Atlas of Fine Needle Aspiration Cytology

Atlas of Fine Needle Aspiration Cytology Second Edition Henryk A. Domanski Editor

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Atlas of Fine Needle Aspiration Cytology

Henryk A. Domanski Editor

Atlas of Fine Needle Aspiration Cytology Second Edition

Editor Henryk A. Domanski Department of Pathology Skåne University Hospital Lund Sweden

ISBN 978-3-319-76979-0    ISBN 978-3-319-76980-6 (eBook) https://doi.org/10.1007/978-3-319-76980-6 Library of Congress Control Number: 2018954717 © Springer International Publishing AG, part of Springer Nature 2019 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, express 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 Switzerland AG The registered company address is: Gewerbestrasse 11, 6330 Cham, Switzerland

To my mother Romana Domańska with love and thanks

Preface to Second Edition

When we began work on the first edition of this atlas, we had hoped to put together a practical atlas/handbook for pathologists, cytopathologists, and trainees in practice looking for a guide in everyday work and diagnosis in cytopathology. As in the first edition, the purpose of this volume is still to provide a practical and “bench-useful” guide for modern cytological diagnosis of surgical pathology. The second edition of the atlas is organized on the basis of the current classifications used in surgical pathology and provides a comprehensive and well-illustrated review of the FNAC diagnoses of the most common entities in the major organ systems. Cytological criteria, differential diagnoses, and correlations between cytology and the diagnostic use of ancillary techniques applicable to FNAC are detailed on an entity-by-entity basis in order to facilitate the diagnostic work-up in the FNA samples. The text of all chapters has been updated and many illustrations have been improved. Some new co-authors have joined our team and have made great contributions. Dr. Elwira Bakuła-Zalewska has revised and updated Chap. 4, “Salivary Glands,” and added a new Chap. 6 specifically on the FNA of the parathyroid. Drs. Matthew W. Rosenbaum and Martha B. Pitman have written a new Chap. 12, “Pancreas” which replaced Chap. 11, “Pancreas” from the first edition. My dear daughter Katarina Bartuma, who works as an ophthalmologist in Stockholm, has also helped us to update Chap. 18, the “Orbit and Ocular Adnexa.” Once again, we hope that the present volume will continue to assist surgical pathologists, cytopathologists, and trainees but will also be of interest to clinicians involved in the diagnosis and therapy of patients with mass lesions. Lund, Sweden

Henryk A. Domanski

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Acknowledgments

As with the first edition I would like to express many thanks to all the co-authors and contributors to the second edition. This atlas would not exist without their expertise and valuable contribution. I would like to thank my wife Anna Domanski CT (MIAC); my daughter Katarina Bartuma, MD, PhD; and my friends Elwira Bakuła-Zalewska, MD, PhD; Måns Åkerman, MD, PhD; Xiaohua Qian, MD, PhD; Mats Ehinger, MD, PhD; Nastaran Monsef, MD, PhD; Fredrik Mertens, MD, PhD; Jerzy Klijanienko, MD, PhD; Donald Stanley, MD; and Beata BodeLesniewska, MD, PhD, for their continuous support, friendship, and contribution during the preparation of the current edition. I wish to thank Donald Stanley, MD, for his valuable medico-linguistic expertise and all of my colleagues and staff at the Department of Pathology, Skåne University Hospital, for their support. I wish to thank the editorial staff of Springer for excellent administrative assistance and patience during our work on the second edition of the atlas.

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Contents

  1 Introduction���������������������������������������������������������������������������������������������������������������    1 Henryk A. Domanski and Fredrik Mertens   2 Image-Guided Fine-Needle Aspiration Cytology���������������������������������������������������   43 Mats Geijer and Henryk A. Domanski   3 Breast �������������������������������������������������������������������������������������������������������������������������   57 Fernando Schmitt, Rene Gerhard, Donald E. Stanley, and Henryk A. Domanski   4 Salivary Glands and Head and Neck�����������������������������������������������������������������������  105 Elwira Bakuła-Zalewska, Henryk A. Domanski, and Gabrijela Kocjan   5 Head and Neck: Thyroid�������������������������������������������������������������������������������������������  159 Paul A. VanderLaan and Jeffrey F. Krane   6 Head and Neck: Parathyroid �����������������������������������������������������������������������������������  205 Elwira Bakuła-Zalewska   7 Lung ���������������������������������������������������������������������������������������������������������������������������  219 Henryk A. Domanski, Nastaran Monsef, and Anna M. Domanski   8 Mediastinum and Endobronchial Ultrasound-Guided Transbronchial Needle Aspiration�������������������������������������������������������������������������������������������������������������������  265 Henryk A. Domanski, Nastaran Monsef, Anna M. Domanski, and Włodzimierz Olszewski   9 Lymph Nodes�������������������������������������������������������������������������������������������������������������  287 Mats Ehinger and Måns Åkerman 10 Spleen �������������������������������������������������������������������������������������������������������������������������  363 Mats Ehinger and Måns Åkerman 11 Liver ���������������������������������������������������������������������������������������������������������������������������  369 Beata Bode-Lesniewska and Henryk A. Domanski 12 Pancreas���������������������������������������������������������������������������������������������������������������������  403 Matthew W. Rosenbaum and Martha B. Pitman 13 Kidney and Adrenal Gland���������������������������������������������������������������������������������������  433 Xiaohua Qian 14 Soft Tissue�������������������������������������������������������������������������������������������������������������������  465 Henryk A. Domanski, Xiaohua Qian, Måns Åkerman, and Donald E. Stanley 15 Skin and Subcutis �����������������������������������������������������������������������������������������������������  553 Henryk A. Domanski and Donald E. Stanley

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16 Bone�����������������������������������������������������������������������������������������������������������������������������  599 Henryk A. Domanski, Xiaohua Qian, and Donald E. Stanley 17 Pediatric Tumors�������������������������������������������������������������������������������������������������������  653 Jerzy Klijanienko and Philippe Vielh 18 Orbit and Ocular Adnexa�����������������������������������������������������������������������������������������  679 Jerzy Klijanienko, Katarina Bartuma, and Henryk A. Domanski Index�����������������������������������������������������������������������������������������������������������������������������������  695

Contents

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Introduction Henryk A. Domanski and Fredrik Mertens

 he Role and Evaluation T of Fine-Needle Aspiration Cytology in Clinical Examination Fine-needle aspiration cytology (FNAC) has been used as a tool to obtain specimens for the morphological diagnosis of numerous lesions in a variety of locations for more than 80 years. Despite early attempts to use thin-needle aspiration for diagnosis of neoplasm and inflammatory conditions [1, 2], first large-scale studies published in the early 1920s and 1930s by Martin, Ellis, and Steward [3, 4] are considered to be the beginning of the modern era of FNAC. In 1950s–1960s, FNAC became a widely used diagnostic tool particularly in Europe, pioneered by some physicians and pathologists in Sweden [5, 6]. Today, this diagnostic modality is more powerful than ever in making rapid preliminary diagnoses in neoplastic and nonneoplastic conditions, guiding further work-up of the patient or even allowing the initiation of definitive treatment. In many clinical situations, FNAC can render a definitive diagnosis either from aspiration smears alone using well-defined cytological criteria (see Fig. 1.1) or from aspiration smears combined with clinical data, radiological findings, and the results of ancillary studies (see Figs. 1.2 and 1.3). The use of ever more sophisticated ancillary methods on aspiration specimens, such as molecular/ genetic analysis and immunocytochemistry, allows a diagnosis of tumors that can be used for predicting prognosis and tailoring individualized “targeted” oncological therapy [7–11].

Nevertheless, considerable differences exist between various diagnostic centers with regard to the role that aspiration cytology plays in the work-up of patients. Many physicians are skeptical about the use of cytological diagnosis due simply to the small amount of diagnostic material obtained. Consequently, in some centers, cytological material is not collected or processed in an optimal or standardized way. In such places, there has never been an understanding of the potential of cytological diagnosis, and clinicians have had to rely on other diagnostic modalities. In diagnostic centers where there is a tradition of diagnostic procedures using reliable FNAC, the technique of aspiration has been optimized and taught to generations of cytologists, radiologists, and clinicians. Careful attention has been paid to specimen handling, and strict morphological criteria have been applied to the microscopical examination.

