: 1. PDAC 2. NET Tips and Clues: • One should exercise caution in diagnosing malignancy when there is clinical suspicion

8.4 Pancreas

•

•

•

•

of CP. In particular, if there is no discrete mass lesion and there are chronic features such a calcifications or ductal stones, the diagnosis of malignancy should be made only if there are unequivocal cytologic features of malignancy (e.g. three dimensional cell clusters, marked architectural and nuclear atypia, abnormal mitoses and coagulative necrosis). A helpful finding amongst reactive cells is that they often occur in cohesive sheets rather than singly. Hence, the presence of dispersed single atypical cells is a corroborative feature that, together with other features, suggests malignancy. In CP, the degree of nuclear atypia usually falls short of what can be called suspicious or malignant. However, potential false positives have been documented, and the clinical team must accept that there is still a small chance of morphologic overlap [67]. It is prudent for the pathologist to have a high threshold for the diagnosis of PDAC because of the graveness of the prognosis and the significant morbidity associated with treatment. Table 8.8 shows some IHC markers and molecular tests that have been shown to be helpful in distinguishing benign/reactive pancreatic epithelial cells from PDAC [68–74]. Other markers not mentioned in the table, which have been found to be preferentially expressed in PDAC, include Claudin 18, Maspin, MUC1 and mCEA [71, 75, 76]. The UroVysion FISH test which contains labelled chromosome enumeration probes (CEPs) to the pericentromeric regions of chromosome 3 (CEP 3), 7 (CEP 7) and 17 (CEP 17) and to chromosomal band 9p21 has also been found to be specific for PDAC if polysomy is present [77].

8.4.6.3 Autoimmune Pancreatitis (IgG4-Related Disease) AIP is part of the spectrum of IgG4-related disease which also includes Riedel thyroiditis, IgG4-related sialadenitis, sclerosing cholangitis and other conditions. The affected organs usually show features of both chronic inflammation (lymphoplasmacytic infiltrates) and fibrosis

195

(sometimes in a storiform pattern) with accompanying obliterative thrombophlebitis [78]. These manifestations are associated with a relative increase in IgG4-positive plasma cells within affected tissues, in terms of both absolute numbers (IgG4-positive plasma cell counts per high power field) and raised IgG4- to IgG-­ positive plasma cell ratios. This is also accompanied by raised serum IgG4 levels. Cytologic samples are not ideal for the diagnosis of AIP due to the substantial amount of architectural information required and the quantitation of IgG4-positive plasma cells [79–81]. Thus, histologic evaluation would be more useful for tissue diagnosis. Cytologic features of AIP include [31, 79, 80]: • Relative paucity of epithelial cells. Ductal cells may show mild nuclear atypia. • Cellular stromal fragments admixed with chronic inflammatory cells (lymphocytes and plasma cells); with frequent crush artefact (Fig. 8.15). • Stromal fragments containing atrophic acinar cells. • A background of scattered single lymphocytes and sometimes plasma cells. Eosinophils may also sometimes be present. • IHC (cell block) showing more than 10 IgG4-­ positive plasma cells per HPF—based on the consensus criteria for small biopsies of the pancreas [78, 82]. A ratio of IgG4/IgG-­ positive plasma cells should also be >40% [82]. CD138 may also be helpful in demonstrating the presence of plasma cells. In practice, the cytologic diagnosis of AIP is challenging for several reasons: 1. The findings are often heterogeneous within the pancreas, and sampling is an issue. 2. The cell block may not yield sufficiently cellular tissue to perform quantitative IHC (IgG4, IgG, CD138) on. 3. The reactive changes in the pancreatic ductal cells, which may mimic neoplasia. 4. On imaging, a localised picture is sometimes seen, which may suggest neoplastic disease.

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Table 8.8  Ancillary Tests in PDAC vs. Benign Ductal Cells

Diagnosis Reactive/benign pancreatic ductal cells (e.g. CP) PDAC

IHC S100P, IMPs, SMAD4 TP53, Mesothelin – + (no loss of nuclear staining) + – (loss of nuclear staining in approximately 50% of PDAC—specific but not sensitive)

In cases in which there is no clinical suspicion of AIP, this condition may be a potential false-­positive diagnosis for neoplasms and even PDAC, both on radiologic and cytologic grounds. Deshpande et al. found that in a series of 11 cases of AIP that had cyto-histologic correlation, 8 cases were diagnosed as atypical (5 cases) or neoplastic (1 SPN, 1 suspicious for PDAC, 1 mucinous lesion) [79]. None of the 11 cases were prospectively diagnosed as AIP. Holmes et al. described a series of 20 aspirates of histologically confirmed AIP, of which 1 was diagnosed as PDAC [80]. In another of their cases, NET could not be excluded; and this is a phenomenon which we have also encountered in one of our resected cases, in which islet cells populated a large part of the aspirate, leading to an erroneous cytologic diagnosis of ‘possible NET’ (Fig. 8.15). Tips and Clues: • Routinely check the imaging findings—if the appearance is that of a diffusely enlarged, sausage-­ shaped pancreas, the possibility of AIP should be considered. Other imaging findings are presented in Table 8.6. • Caution should be exercised in making the diagnosis of an epithelial neoplasm if the radiologic findings are not those of a discrete lesion. • If the clinical suspicion of AIP is raised, look for chronic inflammatory cells, in particular, plasma cells—these may be few in number, but significant if identified.

Molecular

Clusterin B +

–

NA

CDKN2A (p16) homozygous deletion/loss of heterozygosity (LOH)/mutation); SMAD4 homozygous deletion/LOH); p53 (LOH/mutation) FISH UroVysion test positive for polysomy [77]

8.4.6.4 Serous Cystadenoma SCA is an entity that is more readily diagnosed on imaging, particularly in the most common form, the serous microcystic adenoma, in which there may be a ‘honeycomb’-like appearance with central scarring or calcifications (Table 8.7). The macrocystic or oligocystic variant may closely resemble other cystic lesions such as MCNs, especially as both lesions have similar epidemiological profiles and tend to favour the pancreatic body and tail. The cytologic features of SCA include: • Sparse cellularity. • Flat sheets or smaller clusters of relatively bland and uniform cuboidal cells with pale cytoplasm, which may appear vacuolated or clear (Fig. 8.16). • Cell block: Lesional cells contain glycogen and IHC for alpha-inhibin is positive (Fig. 8.16). In practice, however, the cytologic diagnosis of SCA is extremely challenging and a cytologic diagnosis of SCA requires corroborative findings from imaging and, ideally, cyst fluid biochemistry (Table 8.7). The cytologic diagnosis is challenging for several reasons: • Sparse cellularity, sometimes with no lesional cells. • Lesional cells appear relatively nondescript and may not be readily recognised as lesional due to their paucity and bland appearance.

8.4 Pancreas

197

a

b

c

d

Fig. 8.15  Autoimmune pancreatitis mimicking NET. (a) Clusters of endocrine cells giving rise to the appearance of a NET (Pap, 200×); (b) Cellular stromal tissue fragments (Pap, 40×); (c) Cell block showing a cellular stromal frag-

ment with crush artefact. Definite plasma cells are difficult to discern (HE, 40×); (d) Resection specimen showing a hyperplastic islet and surrounding fibrosis with marked lymphoplasmacytic infiltrates (HE, 200×)

• Sparse material in cell block, which may not be sufficient for ancillary testing. • Potential for false-positive diagnosis of mucinous cyst when needle route is transgastric. This is a documented pitfall, and an error that I have made as well, in a case of a serous macrocystic cystadenoma in a female patient. Transgastric aspirates may yield a significant amount of mucin and bland columnar mucinous cells, thereby mimicking a low-grade NMC. • Scant cyst fluid, which may be insufficient biochemical or mutational analysis.

• IHC using alpha-inhibin on cell block can be helpful, as well as histochemical stains for glycogen (PAS/DPAS). • Knowing the needle route will also potentially mitigate a false-positive diagnosis of NMC if the material is from gastric contaminant.

Tips and Clues: • It is especially important in the context of all cystic lesions, to be aware of the radiologic features (Table 8.7), in particular, the EUS imaging findings. If these suggest SCA, the above subtle cytologic features should be actively sought, and appropriate ancillary tests performed.

8.4.7 P  itfalls (False Positives, False Negatives and Mimics) Some diagnostic pitfalls have already been discussed in the previous section on specific challenges. Here I will focus on some potentially treacherous areas—potential causes of error: 1. False positives 2. False negatives 3. Mimics

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8  Approach to Endoscopic Ultrasound-Guided FNA of the Gastrointestinal Tract and Pancreas

a

c

b

d

Fig. 8.16  Serous cystadenoma. (a) A sheet of relatively bland cuboidal cells with round nuclei and moderate amounts of pale cytoplasm (HC, 200×); (b) A sheet of cells with moderately to fairly abundant pale cytoplasm and mild variation in nuclear size (Pap, 200×); (c) Cell

8.4.7.1 False Positives • CP—This has been discussed in the previous section, and the most likely false-positive diagnoses include PDAC and NET. • AIP—As mentioned in the previous section, AIP has been found to be responsible for false-positive cytologic diagnosis including ‘atypical cells’ or specific neoplasms such as PDAC, SPN, mucinous lesions and NET tumour [79, 80]. Some helpful clues and practices have already been mentioned above. • Contaminant gastric mucosa and mucin— Transgastric needle passes may pick up a significant amount of contaminant material from the stomach wall, and hence may be mistaken for lesional tissue from a neoplastic mucinous cyst in an otherwise non-diagnostic specimen.

e

f

block showing hyalinised fibrous stromal fragments associated with bland cuboidal epithelial cells (HE, 100×); (d–f) Cell block from a different case, showing a strip of lesional epithelium containing cytoplasmic glycogen (PAS; DPAS; IHC with alpha-inhibin, 200×)

Applying triple approach, as highlighted above in cystic lesions, and awareness of the needle route may help reduce this risk. • Pancreatic acinar cells—These may sometimes be numerous and can be potentially mistaken for well-differentiated NETs (the differences are described in Sect. 8.4.5.2) or the extremely rare AcCC, which is discussed below, under ‘Mimics’.

8.4.7.2 False Negatives Well-differentiated PDAC—In the clinical context of CP, well-differentiated PDAC may be missed and the neoplastic ductal cells mistaken for reactive epithelial changes. Singly occurring atypical cells, three-dimensional clusters and abnormal mitoses are helpful in favouring malignancy. The IHC and molecular profiles may be of

8.4 Pancreas

some help (Table 8.8), but, ultimately, a sufficient degree of diagnostic certainty has to be met before a definitive cytologic diagnosis can be made, and this threshold varies with experience.

8.4.7.3 Mimics There are a few entities that may be mistaken for other conditions either due to their relative rarity or morphologic overlap with other commoner entities. Acinar Cell Carcinoma AcCC usually occurs in older adults and is more common in males. It does not usually rank high on the list of differential diagnosis owing to its extreme rarity. Radiologically, AcCCs are fairly well-circumscribed masses in contrast to PDACs which are more infiltrative, a feature that may prompt the radiologist to take special note (Table 8.6). Cytologic features (Fig. 8.11): • Aspirates are often highly cellular. • Cells are fairly uniform and are arranged in nests, acinar structures and sometime branching tissue fragments associated with small blood vessels. • The cells have round, eccentric nuclei, often exhibiting prominent nucleoli, and occasional nuclear inclusions. Cytoplasm is variable in amount and may exhibit a finely vacuolated or granular, delicate quality. • Dispersed cells and stripped lesional nuclei may also be present. DDx: 1. Benign pancreatic parenchyma—The acinar structures are more ‘rigid’ and regular in their arrangement and sometimes are helpfully embedded within a fibrous stroma of an entire lobule. If one is fortunate, one may even be able to discern the ductal cells within the intact lobule—a definite benign finding (Fig. 8.8). Benign acinar cells also tend to be smaller and have smaller nuclei and lower N/C ratios than malignant acinar cells. 2. NET—These tumours are far more common than AcCCs and may show similar architec-

199

tural features of acinar-like structures and loosely dispersed cells. The cells often have a plasmacytoid appearance with relatively homogeneous cytoplasm and more well-­ defined cytoplasm, rather than the looser, more delicate cytoplasm of AcCC.  Also, the stippled chromatin in NET is not seen in AcCC. Note that tumours with features of both AcCC and NET may occur, and IHC may be helpful in these instances. 3. SPN—The epidemiology of SPN is quite distinct from that of AcCC, the former occurring in young adult women. Acinar-like structures may also be encountered, but the classical delicate branching stromal cores with bland lesional cells loosely hanging off (Fig.  8.10) are not seen in AcCC. Bare nuclei in SPN also tend to be somewhat more oval than those seen in AcCC. A small discriminatory panel of four ancillary tests may be performed on cell block to distinguish between AcCC, NET and SPN: DPAS (AcCC—zymogen granules), Synaptophysin and Chromogranin (NET—diffuse staining; AcCC— negative or sometimes focal staining) and B-catenin (SPN—strong nuclear staining). An expanded panel may include BCL10 and Trypsin (both positive in AcCC) ; and CD10, PR and Cyclin D1 (all three positive in SPN). Accessory Spleen The clinical presentation of these lesions is usually that of an incidentally discovered rounded mass in the tail of the pancreas—most closely resembling a NET on imaging. It is important to consider this differential diagnosis when a lymphoid-­rich lesion is aspirated in the pancreas, particularly in the region of the tail. Cytologic features (Fig. 8.17): • Cellular smears comprising numerous smallto medium-sized lymphocytes, admixed with occasional histiocytes, plasma cells and eosinophils. Typically, the features of reactive lymph nodes such as tingible body macrophages and lymphohistiocytic aggregates are not seen [76, 77].

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8  Approach to Endoscopic Ultrasound-Guided FNA of the Gastrointestinal Tract and Pancreas

a

c

b

d

e

Fig. 8.17 Accessory spleen. (a, b) Low-power view showing large branching tissue fragments composed of a mixture of lymphocytes and blood vessels (HC, 20×; Pap, 40×); (c) Mixed lymphoid population of small- to medium-sized lymphocytes with a large platelet aggregate

in the centre, the latter a characteristic feature of splenic tissue (HC, 200×); (d) Lymphocytes, plasma cells and an occasional eosinophil (HC, 300×); (e) Cell block showing CD8-positive sinusoidal endothelial cells (CD8; 200×)

• Lymphocytes may form large aggregates and tissue fragments, within which are discernible blood vessels. • Sometimes, platelet aggregates are identified in the background and can be helpful clue to the diagnosis [83]. An example can be seen in Fig. 8.17. • The most useful diagnostic feature is the presence of CD8-positive sinusoidal-lining endothelial cells, which can be demonstrated on the cell block (Fig. 8.17).

fragments, and individual lymphocytes should exhibit a greater degree of cytologic atypia or monotony.

DDx: The main differential diagnoses include benign lymphoid-rich conditions such as a reactive lymph node and lymphoma. Germinal centre material is usually seen in a reactive lymph node; whilst in lymphoma, the cells usually do not form such large, branching, vascular tissue

Metastatic Malignancy to the Pancreas The pancreas may sometimes be the site of metastases. The most common primary sources are colon carcinoma, lung (small and non-small cell) carcinoma, renal cell carcinoma, breast carcinoma, and, in Western populations, melanoma [84–86]. In order not to make an erroneous diagnosis of a primary pancreatic neoplasm, here are some useful tips/clues: 1. Attention to cytomorphology—Certain malignancies have certain recurring cytomorphologic features that provide helpful clues to

References

their origin. Examples include renal cell carcinoma (conventional/clear cell type); colorectal carcinoma and melanoma. Therefore, a population of malignant cells that show unusual morphologic features that are not classical for PDAC should prompt some digging for any relevant history. The specific morphologic features and useful IHC panels for certain metastases are described in Chap. 6. 2. Attention to clinical history and imaging findings—This should be applied in all cases as far as possible, although the information may not always be available.

EUS FNA of Pancreas: Take Home Points

• In pancreatic FNAs, the distinction between cystic and solid lesions is important, because of distinct diagnostic approaches between the two groups. • The commonest solid entities encountered are PDAC and NET. • In cystic lesions, it is important to decide if one is dealing with a neoplastic mucinous cyst. • A basic understanding of EUS imaging findings is helpful. • In cystic lesions, epidemiology, EUS imaging findings and cyst fluid analysis (biochemical and/or molecular) is very helpful (Table 8.7). • Knowing the needle route is important because gastric contaminant epithelium can closely resemble certain lesions, e.g. low-grade neoplastic mucinous cysts.

References 1. Lange P, Kock K, Laustsen J, Arffmann E, Teglbjaerg PS. Endoscopic fine-needle aspiration cytology of the stomach. A new diagnostic procedure. Endoscopy. 1987;19(2):72–3. 2. Vilmann P, Hancke S, Henriksen FW, Jacobsen GK. Endosonographically-guided fine needle aspiration biopsy of malignant lesions in the upper gastrointestinal tract. Endoscopy. 1993;25(8):523–7. 3. Iglesias-Garcia J, Dominguez-Munoz JE, Abdulkader I, Larino-Noia J, Eugenyeva E, Lozano-Leon A, et al. Influence of on-site cytopathology evaluation on the

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203 58. Kadayifci A, Atar M, Wang JL, Forcione DG, Casey BW, Pitman MB, et al. Value of adding GNAS testing to pancreatic cyst fluid KRAS and carcinoembryonic antigen analysis for the diagnosis of intraductal papillary mucinous neoplasms. Dig Endosc. 2017;29(1):111–7. 59. Singhi AD, Nikiforova MN, Fasanella KE, McGrath KM, Pai RK, Ohori NP, et al. Preoperative GNAS and KRAS testing in the diagnosis of pancreatic mucinous cysts. Clin Cancer Res. 2014;20(16):4381–9. 60. van der Waaij LA, van Dullemen HM, Porte RJ. Cyst fluid analysis in the differential diagnosis of pancreatic cystic lesions: a pooled analysis. Gastrointest Endosc. 2005;62(3):383–9. 61. Salomao M, Remotti H, Allendorf JD, Poneros JM, Sethi A, Gonda TA, et  al. Fine-needle aspirations of pancreatic serous cystadenomas: improving diagnostic yield with cell blocks and α-inhibin immunohistochemistry. Cancer Cytopathol. 2014;122(1):33–9. 62. Carr RA, Yip-Schneider MT, Dolejs S, Hancock BA, Wu H, Radovich M, et al. Pancreatic Cyst Fluid Vascular Endothelial Growth Factor A and Carcinoembryonic Antigen: A Highly Accurate Test for the Diagnosis of Serous Cystic Neoplasm. J Am Coll Surg. 2017 May 18. 63. Charville GW, Kao C-S. Serous neoplasms of the pancreas: a comprehensive review. Arch Pathol Lab Med. 2018;142(9):1134–40. 64. Tanaka M, Fernández-del Castillo C, Adsay V, Chari S, Falconi M, Jang J-Y, et  al. International consensus guidelines 2012 for the management of IPMN and MCN of the pancreas. Pancreatology. 2012;12(3):183–97. 65. European Study Group on Cystic Tumours of the Pancreas. European evidence-based guidelines on pancreatic cystic neoplasms. Gut. 2018;67(5):789–804. 66. Tanaka M, Fernández-del Castillo C, Kamisawa T, Jang JY, Levy P, Ohtsuka T, et al. Revisions of international consensus Fukuoka guidelines for the management of IPMN of the pancreas. Pancreatology. 2017;17(5):738–53. 67. Siddiqui AA, Kowalski TE, Shahid H, O’Donnell S, Tolin J, Loren DE, et  al. False-positive EUS-­ guided FNA cytology for solid pancreatic lesions. Gastrointest Endosc. 2011;74(3):535–40. 68. Jhala N, Jhala D, Vickers SM, Eltoum I, Batra SK, Manne U, et  al. Biomarkers in diagnosis of pancreatic carcinoma in fine-needle aspirates: a translational research application. Am J Clin Pathol. 2006;126(4):572–9. 69. Ibrahim DA, Abouhashem NS.  Diagnostic value of IMP3 and mesothelin in differentiating pancreatic ductal adenocarcinoma from chronic pancreatitis. Pathol Res Pract. 2016;212(4):288–93. 70. Chiba M, Imazu H, Kato M, Ikeda K, Arakawa H, Kato T, et  al. Novel quantitative analysis of the S100P protein combined with endoscopic ultrasound-­

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9

Approach to Effusion Cytology

Contents 9.1

Introduction

 206

9.2  pectrum of Conditions and Classification Systems S 9.2.1  Cytologic Classification Systems 9.3 9.3.1  9.3.2  9.3.3  9.3.4 

Technical Notes Collection Method Fluid Volume Optimising Cellular Yield Sample Triage for Ancillary Testing

 208  208  208  210  210

9.4 9.4.1  9.4.2  9.4.3 

Triple Approach Clinical Information Cytology (Gross and Microscopic Evaluation) Ancillary Tests

 211  211  213  229

9.5 9.5.1  9.5.2  9.5.3 

Pitfalls False Negatives False Positives Mimics

 234  234  235  239

References

9.1

 207  208

Introduction

Serous effusions include pleural, pericardial and peritoneal effusions. A fourth potential serous space in which an effusion can occur is the tunica vaginalis cavity; however, this is seldom aspirated, and thus will not be covered in this chapter. Many conditions can give rise to non-specific benign or reactive findings in serous effusions. In some conditions, the reactive changes may be more marked, and it is prudent for the cytopa-

 241

thologist to be cognizant of some of these clinical settings, e.g. cirrhosis, peritoneal dialysis and rheumatoid arthritis (RA), in order to avoid potential false-positive diagnoses. The finding of malignant cells in serous cavities heralds a grave prognosis, signifying late stage disease with limited treatment options. In recent years, some forms of therapy such as ­intraperitoneal chemotherapy have resulted in improved survival in these patients. However, these treatments may also cause some potentially worrying changes in benign cells, some of which will be described here.