H. A. Domanski (*) Department of Pathology, Skåne University Hospital, Lund, Sweden e-mail: [email protected] F. Mertens Department of Clinical Genetics, Skåne University Hospital, Lund, Sweden e-mail: [email protected]

Fig. 1.1  FNA of breast mass: cellular smears with a mixture of benign, branching cell clusters with fragments of myxoid matrix and myoepithelial cells, indicative of fibroadenoma

© Springer International Publishing AG, part of Springer Nature 2019 H. A. Domanski (ed.), Atlas of Fine Needle Aspiration Cytology, https://doi.org/10.1007/978-3-319-76980-6_1

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Fig. 1.2 Combination to preserve FNA specimen for ancillary techniques

Immunocytochemistry has been a special problem as this technique has proven to be more difficult to standardize than immunohistochemistry. Great efforts have to be made in the laboratory to ensure the usefulness of immunological stains in cytology [12–16]. In many institutions, aspirations are routinely performed by a clinician or radiologist without the assistance of a cytopathologist or cytotechnologist. Many non-cytopathologists are well experienced in the method and obtain a material that is perfectly adequate for diagnosis [17–19]. It must be noted, however, that the results of aspiration biopsy are often better when it is performed by an experienced cytopathologist in a puncture cytology clinic (FNA clinic) [16, 20]. In this setting, it is possible to do a rapid, preliminary evaluation (rapid on-site evaluation, ROSE) of the aspirate while the patient waits and then immediately perform additional aspirations if the material is found to be inadequate or

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Fig. 1.3 Rapid on-site evaluation (ROSE) in the puncture cytology clinic (FNA clinic). (a) FNA of a defect in the acromion in a previously healthy 49-year-old man. ROSE: sheets of carcinoma cells (Diff-Quik staining). (b) Cell block preparation from aspiration specimen. Note preserved architecture of the tumor tissue consistent with adenocarci-

noma (H&E). (c) Cytokeratin positivity in a cell block. (d) Thyroid transcription factor (TTF-1) positivity of tumor cells on cell block section indicates lung origin. Subsequent investigation disclosed a mass in the lung

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Fig. 1.4  Equipment for ROSE outside of the puncture cytology clinic. (a) A portable microscope and arrangement for rapid Diff-Quik staining. (b) A box containing FNA equipment, such as syringe holder,

syringes, needles, slides, fixative, and Cytolyt to collect FNA specimen for further liquid-based and cell block preparations and combination to preserve specimen for ancillary studies

if more material for ancillary diagnostic examination are needed (see Fig. 1.3). An expanding role of cytopathology staff members such as pathologists/cytopathologists and cytotechnologists in FNAC performed outside of the dedicated FNA clinic depends mostly on the growing popularity of the ROSE service among radiologists and clinicians performing FNA procedures (see Fig. 1.4) [21–35]. ROSE for FNA samples and touch preparation from core biopsy can be performed in many clinical circumstances and from many body sites (see Fig.  1.5) regardless of palpable or non-palpable lesions [36–40]. In the coming age of telepathology/telecytology, there may be potential for using this technique to ensure FNA specimen adequacy (ROSE) and reliable FNAC diagnosis [41–44]. As FNAC does not always provide adequate cellular or architectural details in certain types of lesions and the FNA

specimen may not be sufficient to prepare a cell block, the combined use of FNAC and core needle biopsy (CNB) can improve obtaining satisfactory amount of material for both routine microscopic examination and ancillary tests [45, 46]. As FNAC and CNB are complementary techniques and both can be performed in the outpatient setting, the procedure of CNB in conjunction with FNAC in the same séance is patient-friendly and cost-effective approach which improves diagnostic accuracy of the FNAC alone (see Figs.  1.6 and 1.7) [46, 47]. The complementary nature of FNAC and CNB in the outpatient setting has been underlined in some serial reports of musculoskeletal neoplasms sampled by those two modalities [48–53]. In one study from Lund, Sweden, a series of 130 consecutive patients with musculoskeletal lesions were evaluated by FNAC and CNB performed simultaneously by the cytopathologist showing a high diagnostic accuracy and speed of diagnosis using this double approach [47].

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Fig. 1.5  ROSE in the department of radiology. (a) A 54-year-old man with a history of colonic adenocarcinoma 4 years ago. A single suspect metastatic lesion was detected during routine checkup. Diagnostic CT before FNAC showing a small mass surrounding the rib. (b) In the prone position, the lesion was aspirated through a trocar. (c) First FNA

smears were air dried and Diff-Quik stained for immediate microscopic examination. Additional two FNA passes were performed to obtain material for (d) alcohol fixed smears (H&E) and (e) cell block preparation confirming diagnosis: metastasis of colonic adenocarcinoma (cell block; H&E)

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Fig. 1.6  The procedure of CNB in conjunction with FNAC in the same séance in the puncture cytology clinic. (a) FNA of soft tissue mass with clinical suspicious of sarcoma. Smears from two FNA passes stained for immediately microscopic examination disclosed scanty and paucicellular specimen containing scattered pleomorphic spindle cells and

fragment of collagenous matrix. (b) Local anesthesia was administrated immediately after aspiration, and (c) four CNB passes were performed providing (d) cores of tumor tissue satisfactory for diagnosis. The combination of those both sampling techniques provided a precise diagnosis of high-grade myxofibrosarcoma

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Terminology and Reporting Two similar designations, FNAC and fine-needle aspiration biopsy (FNAB), have been used to describe the technique of aspirating cells and tissue fragments through a thin needle (up to 0.8  mm OD). Aspiration can be achieved using the capillary pressure of the needle itself or by applying a partial vacuum using a syringe [54–57]. Diagnostic results can be reported in a manner similar to other types of cytological examination: (1) unsatisfactory (inadequate), (2) negative for malignancy (benign), (3) atypical, probably benign (atypical/undetermined), (4) suspicious of malignancy (probably malignant), and (5) positive for malignancy (malignant). For example, these different levels of diagnoses can be applied to mammary FNAC (see

Figs. 1.7, 1.8, 1.9, and 1.10) [58, 59]. Uncertain diagnosis of malignancy is often reported as “cannot rule out malignancy” or “suspicious of malignancy.” The diagnosis of a specific pathological entity can also be given in many cases by FNAC.  In some cases, only a descriptive diagnosis can be reached. Even such a diagnosis can be clinically relevant, guiding the subsequent work-up of the patient [60] (see Figs. 1.11 and 1.12). Recently, stanardized cytology reporting concerning numerousm areas of FNA cytology such as FNA of the thyroid, salivary glands and breast have been elaborated and published. These cytology reporting systems provide a uniform diagnostic terminology, guidance for appropriate clinical management and optimize communication between pathologist and clinician and a cytologic and histologic correlation of cases.

Fig. 1.7  FNA from fibroadenosis with a sheet of apocrine cells: C2 category, benign (negative for malignancy) (H&E)

Fig. 1.8  FNA smears from fibroadenoma: C3 category, atypical, probably benign (H&E)

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Fig. 1.9 (a) FNA smears from tubular carcinoma: C4 category, suspicious of malignancy. (b) FNA smears from lobular carcinoma: C4 category, suspicious of malignancy (H&E)

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Fig. 1.10  FNA smears from ductal breast carcinoma: C5 category, malignant (positive for malignancy) (H&E)

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Fig. 1.11  Alcohol-fixed (a), (H&E) and air-dried (b), (MGG) FNA smears from an enlarged lymph node of a previously healthy 25-year-­ old man. Morphology of granulomatous lymphadenitis. Although the

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Fig. 1.12  FNA smears from a 5 cm subcutaneous swelling in the foot of a 40-year-old man. Granulomatous inflammation indicative of an inflammatory/reactive process (a). (MGG) Further investigation with

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pathogenesis of granuloma is not apparent on the smear, a descriptive report excludes malignancy and guides further work-up

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incisional biopsy of the foot lesion from the previous figure disclosed sarcoidosis (b) (H&E)

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FNAC Techniques and Preparation of Samples FNAC Techniques Depending on the clinical situation, the cytopathologist, surgeon, or radiologist may perform the FNA. The FNA procedure should be clearly explained to the patient to assure patient’s cooperation during FNA procedure. The patient should be placed in a comfortable position that allows easy access to the lesion that needs to be aspirated. For FNA of palpable, superficial lesions, the skin over the area of the procedure is cleaned with an alcohol or with another antiseptic solution. Preparations as for minor surgical procedures are necessary for guided FNA of deeper, non-palpable lesions. For palpable lesions a 27- to 22-gauge (0.4–0.7 mm) needles are suitable, either using a capillary technique without aspiration [56, 57] or a plastic disposable 10- or 20-ml

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syringe attached to a plastic or metal syringe holder (FNA with aspiration) (see Fig.  1.13). For guided FNAs of non-­ palpable, deeper lesions, longer needles are utilized. A large variety of specialist needle types and sizes are available. The nodule is immobilized between the fingers, and the needle tip is rapidly directed through the skin into the nodule. Once the needle enters the mass, the needle is continuously aspirated, while the needle is rapidly moved back and forth to obtain the sample. Suction is then relieved and the needle is withdrawn and detached from the syringe. Air is then aspirated into the syringe, the needle is replaced onto the syringe, and the material is expelled from the needle onto the glass slides (see Fig. 1.14a). The material is gently but rapidly smeared on the slides (see Fig. 1.14b) and immediately dipped in fixative (see Fig. 1.14c) or left to air dry. For liquid based preparation the aspirate is collected directly in specially developed liquid fixative (see Fig. 1.14d).