© Springer Nature Singapore Pte Ltd. 2021 M. E. Nga, Practical Diagnostic Approaches in Non-Gynaecologic Cytology, https://doi.org/10.1007/978-981-15-2961-0_9

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207

In this chapter, the triple approach will be covered, and we will focus on several commonly encountered diagnostic dilemmas such as mesothelial cells vs. carcinoma and benign/reactive mesothelial cells vs. mesothelioma. In many instances, IHC can prove to be a great help in resolving these differentials; hence, recommended IHC panels and recent updates will also be provided.

9.2

Spectrum of Conditions and Classification Systems

The spectrum of conditions that one may encounter in serous effusions generally can be divided into two large categories: benign processes (e.g. inflammatory/infectious conditions) and malignant conditions (late stage malignancy or primary malignancies such as mesothelioma or primary effusion lymphoma). Chart 9.1 shows the common and/or important entities that one may encounter in effusion cytology. The mechanism of fluid accumulation also has some bearing on the differential diagnosis. Serous effusions may be transudative or exuda-

tive. Effusions associated with increased hydrostatic pressure or a relative decrease in oncotic pressure within the vascular system are transudative effusions; whilst increased capillary permeability is associated with exudative effusions. The specific gravity, protein content, LDH levels and cellular contents of transudative effusions are lower than those of exudative effusions. Light’s original criteria has been used to separate the two main types of effusions and includes effusion protein/serum protein level ratio (cutoff 0.5), pleural fluid LDH greater than 200 IU and effusion LDH/serum LDH level ratio (cutoff 0.6) [1]. In general, transudative effusions are seen in the background of congestive cardiac failure, cirrhosis and other low protein states (e.g. end stage renal disease). Exudative effusions are seen in malignancy and inflammatory conditions such as pneumonia, abscess, fistula and autoimmune disease. This is not a hard and fast rule because malignant effusions can sometimes be associated with a transudative picture, and I have encountered such cases uncommonly.

Serous effusions

Benign conditions Non-specific cytologic findings • Reactive mesothelial cells • Inflammatory yield (Acute, chronic, mixed) Possible causes: Transudative: • Hypoalbuminaemia • Hepatic failure/Dysfunction • Renal failure/Dysfunction • Peritoneal dialysis • Congestive cardiac failure • Pulmonary embolism • Postoperative Exudative: • Pancreatitis • Infection e.g. pneumonia; tuberculosis • Autoimmune conditions • Meigs syndrome*

Specific cytologic findings • Rheumatoid effusion • Systemic lupus erythematosus • Empyema • Fistula with gastrointestinal tract • Lymphocytic effusion (e.g. tuberculous effusion, metastatic malignancy) • Eosinophilic effusion (e.g. (haemo)pneumothorax, surgery, pulmonary infarct, parasitic/fungal/viral infection, allergy, autoimmune disease, peritoneal dialysis, malignancy) • Chylous effusion (e.g. traumatic disruption of thoracic duct; haematolymphoid malignancy)

Key: * Although some regard pleural effusions in Meigs syndrome as transudative, a meta-analysis of the literature has shown that majority of cases are exudative [15].

Chart 9.1  Spectrum of conditions encountered in serous effusions

Malignant conditions Metastatic / Disseminated malignancy • Metastatic carcinoma • Metastatic melanoma • Metastatic sarcoma/small round blue cell tumour • Haematolymphoid malignancy • Others, e.g. Germ cell tumours

Primary malignancy • Malignant mesothelioma • Primary effusion lymphoma

9.3 Technical Notes

9.2.1 Cytologic Classification Systems Work is ongoing on a classification system for serous effusions—the International System for Reporting Serous Fluid Cytopathology [2]. This is a collaborative effort between the International Academy of Cytology (IAC) and the American Society for Cytopathology (ASC). The proposed system will comprise a five-tier system with the following categories:

208

1. 2. 3. 4.

Collection method Fluid volume Optimising cellular yield Sample triage for ancillary testing

9.3.1 Collection Method

Peritoneal washings, e.g. performed during laparotomy, yield a different cytologic picture from samples obtained from paracentesis. In washings, the architecture is that of flat, sometimes folded sheets of bland, uniform mesothelial –– Non-diagnostic (ND) cells with very regularly spaced nuclei (Fig. 9.1). –– Negative for malignancy (NFM) The mesothelial cells exhibit no to minimal –– Atypia of undetermined significance (AUS) variation in nuclear size and generally smooth –– Suspicious for malignancy (SFM) nuclear membranes, because they are not ‘reac–– Malignant (MAL) tive’ mesothelial cells, but, rather, just quiescent cells that are mechanically shed from peritoneal At the time of writing, the formal document surfaces during the washing process. Therefore, for practice and reporting guidelines surrounding these should not be described or diagnosed as this classification is being prepared [2]. ‘reactive mesothelial cells’ in cytology reports, Farahani and Baloch, in a recent meta-­ rather a simple description as ‘benign mesotheanalysis, have looked at the performance of an lial cells’ and an accompanying diagnosis of identical five-tier system [3]. They were able ‘No malignant cells seen’ is adequate in the to classify 97% of cases into one of the five negative sample. At times, the nuclei within the categories; and the risk of malignancy was: flat sheets may exhibit flower-like outlines, and 17.4% for ND; 20.7% for NFM; 65.9% for these have been coined ‘daisy cells’ [5]. AUS, 81.8% for SFM, and 98.9% for maligIn contrast, in peritoneal effusions, e.g. ascitic nant [3]. fluid samples, the mesothelial cells tend to form smaller clusters or be seen as dispersed cells, sometimes admixed with inflammatory cells, and these may exhibit mild reactive changes of 9.3 Technical Notes nuclear or cellular enlargement and more promiFluid samples require more processing steps than nent nucleoli (Fig. 9.1). do direct needle aspirates using conventional cytology, and technical points to note include the method of collection (e.g. peritoneal washing vs. 9.3.2 Fluid Volume paracentesis), the fluid volume, sample preservation, cell concentration method, stains used as In the upcoming International System for well as methods for cell block preparation. Reporting Serous Fluid Cytopathology, the For a detailed discussion on the processing issue of optimal volume of fluid for cytologic aspects of serous fluid samples, the reader is evaluation will be addressed [2]. In the current directed to more comprehensive texts [4]. literature, several centres have evaluated pleural A focused discussion of salient technical fluid volume for accuracy in diagnosing maligpoints that may impact cytologic evaluation of nancy. In two such studies, one prospective and fluid samples is provided here. one retrospective, the optimal volume for the

9  Approach to Effusion Cytology

209

a

b

c

d

e

f

Fig. 9.1  Benign mesothelial cells. (a, b) Flat sheets of bland, ‘quiescent’ mesothelial cells exfoliated from a peritoneal washing (HC and Pap, 200×); (c, d) Singly occurring reactive mesothelial cells with peripheral ‘lacy skirts’; with some inflammatory cells in the background

g (HC and Pap, 400×); (e, f) Collagen balls, which are sometimes seen in peritoneal washings; (g) Collagen core—a group of benign mesothelial cells with a central collagen core (HC, 400×)

9.3 Technical Notes

detection of malignancy ranged from 60 to 75 mL [6, 7]. Interestingly, in Swiderek et al.’s prospective study, it was recommended that an optimal minimal volume of 150  mL be used if cell block preparations are required [7]. In my own practice, based on anecdotal examples, I find that the cell block yield may not necessarily be a factor of fluid volume per se, rather, it is a function of fluid cellularity. For example, a highly cellular sample as small as 20  mL may yield a robust cell block with plenty of material for ancillary testing, whilst a larger volume sample that is more paucicellular may not yield sufficient lesional cells.

9.3.3 Optimising Cellular Yield At times, smears may be significantly haemodiluted, rendering them suboptimal for direct processing, due to blood elements obscuring mesothelial or lesional cells. In our laboratory, we apply a lysis buffer (containing ammonium chloride, EDTA and potassium hydrogen carbonate) that lyses erythrocytes and hence effectively reduces the obscuring effects of blood. This method enables the evaluator to see nucleated cells much more readily, including mesothelial cells, leukocytes and epithelial cells. A note of caution: in such samples, because leukocytes within the blood remain in the sample and may be concentrated after the removal of erythrocytes, very haemorrhagic smears may appear to have a high proportion of neutrophils, simply due to leukocytes found in blood. These should not be reported as inflammatory smears. A useful practice to avoid overinterpreting these smears as inflammatory is to also prepare a concomitant set of smears without the lysis buffer step. The untreated smears can thus be reliably examined for the presence of acute inflammation, as opposed to the treated smears which may artificially concentrate the neutrophils from blood.

210

9.3.4 Sample Triage for Ancillary Testing Serous effusion fluids are suitable for ancillary testing using several main modalities: 1. Immunohistochemistry histochemistry 2. Flow cytometry 3. Molecular testing 4. Biochemical analysis 5. Microbiology

(IHC)

and

9.3.4.1 Immunohistochemistry and Histochemical Testing Sample: Cell Block Protocols for specimen processing for cell blocks vary between laboratories. In our laboratory, after centrifugation, the plasma-thrombin method is used to create a solid cohesive pellet for the creation of cell blocks. Cell blocks are preferable to additional smears/cytospin preparations for immunohistochemistry for several reasons: • The fixation and processing conditions are similar to those of histologic tissue biopsies; hence, the validation process for IHC/histochemical staining is more straightforward. • Cell blocks may sometimes show helpful architectural features such as papillary structures or acinar formations. These can aid in the diagnosis and also be more readily compared to histology samples from the same patient when required. • The immunostaining profile of the same cell group can be compared in consecutive ­sections, something which is not possible in separate, independently prepared smears. • This method preserves lesional tissue in a form that can be stored and used at a later date for additional ancillary tests. It is my practice to include a brief semi-­ quantitative description of the number of lesional

9  Approach to Effusion Cytology

211

cells seen (e.g. sparse, moderate numbers), because this information, when included in the cytology report, is helpful as a rough gauge as to whether the sample is adequate for additional ancillary tests after the report is issued. Because patients with metastatic carcinoma in serous cavities are no longer candidates for curative treatment, targeted therapy is one of the main treatment options, and this often requires either IHC or molecular testing, e.g. EGFR mutation status, ROS1 and ALK rearrangements in lung cancer.

9.3.4.2 Flow Cytometry Sample: Fresh fluid (adding RPMI solution may increase the viable period, and optimal collection protocols should be worked out with the haematology laboratory). Fresh effusion fluid samples may be sent to the haematology laboratory for flow cytometry, in cases of suspected lymphomatous involvement. This is helpful in establishing the type of lymphoid cells present as well as the presence of a clonal cell population. 9.3.4.3 Molecular Tests Sample: Smears and Cell Block These are suitable on previously stained smears as well as cell block material. In fact, smears may be advantageous because the material has not been subjected to formalin fixation. Regardless of the sample type that is used, molecular tests should be validated for the type of sample used. PCR-based methods and next-generation sequencing (NGS) work well on cytologic smears, prepared by conventional and liquid-­based cytology [8, 9]. In general, 100–200 cells and a tumour cell proportion of at least 20% of the nucleated cells are considered adequate for NGS [10]. Cell blocks are also suitable for molecular testing with PCR-based methods and NGS. FISH may be performed on either smears or cell blocks. Serous fluid samples may also be prospectively kept frozen for bio-banking and future ancillary tests. This usually entails freezing of fluid sediment for storage [9].

9.3.4.4 Biochemical Analysis Sample: Fresh Fluid This is not within the specific area of expertise of the cytopathologist; however, in many scenarios, biochemical analysis of fluid may prove helpful. One of the chief applications is in the distinction between transudates and exudates, in which LDH levels and fluid/serum ratios can be measured, as mentioned above. Biochemical analysis is also helpful in suspected chylothorax, in which lipid levels (triglycerides, chylomicrons) may be evaluated. In suspected tuberculous effusions, adenosine deaminase (ADA) and IFN-γ levels have been shown to be sensitive and specific markers [11, 12], as compared to the demonstration of acid-­ fast bacilli (AFB) which is of low sensitivity in cytologic preparations.

9.3.4.5 Microbiology (Culture and Sensitivity) Sample: Fresh Fluid The literature shows that microbiologic cultures on pleural fluid have a very low yield in terms of clinically significant positive culture growth (less than 2% of samples); hence, these should only be ordered in situations where clinical suspicion is high [13, 14].

9.4

Triple Approach

As in previous chapters, this entails the three elements of: 1. Clinical information 2. Cytology (Gross and Microscopic Evaluation) 3. Ancillary tests

9.4.1 Clinical Information It should be appreciated from the outset that serous effusions may be associated with conditions that are systemic or multi-organ in

9.4 Triple Approach

distribution, rendering clinicopathologic correlation highly relevant in many instances.

9.4.1.1 Epidemiology –– Age and gender are helpful, particularly in the consideration of malignancy from an unknown primary source. –– In the context of metastatic carcinoma, the site of the effusion is also important in ­ascertaining the likely primary origin and should be considered hand in hand with gender. • In the pleural cavity: In males, lung and gastrointestinal tract origin are high on the list, while pancreatic and urothelial carcinoma are a little lower down. In females, breast, lung, ovarian, gastrointestinal and other Mullerian tract malignancies should be considered. • In the peritoneal cavity: In males, pancreatic, GIT, lung and prostate carcinomas should be considered. In females, ovarian, breast, other Mullerian tract malignancies, GIT and lung malignancies are common sources. –– In children, malignant effusions are most frequently seen in the context of haematolymphoid malignancies, germ cell tumours and ‘small round blue cell tumours’, e.g. Ewing sarcoma. –– Autoimmune conditions such as systemic lupus erythematosus (SLE) tends to occur more frequently in women; hence, associated effusions occur more frequently in women. 9.4.1.2 Site –– As mentioned above, site is an important consideration in metastatic carcinoma (i.e. pleural vs. peritoneal) and should be evaluated in tandem with gender. –– In non-malignant effusions, some conditions tend to occur more frequently in specific sites, and a few examples are listed here: • Tuberculous effusion—more likely in pleural effusions. • Meigs syndrome—ovarian fibroma/ fibrothecoma associated with ascites and pleural effusion, interestingly affects the right pleural cavity more frequently than it

212

does the left. While some believe the effusions to be transudative, there is evidence in the literature that many cases are, in fact, more frequently exudative effusions [15]. • Acute pancreatitis is more often associated with left-sided pleural effusions; however, the effusions can be bilateral.

9.4.1.3 Clinical History –– Small effusions may not cause appreciable clinical symptoms; however, larger effusions, depending on the location, may cause discomfort. Dyspnoea may be caused by a large pleural effusion or even a sizeable peritoneal effusion. Widening abdominal girth may also be noticed. –– Constitutional symptoms such as fever may be associated with infection, autoimmune conditions and malignancy. Loss of weight and appetite may also be seen in malignancy or in infection such as tuberculosis. –– Effusions can be caused by systemic or localised diseases. A history of certain conditions may be helpful in ascertaining the likely cause of the effusion. –– Systemic conditions that may be associated with effusions: • Rheumatoid arthritis. • SLE. • Congestive cardiac failure. • Renal failure (± peritoneal dialysis). • Liver failure and cirrhosis (in cirrhosis, spontaneous bacterial peritonitis is a known complication with a significant mortality rate). • Immunocompromised state (e.g. higher risk of haematolymphoid malignancies such as post-­ transplant lymphoproliferative disorder, primary effusion lymphoma). –– Other relevant history may help to narrow down the aetiology: • Tuberculosis • Pneumonia • Malignancy • Recent surgery, e.g. thoracic surgery, may be associated with an eosinophilic effusion, or a chylous one, in the event of damage to the thoracic duct.

213

• (Haemo)pneumothorax, pulmonary embolism or pulmonary infarction, which may be associated with ipsilateral eosinophilic effusions. • History of instrumentation or severe retching when there is suspected oesophageal rupture causing a connection between the pleural cavity and the gastrointestinal tract (Boerhaave syndrome). • Ovarian masses that may be associated with Meigs syndrome (mentioned above). –– Social and occupational history can be extremely important. For example, in a case of suspected malignant mesothelioma, a history of exposure to asbestos—either direct or through contact with family members—is very relevant, not only for the diagnostic workup, but also for insurance or other claims, e.g. worker’s compensation. –– A history of smoking is also important, because lung cancer is one of the main causes of malignant pleural effusion, although in fluids, metastatic adenocarcinomas outnumber the smoking-related cancers (squamous cell carcinoma, small cell carcinoma). –– Drug history is also relevant as certain medication can cause pleural effusions, e.g. methotrexate, phenytoin [16].

9.4.1.4 Physical Examination –– The presence of generalised oedema is a helpful sign and is more frequently associated with transudative causes of effusions, e.g. hepatic, renal or congestive cardiac failure. Jaundice may or may not be present in the setting of hepatic dysfunction. –– A cachexic patient, as always, raises concern for advanced malignancy. –– When the effusion is of sufficient size, the usual clinical signs of pleural, pericardial or peritoneal effusion are present, which can be elicited on physical examination of the chest and abdomen. –– Lymphadenopathy may be significant in certain clinical settings, e.g. lymphoma, autoim-

9  Approach to Effusion Cytology

mune disease, infection or metastatic malignancy. –– Occasionally, one may be able to identify the primary source in the setting of metastatic malignancy, e.g. metastatic malignant melanoma.

9.4.1.5 Imaging –– Imaging investigations include plain radiographs, ultrasound, CT or MRI scans. Ultrasound may also aid in the aspiration of loculated effusions [16]. –– In the context of a cytologic picture where malignancy is suspected, it is particularly important to evaluate the imaging findings that may point to malignancy. E.g. pleural nodules or thickening in the context of suspected mesothelioma, pulmonary masses (single or multiple) in the context of lung or other carcinoma, and tumour masses within the abdominopelvic organs in the case of malignant ascites or pleural effusions. –– A relatively uncommon but specific cause of pleural effusion (usually on the right side) and ascites is Meigs syndrome—ovarian fibroma/fibrothecoma. Abdominal imaging may be useful in this context, where an ovarian mass, usually an oedematous solid tumour, is seen on a background of ascites. Since its original description as an ovarian fibroma, the definition has been expanded by some authors to epithelial tumours as well, even including ovarian epithelial malignancies [17, 18].

9.4.2 Cytology (Gross and Microscopic Evaluation) Both gross and microscopic evaluation should be performed, and the macroscopic features of the fluid sample should be specifically documented in the cytology report—volume and gross appearance (Table 9.1).

Smells like urine

Clear

Yellowish (looks like urine)

Thin

Nil

Slightly thicker than serous fluid

Clear to more opaque

Greenish or brownish green

Bile-stained effusions  – Acute pancreatitis, biliary tract or gastrointestinal tract perforation Urinothorax (Ipsilateral obstructed kidney)

Nil

Thick

Turbid

Whitish, yellowish or greenish metallic/ opalescent

Collagen vascular disease, e.g. rheumatoid arthritis (RA)

Nil

Foul smelling

Thin to thick

Thick, purulent fluid

Nil

Purulent material (neutrophils, necrotic debris, sometimes bacterial organisms) Malignant pigment-­ containing melanoma cells Spindle-shaped, bizarre histiocytes; multinucleated histiocytes and granular necrotic debris [19] Clumps of bile material (yellowish-green on alcohol-fixed smears, dark green on air-dried smears) Non-specific inflammatory yield [20]

Lipid-filled macrophages; cholesterol crystals

Numerous papillary-like scalloped cell clusters, or single atypical mesothelial cells Lipid globules (positive with oil red O), lymphocytes

Nil

Nil

Cytology Wide range, depends on specific condition

Odour Nil

Turbid

Turbid

Yellowish to greenish

Dark brown to blackish

Milky

Whitish; opalescent, greenish hue

Slightly thicker than serous fluid

Somewhat mucoid or sticky (similar to synovial fluid) Slightly thicker than serous fluid

Clear

Yellowish or like honey; Colour may be obscured by blood (haemorrhagic effusion) Whitish; at times greenish Milky

Viscosity Thin usually

Turbidity Blood-­ stained

Colour Reddish-brown due to blood

Metastatic melanoma

Chylous effusion  – Traumatic disruption of the thoracic duct; haematolymphoid malignancy Pseudochylous effusion  – Long-standing effusions; tuberculous effusions; rheumatoid arthritis Purulent effusion (e.g. empyema)

Type of effusion/clinical conditions Haemorrhagic effusion  – Malignancy, pulmonary embolism/infarction, asbestos-related effusion, traumatic tap Malignant mesothelioma

Table 9.1  Specific Gross Appearances of Effusion Fluid

Resolves when urinary tract obstruction is relieved

–

RA—usually unilateral pleural effusion Potential false-positive pitfall

Cell block and IHC are helpful

Supernatant clears after centrifugation. Rich in cholesterol; low triglyceride and chylomicron levels –

Fluid remains milky after centrifugation. Rich in triglycerides and chylomicrons; normal cholesterol levels

Viscosity is caused by hyaluronic acid produced by the malignant mesothelial cells

Remarks Rarely, endometriosis can occur in body cavities, giving rise to haemorrhagic to chocolate brown effusion fluid

9.4 Triple Approach 214

9  Approach to Effusion Cytology

215

9.4.2.1 Gross Evaluation Several gross characteristics of the fluid may be evaluated and documented: 1. Colour (straw-coloured, whitish, haemorrhagic-­ brownish, blackish, greenish, etc.) 2. Turbidity (clear vs. turbid fluid) 3. Viscosity 4. Odour (if any, this usually declares itself quite readily) Table 9.1 shows some of the cytologic findings and possible aetiologies associated with certain gross appearances of serous effusion fluid. These features may not always be readily apparent in these conditions; however, if a characteristic gross appearance is noted, it is worth considering these specific conditions. One of the more unusual and characteristic gross appearances is a milky appearing fluid, which may indicate a chylous or pseudochylous effusion—the two can be distinguished by biochemical analysis and observing the gross

appearance after centrifugation (Table 9.1) [16, 21].

9.4.2.2 Microscopic Evaluation Chart 9.2 shows an overall morphology-based approach to effusion fluid cytology, in which an early point to note whether the cells appear bland or atypical/suspicious. In effusions, atypical cells often exhibit epithelioid morphology. An algorithmic approach to atypical epithelioid cells is shown in Chart 9.3. Some of the key questions asked in this approach are elaborated on here. Here is an outline of the discussion below: (a) (b) (c) (d)

Key cytologic distinctions Cellular patterns in malignant effusions Cellular patterns in benign effusions Background findings

(a)  Key Cytologic Distinctions As outlined above, the initial cytologic evaluation starts with whether the picture is that of bland cells or atypical/suspicious cells (Chart 9.2).

Cytomorphologic features

Bland cells

Predominantly inflammatory cells • Acute inflammatory yield (including empyema) • Chronic inflammatory yield • Mixed inflammatory yield • Eosinophilic effusion • Lymphocytic effusion

Bland epithelioid cells 1. Mesothelial cells • Benign/ Reactive mesothelial cells Note: Some mesotheliomas appear cytologically bland (correlation with imaging is helpful) 2. Epithelial cells • Endosalpingiosis • Endometriosis

Atypical cells (Ancillary tests not available/noncontributory)

Malignant cells

Epithelioid or Other?

Nature of malignancy? (IHC; Flow cytometry)

Epithelioid cells

Mesothelial or Epithelial? (see Chart 9.3)

Other cell appearances • Haematolymphoid cells • Spindle

Dual cell population (malignant cells + reactive mesothelial cells)

Single malignant cell population

Metastatic malignancy • Carcinoma • Melanoma • Sarcoma Haematolymphoid malignancy Malignant mesothelioma

Chart 9.2  Broad morphologic approach to effusion cytology

9.4 Triple Approach

216 Atypical Epithelioid Cells

Positive: Calretinin D240, Mesothelin WT1 *, CK5/6 **

Mesothelial or Epithelial? (Apply IHC if morphology not definitive)

Positive: EP4, MOC31, TAG72

Mesothelial

Epithelial Minimal atypia

Reactive vs Malignant? Favour Mesothelioma BAP1, MTAP, Desmin: –ve EMA, GLUT1, IMP3, CD146: +ve FISH p16INK4a: Homozygous deletion

Endosalpingiosis Endometriosis

Key: * WT1 is also positive in ovarian serous carcinoma ** CK5/6 is also positive in squamous cell carcinoma *** GATA3 stains lymphocytes and a proportion of mesothelial cells

Cytologically atypical/malignant

Malignant cells – Source? (IHC) Lung

Adenocarcinoma: TTF1, Napsin A Squamous cell carcinoma: p40, p63 Small cell carcinoma: TTF1, Synaptophysin, CD56, Chromogranin

Breast

GATA3***, Mammaglobin

Mullerian

PAX8 (WT1 for ovarian serous; Napsin A and HNF1B for clear cell carcinoma), oestrogen receptor

GIT

CK7,CK20, Villin, CDX2, SATB2

Prostate

PSA, PSAP, PSMA, NKX3.1

Chart 9.3  Approach to atypical epithelioid cells

When there are atypical cells with epithelioid features, several questions may then be asked: 1 . Are the cells mesothelial or epithelial? 2. If mesothelial, are they reactive or malignant? The morphological aspects of these questions will be addressed here, while the ancillary tests are summarised in Chart 9.3 and discussed in more detail in Sect. 9.4.3. 1.  Mesothelial or Epithelial?

Most of the time, the main differential diagnoses include mesothelial cells (reactive or malignant) vs. metastatic adenocarcinoma. Occasionally, macrophages may appear somewhat atypical as well, owing to their large size and occasional tendency to form aggregates. The salient morphologic differences between mesothelial cells, adenocarcinoma and macrophages are summarised in Table 9.2 and illustrated in Fig.  9.2. Some basic distinguishing immunopanels are presented in Chart 9.3, and expanded on in Sect. 9.4.3. An important point that is worth noting is that sometimes in various malignancies including metastatic adenocarcinoma, the classical dual cell population that is often highlighted may not always be present. Hence, even if a single-cell

population is present, it should still be evaluated for nuclear features of malignancy, e.g. nuclear membrane irregularity, raised N/C ratios, abnormal chromatin pattern. This ensures that a single population of malignant cells is not missed, particularly if the features of malignancy are more subtle, such as when the cells occur in a dispersed cell pattern. 2. Reactive or Malignant Mesothelial Cells?

When a cell population of mesothelial cells with some degree of atypia is encountered, the main differentials are reactive vs. malignant mesothelial cells. There are two main histologic types of malignant mesothelioma (MM)—epithelioid and sarcomatoid. Sarcomatoid MM usually does not exfoliate malignant cells into the fluid, and hence, one may simply be left with a picture of chronic inflammation, e.g. a lymphocytic effusion. On the other hand, epithelioid MM does usually exfoliate abundant malignant cells. The initial impression is often that of ‘too many cells’ and ‘too large cells’ (Fig.  9.3). The cells from epithelioid MM are often not as pleomorphic as those in adenocarcinoma, and hence may be challenging to differentiate from reactive ­

Macrophages

Adenocarcinoma

Mesothelial cells (reactive)

Single or sometimes loose aggregates of cells without true cohesion

Architecture Usually small clusters (less than 50 cells). Single or small loose groups. Cell clusters tend to have ‘knobby’ or scalloped outlines (Fig. 9.2b) The presence of collagen cores in fluid and cell blocks favours mesothelial origin (benign or malignant), as seen in Fig. 9.1g [22]. Collagen balls may also be seen, particularly in peritoneal fluid (Fig. 9.1e, f) [23] Rounded, three-dimensional balls of cells; sometimes papillary-like clusters; occasionally single cells ‘Community borders’ of cell balls, unlike scalloped borders in mesothelial cells (Fig. 9.2c) In cell blocks, the cell clusters may form ‘hollow rings’

Remarks Windows are not a specific feature of mesothelial cells and can be seen in adenocarcinoma (Fig. 9.2e)

Several patterns seen:  1. Dual cell population—benign mesothelial and malignant glandular cells  2. Single malignant cell population, often seen as dispersed cells May sometimes be a differential diagnosis for clear cell carcinoma, e.g. renal cell carcinoma

Other features May have windows between two adjacent cells (Fig. 9.2d) and also ‘cell-in-cell’ formations (seen in both benign and malignant mesothelial cells); multinucleation may be present

Cells are generally more pleomorphic than mesothelioma cells

Cytoplasm may contain ingested material, e.g. haemosiderin, melanin pigment

Cytomorphology Nuclei central; mitoses may be readily seen. Two-toned cytoplasm—denser inner cytoplasm and peripheral ‘lacy skirt’ (Fig. 9.1). Several types of vacuoles may be seen:  – Small punched out perinuclear lipid vacuoles [24]  – Peripheral glycogen vacuoles  – A single large empty degenerative vacuole containing water  – A single large vacuole containing granular hyaluronic acid, imparting a signet ring-like appearance (Fig. 9.6) Eccentric nuclei, nuclear features of malignancy present. N/C ratio variable. Cytoplasm may be dense or vacuolated. Sometimes, a small droplet of mucin is discernible within vacuoles (Fig. 9.6e)

Large cells, nuclei usually smaller than mesothelial cells and epithelial cells. Oval/ bean-shaped nuclei, finely foamy cytoplasm

Table 9.2  Salient Morphologic Features—Mesothelial cells, Adenocarcinoma, Macrophages

217 9  Approach to Effusion Cytology

9.4 Triple Approach

218

a

b

d

e

c

Fig. 9.2  Mesothelial cells vs. adenocarcinoma. (a) Low-­ power view showing two cell populations—mesothelial cells (lower left quadrant) and malignant glandular cells with large vacuoles (centre right) (Pap, 100×); (b) Cell ball in mesothelioma with scalloped (‘knobby’) borders (Pap, 100×), compared to (c). Metastatic breast adenocar-

cinoma with smooth ‘community borders’ (Pap, 100×); (d) Dual cell population of smaller mesothelial cells (with prominent windows) and larger adenocarcinoma cells (Pap, 300×); (e) Metastatic adenocarcinoma showing windows that are similar to those seen in mesothelial cell groups (Pap, 600×)

­ esothelial cells. There may be some morphologic m overlap within the two groups, including nuclear enlargement, prominent nucleoli, bi- or multinucleation and readily identifiable mitotic figures. Thus, in the distinction between reactive mesothelial cells and MM, knowledge of radiologic and thoracoscopic findings can be extremely helpful. Here are some helpful morphologic clues favouring MM:

as high as 200 within a cluster is a very suggestive feature of mesothelioma [25]. Large cell clusters by themselves are a worrying feature, whether for metastatic carcinoma or MM. A word of caution—in pericardial fluid, larger cell clusters may be seen in benign conditions. The presence of papillary structures formed by mesothelial cells is also a worrying feature for mesothelioma [22]. Larger cell size—malignant mesothelial cells are often larger than benign cells. Raised N/C ratios in the mesothelial cells. The presence of macronucleoli in the mesothelial cells [22]. In pleural fluid in mesothelioma, there may be small, degenerate cells with orangeophilic

•

• • The gross appearance of fluid that is more viscous and somewhat sticky (akin to synovial fluid), due to the production of hyaluronic acid, in the appropriate clinical context. • Many and larger cell clusters—the presence of numerous cell clusters with cell numbers up to

• • •

9  Approach to Effusion Cytology

219

a

b

c

d

Fig. 9.3  Malignant mesothelioma, epithelioid type. (a) Low-power view showing ‘too many cells’ forming ‘too large clusters’; note the scalloped outlines of the cell clusters and the lack of marked nuclear pleomorphism (HC, 40×); (b) Cell-in-cell pattern, which may be seen in both

mesothelioma and benign mesothelial proliferations (Pap, 300×); (c) Small, ‘pseudoparakeratotic’ cells with pyknotic nuclei and orangeophilic cytoplasm, seen in pleural effusions harbouring MM (Pap, 200×); (d) EMA showing strong membrane reactivity (IHC EMA, 300×)

cytoplasm, somewhat resembling parakeratotic cells (so-called ‘pseudoparakeratotic cells’, Fig. 9.3c). These are a helpful clue and should alert one to the possibility of malignant mesothelioma [26]. A word of caution—in peritoneal fluid, these ‘pseudoparakeratotic cells’ may be seen in benign conditions, and I have personally observed these cells in the context of postintraperitoneal chemotherapy (IPCHT) without associated malignant cells [27].

(b)  Cellular Patterns in Malignant Effusions In malignant effusions, there are certain morphologic features that may help point to specific malignancies. Here are some architectural and cytomorphologic clues that may suggest a specific type of malignancy.

In addition to clinical and cytomorphologic features, IHC is also very helpful in distinguishing between reactive and malignant mesothelial cells—an update of the many IHC markers that have been used in this context is provided in Sect. 9.4.3 and summarised in Table 9.4.

1.  Singly Occurring (Discohesive) Malignant Cells

• Metastatic breast carcinoma –– Breast carcinoma may present with singly occurring cells that closely resemble mesothelial cells, with dense cytoplasm and relatively central nuclei (Fig.  9.4a). Sometimes, small clusters of cells occur with ‘windows’ between cells (Fig.  9.2e), which is another feature of mesothelial cells. Careful attention to nuclear features

9.4 Triple Approach

220

(coarse chromatin, irregular nuclear memmay be preserved or increased), macronubranes and raised N/C ratios) will help to cleoli and irregular nuclear membranes. In distinguish these from reactive mesothelial these instances, IHC on cell block can be cells. IHC is helpful in this distinction very helpful (see Chart 9.3 and Table 9.4). (Chart 9.3). • Haematolymphoid malignancy –– Other ways that breast carcinoma cells –– The cells in haematolymphoid malignanexfoliate are in large, rounded, three-­ cies are discohesive in fluids just as they dimensional balls with smooth ‘commuusually are in aspiration cytology samples nity’ borders, sometimes likened to cannon (Fig.  9.4). Haematolymphoid cells show balls (Fig. 9.2c). similar cytomorphologic features to their –– Lobular carcinoma tends to present also as aspiration cytology counterparts, with one single cells with a small intracytoplasmic difference—lymphoglandular bodies are lumen that may contain a mucin droplet usually not readily identified in effusion (Fig. 9.6e). In addition, lobular carcinoma fluid cytology smears. cells may also characteristically arrange –– The commonest haematolymphoid maligthemselves in chains, not unlike those seen nancies involving body cavities include in small cell carcinoma (Fig. 9.5b). T-lymphoblastic lymphoma, large B-cell • Melanoma lymphoma (LBCL), B-lymphoblastic –– Metastatic melanoma, particularly if amellymphoma, chronic lymphocytic leukae­ anotic, may closely resemble metastatic mia/small lymphocytic lymphoma, mantle carcinoma, due to the presence of single cell lymphoma, follicular lymphoma, analarge malignant cells in the fluid (Fig. 9.4b). plastic large cell lymphoma and Hodgkin Nuclei may be fairly eccentric, resulting in lymphoma [28]. Burkitt lymphoma may a plasmacytoid appearance. Sometimes, also involve body cavities, often the peritointranuclear inclusions may be seen neal cavity. (Fig. 9.7a). None of these features are spe–– Primary effusion lymphoma (PEL), a rare, cific to melanomas—the most suggestive aggressive type of B-cell lymphoma, is feature, which may not always be present, associated with human herpes virus 8 is the presence of fine, ‘dusty’ melanin pig(HHV-8) and clinically presents as body ment (dark brown in the alcohol-fixed cavity effusions (pleural, pericardial or smear) within the malignant cells peritoneal) without a mass-forming tumour (Fig.  9.7a). A note of caution—pigment-­ or lymphadenopathy. Fluid cytology feafilled macrophages should not be mistaken tures large, obviously malignant cells with for malignant cells—these usually have prominent nucleoli and fairly abundant smaller nuclei, low N/C ratio and more basophilic cytoplasm [29]. A perinuclear abundant brown pigment (Fig. 9.7). pale hof may be seen, giving rise to a some• Malignant mesothelioma (MM) what plasmacytoid appearance. The cells –– Although the classical cytologic picture is are positive for HHV8 on IHC, but do not that of numerous papillary cell clusters, show reactivity with B-cell markers. This is MM may sometimes present in a dispersed a diagnosis that should be considered in cell pattern (Fig. 9.4c) which is not immepatients who are immunocompromised, diately recognisable as malignant on lowe.g. HIV infection or transplant recipients. power magnification. Even when a dispersed cell pattern is observed, the pic- 2.  Chains/Single File Rows of Cells ture is that of ‘too many cells’. Closer • Small cell carcinoma examination should reveal the presence of –– Metastatic small cell carcinoma tends to atypical/malignant cytologic features such occur in chains that are sometimes reminisas enlarged cells and nuclei (N/C ratios cent of vertebral bodies (Fig. 9.5a). Other

9  Approach to Effusion Cytology

221

a

b

c

d

Fig. 9.4  Malignancies featuring single dispersed cells. (a) Metastatic breast ductal carcinoma showing dispersed mesothelial-like cells; only a single epithelioid cell population is seen (HC, 300×); (b) Metastatic melanoma featuring mainly single malignant cells; note the size

a Fig. 9.5  Malignancies with specific architectural clues. (a) Metastatic small cell carcinoma arranged in a single-­ file pattern resembling vertebral bodies (Pap, 300×); (b) Breast lobular carcinoma characteristically arranged in chains (Pap, 300×); (c) Metastatic small cell carcinoma

comparison with the circled group of mesothelial cells in the bottom left (HC, 300×); (c) Mesothelioma with a single cell pattern—note the perinuclear lipid vacuoles (HC, 300×); (d) T-cell lymphoma involving the pleural fluid, presenting as dispersed cells (Pap, 400×)

b cell showing a tight cell ball with nuclear moulding (Pap, 300×); (d) Metastatic ovarian serous carcinoma showing a papillary cluster with psammoma bodies—note that psammoma bodies are colourless and refractile in the air-­ dried smear (Pap and HC, 600X)

9.4 Triple Approach

c

222

d

Fig. 9.5 (continued)

cytomorphologic features include nuclear moulding, high N/C ratios, small cell size and granular chromatin. Sometimes, the cells are relatively few in number and can potentially be missed on rapid screening. • Lobular breast carcinoma –– When in fluid, metastatic lobular carcinoma may occur in rows or chains, as described above (Fig. 9.5b) [21]. • Mesothelioma –– Malignant cells in mesothelioma can be seen to form chains in fluids, this feature has been described as one that suggests malignancy [21]. 3.  Papillary Clusters (± Psammoma Bodies)

• Mesothelioma –– Papillary-like clusters, some with central cyanophilic collagen cores, may be seen in mesothelioma, both in smears and in cell blocks [21, 22]. Reactive mesothelial proliferations may also exhibit collagen cores (Fig. 9.1g). The finding of these cores is a feature that favours mesothelial origin over glandular origin, as is the presence of scalloped borders (as opposed to smooth community borders, Fig. 9.2b). • Lung adenocarcinoma –– Occasionally, papillary-like or micropapillary clusters of malignant glandular cells may be seen in effusion fluids, from a lung adenocarcinoma [30]. There usually is no

demonstrable collagen core centrally. IHC on cell block (TTF1, Napsin A) and correlation with clinical and radiologic findings are helpful in the diagnostic confirmation of lung origin. • Ovarian adenocarcinoma –– Papillary-like groups of malignant cells, sometimes with central psammomatous calcification, may be seen in metastatic ovarian serous carcinoma (Fig. 9.5d). The cytology is usually pleomorphic and readily recognisable as malignant. Some of the malignant cells may also appear extremely large, with large cytoplasmic vacuoles (Fig. 9.7b). On IHC, an important point to note is that both serous ovarian carcinoma and mesothelial cells are positive for WT1; hence, interpretation needs to be paired with cytomorphologic evaluation. Psammoma bodies in effusion cytology may be seen in both benign and malignant conditions. Within pleural and pericardial fluid, they are far more likely to be associated with malignancy; however, in the peritoneal fluid, they may be seen in benign conditions including ovarian cystadenoma/ cystadenofibroma, papillary mesothelial hyperplasia, endosalpingiosis and endometriosis [31]. 4.  ‘Signet Ring Cells’

A spectrum of cell types ranging from benign to malignant cells can cause a ‘signet ring’ appearance.

9  Approach to Effusion Cytology

223 Table 9.3  Cells with a Signet Ring Appearance in Effusion Fluids Benign Macrophages

Mesothelial cells

Malignant Gastric adenocarcinoma

Breast carcinoma

Cytomorphologic features (Fig. 9.6)

Remarks

Small, bland, eccentric, compressed nucleus with large degenerative cytoplasmic vacuole 1. Single or few large, empty appearing vacuoles indenting nucleus which appears otherwise bland 2. Occasionally, large vacuoles containing coarsely granular material are also seen

Recognisable because of the small, bland appearing nucleus; the vacuole is usually clear and does not contain bubbly mucin 1. Large, empty vacuoles contain water and are a feature of hydropic degeneration 2. Vacuoles filled with coarse granular material are due to accumulation of hyaluronic acid

Nucleus eccentric and compressed but shows malignant features, cytoplasm bubbly or containing a vacuole with intracytoplasmic mucin

Cytoplasm contains mucin—positive for DPAS and mucicarmine. Signet ring cells can be quite small, even smaller than a benign mesothelial cell—hence, it is not the cell size that indicates malignancy, it is the nuclear features This may be seen in both ductal and lobular carcinoma, but are more frequent in the latter—the lumina tend to be more well defined and smaller than those seen in gastric adenocarcinoma

Nucleus eccentric and indented by intracytoplasmic lumen containing a cyanophilic or pinkish mucin droplet (Pap stain)

The morphologic features are summarised in Table  9.3. Several different entities are shown in Fig. 9.6, all with the appearances of signet ring cells. 5. Additional Specific Cytomorphologic Clues in Metastatic Malignancies

There are a few other lesions in which the cytomorphological appearance provides a clue to the possible site of origin. • Pancreatic adenocarcinoma and ovarian carcinoma –– Very large, bizarre cells with very large cytoplasmic vacuoles are often seen (Fig. 9.7b, c). • Ovarian clear cell carcinoma –– Hyaline, magenta staining cytoplasmic and extracellular globules may be seen within tumour cell clusters (Fig. 9.7d) [32, 33]. • Renal cell carcinoma –– Clear cell renal cell carcinoma (RCC) uncommonly exfoliates into peritoneal fluid and may be initially missed because of the relative paucity of cells. The cells may form small clusters or sometimes papillary groups. The cells are fairly large and polygonal and contain nucleolated nuclei

with relatively abundant foamy or bubbly cytoplasm, not unlike that of macrophages (Fig. 9.10a). If a history of RCC is known, careful attention should be paid to cohesive cell groups within effusions, and nuclei should be examined for the presence of prominent nucleoli. IHC on the cell block can be very helpful (PAX8, CD10, RCC). Other variants, e.g. papillary RCC may not exhibit the classical clear cell (vacuolated) morphology, but, rather, more closely resemble papillary adenocarcinomas. • Small round blue cell tumours –– Most of the tumours that fall into this category are sarcomas of childhood or early adulthood, e.g. Ewing sarcoma, rhabdomyosarcoma, synovial sarcoma. Sometimes, lymphomas and small cell carcinomas are also included in the differential. Ewing sarcoma typically occurs in children and younger adults and can involve serous effusions as a result of spread or metastases; and, even, rarely, as a primary site of origin from the visceral pleura [34]. The cells tend to form small clusters and exhibit high N/C ratios with variably prominent nucleoli and are slightly larger than the cells of small cell

9.4 Triple Approach

224

a

b

c

d

e

f

g

h

i

Fig. 9.6  Cells with a signet ring appearance. (a) A benign mesothelial cell with a large vacuole containing coarsely granular hyaluronic acid (HC, 600×); (b, c) Malignant mesothelioma with occasional large degenerative water-­ filled vacuoles (Pap and calretinin IHC, 400×); (d) Macrophages with large, clear cytoplasmic vacuoles and indented but bland nuclei; note the bubbly cytoplasm around the large vacuole (Pap, 600×); (e) Lobular breast

carcinoma and diffuse large B-cell lymphoma [35]. IHC shows reactivity with CD99 (diffuse and strong) and nuclear FLI1. Molecular or FISH testing often reveals the presence of a fusion translocation involving the EWSR1–FLI1 genes. (c)  Cellular Patterns in Benign Effusions In non-malignant effusions, there may be cytologic features that are characteristic of specific conditions (e.g. rheumatoid pleuritis); or a predominant cellular pattern that is non-specific and due to a variety of causes. Below are some notable examples of both.

carcinoma in peritoneal fluid, showing a mucin droplet within a cytoplasmic vacuole (Pap, 600×); (f–i) Metastatic signet ring cell gastric carcinoma with eccentric nuclei— almost all the cells shown are malignant; note that the cells may sometimes not exhibit definite cytoplasmic vacuoles and that they may be very small (Pap, 400× and HC, 400×)

1.  Rheumatoid Pleuritis

It is worth recognising the conglomerate of findings in rheumatoid pleuritis, because it is a potential false positive diagnosis, with features that may be mistaken for malignant epithelial, mesenchymal or even mesothelial cells. There are three main cytologic findings in rheumatoid effusions [36]: • Spindle cell macrophages—these may appear quite atypical in shape and mimic malignant cells (e.g. squamous cells or sarcomatoid cells); however, the nuclei are bland, and they may be multinucleated.