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Fig. 1.13 (a) FNA procedure without aspiration (capillary technique): The needle tip is passed through the skin, inserted into the lesion, and moved back and forth to dislodge cells. (b) FNA of cutaneous basal cell carcinoma performed by a pathologist. Syringe attached to a Cameco

syringe holder: vacuum in the syringe helps to dislodge cells and to obtain a cellular specimen. (c) Fluoroscopy-aided FNA of skeletal chondrosarcoma. The equipment and aspiration technique is entirely the same as in the aspiration of palpable masses

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Fig. 1.14 (a) Air is aspirated and the material is expelled on glass slides. (b) The material is gently but rapidly smeared on the slides. (c) Slides with smears are immediately dipped in fixative or left to air dry. (d) For liquid based preparation the aspirate is collected directly in specially developed liquid fixative

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Conventional Preparation of FNA

details can best be appreciated in either air-dried or wetfixed specimens, it needs to be pointed out that results of The preparation of aspiration specimens has been well fixation and staining may vary among laboratories (see described in many textbooks. The principle is to spread a Fig. 1.20). There is a relative lack of standardization in hansuitable amount of aspirate (usually about one drop) thinly dling of FNA specimens, compared to the routines followed and evenly over a microscopic slide that is then stained and for histopathological specimens. The choice of the fixation mounted. The result should be comparable to a sectioned his- and staining determines largely by the tradition of a particutological slide with regard to specimen thickness and lar laboratory and by local practice pattern. The standardevenness. ization of sampling, fixation, and staining procedures in With regard to fixation, there are two common methods: aspiration cytology, as well as of immunocytochemical air drying or wet fixing using either 95% ethanol or ethanol-­ methods, is one of the important challenges to be overcome based Cytospray as a fixative. Other liquid fixatives such as if the role of FNAC in morphologic evaluation of tumors is methanol, Saccomanno’s fixative, acetone, isopropyl alco- to expand. hol, and acetone/methanol or other combinations of fixative Air-dried smears can be stained with Diff-Quik or are less frequently used in the routine preparation of aspira- May-­ Grünwald-­ Giemsa (MGG) and wet-fixed smears tion smears. Where both air-dried and wet-fixed slides have with hematoxylin and eosin (H&E) or Papanicolaou been shown to provide comparable results, each of these (Pap). Diff-Quik staining is often used for rapid on-site techniques has characteristic advantages and limitations. evaluation (ROSE) (see Fig. 1.21) of smears but in many Although wet fixation usually better demonstrates such centers has replaced MGG as the routine stain for airdetails as nuclear pattern, chromatin structure, and nucleoli dried smears. MGG staining gives sometimes a slightly (see Figs.  1.15 and 1.16), air-dried specimens give better better micromorphology compared to Diff-Quik (see information on cytoplasmic details (see Fig. 1.17), as well Fig. 1.22). as the extracellular matrix (see Fig.  1.18) and the backAccording to our experience, the best results are obtained ground material (see Fig.  1.19). Despite the general rules when microscopic evaluation is based on both wet-fixed and with regard to which diagnostic features and morphological air-dried smears.

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Fig. 1.15  FNA smears from metastatic malignant melanoma. Wet-fixed and H&E-stained smears (a) better appreciate nuclear details, such as inclusions, compared to air-dried and MGG-stained smears (b)

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Fig. 1.16  FNA smears from thyroid papillary carcinoma. Nuclear details such as nuclear grooves (a) and nuclear inclusions (b) are better appreciated in wet-fixed and H&E-stained slides

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Fig. 1.17  FNA smears from metastatic pancreatic adenocarcinoma. Note mucin vacuoles in the tumor cells, better appreciated in air-dried and MGG-stained smears (a) than in liquid-based preparations and H&E-stained smears (b)

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Fig. 1.18  FNA smears from myxopapillary ependymoma. Note distinctive hyaline globules in the MGG staining (a) compared to shadows appearing at the same locale in H&E staining (b)

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Fig. 1.19  Distinctive “tigroid” background of retroperitoneal metastatic seminoma in the air-dried and MGG-stained smears (a). Myxoid background matrix in the air-dried and MGG-stained smears from metastatic extraskeletal myxoid chondrosarcoma (b)

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Fig. 1.20  FNA smears from metastatic gastric adenocarcinoma. Compared to Fig. 1.17, mucin vacuoles in the tumor cells are better appreciated in H&E (a) than in MGG staining (b)

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Fig. 1.21  Arrangement for rapid Diff-Quik staining

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Fig. 1.22  FNA smears from high-grade osteosarcoma. MGG staining (a) provides slightly better micromorphology compared to the rapid Diff-­ Quik staining (b)

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Cell Blocks Smears can be difficult to prepare reliably as a result of the nature of the aspirate (cystic components, blood contamination) or due to inexperience in the person making the smear. Of the various techniques devised to make best use of aspirates, we and others have found that the preparation of cell blocks (CB) can be very helpful [61–63].There is usually adequate material for many sections from the resulting paraffin block, and the microbiopsies show a preserved tissue architecture, which can aid diagnosis. Since Koss described the plasma-thrombin method [64], several other techniques of preparing CB have been reported. CB preparation advantages are twofold: (1) better visualization of the tumor

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tissue pattern than is possible in FNA smear (see Figs. 1.23, 1.24, and 1.25) and (2) an opportunity of performing immunocytochemical and other special stains on several slides of comparable quality. It should be pointed out that CB quality derived from aspiration specimens is not exclusively a function of the quantity and quality of the cell material obtained. Some techniques for preparing CBs include a centrifugation of the specimen that can partly damage the cells, resulting occasionally in poor morphology, compared to traditional preparing techniques. A processor recently developed by the Hologic company, in which centrifugation has been replaced by filtration, seems to better preserve cells and tissue fragments, resulting in slightly better morphology (see Fig. 1.26).

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Fig. 1.23  FNA smears from high-grade osteosarcoma (a) (H&E) showing obvious features of high-grade sarcoma. Cell block prepared from the aspiration smears facilitating examination of tumor tissue architecture and occurrence of osteoid (b) (H&E)

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Fig. 1.24  FNA smears from colloid goiter (a) (MGG). Cell block prepared from the aspiration smears facilitating a diagnosis of benign goiter (b) (H&E)

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Fig. 1.25  FNA smears from a cystic metastasis of squamous carcinoma to the neck (a) (H&E). Cell block prepared from the aspiration smears confirming keratinizing squamous carcinoma (b) (H&E)

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Fig. 1.26  FNA smears from a giant cell tumor of the fibula. MGG preserved histo- and cytomorphological details. Low-power (c) and staining (a) and H&E staining (b): cell block prepared from the aspira- high-power view (d) (H&E; Cellient; Hologic; Bedford, MA, USA) tion using filter technique shows good histomorphology with well-­

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Liquid-Based Preparations The preparation of exudates, urine, and bronchial and bladder washings has always been “liquid based,” and results in these situations have been satisfactory. It has become popular to prepare even cervix cytology and aspirates through a liquid medium. The liquid-based ThinPrep (TP; Hologic, Marlborough, Mass., USA) and SurePath (SP; BD TriPath, Burlington, N.C., USA) methods have become widely used for both gynecological and non-gynecological specimens including FNAC. Liquid-based preparation (LBP) was designed to improve traditional preparations techniques [65–67], but the result for FNAC can be occasionally problematic. Diagnostic criteria may be different in liquidbased specimens compared to conventional smears because of somewhat altered morphology, and because the smear background and extracellular elements, which is sometimes an important part of the diagnosis, is frequently lost after processing through Cytolyt [68]. Architectural changes such as discohesion of cells, smaller cell clusters and sheets, breakage of papillae, as well as generally smaller and occasionally spindled cells with attenuated

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chromatin detail and more prominent nucleoli are all common findings in LBP aspirates [66, 67, 69–72]. Gerhard et al. [73] reviewed the published literature of LBP applied to breast FNAC.  They concluded that LBP has similar accuracy to conventional smears for the FNAC diagnosis of breast lesions and can be safely used in the preparation of breast aspirates. This technique is easier for collection of samples and can provide appropriate ancillary tests. Characteristic cytologic features of breast aspirates prepared using LBP require appropriate training, however, to prevent misinterpretation [73]. In our experience in Lund, cells and tissues obtained from epithelial neoplasms are usually better preserved than those of mesenchymal origin after liquid-based processing (see Figs.  1.27 and 1.28). Nevertheless, this method can be very useful, especially in the area of immunocytochemistry [68] (see Fig. 1.29) and molecular biological analyses [65, 74–79]. Cell blocks can be prepared with residual specimens preserved in a liquid-­ based cytology medium and immunocytochemical stains, and molecular testing can be successfully performed. These are important adjuncts in order to reach a definitive diagnosis.

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Fig. 1.27  FNA smears from Ewing’s sarcoma. Air-dried and MGG-­ deposition. The same cell specimen prepared by ThinPrep (b) (H&E). stained smears (a). Note double cell population and apparent vacuoliza- The double cell population and cytoplasm vacuolization are not tion of cytoplasm in the larger light cells, indicative of glycogen apparent

1 Introduction

a

17

b

Fig. 1.28  Smears from papillary thyroid carcinoma prepared as ThinPrep. Note well-preserved monolayer morphology (a) (H&E) and typical intranuclear inclusion (b) (H&E)

a

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Fig. 1.29  Sister Mary Joseph nodule. FNA smears of umbilical nodule in a patient with a history of urothelial carcinoma of the bladder. MGG-­ stained, air-dried smears (a), and ThinPrep slides stained with H&E

indicative of the metastatic urothelial carcinoma (b). Immunostains for keratin performed on ThinPrep preparation of residual specimen showing tumor cells with distinctive keratin positivity (c)

18

Ancillary Techniques in FNAC Ancillary techniques are used extensively today as a diagnostic help in FNAC.  Essentially all the methods used for surgical biopsy specimens can be applied to cytological material. Of these methods, immunocytochemistry (IC) is the one most often applied to FNA specimens. Molecular genetic methods are becoming increasingly important to verify or rule out a specific diagnosis for prognostic information and therapeutic drug selection [9]. Particular genetic changes have been described in many types of tumor, and these are steadily increasing. Several genetic and cytogenetic molecular methods, including cytogenetics (chromosome banding), fluorescence in situ hybridization (FISH), polymerase chain reaction (PCR), single nucleotide polymorphism (SNP) array, and next-generation sequencing (NGS), can be applied to cytological material. Experience has shown that chromosome banding analysis of cells from FNA specimens is difficult; it is not always possible to aspirate a sufficient number of tumor cells for successful culture [80, 81]. Molecular cytogenetic or molecular genetic techniques have

Fig. 1.30  Ultrastructure of alveolar soft part sarcoma. Tumor cells contain cytoplasmic crystals that reveal periodic linear, lamellar structure. (Image courtesy of Ms. Catarina Crammert, Department of Pathology, Skåne University Hospital, Lund, Sweden)

H. A. Domanski and F. Mertens

proved to be better suitable for fine-needle aspirates as they do not require cell culturing and thus fewer cells. A number of reports on the diagnostic usefulness of molecular genetic techniques applied to FNA aspirates have been published, and the importance of molecular genetic examination of FNA samples from a variety of neoplasm constantly increases [8, 9, 81–92]. Electron microscopy (EM) applied to cytological specimens can reveal certain differentiated structural features and clarify the origin of cells that lack definite signs of differentiation by light microscopy [93–96] (see Fig. 1.30). Flow cytometry is one of the techniques established in the area of cytology, playing an important role as a diagnostic adjunct in the examination of FNAC specimen from hematopoietic organs, mainly in the diagnosis and classification of leukemia and lymphoma [97–104]. One cannot forget that ancillary studies play a supportive and complementary role in the diagnostic process and must always be subordinate to the routine microscopic examination of smears and the clinical information regarding the case.

1 Introduction

19

Immunocytochemistry The most common ancillary technique used to complement routine microscopic examination of aspirated material is immunocytochemistry (IC). IC was designed to improve routine cytology to aid pathologist/cytopathologist in the differential diagnosis of neoplasm and to render confident and accurate diagnoses on limited tissue samples. As the technique evolved, it has become increasingly important for selection of therapy in some tumor type and to provide prognostic and predictive information [12, 105–107]. IC may be applied to any of the several types of preparations: to direct smears, cell-transferred direct smears [108, 109], cytospin preparations [110], liquid-based preparations, or cell blocks. Compared to histology, the results of immunostains of ­cytological material, however, can be difficult to standardize and can vary depending on the type of specimen fixation and preparation. In addition, IC controls are difficult to prepare, control, and maintain [16]. In our experience, the results of IC examinations of aspirates are dependent not only on the type of specimen preparation but also on the quality and quantity of the material, background material, and the presence of necrosis. Direct smears and slides from cytocentrifugation have heretofore been commonly used techniques to prepare FNA material for IC. IC performed on direct smears can occasionally be difficult to evaluate due to trauma inherent in the

a

technique. Nuclei can be stripped of their cytoplasm, and the background material can contain the fragments and remnants of many different cells (see Figs. 1.31 and 1.32). One problem occurring occasionally in the interpretation of IC results on direct smears and slides from cytocentrifugation is misinterpreting background/cytoplasmic staining as nuclear staining (see Figs. 1.31, 1.32, and 1.33). Liquid-based cytological preparations have recently become a popular technique for preparing FNA material for immunostaining as well (see Fig.  1.34) [111–113]. In the author’s experience, results of such preparations are promising (see Figs. 1.35 and 1.36). Cell block preparations using paraffin-embedded cells are becoming increasingly popular in many busy FNA clinics, allowing both examination of the tissue architecture and making immunostaining more reliable (see Figs. 1.37, 1.38, and 1.39). An advantage of the preparation of cell blocks is that the processing of these slides for immunostaining ­follows the same procedures as for histology specimens (see Fig. 1.39) [86, 113–123]. Advantages and disadvantages of preparation methods of FNA aspirates for IC are presented in Table 1.1. It bears reemphasizing that IC must be used as a complement to the routine cytological examination of the aspirate. Traditional light microscopic examination of air-dried or alcohol-fixed and routine-stained smears or liquid-based prepared smears is still the basis of cytological diagnosis.

b

Fig. 1.31  Slides with direct FNA smears from breast carcinoma processed for estrogen IC (a). The results may be difficult to interpret due to suboptimal technique including heavy background staining (b)

20

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H. A. Domanski and F. Mertens

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Fig. 1.32  Immunostains with S-100 and HMB45 on direct smears (a) from metastatic malignant melanoma with suboptimal results of both S-100 (b) and HMB45 (c): difficult to interpret due to technically suboptimal conditions

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Fig. 1.33  Slides with cytocentrifuged smears (cytospin) from breast carcinoma processed for estrogen IC (a). As with direct smears, results may be difficult to interpret due to suboptimal technical conditions (b)

1 Introduction

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b

Fig. 1.34  Slides with ThinPrep-prepared smears from breast carcinoma processed for estrogen IC (a). The result of IC is comparable of that of histological specimen (b)

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Fig. 1.35  The result of immunostains for desmin of the specimen aspirated from rhabdomyosarcoma prepared as centrifuged smears (a) and in ThinPrep processor (b)

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H. A. Domanski and F. Mertens

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Fig. 1.36  Alcohol-fixed and H&E-stained FNA smears from a breast nodule in a patient with a history of lung carcinoma (a). Smears prepared in the ThinPrep processor showing a sheet of mammary epithe-

a

lium with admixture of carcinoma cells (b). Immunostains performed on ThinPrep showing clear positivity for TTF-1, indicative of metastatic lung carcinoma (c)

b

Fig. 1.37  Slides with cell block section from breast carcinoma processed for estrogen IC (a). The result is similar to immunohistochemical staining on histological sections from biopsy specimen (b)

1 Introduction

23

a

b

Fig. 1.38  Cell block section (a) and immunostains with EMA (b) on cell block section from monophasic synovial sarcoma. The result is similar to histological sections of biopsy specimen

a

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Fig. 1.39  Processing of the cell block slides (a) for immunostains follows the same procedure as for histology specimens with appropriate controls (b) and the result is similar to immunostains on biopsy

s­ pecimen (Ewing sarcoma-cell block sections showing positive immunostains for CD99)