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225

a

b

c

d

Fig. 9.7  Metastatic malignancies with specific cytomorphologic clues. (a) Metastatic melanoma in pleural fluid—contrast the fine, dusty pigment in the malignant cells with the far more obvious, coarser pigment in the macrophages (Pap, 600×); inset shows a melanoma cell with an intranuclear inclusion (Pap, 600×); (b) Large cells

containing large vacuoles in metastatic ovarian carcinoma in peritoneal fluid (Pap, 400×); (c) Large cytoplasmic vacuoles and low N/C ratios in metastatic pancreatic adenocarcinoma in peritoneal fluid (HC, 300×); (d) Ovarian clear cell carcinoma showing striking hyaline globules (HC, 200×)

• Multinucleated histiocytic cells—unlike most other reactive effusions in which multinucleated cells are usually mesothelial in nature, multinucleated cells in rheumatoid effusions are usually histiocytic. • Dirty, coarsely granular background—this includes necrotic cellular debris.

synovial fluid, and subsequently in the pleural fluid. They are not specific for RA, and their presence does not exclude the possibility of other inflammatory conditions. If a history is forthcoming, one should be cognizant of the degree of atypia that may be seen so as to avoid an overcall of malignancy. A word of caution is advised as lung imaging may sometimes reveal pulmonary nodules in patients with rheumatoid arthritis (RA). If the history of RA is not given, then one should be alert to this combination of features and perform clinicopathologic correlation.

The cytologic findings reflect the necrotising granulomatous inflammation that involves the serosal membranes, similar to that seen in synovial tissue. Other features such as cholesterol crystals and ‘ragocytes’ or RA cells may also be noted. RA cells are neutrophils or sometimes monocytes containing lipid-rich rounded cytoplasmic inclusions [37]. They were initially described in the

2.  Systemic Lupus Erythematosus (SLE)

Body cavity effusions are not uncommon in SLE, and the predominant finding is that of

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226

a

b

c

d

Fig. 9.8  Specific benign conditions. (a, b) Oesophageal rupture. (a) Pleural fluid sample showing vegetable material (HC, 200×); (b) A piece of degenerate skeletal muscle from ingested meat, from a case of oesophageal rupture— note also the presence of Candida yeast forms in the back-

ground (Pap, 600×); (c) LE cell in pleural effusion fluid from a patient with SLE (HC, 600×); (d) Intracellular bacteria in a pleural fluid sample with an acute inflammatory yield (HC; 600×)

n­eutrophils and sometimes necrotic material, with ‘LE cells’ and possibly tart cells. LE cells are neutrophils (a broader definition also includes macrophages) that have engulfed the homogenised, glassy nucleus of a non-viable cell (Fig. 9.8c), while in a tart cell, the engulfed non-viable nucleus still shows some chromatin structure [38]. Reactive mesothelial cells which show some degree of cellular enlargement may also be seen in SLE.

eosinophil-­ rich effusions, particularly in the pleural cavity:

3.  Eosinophilic Effusions

• Trauma-related effusions—(Haemo) pneumo­thorax. • Iatrogenic situations e.g. post-cardiothoracic surgery, post thoracocentesis, peritoneal dialysis (peritoneal effusion). • Pulmonary embolism and infarct. • Infections, e.g. parasitic and sometimes fungal infections. • Allergic conditions. • Systemic immune-related conditions, e.g. Churg–Strauss syndrome.

In such effusions, numerous eosinophils are present, and sometimes Charcot–Leyden crystals can be seen in the background [39]. A number of non-­ Eosinophilic effusions may also be seen in neoplastic conditions are associated with more common conditions such as malignancies,

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tuberculosis and pneumonia. Thus, whilst the diagnosis is usually benign, attention to clinical correlation may be rewarding in this context. 4.­  Lymphocytic Effusions

Lymphocytes are a non-specific finding and may be seen in inflammatory, infectious and malignant conditions. There are a few things worth noting when this picture predominates. Firstly, the lymphocytes should be examined for cytologic features of malignancy, e.g. monotonous cells with abnormal chromatin, or features that suggest haematolymphoid malignancy. In benign appearing lymphocytic effusions, the presence or absence of benign mesothelial cells should be noted. If absent or sparse, the possibility of a tuberculous effusion should be considered, and it is prudent to include a comment on this association, so that the clinician team may be alerted to perform appropriate investigations. The histologic correlate is tuberculous pleuritis, where there is fibrinous pleuritis with underlying necrotising granulomatous inflammation, and the exfoliation of mesothelial cells is prevented by the surface fibrinous exudate. In most instances of benign lymphoid-rich effusions, T cells outnumber B cells. If there is a suspicion of lymphoma, flow cytometry may be performed on the fluid sample, and a cell block for IHC or molecular tests can prove very helpful. 5.  Acute Inflammatory Yield

Neutrophils are seen in serous fluids as a result of inflammatory conditions, parapneumonic effusions, direct infection within the body cavity (e.g. empyema) or in association with infarction of adjacent organs. Therefore, their presence does not always connote direct infection within the body cavity. In the presence of direct bacterial infection, there is usually accompanying necrosis, and the fluid appears grossly purulent and foul-smelling. Bacterial organisms may also be seen in the background, and sometimes within neutrophils (Fig. 9.8d), and these should be documented in the cytology report, for correlation with relevant microbiologic investigations.

9  Approach to Effusion Cytology

(d)  Background Findings As a matter of routine, one should always evaluate the background of any cytologic sample. In the case of fluids, there are some background findings that may suggest specific diagnoses: • Mucin –– Intra-abdominal mucinous neoplasms (ranging from low- to high-grade neoplasms/malignancies) may produce copious amounts of mucin, resulting in so-called pseudomyxoma peritonei (Fig. 9.9). Two things are important to note in such instances—firstly, that the gross appearance of the fluid is gelatinous and should be documented in the cytology report. Secondly, while extracellular mucin is abundant and obvious at low-power microscopy, there may be a paucity of actual neoplastic mucinous cells within the cytology smear, and one may mostly encounter muciphages and mesothelial cells—hence a careful search should be made for epithelial cells. Possible sources of such neoplasms include the appendix, rest of the gastrointestinal tract, ovary, pancreatobiliary tract and urachal neoplasms. SATB2 IHC may be helpful in suggesting an appendiceal or colonic source [40]. • Food (digestive tract contents) –– Spontaneous oesophageal rupture, which may be preceded by retching or vomiting, is known as Boerhaave syndrome. As a result, gut contents are present in the pleural fluid and can be recognised as vegetable (plant material), meat (appearing as skeletal muscle), yeasts such as Candida species (gut commensals) and, sometimes, clumps of bacterial organisms (Fig.  9.8a, b) [41]. Benign squamous cells from the oesophageal mucosa may also be present. If this picture is encountered, it is a true medical emergency, and a senior clinician should be informed immediately because of the high mortality rate and the necessity for immediate investigation and intervention.

9.4 Triple Approach

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a

b

c

d

Fig. 9.9  Pseudomyxoma peritonei. (a) Peritoneal fluid showing a loose group of cells caught within extracellular mucin (Pap, 200×); (b) Cell block with IHC for EP4, showing negative staining amongst the epithelioid cells (EP4, 200×); (c) Cell block with IHC for CD163 showing numerous macrophages (CD163, 200×); (d) Cell block

showing reactivity for WT1  in some of the epithelioid cells, indicating that they are reactive mesothelial cells (WT1, 300×). Extracellular mucin, even without demonstrable epithelial cells, is an abnormal finding and suggests the presence of a mucin-producing neoplasm

Pneumothorax and subcutaneous emphysema may also be present clinically in Boerhaave syndrome. • Finely granular background—hyaluronan/ hyaluronic acid –– This may sometimes be encountered in MM and is due to hyaluronic acid production by the malignant cells [24]. The presence of abundant hyaluronan is also responsible for the viscous gross appearance of the fluid (Table 9.1). • Dirty granular background –– This is seen in rheumatoid disease, in which the background contains granular to clumpy, amorphous material which may appear pink-

ish/orangeophilic or greenish in alcohol-fixed samples. The low-power view may be somewhat alarming, particularly if other reactive cellular changes are present; hence, clinical correlation is very helpful. Rheumatoid pleuritis has been discussed above. • Cholesterol crystals –– The presence of cholesterol crystals is often a non-specific degenerative finding and occurs in the setting of long-standing effusions in chronic conditions such as tuberculosis and rheumatoid arthritis. In particular, their presence may correlate with the gross impression of a pseudochylous effusion (Table 9.1).

9  Approach to Effusion Cytology

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9.4.3 Ancillary Tests Ancillary tests are instrumental in the evaluation of serous effusions, particularly when there is suspicion of malignancy. As emphasised in previous chapters, in cytology, one aims to apply the most discriminatory IHC markers in order to conserve tissue, so that there is optimal use of limited tissue for diagnostic, prognostic and predictive test panels. When evaluating IHC markers, it is extremely important to ensure that the cells in question are seen in the cell block section that is being evaluated. This is particularly so when the IHC result is expected to be negative (e.g. negative mesothelial markers in atypical cells suspected to be epithelial). Absence of lesional cells does not equate to a negative result—rather, the IHC marker should be reported as ‘non-contributory’, because lesional cells are NOT present for evaluation. The following discussion focuses on two common diagnostic dilemmas discussed earlier (Sect. 9.4.2.2) and provides an update on the discriminatory IHC markers and molecular tests that can be applied to mesothelial cells.

9.4.3.1 Mesothelial or Epithelial Cells? Ancillary tests, particularly IHC, are very helpful in answering a few questions that were highlighted above. We will focus on two main questions here: • Atypical epithelioid cells: Are they mesothelial or epithelial? • If epithelial, where is the source/origin? (i.e. metastatic carcinoma, if benign conditions such as endosalpingiosis are excluded). The morphological aspects of this question have been discussed above and summarised in Table 9.2. When applying IHC, there are two approaches to tackling these questions. One is a sequential approach which first involves applying mesothelial and epithelial lineage markers. If IHC shows an epithelial immunophenotype, then source-­ specific markers for metastatic carcinoma can be applied. If IHC points to mesothelial cells, then the question is whether these are reactive or

malignant, and this relies on a combination of cytomorphology (discussed in the previous section) and ancillary markers (discussed below). The other approach—one which I prefer, is to apply a limited immunopanel which will answer both the questions upfront: a mesothelial marker, and epithelial marker and a source-specific marker, based on clinicopathologic correlation. A sample case is provided below, with a panel of only three to four discriminatory markers being sufficiently diagnostic in most cases. In order to be able to judiciously apply source-specific markers, knowledge of the clinical and imaging findings is crucial. In instances when the primary malignancy is known and there is documentation of previous relevant histology or cytology, IHC may not even be required, but rather, a comparison between previous and current morphology is sufficient to render a diagnosis of metastatic malignancy. It is also getting increasingly common to encounter patients with more than one known malignancy, in which case a combination of morphologic comparison and IHC will usually provide the answer.

Sample Case

Brief history: A 67-year-old lady with a history of breast carcinoma, now with two left lung masses. Left pleural effusion; tap performed. –– Cytology shows atypical dispersed cells which are highly suspicious for malignancy; however, they closely resemble mesothelial cells (Fig. 9.4a). –– Differential diagnoses: Mesothelial cells (reactive vs. malignant); metastatic breast carcinoma; other metastatic carcinoma. Perform clinicopathologic correlation: • Look up previous breast carcinoma histologic type and grade, and stage of tumour. • Check radiologic findings and impression—note any likely site of new primary malignancy.

9.4 Triple Approach

If the clinical and cytologic findings are suggestive of metastatic breast carcinoma, perform a limited IHC panel of three markers: • Mesothelial: Calretinin (see below for other useful markers). • Epithelial: EP4 (see below for other useful markers—note that MOC31 is preferred for lobular breast carcinoma, as EP4 may be negative). • Breast origin: GATA3. If the cytologic features differ somewhat from the breast primary, addition of TTF1 may be considered, as the lung is one of the commonest primary sites for metastases to the pleural cavity. Other potential IHC panels may be applied depending on clinical suspicion and imaging. While awaiting the initial IHC results, it may also be prudent to check with the managing clinician/oncologist if other markers, e.g. hormonal markers and HER2, are required and perform them after a breast origin is established.

Some of the helpful origin or lineage-specific markers that can be performed to ascertain or confirm the site of origin are summarised in Chart 9.3. In the Lymph Node chapter, Tables 6.2 and 6.3 also show some useful IHC panels that aid in identifying specific primary sites. Practical Tips: • In a malignant effusion with an unknown primary site, a targeted search for clinical information is helpful: –– Radiologic findings (e.g. lung mass, breast imaging, pancreatic, GIT or uterine adnexal masses, etc.). –– Tumour markers (e.g. CA19-9, CEA, PSA). One should be aware that in many instances, tumour markers may also be raised in non-malignant conditions. • The most likely type of metastatic carcinoma in effusion fluids is adenocarcinoma; with

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lung, gastrointestinal tract, pancreas, breast and Mullerian tract malignancies being the most frequent sources. • In malignant pleural effusions where there is no history of known malignancy and no localising imaging findings, lung adenocarcinoma is among the commonest primary sites; hence, TTF1 and Napsin A should be considered in the IHC panel. • Gender and site of effusion (pleural vs. peritoneal) are important considerations (refer to Sect. 9.4.1). Suggested IHC Panels: • Below are some examples of fairly discriminatory markers that differentiate between mesothelial and epithelial cells. When selected carefully, a panel of three markers is usually sufficiently discriminatory to answer both questions above—one mesothelial marker, one epithelial marker and one site-specific marker: –– For mesothelial cells: Calretinin, WT1, CK5/6, D240, Mesothelin. –– For epithelial cells: EP4, MOC31, Tag72, CEA, CD15. –– Site-specific markers: Refer to Chart 9.3 (TTF1, Napsin A, GATA3, PAX8, CDX2, SATB2, Villin, PSA, NKX3.1). • For adenocarcinoma vs. squamous cell carcinoma: –– For squamous cell carcinoma: p40, p63, CK5/6 (note: CK5/6 positive in mesothelial cells too). –– For adenocarcinoma: mucin histochemical stains, TAG72, site-specific markers such as TTF1, PAX8, etc. –– Note that EP4 and MOC31 are positive in both adenocarcinoma and squamous cell carcinoma. Cautions and Caveats: • Always ensure that internal and external controls are showing an appropriate pattern of reactivity. • AE1/3 or pancytokeratins are of no use in the distinction between mesothelial and epithelial cells; they are positive in both. Similarly, CK7

231

•

•

•

•

•

•

is positive in mesothelial cells most of the time; while CK20 is usually negative. If CK7 is applied to ascertain site of origin of carcinoma, it is necessary to ensure that the cytologically malignant cells are evaluated, and not bystander mesothelial cells. WT1 should be avoided if the differential diagnosis is between mesothelial cells and ovarian serous carcinoma—it is positive in both. Napsin A is also positive in macrophages, and hence interpretation in cell blocks must be performed with caution. EP4 is often negative in lobular breast carcinoma (Pai et al. showing only 27.5% of cases with immunoreactivity, n = 44); while MOC31 is more reliable (89% positive) [42]. Hence, a suggested panel to confirm metastatic lobular carcinoma includes MOC31 and GATA3. Mesothelial cells may occasionally exhibit weak reactivity with epithelial markers, in particular, EP4; hence, application of more than one mesothelial and epithelial marker may be required, coupled with careful attention to cytomorphology. GATA3 is also positive in lymphocytes and may show reactivity in a proportion of mesothelial cells; hence, cytomorphology must be evaluated in tandem with IHC.  Other cell types besides breast that are positive for GATA3 include urothelial cells and some endocrine type tumours, e.g. parathyroid lesions and paraganglioma. HBME1 is generally not discriminatory between mesothelial and epithelial origin, because it is frequently positive in both cell types.

9.4.3.2 Benign/Reactive Mesothelial Cells vs. Mesothelioma The distinction between benign/reactive mesothelial cells (BRMs) and epithelioid MM can be more challenging than the distinction between mesothelial and epithelial cells. This is because experience with the diagnosis of MM is limited in many parts of the world and also because of a fair degree of morphological overlap between BRMs and MM. Furthermore, the implications of

9  Approach to Effusion Cytology

a diagnosis of MM go beyond the medical aspects and also impact on occupational health, worker’s compensation and insurance claims. Lastly, the diagnosis of MM is a grave one, heralding a poor prognosis in most cases, and hence should only be made when a definite malignant threshold is met. In sarcomatoid MM, even with ancillary tests, the diagnosis can be extremely challenging on both histology and cytology. A few markers that have shown some promise in recent years include BAP1 IHC (however, the sensitivity is less than 50%), and FISH for homozygous deletion of 9p21. However, the cytologic diagnosis of sarcomatoid MM remains challenging at the current time. To make the distinction between BRMs and MM, one should look at all available clues: 1. Clinical and radiological findings—e.g. exposure to asbestos, whether occupational or domestic; imaging findings in the thoracic cavity. 2. Morphology—this has been discussed above under the Sect. 9.4.2. 3. Ancillary tests—There is an ever increasing array of IHC markers, as well as some molecular tests that are helpful in the distinction between benign/reactive mesothelial cells from malignant mesothelial cells. These tests are discussed here and summarised in Table 9.4. IHC Markers • EMA (MUC1, clone E29; cytoplasmic and/or membranous stain) –– Reactivity pattern: Strong membranous staining (with or without weaker cytoplasmic staining) favours MM. –– This is arguably one of the most helpful and accessible markers available in the distinction between BRMs and MM. Creaney et al. documented the importance of using the optimal clone of the MUC1 antibody, by testing seven MUC1 clones and finding EMA to be the most consistently specific and sensitive clone [43]. The positive staining pattern is strong membranous staining

9.4 Triple Approach

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Table 9.4  Ancillary Tests for Benign vs. Malignant Mesothelial Cells BRMs

MM

Remarks

IHC Desmin

+

−

BAP1

+

−

MTAP

+

−

EMA (Clone E29)

−

GLUT1 IMP3 CD146

− − −

+ve with strong membrane staining + + +

p53

‘Normal’ pattern— weak, focal reactivity

Strong reactivity in majority of lesional cells

Relatively high sensitivity and specificity Sensitivity limited, especially in sarcomatoid MM; while specificity close to 100% Sensitivity limited; specificity close to 100%. More data required on sarcomatoid MM EMA is also positive in metastatic carcinoma Also positive in carcinoma Also positive in carcinoma Also positive in non-mesothelial malignancies Interpretation is subjective and may be difficult; hence of limited helpfulness

Intact

Homozygous deletion

Molecular FISH 9p21 locus (p16INK4a or CDKN2A) gene)

with some cytoplasmic reactivity (Fig.  9.3d). It should be noted that metastatic carcinoma may also be positive for EMA; hence, accompanying mesothelial stains such as Calretinin, WT1, etc. should also be performed. Hasteh found the sensitivity and specificity to be 100% and 91%, respectively [44]. • BAP1 (nuclear stain) –– Reactivity pattern: Loss of nuclear reactivity favours MM. Stromal and inflammatory cells are positive internal controls (nuclear staining). –– BAP1 (BRCA1-associated protein 1) has been found to show loss of expression on IHC in epithelioid mesothelioma (56– 81%), but much less so in sarcomatoid MM (as low as 15%) [45]. More importantly, it has been found to be reliably expressed in BRMs, and hence loss of expression is highly specific for malignancy (close to 100%) [45, 46]. Cytological samples have also been tested, yielding similar results on cell blocks to histologic biopsies, with a specificity of 100% [46, 47]. Reassuringly,

Sensitivity limited; also lost in sarcomatoid MM. Specificity approaching 100%

Kinoshita et al. found that BAP1 reactivity was retained in all their biopsy samples of fibrous pleuritis, whilst it was lost in 36.7% of cases with a sarcomatoid mesothelioma component [48], attesting to the limited sensitivity but high specificity. A note of caution is that loss of BAP1 expression may be seen in other non-mesothelial malignancies; hence, the mesothelial lineage of the lesional cells should be confirmed using a reliable IHC panel. • MTAP (nuclear and cytoplasmic stain) –– Reactivity pattern: Loss of nuclear and cytoplasmic reactivity favours MM. –– Methythioadenosine phosphorylase (MTAP) is a gene which is located in the 9p21.3 locus and may be lost due to homozygous deletion of 9p21  in certain malignancies, including MM. The loss of MTAP monoclonal antibody expression on IHC has been found to correlate with 9p21 deletion (kappa coefficient of 0.69) [49]. –– Kinoshita et  al. showed a sensitivity of 42% and a specificity of 100% in the distinction of MM from BRMs using MTAP