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H. A. Domanski and F. Mertens

Table 1.1  Advantages and limitations of different preparation methods of fine-needle aspirates for immunocytochemistry Preparation method Direct smear

Advantages No specific preparation; universally accepted method for cytochemistry and immunocytochemistry

Destaining of archive slides; cell-­transferred direct smears

Can be used retrospectively

Cytospin preparation

Easy preparation. Universally accepted method for cytochemistry and immunocytochemistry

Cell block preparation: manually

Similar to a small histologic biopsy. Easy to perform controls and compare with subsequent IC on histological samples. Material can be saved for further evaluation

Cell block preparation: automatic

Standardized preparation procedure. Similar to a small histologic biopsy. Easy to perform controls and compare with subsequent IC on histological samples. Material can be saved for further evaluation

Liquid-based cytology

“Clean” background. Monolayer of cells. Material can be saved for further evaluation

Disadvantages Can be difficult to evaluate due to cytoplasmic background and stripped nuclei. In our experience, nuclear antibodies suitable Not sufficiently evaluated; in our experience, gives a higher percentage of false-negative results Risk for false-­ negative results due to focal expression of antibodies. Occasional difficulty in the interpretation of results Time-consuming. Can be difficult to obtain sufficient specimen from lesions with abundant collagenous matrix Expensive compared to other techniques. Can be difficult to obtain sufficient specimen from lesions with abundant collagenous matrix Not yet sufficiently evaluated with regard to all antibodies

 ytogenetic, Molecular Cytogenetic, C and Molecular Genetic Analysis Chromosome banding analysis of tumor cells has been performed for more than 40 years, and cytogenetic information is available on around 65,000 neoplasms [124]. From these studies, it has become apparent that most tumor types display nonrandom patterns of chromosome aberrations. Furthermore, it is also recognized that different chromosome aberrations play different roles during tumor development: some are primary, tumor-initiating events, whereas others are secondary aberrations, occurring later in tumor development and possibly influencing tumor progression. Particularly the primary aberrations are often strongly, sometimes specifically, associated with a certain morphologic entity (Table 1.2; see Figs. 1.40, 1.41, and 1.42) [125]. Thus, they may serve as excellent diagnostic markers. Many primary aberrations are balanced translocations, resulting in fusion genes that can be detected by reverse transcriptase PCR (RT-PCR) or FISH [126]. It is not only individual chromosome aberrations that may be of diagnostic importance, however. Simply finding clonal aberrations strongly suggests that a lesion under study is neoplastic rather than reactive, and the general correlation that exists between malignancy grade and genetic complexity makes it highly unlikely that a karyotype with gross aneuploidy and multiple structural aberrations should derive from a benign tumor [124]. Whereas the significance of individual chromosome aberrations as well as patterns of aberrations for diagnostic purposes is well recognized, the prognostic impact of acquired genetic changes remains less well investigated. With the development of novel therapeutic approaches that target specific proteins or pathways of cellular signalling, it is likely that many more studies in this area will be seen. Tests for a number of genetic aberrations that may occur in solid tumors are already routinely done to stratify patients with respect to further treatment. To mention one example, gastrointestinal stromal tumors (GIST) typically show activating mutations in KIT or PDGFRA, two genes encoding tyrosine kinases; type and location of the activating mutations have been shown to have a strong impact on the response to treatment with kinase inhibitors [127]. A detailed

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Table 1.2  Examples of characteristic balanced chromosome rearrangements and their corresponding gene fusions in lymphomas and solid tumors Chromosome rearrangement Lymphomas t(2;5)(p23;q35) t(8;14)(q24;q32) t(14;18)(q32;q21) t(14;18)(q32;q21) Benign solid tumors t(1;2)(p13;q37) t(2;3)(q13;p25) t(3;12)(q28;q14) Malignant solid tumors t(X;1)(p11;q23) t(X;18)(p11;q11) t(2;3)(q13;p25) t(7;16)(q33;p11) t(7;17)(p15;q11) t(9;22)(q31;q12) t(11;19)(q21;p13) t(11;22)(p13;q12) t(11;22)(q24;q12) t(15;19)(q14;p13)

Gene fusion

Tumor type

NPM1/ALK IGH/MYC IGH/MALT1 IGH/BCL2

Anaplastic large T-cell lymphoma Burkitt lymphoma/leukemia Extranodal marginal zone B-cell lymphoma Follicular B-cell lymphoma, diffuse large B-cell lymphoma

COL6A3/CSF1 PAX8/PPARG HMGA2/LPP

Tenosynovial giant cell tumor Follicular thyroid adenoma Conventional lipoma

PRCC/TFE3 SS18/SSX1,SS18/SSX2,or SS18/SSX4 PAX8/PPARG FUS/CREB3L2 JAZF1/SUZ12 EWSR1/NR4A3 CRTC1/MAML2 EWSR1/WT1 EWSR1/FLI1 BRD4/NUTM1

Papillary renal cell carcinoma in children and adolescents Synovial sarcoma Follicular thyroid carcinoma Low-grade fibromyxoid sarcoma Endometrial stromal sarcoma of the uterus Soft tissue chondrosarcoma with abundant myxoid matrix in adults Mucoepidermoid carcinoma of the salivary glands Desmoplastic small round cell tumor Ewing family of tumors Poorly differentiated “midline” carcinoma

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Fig. 1.40  Myxoid liposarcoma with the specific t(12;16)(q13;p11), which results in the FUS/DDIT3 fusion gene

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Fig. 1.41  Aggressive, undifferentiated midline carcinoma with the specific t(15;19)(q14;p13), which results in the BRD4/NUT fusion gene

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Fig. 1.42  Parosteal osteosarcoma with a supernumerary ring chromosome as the sole aberration. This karyotypic feature separates parosteal osteosarcomas from high-grade osteosarcomas. Supernumerary ring

chromosomes may be found in a variety of other low-grade malignant mesenchymal tumors, such as atypical lipomatous tumor and dermatofibrosarcoma protuberans

1 Introduction

27

description of the spectrum of chromosome aberrations occurring in human neoplasms, as well as a detailed account of their significance for diagnostic and prognostic purposes, can be found in [128]. The principles for handling FNA samples for cell culturing and cytogenetic analysis are similar to those for surgical biopsies. It is vital that the samples are handled under sterile conditions and that cell culturing is initiated as quickly as possible. Only a few large-scale attempts to obtain tumor karyotypes from FNA specimens have been made, and the general impression is that the results are clearly inferior to those on samples from surgical biopsies. For instance, in bone and soft tissue neoplasms, tumor-representative karyotypes were found in two-thirds of surgical biopsies but in only one-fourth of FNA specimens, strongly indicating that the number of cells obtained by FNA sampling is too small for reliable culturing [80]. Other drawbacks related to the fact that chromosome banding analysis requires living, dividing cells is that tumor cells of different lineages (e.g., epithelial and mesenchymal) require different culturing conditions and that some tumor cells must be cultured for many days before there is a sufficient number of mitotic cells to analyze [129]. None of the abovementioned shortcomings applies to directed genetic studies. For FISH analysis, which can be performed on smears as well as on cells that have been centrifuged and fixed on slides, typically around 100 interphase nuclei will suffice. By using locus-specific nucleotide probes, a variety of chromosome aberrations can be demonstrated: translocations and other structural rearrangements of specific genes, deletions, amplification, and aneuploidy (see Figs. 1.43, 1.44, 1.45, and 1.46) [130–132]. With few exceptions, the material should be sufficient for analyzing at least two to three different chromosome aberrations in a single-­ FNA specimen. A growing number of commercial probe sets

for clinically relevant chromosome aberrations are now available and should be used whenever possible. Various PCR approaches (e.g., for fusion genes, specific mutations, allelic imbalances, or gene expression levels) can also be used with good results on FNA specimens [133–136]. Compared to FISH, the PCR technologies are more sensitive, but this is also a potential drawback; high sensitivity also means that there is considerable risk of false-positive results due to contamination. Furthermore, when using RNA as the starting material for reverse transcriptase PCR (RT-PCR), it should be kept in mind that RNA is more sensitive than DNA/nuclei to degradation, making the time span from sampling to analysis more important for RT-PCR than for FISH or genomic PCR. Thus, both RT-PCR and genomic PCR protocols should always include negative and positive controls (see Fig. 1.47). New genetic technologies that within the next few years may replace some of the current approaches include various array-based methods to identify chromosomal imbalances (see Fig. 1.48) and deep sequencing of DNA or RNA for mutation and gene fusion detection. The number of reported studies is still low, but preliminary data are promising [137, 138].