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•

•

•

•

9  Approach to Effusion Cytology

IHC, evaluating only epithelioid or bipha–– This is a cell adhesion molecule which has sic MMs [47]. been shown to be expressed in certain Desmin (cytoplasmic stain) malignancies such as melanoma and pros–– Reactivity pattern favouring MM: Loss of tate cancer. Several authors have shown cytoplasmic reactivity (i.e. no reactivity). that the expression of CD146 on the cell –– Hasteh et al. demonstrated a sensitivity and membranes of mesothelial cells favours specificity of 84% and 94%, respectively, MM over BRMs, with a sensitivity ranging using desmin to distinguish between BRMs from 71 to 94%, and specificity ranging and MM (52 cases of MM, 64 cases of from 98 to 100% [51, 52]. BRMs) [44]. I have found desmin to be a • p53 (nuclear stain; less discriminatory fairly reliable marker in highlighting overall) benign mesothelial cells. –– Reactivity pattern: Strong reactivity in GLUT1 (membrane staining, sometimes majority of cells favours MM. cytoplasmic) –– This has been reported as variably positive –– Reactivity pattern: Positive staining favours in MM, however, to date, has not found to MM. reliably distinguish between benign and –– This is the glucose transporter 1 protein, malignant mesothelial proliferations [45]. which has been found to be reactive in MM The interpretation of this marker is also favourably over BRMs; however, the sensisomewhat challenging, because the prestivity and specificity are both limited (41% ence of wild-type p53 gene correlates with and 88%, respectively), in a study of 43 some degree of reactivity in benign mesoMM and 15 BRMs cases by Hasteh et al. thelial cells, hence rendering the interpreta[44]. GLUT1 also stains positively in metation somewhat challenging in cell block static carcinoma (adenocarcinoma and material. squamous cell carcinoma); hence, it would be prudent to include mesothelial and epi- Molecular Tests thelial markers in the panel if there is • Homozygous deletion of p16 by FISH (9p21 uncertainty [50]. A compounding difficulty deletion) is that the marker also stains erythrocytes, –– The 9p21 locus contains several important thus rendering morphologic interpretation genes involved in cell cycle progression, challenging. including p16INK4a (CDKN2A) and MTAP genes. It has been shown recently that both IMP3 (cytoplasmic stain) epithelioid and sarcomatoid mesothelio–– Reactivity pattern: Cytoplasmic reactivity mas, particularly arising in the pleura, favours MM. exhibit homozygous deletion of p16 by –– This is insulin-like growth factor-II mRNA-­ FISH.  In their literature analysis, Churg binding protein, which is favourably et al. found the deletion rate of 45–85% in expressed in malignant cells in effusions— epithelioid MM (average 64%) and including MM and metastatic carcinoma; 33–100% in sarcomatoid MM [45]. and more likely to be negative in BRMs. However, this test did not fare so well in Ikeda et al. found a sensitivity rate of 77% peritoneal epithelioid MMs, with a mean and specificity of 94% for malignancy deletion rate of only 38%. An important (MM or carcinoma) in 171 pleural and finding, however, is the extremely high peritoneal fluid samples [50]. Again, mesospecificity, of practically 100% in the thelial vs. epithelial lineage should be recent literature, based on more than 200 ascertained if IMP3 is used. cases of BRMs which were all found to CD146 (cell membrane stain) have two preserved gene copies. –– Reactivity pattern: Positive staining favours MM.

9.5 Pitfalls

–– This test also provides prognostic information, because the presence of this deletion is associated with a worse prognosis. The commercially available Urovysion test has been applied successfully for this purpose, because of the inclusion of the p16 INK4A gene probe (at the 9p21 gene locus) in test [53]. • Telomerase –– Several groups have shown increased telomerase activity in MM as compared to BRMs, using IHC with hTERT (telomerase reverse transcriptase), protein immunoassay or PCR-based telomere amplification assessment methods [54–56]. The most reliable assay is the telomere repeat amplification protocol (TRAP) assay; however, this has yet to become part of the routine diagnostic workup [24]. • Multigene classifier –– Churg et  al. has described a highly sensitive and specific molecular tool combining a multigene expression assay (117 genes) and a specific computational algorithm, which essentially classifies mesothelial proliferations into malignant and benign groups [57, 58]. These results are promising but require larger study cohorts for validation for routine use.

9.5

Pitfalls

As with other specimen types, it is helpful to be aware of some of the pitfalls in the evaluation of serous effusions. We will look at some of the potential traps for false-negative and -positive diagnoses, as well as mimics that may cause difficulties in interpretation.

9.5.1 False Negatives In the well-processed cytologic sample, malignant cells may be missed because of quantitative (few malignant cells, e.g. Hodgkin lymphoma involving body cavity) or qualitative reasons (e.g.

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small malignant cells or single malignant cell population composed of dispersed cells). Here are some malignancies which may be missed for qualitative reasons.

9.5.1.1 Breast Carcinoma The cells of both ductal and lobular carcinoma may occur as a dispersed, single cell population in fluids and may sometimes resemble reactive mesothelial cells (Fig. 9.4a), as mentioned above in the Sect. 9.4. Furthermore, in lobular carcinoma, the cells may be fairly modest in size and hence easy to miss. Thus, if a clinical history is forthcoming, smears should be screened carefully. Another potential cause for missed diagnosis is the tendency for lobular carcinoma to be negative for EP4 on cell blocks [42]. Therefore, if metastatic lobular carcinoma is suspected, MOC31 is recommended instead as an epithelial marker, together with GATA3, and also hormonal markers/HER2 neu if requested by the clinician. Tips and Clues: • Even in a single-cell population that looks like reactive mesothelial cells, one should routinely take careful note of nuclear features in order to pick up subtle features of malignancy, e.g. raised N/C ratios, coarse chromatin, prominent nucleoli and mild nuclear membrane irregularities. • In metastatic lobular carcinoma, screen carefully for small single cells with small intracytoplasmic lumina which contain a small mucin droplet (Fig. 9.6e). This appearance is highly suggestive of malignancy. • Another clue to the presence of metastatic lobular carcinoma is the occurrence of small malignant cells arranged in chains (Fig. 9.5b). • IHC on cell block usually provides confirmation of the presence of metastatic breast carcinoma, using a combination of epithelial markers (see note of caution above), mesothelial markers (if necessary) and breast markers. IHC should be applied judiciously, so that tissue can be preserved should the clinical team request for other markers (e.g. hormone receptors, etc.) .

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9.5.1.2 Small Cell Carcinoma Small cell carcinoma cells may be missed because they tend to be relatively few and inconspicuous, forming small clusters in between much larger mesothelial cells. Tips and Clues: • If there is clinical suspicion of metastatic small cell carcinoma, an active search should be made for small malignant cells. One should look for small tight groups of malignant cells with dark nuclei, granular chromatin and nuclear moulding, as well as linear chains of malignant cells (Fig. 9.5a). • If clinical suspicion is high, IHC may be performed on the cell block (TTF1, Synaptophysin or CD56), because this can increase the sensitivity of cytologic evaluation, picking up rare malignant cells which may not be immediately appreciable on the smears.

9.5.1.3 Malignant Mesothelioma: Dispersed Cell Pattern The cells of MM may sometimes appear deceptively bland, particularly when they present as singly dispersed cells. Thus, the clinical index of suspicion is important and should always be taken seriously, so that closer attention may be paid to pick up subtle clues, and relevant ancillary tests can be performed. Tips and Clues: • Subtle clues that point towards malignancy should not be overlooked, e.g. the appearance of ‘too many cells’, mild cellular and nuclear enlargement, nuclear membrane irregularity, prominent nucleoli and raised N/C ratios. • A host of IHC markers have been shown to be helpful in distinguishing benign/reactive mesothelial from malignant mesothelial cells including EMA, BAP1, MTAP and Desmin. These have been discussed in detail in Sect. 9.4.3 and summarised in Table 9.4.

9.5.1.4 Renal Cell Carcinoma The occurrence of metastatic renal cell carcinoma in body cavity effusions is rare. In a retro-

9  Approach to Effusion Cytology

spective study looking at more than 400 cases with nephrectomies, Renshaw et  al. only found malignant effusions in no more than 2% of patients [59]. Within their series of eight malignant effusions from seven patients, none of them were from sarcomatoid RCC—all were either conventional clear cell or papillary RCC, both of which show similar cytologic features. In my own practice, I have found that in metastatic clear cell RCC, the cells have been few and only occur in very small groups of 2–3 cells and resemble macrophages due to the abundance of pale vacuolated cytoplasm. Tips and Clues: • A high index of suspicion is helpful—pay attention to small groups of cells hiding in the midst of mesothelial cells, macrophages and other inflammatory cells (Fig.  9.10a). Even though the N/C ratios of the malignant cells may be low, the nuclei are usually of sufficient atypia to distinguish them from macrophages. • IHC may be performed on the cell block (RCC, PAX8 and CD10 for RCC vs. CD163 for macrophages) and aid in picking up sparse malignant cells.

9.5.2 False Positives Sometimes, benign conditions may lead to markedly reactive changes in mesothelial or inflammatory cells (e.g. macrophages) that may raise the question of malignancy. Of course, malignancy may co-exist in some of these conditions, e.g. rheumatoid arthritis; hence, the application of IHC to cell block material should be performed if there is uncertainty.

9.5.2.1 Rheumatoid Pleuritis The cytomorphologic features of rheumatoid pleuritis have been described above in Sect. 9.4.3. Essentially, the spindle cell macrophages may be mistaken for metastatic malignancy, including sarcoma, sarcomatoid carcinoma or squamous cell carcinoma (tadpole or fibre cells).

9.5 Pitfalls

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a

b

c

d

Fig. 9.10  Pitfalls—potential false positives and negatives. (a) Renal cell carcinoma in peritoneal fluid, showing a few large polygonal cells with abundant vacuolated cytoplasm, superficially resembling macrophages (HC, 300×); (b) Atypical reactive mesothelial cell in peritoneal dialysate (Pap, 400×); (c, d) Potential false positives—

peritoneal fluid, after intraperitoneal chemotherapy for advanced gastric carcinoma, showing ‘exploding mitoses’ and multinucleated mesothelial cells; the inset in d. shows WT1 IHC which is positive in a mitosing cell nucleus (HC, 400×; Pap, 600×)

Tips and Clues: • Other morphological clues in the usual cytomorphologic triad should be sought, if atypical degenerate spindle cells are seen. These include multinucleated macrophages and coarsely granular background debris. • The nuclei of the spindle cells are usually pyknotic, with fairly bland chromatin if viable. The cytoplasm of the spindle or bizarre cells, if orangeophilic, may be granular rather than dense and waxy as seen in true keratinised squamous cells. • Clinical clues are, of course, the history of RA, sometimes with lung nodules—such a history should alert one to expect some atypical cytologic features in the effusion fluid.

9.5.2.2 Chronic Kidney Disease, Uraemia and Peritoneal Dialysis In patients with chronic kidney disease with resulting uraemia, as well as in patients with peritoneal dialysis, marked reactive changes may be present in mesothelial cells, raising a concern for malignancy. The cells may occur in three-­ dimensional or even papillary-like clusters and exhibit enlarged, irregular nuclei with prominent nucleoli, raised N/C ratios and even atypical mitotic figures [60–63]. Tips and Clues: • The threshold for the diagnosis of malignancy should be adjusted in this clinical b­ ackground,

9  Approach to Effusion Cytology

237

knowing the marked reactive features that may be encountered. • Where doubt exists, cell block with IHC should be performed.

9.5.2.3 Cirrhosis Chronic liver disease, including cirrhosis, may give rise to both architectural and cytologic atypia amongst mesothelial cells, usually seen in ascitic fluid. The cells may occur in papillary or acinar clusters and exhibit enlarged nuclei, prominent nucleoli, multinucleation and abnormal chromatin patterns, as well as prominent cytoplasmic vacuolation (Fig. 9.11) [21, 64, 65].

a

c

Tips and Clues: • Caution should be exercised when considering a malignant cytologic diagnosis in peritoneal fluid in the background of cirrhosis. Clinical and radiologic correlation should be performed in such instances. • When in doubt, IHC on cell block is helpful.

9.5.2.4 Pancreatitis In acute pancreatitis, the peritoneal surface may be irritated by enzymatic leakage, resulting in marked reactive mesothelial cell changes (irregular nuclear membranes, prominent nucleoli) that may mimic malignancy [66]. Analysis of the fluid usually yields a high amylase level, and cytology,

b

d

Fig. 9.11  Pitfalls—cirrhosis and endometriosis. (a, b) Ascitic fluid in a case of cirrhosis showing reactive mesothelial cells with nuclear enlargement and binucleation (HC, 200×; Pap, 600×); (c–e) Endometriosis in a peritoneal fluid sample—(c) A tight cluster of epithelial cells

e with bland nuclear features, inset showing an aggregate of haemosiderophages (Pap, 400×); (d, e) EP4 and CD10 IHC on cell block highlight epithelial and stromal cell components, respectively, in the same cell group (EP4 and CD10 on cell block)

9.5 Pitfalls

in addition to reactive mesothelial cells, often yields numerous neutrophils. Caution should be exercised in rendering a malignant diagnosis in this setting, unless unequivocal cytologic features of malignancy are present, as well as corroborative imaging and clinical findings.

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to have an idea of the morphologic features of the malignant cells. • When one encounters atypical cells that are difficult to characterise, there should be a low threshold for IHC on cell block (applying epithelial and mesothelial markers). • A helpful practice is to work with the clinical 9.5.2.5 Post-intraperitoneal team to ensure that the relevant history of Chemotherapy malignancy with IPCHT is conveyed to the Many reactive cell changes occur as a result of cytology laboratory, so that the pathologist chemotherapy, which can give rise to sometimes may be alerted to the more marked reactive alarming appearances. In advanced intra-­ changes that can be expected. abdominal malignancies, e.g. gastric adenocarcinoma with documented peritoneal involvement, 9.5.2.6 Endosalpingiosis and Endometriosis one of the modes of treatment is intraperitoneal chemotherapy (IPCHT), in which the peritoneal In endosalpingiosis and endometriosis, benign cavity is directly exposed to chemotherapeutic epithelial cells may be identified in peritoneal effusions. Rarely, endometriosis may also involve agents [67]. In our institution, we have reviewed and pub- the pleural cavity [68]. The number of lesional lished a series comprising 39 peritoneal fluid epithelial cells is usually not high. Thus, the mere presence of epithelial cells in samples from patients undergoing IPCHT, showing the occurrence of frequent mitotic features in an effusion does not always equate to malignancy—malignant morphologic features benign mesothelial cells (confirmed on IHC on ­ cell block, Fig. 9.10) and other features such as are still required in order to make a definitive multinucleation and nuclear membrane irregular- malignant diagnosis. ity [27]. These cases can be particularly challenging because of the clinical setting of known Tips and Clues: malignancy, as well as the significant morpho- • These possibilities should always be considered in a female patient in which relatively logic overlap between residual malignant cells bland epithelial cells are seen in the peritoneal and reactive mesothelial cells. fluid. The only feature that was found to be more frequent in IPCHT cases with IHC-confirmed • In endosalpingiosis, the cells may appear as tight, crowded papillary-like clusters with malignant cells was raised N/C ratios. A notable high N/C ratio cells, uniform nuclei and fairly feature in IPCHT cases was that in the reactive scant cytoplasm [69]. Psammoma bodies may mesothelial cells, mitotic figures showed an also be identified [70]. Always actively look unusual ‘exploding mitoses’ morphology, which somewhat resembled the cartwheel appearance for cilia, which is a good sign that the cells are of fireworks or a starburst pattern (Fig. 9.10c, d). likely to be benign, if the nuclear features are We also noted the presence of increased numbers otherwise bland. of eosinophils in the background, as well as scat- • In endometriosis, the endometrial cells may tered small, orangeophilic cells, similar to the be few, or even absent, with the only clue pseudoparakeratotic cells seen in MM [27]. being haemosiderophages. An example is shown in Fig. 9.11c–e. The endometrial cells Tips and Clues: occur in rounded groups or sometimes larger • It is helpful to know the histologic type of clusters with a central stromal core and usumalignancy that was diagnosed initially, so as ally exhibit bland nuclear features, and

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s­ ometimes may show psammoma bodies [71, 72]. Because these cells are usually not ciliated, at times the differential diagnosis of well-­differentiated Mullerian epithelial neoplasms needs to be considered. On IHC, PAX8 and CD10 may be helpful, particularly the latter in delineating the stromal component (Fig. 9.11e). Looking at the clinical history is also helpful in these instances, for evidence of endometriosis.

9.5.3 Mimics This section includes mimics that may result in false-negative/positive diagnoses or misclassification of malignancies that show morphologic overlap.

9.5.3.1 Metastatic Squamous Cell Carcinoma vs. Adenocarcinoma Metastatic squamous cell carcinoma (SCC) is rare in serous effusions. In two series of metastatic SCC in effusion fluids from the United States, the case numbers amounted to 36 (over a 22-year period) and 49 (over 17  years) respectively, attesting to its rarity [73, 74]. In their series, Huang et  al. found that squamous pearls and tadpole cells were identified in only 13% and 17% of cases, respectively, whilst 52% of cases exhibited cytoplasmic vacuoles, a feature that is commonly present in adenocarcinoma [74]. LePhong et al. reviewed 49 cases with accompanying cell blocks and often histologic correlation and found that on cytologic smears alone, only 39% were initially recognised as squamous on cytology, whilst 29% were read initially as non-small cell carcinoma (including 2 that were called adenocarcinoma) [73]. In fluids, non-keratinising SCC may form cell balls that are virtually indistinguishable from those seen in adenocarcinoma. Figure 9.12 shows two different cases of metastatic non-keratinising

9  Approach to Effusion Cytology

SCC, one presenting as cell balls and the other as single cells, both of which could easily be mistaken for metastatic adenocarcinoma, without the supporting history or IHC picture. Tips and Clues: • Knowledge of a history of SCC is very helpful, so that appropriate IHC may be performed on the cell block (p40, p63). The most common sources are lung SCC, head and neck SCC and uterine cervical SCC, the latter usually presenting in peritoneal effusions.

9.5.3.2 Mesothelioma vs. Adenocarcinoma The distinction between epithelioid MM and adenocarcinoma can pose a challenge on ­morphology alone, due to overlapping features—­ three-­ dimensional cell balls, windows, cytoplasmic vacuoles and cell-in-cell formations can be seen in both malignancies. In most instances, adenocarcinoma is by far more common than MM; however, epidemiology alone is not a sufficiently reliable parameter. Tips and Clues: • Morphologic clues are shown in Table 9.2. • IHC is extremely helpful, and the most discriminatory markers have been presented in Chart 9.3 as well as Sect. 9.4.3. In my practice, I find the mesothelial markers Calretinin and WT1 highly reliable, with the caveat that WT1 should not be used if ovarian serous carcinoma is suspected. For epithelial markers, I favour EP4 and MOC31. • Some cautionary notes in the interpretation of IHC are also presented in Sect. 9.4.

9.5.3.3 Signet Ring Cells Several cell types may present with signet ring-­ like morphology, superficially resembling metastatic signet ring cell carcinoma, which signals late-stage gastric adenocarcinoma. These are discussed in Sect. 9.4.2 and summarised in

9.5 Pitfalls

240

a

b

c

d

Fig. 9.12  Pitfalls—two cases of metastatic squamous cell carcinoma from the uterine cervix. (a, b) Metastatic non-keratinising squamous cell carcinoma forming balls in peritoneal fluid—note the small cytoplasmic vacuoles (HC, 600×; Pap, 100×); (c, d) A different case of meta-

static SCC occurring as single cells with high N/C ratios, and irregular, hyperchromatic nuclei somewhat resembling those seen in high-grade intraepithelial neoplasia of the cervix, inset shows p40 IHC on the cell block (HC, 400×; Pap, 400×)

Table 9.3. Benign cells include both macrophages and mesothelial cells, the latter containing degenerative water-filled clear vacuoles. Some examples are illustrated in Fig. 9.6.

• Nuclear features should be carefully evaluated in order to ascertain if the cells are benign or malignant—one should not be overly concerned by the resemblance to signet ring cells. • IHC may be helpful to ascertain the type of cell. In addition, a mucin stain can be very useful as well, in highlighting cytoplasmic mucin which is not a feature of mesothelial cells.

Tips and Clues: • Morphological features of the various benign and malignant entities with signet ring cell morphology are summarised in Table 9.3.

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Take Home Points

• Body cavity effusions may reflect the presence of systemic disease; hence, it is ideal to know the clinical setting as far as possible, in particular, the presence of autoimmune conditions or malignancy. • Mesothelial and epithelial cells can show considerable morphologic overlap; hence, clinical context and targeted IHC panels are important in distinguishing between the two. • In malignant effusions, ancillary testing is ideally done on cell blocks rather than on additional smears/cytospins because they provide a means to archive tissue for predictive and prognostic ancillary tests. • Cells behave differently within a fluid environment and hence will appear different in fluids compared to FNAs—e.g. SCC may appear morphologically similar to adenocarcinoma. • In the evaluation of IHC results, one must always look for the lesional cells on the section—even if the expected result is negative, to ensure that the section contains representative material before making an interpretation.

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Index1

A Abscess EUS FNA, 158c, 161, 207 lymph node, 89, 90, 158c Accessory spleen, see “Spleen, accessory” Acinar Brunner gland adenoma, 174f effusions, 210, 237 EUS FNA acinar structures, 184 lymph node metastases, 91–93, 105, 105f pancreatic acinar cells, 159, 171f, 173, 178f, 178–179, 184, 185, 194, 198 salivary gland acinar patterns, 148, 152 sample cytology report, 16, 17 solid pseudopapillary neoplasm, 186f triple approach, 23, 23f Acinar cell carcinoma (AcCC) clinical findings, 175, 180 cytologic diagnosis of pancreatic lesions, 177t cytomorphology, 186, 187f, 187, 199 DDx, 199 DDx with benign pancreas, 159, 179, 199 EUS FNA of pancreatic lesions, 175c IHC, 190, 199 imaging, 182t Acini pancreatic, 159, 178f, 184 Acinic cell carcinoma (ACC) clinicoradiological correlation, 131c cytomorphology, 132f DDx secretory carcinoma, 149f false-negative diagnoses, 152t IHC, 137 lymphoid-rich lesions, 141c neoplasms readily diagnosed on cytology, 128t oncocytic/oncocytoid neoplasms, 126, 133t, 134, 145c salivary gland aspirates DDx, 125c Adenoid cystic carcinoma (AdCC) ancillary tests, 137, 138t, 138 basaloid neoplasms/appearance, 131c, 132f, 133t, 143, 144, 144c, 153

cytologic diagnosis, 128t cytomorphology, 135f, 144c false-negative and positive diagnoses, 152t isolated cells, 136 Milan malignant category, 127 pitfalls, 151 salivary gland, main entities, 125c sample case, 144 stromal features, 134t Adenosine deaminase (ADA), 211 Adnexal lesions basaloid lesions in lymph nodes, 144 clinical findings in lymph node FNA, 89 DDx in lymph node FNA, 82c, 84, 103, 105, 125c, 129, 153 false-positive, lymph nodes, 108 imaging pelvis in effusion, 230 Adrenocortical carcinoma, 30, 91t AE1/3, 230 ALK anaplastic large cell lymphoma, 117 inflammatory myofibroblastic tumour, 173 lung carcinoma, 211 Alpha-inhibin adrenocortical carcinoma, 91t serous cystadenoma pancreas, 183c, 196, 198f American Thyroid Association (ATA), 45, 48, 50 Amylase ancillary testing in pancreas, 160, 189 pancreatic cystic lesions, 28t, 179, 191t, 192c peritoneal fluid, 237 Amyloid DDx hyalinising trabecular tumour, 76 medullary thyroid carcinoma, 63f thyroid FNA, 62, 64 Anaplastic large cell lymphoma (ALCL) age group, 87 cohesive-appearing, 104–105, 117 cytomorphology, 95t, 99 DDx for Hodgkin lymphoma, 115 effusions, 220 pattern-based approach to lymph node FNA, 95c

Page numbers followed by “c” “f” “t” denote “Chart” “Figure” and “Table” respectively 1 

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246 Anaplastic thyroid carcinoma (ATC), 66, 74 Androgen receptor (AR), 138t Angioimmunoblastic T-cell lymphoma (AITL) cytomorphology, 115, 116f DDx for Hodgkin lymphoma, 94t, 115 lymphomas that may be missed, 85t Apoptosis, 24 Artefacts(s) autoimmune pancreatitis, 196, 197f blood clots, 48 lymph node cytology, 83, 85, 114 pancreatic cytology, 176t thyroid cytology, non-diagnostic, 44t thyroid cytology, nuclear inclusions, 57, 60f ultrasound gel, 25, 25f Atypia of undetermined significance (AUS) approach to AUS/FLUS, 45, 71c atypical lymphoid cells, 45, 69c, 71, 71c cytologic criteria, thyroid, 56, 70–71, 72f, 74 effusions, 208 FVPTC/NIFTP, 47, 78 Hurthle cell-rich lesions, 69 Milan system, salivary gland, 126 salivary gland cystic lesions, 136, 140c, 140 salivary gland, lymphoid lesions, 142–143 the Bethesda System for Reporting Thyroid Cytopathology, 44t, 45 thyroid entities not readily diagnosable, 47t treatment-related atypia, thyroid, 75 Atypical lymphoid cells, 70, 70c, 71, 71c Australasian Classification (ASC) for thyroid cytology, 44t Autoimmune pancreatitis (AIP) cytologic diagnosis, 177t cytomorphology, 184, 189, 192, 195–196, 197f false-positive diagnosis, 181, 198 imaging, 181, 182t specimen triage, 179 Autoimmune thyroiditis, 4, 49 B BAP1, 231, 232, 235 Bare nuclei acinar cell carcinoma, 199 gastrointestinal stromal tumour, 166t paraganglioma, 166t parathyroid, 77f salivary gland, 144c Bare oval nuclei, 132f, 134t, 136, 144, 147, 147f Basal cell adenocarcinoma (BCAC) ancillary testing, 137 benign vs. malignant, 124, 125c, 127, 146 cytomorphology, 133t salivary gland aspirates, 125c SUMP, 126 Basal cell adenoma (BCA) ancillary testing, 139 basaloid cells, 133t, 144c, 144, 148 benign vs. malignant, 127

Index salivary gland aspirates, 125c stromal features, 134t SUMP, 126 Basal cell carcinoma (BCC), 133t Basal cell neoplasm (BCN) ancillary tests, 137–139 basaloid neoplasms, 131, 137, 139, 143, 144c cytomorphology, 135f nuclear palisading, 24 salivary gland, challenging areas, 144, 148, 149, 152t, 152 stromal features, 134t SUMP, 126 Basaloid cells cytomorphology, 131 pilomatrixoma, 109 Basaloid neoplasms ancillary tests, 137–139 approach, 139, 143–144, 144c background cells, 136 cytomorphology, 132f, 135f DDx, 133, 133t stromal features, 134t, 135 SUMP, 126, 127 Basaloid squamous cell carcinoma (BSCC), 133t BCL10, 190, 199 Benign follicular nodule (BFN) architectural features, 53, 53c, 54, 55f cellularity, 52 colloid in, 51 DDx AUS/FLUS, 56 Hurthle cell-rich lesions, 68, 69c molecular alterations, 46 nuclear morphology, 58t, 59f, 65 peripheral ‘fireflares’, 61f specific diagnoses, 45 thyroid, main entities, 42c β-HCG, 93t Bethesda System for Reporting Thyroid Cytopathology (TBSRTC) AUS/FLUS, 70–71 colloid only, 51 cellularity, 52, 73 diagnostic categories, 44t, 44–46 FVPTC, 47 Hurthle cell-rich lesions, 69, 69c imaging, 50 major classification systems in cytology, 5, 43 NIFTP, 43, 47 thyroid entities that are not readily diagnosed, 47t Bile hepatocytes, 24, 99 bile-stained effusion(s), 214t Blebs, 24 Boerhaave syndrome, 213, 227, 228 Branchial cleft cyst lymph node cytology, 84, 87, 89, 90, 95c, 100 gross aspirate appearance, 90 salivary gland cytology, 141, 143 BRCA1, 180, 232

Index BRCA2, 180 Breast carcinoma cytomorphology, 91t–94t, 100, 184 effusions, 211, 216c, 218f, 219–220, 221f, 234 effusions, sample case, 229–230 IHC, 91t–93t, 231 lobular carcinoma (see “Lobular carcinoma, breast”) metastases to lymph nodes, 88–91, 91t–93t metastases to thyroid, 66 metastatic to pancreas, 200 signet ring appearance, 223t, 224f single file architecture, 23f, 24 triple approach, 29 British Thyroid Association (BTA), 5, 44t Brunner gland adenoma/hyperplasia, 173–174, 174f Burkitt lymphoma (BL) age, 87 cytomorphology, 94t, 106f effusions, 220 lymphoma diagnosis, 85t C CA19-9, 181, 193, 230 Calcification(s) ovarian carcinoma, 222 pancreatitis, 182t, 187, 195 pilomatrixoma FNA, 109 serous cystadenoma, 191t, 192c, 196 solid pseudopapillary neoplasm, 191t, 192c thyroid imaging, 50, 51 Calcitonin, 46, 47t, 65t Calretinin adrenocortical carcinoma, 91t mesothelial cells, 216c, 224f, 230, 232, 239 CAM5.2 DDx epithelioid gastrointestinal stromal tumour, 171 lymph node cytology, 92t neuroendocrine tumours, 171 small cell carcinoma, 93t Carbimazole, 50, 71, 75 Carbonic anhydrase IX, 191t Carcinoma ex pleomorphic adenoma (CEPA), 127–129, 151, 152t Carney triad, 162 Caveat(s) cytology report, 14 effusion cytology, 230, 239 EUS FNA, 170 lymph node cytology, 110 lymph node, reactive, 98 CD10 endometriosis, 237f, 239 renal cell carcinoma, 91t, 223, 235 solid-pseudopapillary neoplasm, 191t, 199 CD15, 230 CD30, 93t, 117 CD56

247 neuroendocrine tumours, 91t, 190 small cell carcinoma, 93t, 216c, 235 CD117 adenoid cystic carcinoma, 138 gastrointestinal stromal tumour, 164, 166t, 169t, 171 germinoma, 92t CD138 plasma cells, 92t autoimmune pancreatitis, 195–196 CD146, 216t, 232t, 233 CD163, 118, 235 CDKN2A, 232t, 233 CDKN2A (p16), 196t CDX2, 92t, 230 CEA lymphoepithelial cyst, 193 medullary thyroid carcinoma, 47t, 65t pancreatic cyst fluid analysis, 28t, 160, 179, 189–190, 191t, 192c, 193 serum tumour markers, 181, 230 Cell-in-cell, 217t, 239 Centroblast(s), 98, 104, 110, 114 Centrocyte(s), 104, 108 Chain(s) lobular carcinoma, 220, 221f, 222, 234 mesothelioma, 222 small cell carcinoma, 220, 221f, 235 Charcot–Leyden crystals, 226 Chief cell(s) cytomorphology, 163, 163f potential false positive, 178 Cholesterol crystal(s) cystic thyroid nodule, 68 longstanding effusions, 228 pseudochylous effusion, 214t, 228 rheumatoid pleuritis, 225 triple approach, smear background, 25f Choriocarcinoma, 93t Chromogranin neuroendocrine tumours, 91t, 169t, 171, 173, 190 pancreatic acinar cell carcinoma, 190, 199 small cell carcinoma, 93t Chronic sialadenitis, 136 Chylous effusion, 214t, 215 Cirrhosis, 206, 207, 212, 237f CK5/6 mesothelial cells, 216c, 230 squamous cell carcinoma, 92t, 93t CK7 colorectal carcinoma, 92t effusions, 216, 230–231 gastric carcinoma, 92t lung adenocarcinoma, 93t CK20 colorectal carcinoma, 92t effusions, 216, 231 gastric carcinoma, 92t lung adenocarcinoma, 93t

Index

248 Classical Hodgkin lymphoma (CHL) age, 87 ancillary testing, 28t, 101t, 102 background eosinophils, 26 cytologic features, 85t, 99, 114 effusions, 220 granulomas in, 104, 105 morphology, 93t tissue fragments, 95c, 105 Clear cell(s) clear cell carcinomas, 91t clear cell sarcoma, 25 DDx macrophages, 217t Mullerian clear cell carcinoma, 216c, 223, 225f renal cell carcinoma, 30, 32, 200, 223, 235 salivary gland neoplasms, 126, 127, 131c, 133t, 135, 146–148, 153 Clear cell carcinoma DDx, 30, 91t Mullerian clear cell carcinoma, 216c, 223, 225f ovary, 225f salivary gland neoplasms, 126 Clusterin B, 196t Collagen balls, 217t Collagen core(s), 217t, 222 Collagen vascular disease, 214t Colloid AUS/FLUS, 91t benign thyroid nodule, 23, 45, 67c, 68 colloid-like mucin, 175c, 188f, 188, 192c crystals, 25 cystic thyroid nodule, 44t cytomorphology, 52f, 54, 56, 62–63 DeQuervain thyroiditis, 64 follicular neoplasm, 45, 73 gross appearance, 6, 17, 21, 51 Hurthle cell-rich lesions, 68–69 lymphoid-rich lesions, 70c metastatic papillary thyroid carcinoma, 91t non-diagnostic nodule, 44 parathyroid lesions, 77 Colloid nodule(s) architecture, 53, 54f the benign cytology report, 13 benign thyroid nodules, 42c, 45 cellularity, 22 cystic nodules, 67c fresh vs. old blood, 26 Hurthle-cell rich lesions, 68 picture using smartphone, 8f Colorectal carcinoma cytologic features, 24, 92t, 105f, 200–201 sample cytology report, 16 Colorectum, 161t, 162 Comment(s) the cytology report, 11–17, 21, 30–31, 48, 49 gastrointestinal stromal tumour, 165, 170 Hurthle cell-rich thyroid lesions, 69 Hashimoto thyroiditis, 74 FVPTC/NIFTP, 78

lymph node cytology, 84, 85, 114 lymphocytic effusion, 227 the provisional report, 36, 37 salivary gland cytology, 142, 143, 148, 149 sample in report, 32, 83, 143, 144, 148, 170 thyroid lymphoma, 69 Congestive cardiac failure, 207, 207c, 212 Contaminant DDx pancreatic neoplastic mucinous cysts, 177t, 183c, 189, 190, 193, 198, 201 gastrointestinal epithelium, 22, 159, 163, 163f, 164, 164f, 177t, 177–179, 183, 190, 192c, 197, 198 (see also “Gastric epithelium/ contaminant”;“Duodenal epithelium/ contaminant”) gastrointestinal mucin, 179, 182, 187–188, 198 pancreatic parenchyma, 159, 178, 178f, 179, 184, 199 subcutaneous fat, 89 Crescentic histiocyte(s), 111, 112f Cyclin D1, 191t, 199 Cystadenoma ovarian cystadenoma, 222 pancreas serous cystadenoma (see “Serous cystadenoma”) salivary gland, 131c, 133t, 140c, 145c Cyst fluid crystals in, 25 EUS FNA specimen triage, 160, 177t gross appearance, 30 pancreatic cyst fluid analysis, 20, 28t, 179, 191t, 190–192, 192c, 201 pancreatic FNA gross evaluation, 184 pancreatic serous cystadenoma, 196 salivary gland cystic lesions, 136 specimen triage, 35 thyroid, aspiration of, 47 thyroid, cytologic features, 68 thyroid, gross appearance, 68 Cyst lining cells thyroid cytology, 57, 68, 72f, 75 Cystic lesions cystic papillary thyroid carcinoma, 73 lymph node cytology, 89, 100 needle gauge, aspiration, 86 pancreas, 21, 159, 160, 175, 175c, 176t, 177t, 179, 190–192, 196, 201 salivary gland cytology, 126, 130, 131c, 136, 137, 139–140, 140c, 149 thyroid, cystic lesions, 44t, 67–68 D D240, 230 Daisy cells, 208 DeQuervain thyroiditis (DQT), 49, 50, 64, 78 Desmin inflammatory myofibroblastic tumour, 173 leiomyoma, 164, 169t mesothelial cells, 216c, 232t, 233, 235

Index Diagnostic certainty the cytology report, 14–15 pancreatic ductal adenocarcinoma, 199 thyroid cytology, 46 Dialysis, 206, 213, 226, 236–237 Diffuse large B-cell lymphoma (DLBCL) cytomorphology, 94t, 100, 103, 116f effusions, 220, 224 lymph node cytology approach, 85t, 86, 95c thyroid lymphoid-rich lesions, 69 tissue fragments, 105 DOG1 gastrointestinal stromal tumour, 166t, 167f–168f, 169t, 171 leiomyoma, 164 salivary gland neoplasms, 138, 138t, 148 DPAS acinar cell carcinoma, 190, 199 clear cell lesions, 91t gastric adenocarcinoma, 223t secretory carcinoma salivary gland, 148 serous cystadenoma pancreas, 191t, 197, 198f Duodenal epithelium/contaminant normal duodenum, 164, 164f E Electronic medical records (EMR), 12, 20 EMA anaplastic large cell lymphoma, 117 mesothelioma, 216c, 219f, 231–232, 232t, 235 Embryonal carcinoma, 93t Empyema, 214t, 227 Endobronchial ultrasound (EBUS) provisional report, 36 specimen adequacy, 29, 33, 83 specimen triage, 26 Endometriosis, 214t, 216c, 222, 237f, 238, 239 Endosalpingiosis, 222, 229, 238 Endoscopic ultrasound (EUS) needle route, 159, 160 sites of EUS FNA, 158 specimen adequacy, 22, 34, 160 specimen triage, 26, 160, 165, 169 Eosinophilic effusion(s), 207c, 212, 226, 238 Eosinophils accessory spleen, 199, 200f autoimmune pancreatitis, 195 background cells, 26 Hodgkin lymphoma, 94t, 115 T-cell lymphoma, 95t,116 Epidermal cyst, 90 Epithelial-myoepithelial carcinoma (EMC) basaloid salivary gland neoplasms, 134t, 144c cytomorphology, 136, 146–148 salivary gland cytology approach, 125c, 127, 133t SUMP, 126 ETV6–NTRK3 gene fusion, 138t, 139 Ewing sarcoma effusion cytology, 211, 212, 223

249 EWSR1–FLI1, 224 Exploding mitoses, 238 Exudative effusions, 207, 207c, 212, 227 F Familial cancer, 21, 180 Familial GIST, see “Gastrointestinal stromal tumour, familial” Familial medullary thyroid carcinoma, 50 Fibroma, 212, 213 Fibrothecoma, 212, 213 Fireflares, 60, 61f Fistula, 207 Flame cells, 60, 61f FLI-1, 224 Flow cytometry cell block vs., 179 effusion cytology, 207c, 210, 227 lymph node cytology, 85, 86, 101t, 103, 113, 137, 143, 161, 179, 190, 192 relevant clinical findings, 20 specimen triage, 27, 34–35 suspected lymphoma, 28t thyroid cytology, 44t, 47t, 48, 65, 66, 70, 70c Follicular adenoma (FA) architecture, 53c, 56 papillary structures, 75 thyroid cytology approach, 42c what cytology can’t do, 4, 42, 46, 47t Follicular carcinoma architecture, 53c, 56 metastatic, 91t molecular aberrations, 46 thyroid cytology approach, 42c what cytology can’t do, 4, 47t Follicular dendritic cell (FDC), 97f, 99, 104, 108 Follicular dendritic cell sarcoma, 99 Follicular lesion of undetermined significance (FLUS) adenomatoid nodule, 47t cytologic criteria, 45, 56, 70–71, 71c, 78 FVPTC/NIFTP, 47 gollicular adenoma, 47t Hurthle cell-rich lesions, 69, 69c hyalinising trabecular tumour, 47t Follicular lymphoma (FL) cytomorphology, 98f, 109 effusion cytology, 219 false negative diagnosis, 113–116 lymph node cytology approach, 95c lymphoid aggregates, 96, 105 spindle appearance, 99 what cytology can’t do, 85t Follicular neoplasm (FN, SFN) architectural patterns, 53, 53c, 55, 55f, 65 cellularity, 52, 73 cytomorphology, 55, 55f, 56, 58t DDx suspicious for malignancy, 46 description, 44t, 45, 56 FVPTC, 47, 71, 78

Index

250 Follicular neoplasm (FN, SFN)  (cont.) Hashimoto thyroiditis  74 Hurthle cell-rich lesions, 69c indeterminate nodules, 47t NIFTP, 46, 47, 71, 73, 75, 78 poorly differentiated thyroid carcinoma, 73 risk of malignancy, 45 Follicular variant papillary thyroid carcinoma (FVPTC) architecture, 53c, 57 Bethesda categories, 45, 47, 70, 78 cytomorphology, 59f relation to NIFTP, 43 tips and clues,75 what cytology can’t do, 47t Food, 227 Foregut cyst, 177t ‘Fresh’ blood, 26, 63 Fukuoka criteria, 194 G Ganglioneuroblastoma, 104, 105 Gastric epithelium/contaminant DDx neoplastic mucinous cysts of pancreas, 164, 190, 197, 198, 201 gastric foveolar epithelium, 159, 163, 163f, 179, 183, 192c honeycomb sheets, 183 needle route, 198, 201 parietal and chief cells, 163f, 177 Gastric wall contaminant smooth muscle, 159, 164f Gastrointestinal contamination (GIT), see “Contaminant, gastrointestinal epithelium” Gastrointestinal stromal tumour (GIST) ancillary tests, 169t cytomorphology, 163–165, 166t, 167f endoscopic findings, 162 epithelioid, 158c, 166t, 167f-168f, 169t, 171 EUS FNA, gastrointestinal tract, main entities, 158c, 174 familial, 162 imaging, 162 location in gastrointestinal tract, 161, 161t risk stratification, 165, 170 specimen triage, 160 GATA3 breast carcinoma, 91t–93t, 216c, 230, 231, 234 effusion cytology, 230 lymphocytes, 231 paraganglioma, 169t, 173 parathyroid lesion, 65t, 77 salivary gland neoplasms, 138t, 138 secretory carcinoma, salivary gland, 148 urothelial carcinoma, 92t GCDFP15, 93, 148 Gene expression classifier (GEC), 44t, 45 Germinal centre(s) accessory spleen, 200 centroblasts in, 98

cytomorphology, 96, 96f, 97f, 99–100, 103, 104 marginal zone lymphoma, 113 nodular lymphocyte predominant Hodgkin lymphoma, 115 progressive transformation of, 82c See also “Lymphohistiocytic aggregates” Germinoma ancillary testing, 92t cytomorphology, 92t granulomas in, 104 tigroid background, 25f, 100 Ghost cell(s), 112, 112f Glomus tumour, 158c, 166t, 169t GLUT1, 191t, 216c, 232t, 233 Glycogen germinomas, 25f, 92t, 100 mesothelial cells, 217t serous cystadenoma, pancreas, 191t, 196, 197 tigroid background, 6, 25, 25f, 100 Glypican 3, 93t GNAS, 179, 189 Goblet cells, 136, 137, 150f, 164 Granular cell tumour, 161t, 162, 169t Granulation tissue acute pancreatitis, 186 cytomorphology, 102f, 106 lymph node cytology, pattern-based approach, 95c Granuloma(s) clinical context, 12, 20 cytomorphology, 37f, 99, 104, 118f–119f germinomas, 92t Hodgkin lymphoma, 94t, 115 lymph node cytology, 82c, 84, 95c nasopharyngeal carcinoma, 104, 118 necrosis with, 26, 100 sample case provisional report, 36, 37f squamous cell carcinoma, 104 Granulomatous thyroiditis, 42c, 45, 49, 64 Graves’ disease cytoplasm, 62 family history, 50 nuclear atypia, 57, 58t, 74–75 physical examination, 50 triple approach, 21 Grocott methenamine silver (GMS), 28t, 101t H Haemopneumothorax, 213, 226 Haemosiderin paravacuolar granules, 60 macrophages, 64, 223 Hallmark cells, 95t, 117 Hashimoto thyroiditis (HT) architecture, 53c, 56 clinical findings, 49–51, 70 cytomorphology, 57, 58t, 61f, 65 false-positive diagnosis, 74 flame cells/’fireflares’, 60 Hurthle cell-rich lesions, 68, 69, 69c