Fig. 1.43  FISH image showing amplification of the COAS1 (yellow) and COAS2 (red) genes in a high-grade leiomyosarcoma. (From Nilsson et al.; with permission) [125]

Fig. 1.44  Interphase FISH image showing hemizygous deletion of the TP53 gene (red) in three nuclei (arrows) from a colorectal carcinoma. Green and blue signals represent control probes for chromosome 17

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a

b Fig. 1.46  Interphase FISH analysis of a neuroblastoma. Relative overrepresentation of distal 17q is a common feature of neuroblastomas, here illustrated by three to four copies of the 17q signal (red) compared to the two signals from the control locus at 17p (green signal)

Fig. 1.45  Interphase FISH analysis of a Ewing sarcoma. Almost all Ewing sarcomas have structural chromosome rearrangements involving the EWSR1 gene, the most common (85% of the cases) being the t(11;22)(q24;q12) resulting in an EWSR1/FLI1 fusion gene. (a) The gene fusion can be detected in interphase nuclei using a dual fusion probe set, here with the probe for the EWSR1 gene in green and the probe for the FLI1 gene in red. The t(11;22) generates two fusion signals (arrows), one at each derivative chromosome. (b) By using a break-apart probe (BAP) only for the EWSR1 gene, all possible translocations and fusions involving the EWSR1 gene can be detected. The normal allele is seen as juxtaposed red and green signals, whereas the allele involved in the translocation is seen as split red and green signals. It should be emphasized that rearrangements of the EWSR1 gene are not specific for the Ewing family of tumors

Negative Control No RT

Negative Control No RNA

Positive Control Type 2

100 bp ladder

Fig. 1.47  RT-PCR using primers specific for the EWSR1 and FLI1 genes on RNA extracted from an FNA sample from a Ewing sarcoma. The analysis reveals a type 1 fusion transcript (exon 7 of EWSR1 fused with exon 6 of FLI1) in the patient sample

Positive Control Type 1

29 Patient Sample Type 1

1 Introduction

300bp

200bp

Fig. 1.48  Single nucleotide polymorphism (SNP) array analysis of a aggressive neuroblastomas, e.g., loss of parts of chromosome arm 1p, neuroblastoma. Copy number probes (red, upper part) show a near-­ amplification of the MYCN locus in 2p, and loss of distal 11q. The cordiploid karyotype with chromosomal imbalances that are typical for responding changes in allele status are shown below in grey

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H. A. Domanski and F. Mertens

Electron Microscopy

Advantages and Limitations of FNAC

Ultrastructural examination of aspiration smears in daily diagnostic work has been replaced to a large extent by other techniques, such as IC, and molecular biologic techniques. The diagnostic value of electron microscopy (EM) is still important [139–141] and is especially valuable in the differentiation of epithelial from mesenchymal neoplasms and for determining their specific histogenesis such as neuroendocrine, melanocytic, myogenic, neurogenic, or fibroblastic/myofibroblastic [142–145]. EM is also a valuable diagnostic adjunct in the evaluation of extracellular or intracellular deposits such as glycogen, amyloid [146], and osteoid [147]. Although electron microscopy cannot identify all cell types, it can reveal certain differentiated structural features and clarify the origin of cells that lack definite signs of differentiation by light microscopy. For example, specific ultrastructural hallmarks for a number of neoplasms (e.g., Birbeck granule in Langerhans cell histiocytosis; premelanosomes in melanoma and clear cell sarcoma; Weibel-Palade bodies in vascular tumors; lipoblasts in liposarcomas) can be of great diagnostic help. It is important to point out, however, that FNAC ultrastructural findings in routine diagnostic work should always be correlated to conventional smears and that interpretation must be made within the context of the overall morphologic pattern of the smears. Compared with the preparation of cytological material for other ancillary techniques, the processing of aspirates for EM is more time-consuming. The results of electron microscopy in aspirated cells and cell fragments compare favorably with results seen in biopsy material.

Fine-needle aspiration (FNA) is an outpatient procedure compared to most cases of incisional/excisional biopsy which are procedures that frequently require an operation room as well as medical staff and all the resources necessary to perform surgery. In addition, most open biopsies require general anesthesia, and there is a risk of complications connected to these procedures. Compared to open biopsy, the FNA procedure allows easy and quick sampling and provides adequate diagnostic specimens in most cases, using minimal resources with negligible risk of serious complications. General or local anesthesia is seldom necessary in sampling for FNA, and the procedure is well tolerated by patients. With thin needles and repeated aspirations, it is ­usually easy to sample material from different parts of large masses and thereby elucidate possible tumor heterogeneity. In rapid on-site evaluation (ROSE) situations, rapid staining with Diff-Quik or rapid hematoxylin (H&E) makes it possible to assess the adequacy of aspiration smears while the patient waits. In such situations, additional aspirations can be performed to obtain diagnostic smears or more material from the lesion for ancillary studies. In many settings, the accuracy of FNAC in distinguishing benign from malignant neoplasm and reactive/inflammatory conditions has been shown to be comparable to that of surgical biopsies, while its accuracy in establishing a specific subtype diagnosis is often inferior to surgical biopsies in some organs. One major disadvantage of FNAC, however, is the occasional difficulty in obtaining sufficient material for ancillary studies. Another disadvantage is the inherent lack of histological architecture in most aspirates. Tumor tissue architecture is generally best evaluated in surgical biopsy or core needle biopsy (CNB) samples. In FNA samples, however, architecture can also be evaluated in the microbiopsies that can be seen in cell blocks (see Fig. 1.49). Though rapid staining and preliminary reporting is more conveniently applied to aspiration smears, imprint preparations from CNB and cryostat sections from open biopsy specimen can be also evaluated quickly. Frozen section procedures are, however, more complicated, and it has to be pointed out that in technically satisfactory FNA smears, cytomorphology is usually superior to that seen in core needle and incisional biopsy specimens when these are prepared as frozen sections. An additional advantage of FNA is that it is both easy to perform and easy to learn. A brief comparison of major advantages and disadvantages of different biopsy techniques is presented in Table 1.3.

FCM Immunophenotyping FCM immunophenotyping has been used for many years as a diagnostic complement of FNAC in cases of suspected non-Hodgkin’s lymphoma. FCM is most important in the differential diagnosis of indolent B-cell lymphomas and reactive lymphadenitis as well as in rendering the exact lymphoma subtype from FNA material. More about this subject is presented in the chapter on lymph node FNAC (see Chap. 9).

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a

b

c

d

Fig. 1.49  FNA smears from Warthin’s tumor (a) and oncocytoma (b) of the parotid gland (MGG). In the absence of lymphocytic infiltration in FNA smears from Warthin’s tumor, it can be extremely difficult to distinguish these two neoplasms. Compared with cell block, this

distinction is easy since the architecture of tissue fragments obtained by FNA is highly diagnostic for Warthin’s tumor (c) (H&E) and oncocytoma (d) (H&E)

Table 1.3  Comparison of fine-needle aspiration cytology with core needle biopsy and open biopsy Characteristics Techniques

Fine-needle aspiration cytology Easy procedure (to perform and learn)

Core needle biopsy Easy procedure (to perform and learn)

Risk of complications Interference with subsequent treatment Speed of preparation Availability of tissue architecture Availability of ancillary techniques Accuracy in differentiating benign vs. malignant Accuracy in malignancy grading Accuracy of histologic subtyping Cost

Very low No

Low No

Open biopsy Complex procedure; most often requires general anesthesia and operating room/operating staff Higher than FNAC and CNB Yes

Fast Yes (cell block)b Yesb High

Slowa Yes Yes High

Slowa Yes Yes High

Low 30–75%c Low

High 75–90%c Low

High >95%c High

Can be speeded by touch preparation and frozen section Depending on quality and quantity of specimen obtained c See Kilpatrick [148] a

b

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Pitfalls, Risks, and Complications

H. A. Domanski and F. Mertens

perform aspirations should be trained in FNA clinics in the technical aspects and must receive immediate feedback on their perFNAC in the routine evaluation of tumor masses requires an formance. For optimal results, close cooperation between understanding of the potentials and limitations of the method. clinicians and the cytopathologist (ROSE) is necessary. The final FNAC diagnosis should be correlated to the diagThe histologic subtyping of neoplasms via FNA smears, nostic level necessary for treatment initiation and/or plan- in a manner similar to what is done in histopathology, is ning of the further diagnostic work-up. more reliable when dealing with well-differentiated tumors The technical problems of FNA techniques often relate to showing specific diagnostic cytomorphologic and immunoinsufficient cytological specimens, which can result from cytochemical criteria. Definitive diagnosis can often be different causes: obtained from FNA smears complemented by ancillary techniques in lesions where cytological diagnostic criteria have 1. The lesion can be missed altogether by the aspirator, and been well characterized (see Fig. 1.52). cells can be aspirated from the tissue surrounding the Conversely, poorly differentiated neoplasms usually replesion. Misinterpretation of reactive changes in the tissue resent a difficult diagnostic group due to a lack of distinctive surrounding a lesion may result in a wrong diagnosis. morphological criteria. In such lesions, definitive diagnosis These problems occur most often when small and deep-­ can be difficult to render from aspiration smears despite the seated lesions are needled. The person performing the use of ancillary techniques and access to clinical/radioaspiration should be experienced enough to evaluate graphic data. Where definitive diagnosis is difficult to render whether the material obtained might be consistent with from FNAC, differential diagnostic possibilities should be the lesion in question. This evaluation is often based, at suggested and core biopsy/surgical biopsy considered as the least partly, on clinical data and on the findings of next diagnostic procedure (see Figs. 1.53 and 1.54). ­palpation. Ultrasound or CT guidance may be of help in With regard to complications occurring during or after the aspiration of small and deep-seated lesions. FNA procedures, the question of tumor cell spread in the 2. If the aspirated mass is cystic, necrotic, or hemorrhagic, needle tract is often brought up [149–155]. The incidence of representative diagnostic areas may be difficult to sample this event is, in fact, exceedingly low [156–158]. adequately, and guided FNA may be required (see Nevertheless, in cases where the risk for tumor cells spread Fig.  1.50). Benign vascular neoplasms most often yield exists, for example, in the aspiration of deep-seated masses predominantly blood and only a few tissue cells. suspected as being sarcomas, tattooing the skin at the aspi 3. Another difficulty is obtaining a sufficient number of ration site may be a good precaution so that the needle tract cells from lesions with an abundance of collagenous or can be removed if surgery is required (see Figs.  1.55 and hyalinized matrix from which cells can be very hard to 1.56). aspirate (see Fig. 1.51). Serious complications such as major hemorrhage, septicemia, acute pancreatitis, rupture of cystic tumors, bile periWhen the cytopathologist performs both the aspiration and tonitis, etc. are extremely rare [159–163]. Other complications examines the smears, the technical considerations and occa- of the aspiration procedure are minor and include hematosional difficulties are minimized. Non-palpable swellings mas and localized tenderness. Infection due to fine-needle should be aspirated by a radiologist with the aid of imaging aspiration can be easily avoided using elementary antiseptic techniques and in conjunction with a cytopathologist. In addi- methods such as cleansing the skin with alcohol or iodine tion, radiologists and other specialists who swabs.

1 Introduction

a

Fig. 1.50  FNA of cystic schwannoma showing clusters of epithelioid/ histiocytoid cells with admixture of dark pigment. The FNA specimen does not allow any distinctive diagnosis (a) (MGG). Cell block of aspiration material (b). Fragment of the tumor tissue with spindle cells and

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epithelioid cells positive for S-100; these morphological features together with radiological findings allow correct diagnosis of schwannoma

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Fig. 1.51  Aspiration smears from an extra-abdominal desmoid showing hyalinized collagenous matrix and a few tumor cells (a) (H&E). In such cases, CNB sampling procedures can be superior to FNA (b) (H&E)

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Fig. 1.52  MRI (a) showing soft tissue mass in the right elbow: FNA of the mass: alcohol-fixed, HTX-stained (b) and air-dried, MGG-stained (c) smears indicative of Hodgkin’s lymphoma. HTX-stained (d) and

immunostained for CD30 (e) sections from a cell block confirm diagnosis of Hodgkin’s lymphoma

1 Introduction

a

Fig. 1.53  FNA smears from ischemic fasciitis showing a cluster of spindle cells, some with pleomorphic hyperchromatic nuclei suggestive of malignancy (a) (H&E). Core needle biopsy performed simultane-

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ously with FNA facilitated characteristic “zonal” architecture of ischemic fasciitis (b) (H&E)

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Fig. 1.54  Inflammatory mammary carcinoma (a). FNA specimen consists of some droplets of fluid containing a few highly atypical cells (b) (MGG). Subsequent core biopsy discloses pleomorphic carcinoma cells growing in the lymphatic vessels (c) (H&E)

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Fig. 1.55  Patient with a huge recurrence of dermatofibroma protuberans in the back. Tattoo with sterile ink of the needle insertion point helps an orthopedic surgeon remove the needle track

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Fig. 1.56  Tattoo point after aspiration from a subcutaneous leiomyosarcoma (a). Operative specimen showing persistent tattoo in the central area of the specimen (b)

1 Introduction

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H. A. Domanski and F. Mertens expression in breast carcinomas: comparison on cell block, needle-­ core, and tissue block preparations. Cancer. 2009;117(4):279–88. 122. Wu M, Kafanas A, Gan L, Kohtz DS, Zhang L, Genden E, et al. Feasibility of immunocytochemical detection of tumor markers (XIAP, phosphohistone H1 and p63) in FNA cellblock samples from head and neck squamous cell carcinoma. Diagn Cytopathol. 2008;36(11):797–800. 123. Chandan VS, Faquin WC, Wilbur DC, Khurana KK.  The role of immunolocalization of CD57 and GLUT-1  in cell blocks in fine-needle aspiration diagnosis of papillary thyroid carcinoma. Cancer. 2006;108(5):331–6. 124. Mitelman F, Johansson B, Mertens F. Mitelman database of chromosome aberrations and gene fusions in cancer 2017 [Available from: http://cgap.nci.nih.gov/Chromosomes/Mitelman]. 125. Heim S, Mitelman F. Nonrandom chromosome abnormalities in cancer: an overview. In: Heim S, Mitelman F, editors. Cancer cytogenetics: chromosomal and molecular genetic abberrations of tumor cells. 4th ed. Chichester: Wiley Blackwell; 2015. p. 26–41. 126. Mitelman F, Johansson B, Mertens F.  The impact of translocations and gene fusions on cancer causation. Nat Rev Cancer. 2007;7(4):233–45. 127. Antonescu CR.  The GIST paradigm: lessons for other kinase-­ driven cancers. J Pathol. 2011;223(2):251–61. 128. Heim S, Mitelman F.  Cancer cytogenetics : chromosomal and molecular genetic aberrations of tumor cells. 4th ed. Chichester: Wiley Blackwell; 2015. p. ix. 632 pages p. 129. Swansbury J.  Introduction. Cancer cytogenetics: methods and protocols. Methods Mol Biol. 2003;220:1–8. 130. Bofin AM, Ytterhus B, Martin C, O’Leary JJ, Hagmar BM.  Detection and quantitation of HER-2 gene amplification and protein expression in breast carcinoma. Am J Clin Pathol. 2004;122(1):110–9. 131. Lin F, Shen T, Prichard JW. Detection of Her-2/neu oncogene in breast carcinoma by chromogenic in situ hybridization in cytologic specimens. Diagn Cytopathol. 2005;33(6):376–80. 132. Zhang S, Abreo F, Lowery-Nordberg M, Veillon DM, Cotelingam JD. The role of fluorescence in situ hybridization and polymerase chain reaction in the diagnosis and classification of lymphoproliferative disorders on fine-needle aspiration. Cancer Cytopathol. 2010;118(2):105–12. 133. Khalid A, Nodit L, Zahid M, Bauer K, Brody D, Finkelstein SD, et  al. Endoscopic ultrasound fine needle aspirate DNA analysis to differentiate malignant and benign pancreatic masses. Am J Gastroenterol. 2006;101(11):2493–500. 134. Smith GD, Chadwick BE, Willmore-Payne C, Bentz JS. Detection of epidermal growth factor receptor gene mutations in cytology specimens from patients with non-small cell lung cancer ­utilising high-resolution melting amplicon analysis. J Clin Pathol. 2008;61(4):487–93. 135. Algeciras-Schimnich A, Milosevic D, McIver B, Flynn H, Reddi HV, Eberhardt NL, et al. Evaluation of the PAX8/PPARG translocation in follicular thyroid cancer with a 4-color reverse-­ transcription PCR assay and automated high-resolution fragment analysis. Clin Chem. 2010;56(3):391–8. 136. Lappinga PJ, Kip NS, Jin L, Lloyd RV, Henry MR, Zhang J, et al. HMGA2 gene expression analysis performed on cytologic smears to distinguish benign from malignant thyroid nodules. Cancer Cytopathol. 2010;118(5):287–97. 137. De Biase I. Commentary. Clin Chem. 2016;62(8):1064. 138. Kameta E, Sugimori K, Kaneko T, Ishii T, Miwa H, Sato T, et  al. Diagnosis of pancreatic lesions collected by endoscopic ultrasound-guided fine-needle aspiration using next-generation sequencing. Oncol Lett. 2016;12(5):3875–81. 139. Turbat-Herrera EA, Herrera GA. Electron microscopy renders the diagnostic capabilities of cytopathology more precise: an approach to everyday practice. Ultrastruct Pathol. 2005;29(6):475–82.