Index lymphoid-rich thyroid lesions, 63f, 64, 69, 70c, 71c, 72f multinucleated giant cells, 64 nuclearpseudoinclusions, 59 HBME1, 231 H-caldesmon, 169t Hepatocellular carcinoma clear cell morphology, 30 endothelial wrapping, 23f intracytoplasmic bile, 99 trabeculae, 23f, 24 HER2, 138t, 230, 234 High power field (HPF) autoimmune pancreatitis, 195 follicular lymphoma grading, 114 neuroendocrine tumour grading, 190 Hilum (lymph node), 89, 90 Histiocyte, crescentic, 110, 111, 112f HNF-1β, 91t HPV-related squamous cell carcinoma, see “Oropharyngeal squamous cell carcinoma” Hodgkin lymphoma (HL) classical (see “Classical Hodgkin lymphoma”) Hodgkin lymphoma, nodular lymphocyte predominant, 85t, 94t, 99, 117 Hodgkin lymphoma, nodular sclerosing subtype (NSHL), 23, 95, 96 Honeycomb chronic pancreatitis, 194 drunken honeycomb, 183, 185f, 186 FLUS diagnosis, 56, 71, 71c gastrointestinal epithelium, 163, 163f, 164, 164f, 183 histo-cytological parallels, 6 Hurthle cell-rich lesions, 68 hyperplastic nodules, 75 morphological features, 53, 54f neoplastic mucinous cysts of pancreas, 186 pancreatic ductal cells, 183, 185f syncytial sheets vs., 53–54 thyroid cytology, architectural patterns, 53c Human immunodeficiency virus (HIV), 88, 220 Hurthle cell(s) cytomorphology, 58t, 59, 61f DDx hyperfunctional follicular cells, 62 DDx medullary thyroid carcinoma, 62 lymphoid-rich lesions, 69 Hurthle cell adenoma cytomorphology, 61f Hurthle cell-rich lesions, 68 IHC, 65t microfollicular/trabecular architecture, 53c thyroid, main diagnostic entities, 42c what cytology can’t do, 46, 47t Hurthle cell carcinoma (HCC) Hurthle cell-rich lesions, 68 IHC, 65t microfollicular/trabecular architecture, 53c Hurthle cell lesions clinical correlation, 78

251 DDx medullary thyroid carcinoma, 62, 65 features favouring neoplasm, 57 Hurthle cell-rich lesions, 67, 68, 69, 69c IHC, 65t microfollicular/trabecular architecture, 53c mimics, 65, 76 thyroid, main diagnostic entities, 42c what cytology can’t do, 46, 47t Hurthle cell neoplasm (HCN) cytological diagnosis, 46 cytological features favouring, 57, 58t general morphological approach, 4 mimics, 65, 74 Hyaline globules, 91t, 223, 225f Hyalinising trabecular tumour (HTT) DDx Hurthle cell lesion, 68 DDx medullary thyroid carcinoma, 65, 76 DDx papillary thyroid carcinoma, 76 IHC, 48, 65t indolent thyroid neoplasms, 47t nuclearpseudoinclusions, 59, 60f thyroid, main diagnostic entities, 42c Hyaluronic acid (hyaluronan), 214t, 217t, 223t, 224f, 228 Hyperechoic chronic pancreatitis, 182t endoscopic ultrasound of gut wall, 162 gastrointestinallipoma, 162 thyroid imaging, 51 Hyperplastic nodule benign follicular nodule, 45 Hashimoto thyroiditis, 50 Hurthle cell-rich nodule, 68, 69, 76 papillary structures, 55, 75 thyroid cytology architectural patterns, 53c thyroid, main diagnostic entities, 42c Hypoechoic endoscopic ultrasound of gut wall, 162 EUS gastrointestinal wall lesions, 161, 162 EUS of pancreatic lesions, 182t lymph node imaging, 89 pancreatic heterotopia, 173 thyroid imaging, 50 I I131, 71, 75 IFN-γ, 211 IgG4-related disease pancreas, 175c, 177t, 187, 195, 196 salivary gland, 130 thyroid (Riedel’s thyroiditis), 42c, 50, 64 Immunoblast(s) angioimmunoblastic T-cell lymphoma, 94t cytomorphology, 97 DDx nodular lymphocyte predominant Hodgkin lymphoma, 115 infectious mononucleosis, 94t Kikuchi-Fujimoto disease, 110 Immunocompromised state, 21, 88, 212

252 IMP3, 232t, 233 Infectious mononucleosis syndrome (IMS), 94t, 110 Inflammatory myofibroblastic tumour (IMT) EUS FNA, 158c, 174 Intraductal papillary mucinous neoplasm (IPMN) cytologic diagnosis of pancreatic lesions, 177t cytomorphology, 185, 187f–189f, 191t, 194 DDx Gastric foveolar cells, 179 EUS FNA pancreatic lesions, 175c gross evaluation, 21 pancreas solid vs. cystic lesions, 183c pancreatic cystic lesions approach, 179, 191t, 180–182, 188–189, 190–192, 192c Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology, 175c, 176t Intraductal tubulopapillary neoplasm (ITPN), 175c Intraperitoneal chemotherapy (IPCHT), 219, 236f, 238 K Ki-67 hyalinising trabecular tumour, 47t, 65t, 76 neuroendocrine tumours, 168, 170, 190 Kikuchi-Fujimoto disease (KFD, histiocytic necrotisinglymphadenitis) benign nodal disease, 84 cytomorphology, 110–111, 112f lymph nodes, main entities, 82c KIT mutation, 162, 168, 169t KRAS cytologic material testing, 26 pancreatic cystic lesions, 28t, 179, 190, 191t, 192, 192c L LDH, 207, 211 LE cells, 226, 226f LEF1, 137 Leiomyoma (LM) cytomorphology, 164, 166t, 167f DDx native gut wall, 159, 164 EUS FNA gastrointestinal tract, 158c, 161, 161t imaging, 162 location in gastrointestinal tract, 161t Leiomyosarcomas (LMS), 161 Light’s criteria, 207 Lipase, 180, 190 Lipoma(s) DDx lymph node FNA, 82c, 84, 89, 90, 105, 109 DDx salivary gland FNA, 126, 129 DDx subcutaneous fat, 15–16 DDx Thyroid FNA, 50 EUS FNA Gastrointestinal tract, 158c, 162, 175c, 176t EUS imaging, 162 gross aspirated material, 90 Liquid-based cytology (LBC), 33, 44–45, 87, 211

Index Liver failure, 212 Lobular carcinoma, breast chains, 220, 221f, 222 cytomorphology, 92t, 93t dispersed cell architecture, 95c, 106, 220, 234 IHC, 231 signet ring appearance/intracytoplasmiclumina, 223t, 224f, 234 Loss of heterozygosity (LOH), 191t, 196 Low-grade mucoepidermoid carcinoma (LGMEC), see “Mucoepidermoid carcinoma, low-grade” Lymph node, shape matted, 88, 89 round, 88, 89 Lymphangioma age, 87 cystic salivary gland lesions, 140c lymphoid-rich salivary gland lesions, 137, 141c, 142f pancreatic lesions, 175c, 176t salivary gland cytology, 126, 131c Lymphoblastic lymphoma (LBL) age, 87 cytologic diagnosis, 85t cytomorphology, 94t, 104 effusion cytology, 220 Lymphocyte, small classical Hodgkin lymphoma, 115 follicular lymphoma, low grade, 114 infectious mononucleosis, 110 low grade B-cell lymphomas, 113–115 nodular lymphocyte predominant Hodgkin lymphoma, 115 predominantly small lymphocytes, 96f, 110 size comparison, 93t–95t, 99, 104 small cell carcinoma, 117 small cell melanoma, 117 Lymphocytic effusion, 216, 227 Lymphoepithelial cyst CEA levels in cyst fluid, 193 cystic salivary gland aspirates, 139–140, 140c dispersed cells, 106 false-negative salivary gland diagnosis, 152t gross aspirate appearance, 181 lymph node cytology, 90 lymph node FNA, pattern-based approach, 95c lymphoid-rich salivary gland lesions, 137, 141c pancreatic lesions, 175c, 176t, 177t, 183c Lymphoepithelioma-like carcinoma (LELC)/ Lymphoepithelial carcinoma cytomorphology, 23f, 118f–119f DDx granulomatous inflammation, 118f–119f lymphoid-rich salivary gland lesions, 141c, 143 malignant salivary gland neoplasms, 127 salivary gland cytology, clinicopathological approach, 131c salivary gland cytology, morphological approach, 133t syncytial architecture, 24

Index See also “Nasopharyngeal carcinoma”;“Undifferentiated carcinoma” Lymphoglandular bodies (LGBs) anaplastic large cell lymphoma, 117 background material in smears, 25 DDxPoorly differentiated carcinoma, 116–117 effusion cytology, 220 lymphoid or not?, 95c morphology, 100, 104 Lymphohistiocytic (LH) aggregates accessory spleen, 199–200 cell sizes, 97, 104 morphology, 94t–95t, 103 tissue fragments in lymph node aspirates, 95c, 97f, 105 See also “Germinal centre” Lymphoma, low grade cytologic diagnosis of, 85t, 113–116 lymph node cytology, caveat, 84 mixed cell population, 98 salivary gland cytology, 142, 143, 151 specimen triage, thyroid cytology, 48, 66 thyroid AUS, 47t thyroid low-grade lymphoma, cytologic diagnosis, 73 M MAML2–CRTC1 gene fusion, 138t Mammaglobin, 93t, 139 Mammary analogue secretory carcinoma, see “Secretory carcinoma” Mantle cell lymphoma, 85t, 93t, 220 Marginal vacuoles, 60 Marginal zone lymphoma (MZL) cell size variation, 98, 108, 113–114 DDx reactive lymph node, 84, 85t, 113–114, 114f lymph node cytology approach, 95c Maspin, 195 Matrix (stroma) chondromyxoid, 134t, 147, 152 myoepithelial neoplasms, 153 nodular fasciitis, 153f pleomorphic adenoma, 134t, 151, 153f salivary gland neoplasms, 131c, 133t, 134t, 134–135, 144c, 151, 152t schwannoma, 152, 169t Matted lymph nodes, 88, 89 Medullary thyroid carcinoma (MTC) amyloid, 62, 63f ancillary tests, 48, 65 architectural patterns, 53c, 56f, 57 cytomorphologic features, 57, 59f, 61f, 62 DDx hyalinising trabecular tumour, 76 diagnosis of, 42, 46, 47t family history, 50 Hurthle cell-rich lesions, 68, 69, 69c nuclear features, 58t, 59f, 60f

253 Medulloblastoma, 24 Meigs syndrome, 207c, 212, 213 Melan A, 91t, 92t Melanin, 92t, 99, 107f, 217t, 220 triple approach, 24 Melanoma architecture, 95c, 107 cytomorphology, 97, 99, 107, 107f effusions, 207, 207c, 213, 214t, 215c, 220, 221f, 225f metastatic to pancreas, 200 plasmacytoid cells, 92t, 184 small cell, 92t, 117, 118, 118f–119f SOX10, 92t Melanophage(s), 118f MEN 2, 50 Mesothelial cells DDx breast carcinoma, 234 peritoneal washings, 209f reactive atypia, 236–238, 236f, 237f reactive vs. malignant, 216c, 216–219, 231–234 reactive, 24, 209f, 217t Mesothelin, 196t, 230 Mesothelioma vs. adenocarcinoma, 239 atypical epithelioid cells approach, 216c cytomorphology, 216–219, 218f, 219f, 220, 221f, 229 effusion cytology diagnoses, 207c, 216c gross fluid appearance, 214t imaging, 213 occupational history, 213 papillary clusters, 222 vs. reactive/benign mesothelial cells, 216c, 216–219, 231–234 sarcomatoid, 216, 224, 231–233, 232t ‘signet ring cell’ appearance, 224f Metachromatic globules, 147, 148 Metaplasia mucinous, salivary gland, 109, 126, 131c, 136 141, 149, 151, 152t squamous, in chronic pancreatitis, 194 squamous, salivary gland, 109, 124, 126, 127, 141, 149, 151, 152t Metastases to pancreas, 200 Metastases to thyroid, 66 Metastatic papillary thyroid carcinoma, 90, 100 See also “Papillary carcinoma, cystic” Metastatic squamous cell carcinoma (SCC), 90, 93t, 143, 239, 240f Methythioadenosine phosphorylase (MTAP), 216c, 232, 232t, 233, 235 Microcalcifications, 50 Microfollicles, 55–56, 55f, 71, 71c Milan System for Reporting Salivary (MSRSGC), 5, 124, 125t, 126, 137, 139, 145 Mitoses, exploding, 235, 236f MOC31, 230, 231, 234, 239 MUC1, 195, 231 Mucicarmine, 148, 223

Index

254 Mucin background, 25 breast lobular carcinoma, 92t, 221, 223t, 224f, 234 cytoplasmic, in triple approach, 24 duodenal goblet cells, 164 effusions, 217t, 227, 228, 228f gastric carcinoma, 92t, 223t gastric contaminant, 163f, 187, 197 low-grade mucoepidermoid carcinoma, 150f, 152t lymph node cytology, 100 mesothelial cells, 240 pancreatic cystic lesions, 188f, 189f, 187–188, 191t, 192c salivary gland cytology, 136, 138t, 139, 141c secretory carcinoma, 148, 149f viscosity, 182, 188f, 187, 188, 192c Mucinous cystic neoplasm (MCN) cystic lesions approach, 191t, 192c, 190–194 cytologic diagnosis of pancreatic lesions, 177t cytomorphology, 184, 188f, 190 DDx Gastric foveolar cells, 179 DDx macrocystic serous cystadenoma, 197 gross evaluation, 21 pancreas solid vs. cystic lesions, 183c pancreas, main entities, 175c Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology, 176t Mucinous non-neoplastic cyst, 190 Muciphages, 136, 137, 227 Mucocoele, 124, 126, 136 Mucoepidermoid carcinoma (MEC) cystic salivary gland lesions, 131c DDx pilomatrixoma, 109 lymphoid-rich salivary gland neoplasms, 131c, 141c oncocytic/oncocytoid lesions, 126, 145c salivary gland, main entities, 125c Warthin-like mucoepidermoid carcinoma, 150f–151f Mucoepidermoid carcinoma, low grade (LGMEC) ancillary tests, 137, 138t AUS in Milan system, 126 cystic salivary gland lesions, 131c, 140, 140c cytomorphology, 146, 149, 150f DDx duct obstruction, 127 DDx mucocoele, 124, 136 DDx secretory carcinoma, 148, 149 diagnostic errors, 154 false-negative diagnosis, 140, 149, 152t goblet cells vs. macrophages, 136 main cell types in salivary gland neoplasms, 131, 133t Mucosa-associated lymphoid tissue (MALT) lymphoma ancillary testing, 48, 66, 69 lymph node cytology, 113 salivary gland cytology, 131, 142, 142f, 143 thyroid, 47t, 66, 69, 73 Mucus retention, 136, 139 Mullerian clear cell carcinoma, 91t, 225f Multinodular goitre, 69, 69c

Multinucleated histiocytes, 109, 214t, 225 Muscularis propria, 162, 164 MYB, 137, 139 MYB–NFIB fusion, 138t, 139 Mycobacterial infection the cytology report, 12, 20 necrosis, 100 personal protective equipment, 86 physical examination of lymph nodes, 88 purulent aspirate, 90 sample provisional report, 37f Mycobacterium avium complex (MAC), 100, 102f Myeloperoxidase (MPO), 111 Myoepithelial carcinoma (MECA) basaloid salivary gland neoplasms, 144c cytomorphology, 153 main cell types in salivary gland neoplasms, 133t plasmacytoid cells, 92t, 99 salivary gland neoplasms, 125c, 127 stromal features, 134t Myoepithelial neoplasm(s) benign vs. malignant, 127, 139, 153 clear cell neoplasms, 126 cytomorphology, 133t, 134t, 139, 152, 153 DDx pleomorphic adenoma, 151–152 SUMP, 126, 145c Myoepithelioma (ME) basaloid salivary gland neoplasms, 144c cytomorphology, 132f main cell types in salivary gland neoplasms, 133t salivary gland neoplasms, 125c stromal features, 134t Myofibroblasts granulation tissue, 102f, 105 nodular fasciitis, 152 N NANOG, 92, 93 Napsin A lung adenocarcinoma, 91t, 93t, 216c, 222, 230 macrophages, 231 Mullerian carcinoma, 91t Nasopharyngeal carcinoma (NPC). cytomorphology, 23f, 119f DDx granulomatous inflammation, 118, 118f dispersed cells, 118f granulomas in, 104 syncytial sheets, 24, 91t See also “Lymphoepithelioma-like carcinoma” and “Undifferentiated carcinoma” Necrosis acinar cell carcinoma, 182t adrenocortical carcinoma, 91t anaplastic large cell lymphoma, 95t bacterial infections, effusion cytology, 227 coagulative necrosis, 187, 195 colorectal carcinoma, 16, 92t, 105f DDx ultrasound gel, 25, 63 diagnostic workup, 12, 21, 26

Index fat necrosis in chronic pancreatitis, 187, 194–195 gastrointestinal stromal tumour, 161, 165 infectious conditions, 104 Kikuchi-Fujimoto disease, 110, 112 lymph node cytology, 101 lymph node imaging, 89 morphology, 25f neoplastic mucinous cysts, pancreas, 191t neuroendocrine tumours, 91t, 166t poorly differentiated thyroid carcinoma, 53c, 73–74 sample provisional report, 37 smooth muscle tumours, 166t Necrotising lymphadenitis (Kikuchi-Fujimoto disease (KFD), histiocytic necrotising lymphadenitis) benign lymph node, 84 cytomorphology, 110–113, 112f lymph nodes, main entities, 82c Needle gauge, 47, 86 Negative findings, 12, 20, 135 Neoplastic mucinous cyst (NMC) false-positive diagnosis, 198 gross evaluation of cyst fluid, 21 integrated diagnostic approach, 180, 191t, 190–194, 192c morphology, 188, 188f, 189f, 193f Papanicolaou Society guidelines, pancreas, 176t Neuroendocrine carcinoma (NEC) pancreatic lesions, 175c Papanicolaou Society guidelines, pancreas, 176t Neuroendocrine tumour (NET) ancillary tests, 169t, 179, 189 clinical information, 180 cytologic diagnosis of, 177t cytomorphology, 91t, 172f, 184–185 DDx accessory spleen, imaging, 199–200 DDx epithelioid GIST, 165, 166t, 171 DDx pancreatic acinar cells, 159, 178, 178f, 198 DDx pancreatic heterotopia, 173 DDx paraganglioma, 166t, 169t, 171–173 EUS FNA gastrointestinal tract, 158c EUS FNA pancreas, 175c false positive diagnosis, 194, 196, 197 Papanicolaou Society guidelines, pancreas, 176t prognosis/grading, 165, 168 solid vs. cystic pancreatic lesions, 183c Neurofibromatosis, 162 9p21, 195, 231–234 NKX3.1, 91, 230 Nodular fasciitis (NF) salivary gland, main diagnostic entities, 125c Milan system, 126 DDx pleomorphic adenoma, 151–153 DDx salivary gland lesions, 129 cytomorphology, 132f, 153, 153f spindle cells, 132f, 133t stroma, 135, 152 Nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL), 85t, 94t, 99, 115 Non-diagnostic (ND)

255 cellular non-diagnostic smear, 22, 22f, 34, 174 the cytology report, 13, 15 EUS FNA, 158, 174, 176t, 194 lymph node cytology, 83 the provisional report, 36 salivary gland cytology, 124, 125t, 126, 131c, 139 thyroid cytology, 44, 44t, 67, 68, 73 Non-Hodgkin lymphoma (NHL) cytomorphology, 98 DDx Kikuchi-Fujimoto disease, 110–113 specimen triage/ancillary testing, 28t, 48, 66, 84, 101t Non-invasive follicular thyroid neoplasm with papillary-­like nuclear features (NIFTP) architectural patterns, 53c AUS/FLUS Bethesda category, 45 diagnosis on cytology, 46, 47, 47t, 75 diagnostic criteria, 43 follicular neoplasm (FN) Bethesda category, 45 malignant Bethesda category, 46 nuclear atypia, 71 suspicious for malignancy (SM) Bethesda category, 45 thyroid classification systems, 44t thyroid cytology main entities, 42c NPM, 117 Nuclear moulding, 24, 93t, 118f, 221, 221f, 222, 222f, 235 Nuclear palisading, 16, 24, 166t O OCT 3/4, 92t, 93t Oesophagus, 161–162, 166t Oestrogen receptor, 216 ‘Old’ blood, 26, 63, 64 Oncocyte(s), 59, 110, 153f Oncocytic cells. epithelial-myoepithelial carcinoma, 146 mucoepidermoid carcinoma, Warthin-like variant, 149, 150f parathyroid cells, 76 salivary gland cytology, 132f, 133t, 134, 137, 139, 143, 144–146, 145c secretory carcinoma, 148–149 thyroid cytology, 58t, 68, 74 Warthin tumour, 109, 146f See also “Hurthle cell(s)” Oncocytic carcinoma, 125c, 126, 133t, 145, 145c Oncocytic/oncocytoid lesions, salivary gland cytomorphology, 132f, 134, 144–146 differential diagnoses, 133t, 145c major cell types of salivary gland neoplasms, 126–127 myoepithelial neoplasms, 153–154 secretory carcinoma, 148–149 Oncocytoma benign salivary gland neoplasms, 126 diagnosis on cytology, 128t main salivary gland neoplasms, 125c oncocytic/oncocytoid lesions, 133t, 145, 145c, 146f

Index

256 Oncocytosis, 126, 133t, 145, 145c Oncotic cells, 188f, 188 Oropharyngeal squamous cell carcinoma (HPV-related SCC, HPV-related squamous cell carcinoma), 89, 90, 92t, 93t, 100, 110, 140, 141c Ovarian carcinoma, 181, 216c, 221f, 222, 225f P p40, 92, 93, 230, 239 p53, 196t, 232t, 233 p63, 92, 93, 122, 230, 239 Pancreatic acinar cells, 159, 178f, 178–179, 184, 185, 198, 199 Pancreatic ductal adenocarcinoma (PDAC) CA19-9, 181 common pancreatic entities, 175c cytomorphology, 184–185 DDx autoimmune pancreatitis, 195–196 DDx chronic pancreatitis, 185f, 188, 194–195, 196t DDx metastatic malignancy, 200 drunken honeycomb sheets, 183, 185f false-negative diagnosis, 198–199 imaging, 182t new onset diabetes, 180 Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology, 176t solid pancreatic lesions, 177t, 182 Pancreatic ductal cells, 173, 178f, 183, 194, 195, 199 Pancreatic heterotopia, 173 Pancreatitis acute, 186–187, 212, 214t, 237 autoimmune (see “Autoimmune pancreatitis”) chronic, 175c, 176t, 177t, 183c, 185f, 187, 192c, 194–195, 196t effusions, 207c, 213, 214t, 237 Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology, 176t Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology (PSGPBC), 176t Papillary structures benign thyroid lesions, 75 cytomorphology, 24, 54–55, 55f DDx granulation tissue, 105 effusions, 213, 214t, 217t, 222f, 223–226 Graves’ disease, 75 lymph node cytology, 91t, 105 NIFTP, 75 pancreatic lesions, 184, 193 secretory carcinoma, 148–149 thyroid cytology, 52, 53c, 64 Papillary thyroid carcinoma (PTC) ancillary tests, 65t, 66 AUS/FLUS, 71, 71c, 78 colloid, in, 62 cystic, 60–62, 61f, 67–68, 73 cytologic diagnosis, 46, 52

cytomorphology, 53–54, 53c, 54f, 55, 55f, 56f, 57, 58t, 59, 59f, 60f, 65 DDx hyalinising trabecular tumour, 76 enlarged lymph nodes, 50 False positive diagnosis, 74, 75 follicular variant, 43, 45, 47, 47t, 53c, 57, 59f, 71c, 75 Hurthle cell-rich lesions, 68, 69, 69c multinucleated giant cells, 64 Warthin-like, 64 Paraganglioma, 162, 171–173, 172f, 231 Paraimmunoblast(s), 93t Parathyroid, 48, 65, 67, 76, 77 Parathyroid cyst, 51, 68 Paravacuolar granules, 60, 61f Parietal cell(s), 163f, 177 PAS pancreatic acinar cell carcinoma, 187f, 190 pancreatic serous cystadenoma, 191t, 200, 198f renal cell carcinoma, 91t PAX8 effusions, 216c, 230, 235, 239 endometriosis, 239 Mullerian carcinoma, 91t, 216c renal cell carcinoma, 91t, 223, 235 sample case, 30, 32f thyroid, 66, 91t PCR, 26, 37 PDGFRA mutation, 168, 169t Peripheral T-cell lymphoma (PTCL) clinical information, 87 cytologic diagnosis, 85t cytomorphology, 95t, 104, 113, 115–116 effusions, 221f Pigment bile, 99 melanin, 92t, 99, 107f, 217t, 220, 225f melanoma, 214t, 225f melanophages, 220 triple approach, 24 Pilomatrixoma (PMX) clinical features, 109 the cytology report, 15 cytomorphology, 109, 112f DDx metastatic carcinoma, 82, 82c, 109 lymph node cytology, 107, 108 salivary gland cytology, 125c, 129, 133t, 144, 144c, 152 PLAG1, 137 PLAP, 92t, 93t Plasmacytoid basaloid salivary gland lesions, 131, 132f, 133t, 134t, 144c breast carcinoma, 91t, 93t DDx plasmacytoid cells, 99, 107f epithelial-myoepithelial carcinoma, 147 infectious mononucleosis, 110 Kikuchi-Fujimoto disease, 110–113 medullary thyroid carcinoma, 57, 58t melanoma, 92t, 117, 220

Index myoepithelial neoplasms, 152 neuroendocrine tumour, 91t, 166t, 171f, 184, 185, 199 paraganglioma, 166t pleomorphic adenoma, 132f, 133t, 134t, 135f, 144c primary effusion lymphoma, 220 Platelet aggregates, 200 Pleomorphic adenoma (PA) ancillary tests, 137 background cells, 136 basaloid neoplasms, 131, 132f, 133t, 143–144, 144c carcinoma ex PA(CEPA), 127, 129, 151, 152, 152t cytologic diagnosis, 128t DDx adenoid cystic carcinoma, 138t DDx epithelial-myoepithelial carcinoma, 146 DDx low-grade mucoepidermoid carcinoma, 146 DDx myoepithelial neoplasms, 152–153 DDx schwannoma, 152 false positive diagnosis, 149, 151, 152t gross aspirated material, 130 longstanding history, 129 Milan system, 126 salivary gland entities, 125c stromal features, 134t, 135, 135f, 153f, 154 Pneumothorax, 228 Polymorphous adenocarcinoma (PLAC), 129, 133t, 134t, 138 Poorly differentiated thyroid carcinoma (PDTC), see “Thyroid carcinoma, poorly differentiated” Post-transplant lymphoproliferative disease (PTLD), 88 Primary effusion lymphoma (PEL), 207, 212, 220 Prolymphocytes, 93 Prostatic carcinoma (prostatic carcinoma) acinar formations, 23, 23f, 91t cytomorphology, 105f effusions, 212 immunohistochemistry, 91t, 216c Provisional diagnosis, 33, 36, 37 Provisional report, 33, 36, 37 PSA, 91t, 230 Psammoma bodies appearance, 24 cystic papillary thyroid carcinoma, 61, 68, 73 endometriosis, 238–239 endosalpingiosis, 238 mesothelial cells, 222 metastatic papillary thyroid carcinoma, 91t ovarian serous carcinoma, 222f, 222 PSAP, 91 Pseudochylous effusion, 28t, 213, 214t, 228 Pseudocyst (PC), 175c, 176t, 181, 182t, 183c, 192c, 193 Pseudoinclusions ‘fake’ pseudoinclusions, 57, 59 FVPTC / NIFTP, 47 Hashimoto thyroiditis, 59, 74 cytomorphology, 54f, 57, 59, 60f papillary thyroid carcinoma, 58t, 59, 60f, 71 medullary thyroid carcinoma, 59, 60f hyalinising trabecular tumour, 59, 60f, 76 Pseudomyxoma peritonei, 21, 227, 228f

257 PSMA, 91 PTH, 65, 77 Pulmonary embolism, 213, 214, 226 Purulent ancillary testing, 48, 67 effusions, 214 gross aspirated material, 28, 35, 37, 67, 101t R Radioactive iodine, 71, 75 Ragocytes, 225 Reactive lymph node cytomorphology, 84, 106, 108 DDx accessory spleen, 200–201 DDx angioimmunoblastic T-cell lymphoma, 116–117 DDx low grade follicular lymphoma, 114–115 DDx marginal zone lymphoma, 98, 113–114 DDx nodular lymphocyte predominant Hodgkin lymphoma, 115 DDx peripheral T-cell lymphoma, 95t diagnostic caveat, 84 false negative diagnosis, 84, 113, 116 false positive diagnosis, 109 LH aggregates, 103 lymph node cytology, main entities, 82c pattern-based approach, 95c salivary gland lymphoid-rich lesions, 129, 141, 141c, 142f thyroid lymphoid-rich lesions, 70c Recommendations, 4, 14, 17, 43, 44t Red blood cells (RBCs) ‘fresh’ blood, 26, 63 ‘old’ blood, 26, 63, 64 Reed–Sternberg cells (RS cells), 94t–95t, 106f, 110, 115, 117 Renal cell carcinoma (RCC) effusions, 217t, 223, 235, 236f immunohistochemistry, 91t metastatic to lymph node, 108 metastatic to pancreas, 199 metastatic to salivary gland, 133t, 144c metastatic to thyroid, 66 morphology, 91t sample case triple approach, 30–32, 32f Renal failure, 212 Rheumatoid pleuritis clinicopathologic correlation, 212, 229 cytologic features, 224–225, 230 effusions, spectrum of conditions, 207c false positive diagnosis, 224, 235 gross appearance of effusion fluid, 214t pseudochylous effusion, 214t Riedel thyroiditis, 50, 64, 195 Rosette(s) cytomorphology, 23, 171f, 172f, 184 neuroendocrine tumours, 91t, 166t, 184 pancreatic acini, 184 paraganglioma, 172f

258 S S100 granular cell tumour, 169t melanoma, 92t myoepithelial neoplasms, 153 salivary gland neoplasms, 138t schwannoma, 152, 169t secretory carcinoma, 148 sustentacular cells, 173 S100P, 196t Salivary duct carcinoma (SDC) cytologic diagnosis, 127 DDx acinic cell carcinoma, 138 IHC, 138t oncocytic/oncocytoid neoplasms, 133t, 145c salivary gland, main entities, 125c Salivary gland neoplasm of uncertain malignant potential (SUMP) basaloid neoplasms, 143–144 cystic salivary gland lesions, 140c low-grade salivary gland malignancies, 146, 148, 149 Milan definition, 126–127 myoepithelial neoplasms, 153 oncocytic/oncocytoid neoplasms, 145c what cytology can’t do, 127 SALL4, 92t, 93t Sarcoidosis, 84, 104 Sarcoma, 25, 235 Sarcomatoid malignant mesothelioma, 216, 224, 231–233, 232t sarcomatoid carcinoma, 173, 235 SATB2, 92t, 227, 230 Schwannoma(s) EUS FNA gastrointestinal tract, 158c, 161, 162, 166t, 169 EUS FNA pancreas, 175c false positive diagnosis, 109 ‘lymph node’ cytology, 82c, 84, 89, 95c, 105 salivary gland cytology, 125c, 126, 129, 133t stroma, 135, 152 Secretory carcinoma (SC) clear/ granular cell lesions, 133t cystic salivary gland lesions, 131c cytologic diagnosis of, 124, 145, 149 DDx low-grade mucoepidermoid carcinoma, 149, 150f, 152t immunohistochemistry, 138t, 139 oncocytic/oncocytoid lesions, 133t, 145c salivary gland, main entities, 125c SUMP, 126 Seminoma, 92t, 93t Serous cystadenoma (SCA) cytologic diagnosis, 177t cytomorphology, 198f gross evaluation, 181–183 imaging, 180, 181 integrated diagnostic approach, 191t, 196–197 pancreas, main entities, 175c, 176t pancreatic cystic lesions, 183c, 192c, 196–197

Index specimen triage, 179 Von Hippel Lindau disease, 180 Sialadenosis, 14, 126, 130 Sialolithiasis, 126, 136, 141 Signet ring cell morphology DDx in effusion cytology, 222–223, 223t, 224f, 239–240 gastric carcinoma, 92t, 95c, 107 mesothelial cells, 217t Simple mucinous cysts (mucinous nonneoplastic cyst), 183c, 192 Skeletal muscle Boerhaave syndrome/oesophageal rupture, 226f, 227 DDx colloid, 52f, 62, 64 SMA, 92, 153, 169t, 173 SMAD4, 176t, 196t SMAD4 homozygous deletion, 196t Small cell carcinoma (SmCC) cell size, 98, 103 chains of cells, 220, 221f cytomorphology, 93t, 99, 107, 117, 118f, 223, 235 dispersed cells, 107f Dx large B-cell lymphoma, 118 effusions, 216c, 223, 235 history of smoking, 213 immunohistochemistry, 93t, 216c lymph node pattern-based approach, 95c Small intestine, 161t Small round cell tumours, 24, 212, 223 Soccer ball chromatin, 93t Solid pseudopapillary neoplasm (SPN) cytologic diagnosis, 177t, 191t cytomorphology, 184, 186f DDx acinar cell carcinoma, 199 epidemiology, 192 false positive diagnosis, 196, 197 imaging, 191t immunohistochemistry, 190, 199 pancreas, main entities, 175c pancreatic cystic lesions, 192c, 193 Papanicolaou Society of Cytopathology Guidelines for Reporting Pancreaticobiliary Cytology, 176t solid appearance, 189–190 specimen triage, 179 SOX10 melanoma, 92t salivary gland neoplasms, 137–138, 138t Spindle cells anaplastic thyroid carcinoma, 58t Brunner gland adenoma/hyperplasia, 173 epithelial-myoepithelial carcinoma, 134t, 136, 147 EUS FNA gastrointestinal tract, main entities, 158c, 166t, 169, 169t, 174 gastrointestinal stromal tumour, 161, 165, 170 gut wall muscle, 164f hyalinising trabecular tumour, 76 inflammatory myofibroblastic tumour, 173 lymphangioma, 141c lymphoid cells, 99

Index medullary thyroid carcinoma, 58t, 59f myoepithelial neoplasms, 132f, 134t, 152–154 nodular fasciitis, 153 pancreatitis, 187 pleomorphic adenoma, 152 rheumatoid pleuritic, effusions, 214t, 224, 235–236 salivary gland cytology main cell types, 131c, 133t, 134 schwannoma, 152 Spleen, accessory cytomorphology, 184, 200f, 200–201 EUS FNA pancreas, main entities, 166t, 177t imaging, 199 location, 181 Squamous cell carcinoma (SCC) basaloid, 133t, 144c cystic metastases, 92t, 90, 100, 139 cytomorphology, nodal metastases, 92t, 93t, 98f, 111f DDx pilomatrixoma, 109 DDx Warthin tumour, 111f, 143 effusions, 239, 240f, 241 false-positive diagnosis, 152t fluctuant lymph node, 89 granulomas, 104 imaging, nodal metastases, 89 oropharyngeal (HPV-related), 89, 92t, 100, 110, 140, 141c ‘purulent’ nodal aspirates, 90 Squamous metaplasia chronic pancreatitis, 194 salivary gland, 110, 124, 126, 127, 141, 149, 152t Stomach contaminant, 182, 198 gastrointestinal stromal tumour, 161t, 162, 165 pancreatic heterotopia, 173 schwannoma, 162 spindle cell tumours, 166t Stone, 136, 182t, 195 Strips (of cells) colorectal carcinoma, 92t differential diagnoses, 24 Stroma autoimmune pancreatitis, 195, 197f BAP1, 232 Brunner gland adenoma/hyperplasia, 173, 174f chronic pancreatitis, 194 cylinder(s), 134t, 135f, 144c endometriosis, 237f, 238–239 evaluation of, 25 fibrillary, 25, 62, 134t, 135f, 144c, 152, 153f fibrovascular stromal cores, 24, 91t globules, 134t, 135, 144c, 147, 147f, 153 hyalinisation, lymph nodes, 90 hyalinising trabecular tumour, 76 magenta, 62, 134t metachromatic, 134t, 135, 135f, 147, 153 mucinous cystic neoplasm, 188f, 190 pancreatic ductal adenocarcinoma, 188 pancreatic parenchyma, 178–179, 184, 199

259 salivary gland neoplasms (see “Matrix (stroma)”) schwannoma, 152 serous cystadenoma, pancreas, 198f solid pseudopapillary neoplasm, 186f, 199 stromal vessels, 55, 55f Subacute granulomatous thyroiditis, see ‘DeQuervain thyroiditis” Sublingual, 128, 136 Submandibular, 82, 82c, 89, 128–131, 136 Submucosa, 162, 166t, 170 Supernatant, 28t, 179, 189, 214t Suspicious for follicular neoplasm (SFN), see ‘Follicular neoplasm” Synaptophysin, 91t, 93t, 169t, 173, 189, 199, 216c, 235 Syncytial carcinoma, 105 cytomorphology, 23f description, 24, 53 nasopharyngeal carcinoma, 92t, 118f, 118 papillary thyroid carcinoma, 53c, 54, 54f, 75, 91t syncytial variant, Hodgkin lymphoma, 95c, 105 Systemic lupus erythematosus (SLE) clinical history, effusion cytology, 212 effusion cytology, 207c, 225–226 epidemiology, 212 histiocytic necrotising lymphadenitis, 113 T Tadpole-shaped cells, 92t, 110, 239 Tag72, 230 Tart cells, 226 T-cell-rich B-cell lymphoma (TCRBCL), 99, 111f Telecytology, 160 Telomerase, 234 Teratoma, 93t Thyroglobulin, 65, 66, 91t, 101t Thyroid carcinoma, 49, 101t Thyroid carcinoma, poorly differentiated cytologic diagnosis, 47t cytomorphology, 56, 56f, 57, 58t, 73–74 immunohistochemistry, 65 malignant Bethesda category, 45 micofollicular/trabecular architecture, 53c, 56, 57 physical examination, 50 Tigroid background, 6, 25, 25f, 92t, 100 Tingible body macrophages (TBMs) Burkitt lymphoma, 94t cytomorphology, 97f LH aggregates/germinal centres, 99, 108, 200 marginal zone lymphoma, 113, 114f Trabecular architecture AUS/FLUS nodule, 71, 71c cytomorphology, 23f, 24, 55f, 56 differentials in thyroid cytology, 53c follicular neoplasm, thyroid, 45, 73 hyalinising trabecular tumour, 76 paraganglioma, 172f secretory carcinoma, salivary gland, 148 Trabeculae, 45, 53, 56, 71, 76

Index

260 Transudates, 211 Transudative effusions, 207, 207c, 212 Trypsin, 190, 199 Tuberculosis adenosine deaminase, 211 cholesterol crystals in effusions, 228 clinical information, 87, 212 effusion cytology, main entities, 207c eosinophilic effusion, 226 lymph nodes, main entities, 82c lymphocytic effusion, 227 physical examination. 88–89 pseudochylous effusion, 214t sample provisional report, 37f U Ultrasound gel, 25, 25f, 63 Undifferentiated carcinoma dispersed cells, 106–107 lymph nodes, 95c pancreas, main entities, 175c See also “Lymphoepithelioma-like carcinoma”;“Nasopharyngeal carcinoma” Uraemia, 236–237 Urinothorax, 214t Uroplakin, 92t Urothelial carcinoma (UC), 92t, 212 UroVysion, 195, 196t, 234 V Vacuolation(s) adenocarcinoma, effusions, 218f, 239 Burkitt lymphoma, 94t cirrhosis, effusions, 237 cystic papillary thyroid carcinoma, 60, 61f, 62, 73, 90 describing cytoplasm, 24, 30, 59 epithelial-myoepithelial carcinoma, 147f gastric signet ring cell carcinoma, 92t germinoma, 92t large B-cell lymphoma, 94t macrophages, 68 marginal vacuoles, 60 mesothelial cells, various types of vacuoles, 217t, 221f, 239, 240 mucin vacuoles, 217t mucoepidermoid carcinoma, low-grade, 150f Mullerian clear cell carcinoma, 91t nuclear, 57 oncotic cells, 188, 188f ovarian carcinoma, 222, 225f pancreatic carcinoma, effusions, 223 pancreatic ductal adenocarcinoma, 185f paraganglioma, 172f

renal cell carcinoma, 91t, 223, 235, 236f salivary gland neoplasms, 133t, 135 secretory carcinoma, salivary gland, 145c, 148, 149f serous cystadenoma, pancreas, 196 signet ring appearance, effusions, 223t, 224f squamous cell carcinoma, effusions, 239, 240f Vascularity lymph node, 89 thyroid, 47 Vegetable, 226f, 227 Villin, 92t, 216c, 230 Viscous fluid effusion fluid, mesothelioma, 218 pancreatic neoplastic mucinous cysts, 21, 179, 191t, 192c pseudomyxoma peritonei, 227, 228f Von Hippel Lindau syndrome, 180 W Warthin tumour (WT) clinical features, 130 cystic salivary gland lesions, 140c, 141c cytologic diagnosis, 128t, 131c cytomorphology, 109–110, 111f, 146f DDx cystic ‘lymph node’, 100, 108 DDx low-grade mucoepidermoid carcinoma, 149–151 DDx oncocytosis/oncocytoma, 146f DDx squamous cell carcinoma, 109, 111f, 141, 143 DDx Warthin-like mucoepidermoid carcinoma, 149, 150f false-negative diagnosis, 150–151, 152t false-positive diagnosis, 151, 152t, 153f gross appearance of aspirate, 130 lymph nodes, pattern-based approach, 95c lymphoid-rich lesions, 26, 137, 141, 141c, 143 oncocytic lesions, salivary gland, 133t, 145, 145c salivary gland, main entities, 125c Windows adenocarcinoma, 218f. 219, 239 mesothelial cells, 217t, 218f, 239 WT1 mesothelial cells, 216c, 222, 228f, 230, 231, 232, 236f, 239 Mullerian carcinoma, 216c, 222, 230 Y Yolk sac tumour, 93t Z Ziehl Neelsen (ZN), 28t, 37, 101t, 102f