1 Introduction 140. Taccagni G, Cantaboni A, Dell'Antonio G, Vanzulli A, Del Maschio A.  Electron microscopy of fine needle aspiration biopsies of mediastinal and paramediastinal lesions. Acta Cytol. 1988;32(6):868–79. 141. Davidson DD, Conces DJ, Goheen MP, Clark SA.  Comparative ultrastructure of needle aspiration biopsy and surgical resection specimens of lung tumors. Ultrastruct Pathol. 1992;16(5): 505–19. 142. Neill JS, Silverman JF.  Electron microscopy of fine-needle aspiration biopsies of the mediastinum. Diagn Cytopathol. 1992;8(3):272–7. 143. Domanski HA. Elastic fibers in elastofibroma dorsi by fine-needle aspiration. Diagn Cytopathol. 2014;42(7):609–11. 144. Domanski HA, Akerman M, Rissler P, Gustafson P. Fine-needle aspiration of soft tissue leiomyosarcoma: an analysis of the most common cytologic findings and the value of ancillary techniques. Diagn Cytopathol. 2006;34(9):597–604. 145. Kindblom LG, Walaas L, Widehn S.  Ultrastructural studies in the preoperative cytologic diagnosis of soft tissue tumors. Semin Diagn Pathol. 1986;3(4):317–44. 146. Shidham VB, Hunt B, Jardeh SS, Barboi AC, Devata S, Hari P. Performing and processing FNA of anterior fat pad for amyloid. J Vis Exp. 2010(44):1747. 147. Walaas L, Kindblom LG. Light and electron microscopic examination of fine-needle aspirates in the preoperative diagnosis of osteogenic tumors: a study of 21 osteosarcomas and two osteoblastomas. Diagn Cytopathol. 1990;6(1):27–38. 148. Kilpatrick SR, editor. Diagnostic musculoskeletal surgical pathology. Clinicoradiologic and cytologic correlations. Philadelphia: Saunders; 2004. 149. Douville NJ, Bradford CR. Comparison of ultrasound-guided core biopsy versus fine-needle aspiration biopsy in the evaluation of salivary gland lesions. Head Neck. 2013;35(11):1657–61. 150. Pavan C, Parisi A, Girelli D. Recurrent needle-tract metastases of hepatocellular carcinoma following fine-needle aspiration. Intern Med J. 2007;37(2):134–6. 151. Ito Y, Asahi S, Matsuzuka F, Nakamura Y, Amino N, Miyauchi A.  Needle tract implantation of follicular neoplasm after fine-­ needle aspiration biopsy: report of a case. Thyroid Off J Am Thyroid Assoc. 2006;16(10):1059–62.

41 152. Ito Y, Tomoda C, Uruno T, Takamura Y, Miya A, Kobayashi K, et al. Needle tract implantation of papillary thyroid carcinoma after fine-needle aspiration biopsy. World J Surg. 2005;29(12):1544–9. 153. Slywotzky C, Maya M.  Needle tract seeding of transitional cell carcinoma following fine-needle aspiration of a renal mass. Abdom Imaging. 1994;19(2):174–6. 154. Yamada N, Shinzawa H, Ukai K, Wakabayashi H, Togashi H, Takahashi T, et  al. Subcutaneous seeding of small hepatocellular carcinoma after fine needle aspiration biopsy. J Gastroenterol Hepatol. 1993;8(2):195–8. 155. Tyagi R, Dey P. Needle tract seeding: an avoidable complication. Diagn Cytopathol. 2014;42(7):636–40. 156. Shields CL, Manquez ME, Ehya H, Mashayekhi A, Danzig CJ, Shields JA.  Fine-needle aspiration biopsy of iris tumors in 100 consecutive cases: technique and complications. Ophthalmology. 2006;113(11):2080–6. 157. Shah KS, Ethunandan M. Tumour seeding after fine-needle aspiration and core biopsy of the head and neck – a systematic review. Br J Oral Maxillofac Surg. 2016;54(3):260–5. 158. Roussel F, Dalion J, Benozio M. The risk of tumoral seeding in needle biopsies. Acta Cytol. 1989;33(6):936–9. 159. Smith EH. Complications of percutaneous abdominal fine-needle biopsy. Rev Radiol. 1991;178(1):253–8. 160. Gomez-Rubio M, Lopez-Cano A, Rendon P, Munoz-Benvenuty A, Macias M, Garre C, et  al. Safety and diagnostic accuracy of percutaneous ultrasound-guided biopsy of the spleen: a multicenter study. J Clin Ultrasound JCU. 2009;37(8):445–50. 161. Virgilio E, Mercantini P, Ferri M, Cunsolo G, Tarantino G, Cavallini M, et  al. Is EUS-FNA of solid-pseudopapillary neoplasms of the pancreas as a preoperative procedure really necessary and free of acceptable risks? Pancreatol Off J Int Assoc Pancreatol. 2014;14(6):536–8. 162. Vadvala HV, Furtado VF, Kambadakone A, Frenk NE, Mueller PR, Arellano RS. Image-guided percutaneous omental and mesenteric biopsy: assessment of technical success rate and diagnostic yield. J Vasc Interv Radiol JVIR. 2017;28(11):1569–76. 163. Marconi L, Dabestani S, Lam TB, Hofmann F, Stewart F, Norrie J, et  al. Systematic review and meta-analysis of diagnostic accuracy of percutaneous renal tumour biopsy. Eur Urol. 2016;69(4):660–73.

2

Image-Guided Fine-Needle Aspiration Cytology Mats Geijer and Henryk A. Domanski

Several factors may make image guidance necessary in fine-­ needle aspiration cytology (FNAC): A lesion may be diffuse and difficult to palpate; the cytopathologist may require image guidance for all deep-seated nonpalpable lesions; there may be sampling issues in a nonhomogeneous lesion with areas of different tissue characteristics (e.g., necrosis versus viable tumor or purely fatty areas versus contrast-­ enhancing areas in liposarcomas); or the lesion may be inaccessible behind bony structures [1, 2] or located inside bone [3–5]. In many cases, the cytologist may simply prefer to use image guidance for a more exact needle placement. In all cases, however, the first step should be a thorough and careful review of all pertinent imaging studies to select the appropriate guiding modality, to evaluate all possible access routes, and to select appropriate needles and devices to reach the lesion. At this stage, the patient should also be considered: Is there coagulopathy or is the patient on an anticoagulant, antiplatelet, or thrombolytic medication? Are there any other absolute or relative contraindications to the procedure or to any imaging modality (e.g., magnetic resonance imaging [MRI])? If needed, the coagulant status should be corrected to acceptable levels.

Imaging Modalities Several imaging modalities are available to assist in the best approach to the target lesion. The choice of imaging modality is dictated by several factors (see Table 2.1) such as personal preference, availability on site and available time slots, ease of performing the biopsy, procedural cost, and radiation dose to the examiner and patient. The use of fluoroscopy has waned in recent years due to the ease of performing ultrasound-­guided biopsies and the introduction of multidetector computed tomography (CT) scanners. Today, fluoroscopy-­guided FNAC is usually reserved for destructive bony lesions in easily accessible areas of the peripheral skeleton, for spinal biopsies using mostly biplane imaging, and for biopsy of high-contrast pulmonary lesions in the lower lung fields where breathing motion may render other modalities virtually useless. Instead, CT has come to replace fluoroscopy for almost all skeletal biopsies, for most lung biopsies, and for many deep-seated extra-abdominal biopsies. Ultrasound is frequently used for breast biopsies and for most intra-abdominal biopsies, due to both the excellent soft-tissue contrast and the ability to direct the biopsy needle from any direction. MRI-guided biopsies are still rare because the strong magnetic field requires special equipment. In breast biopsies, stereotactic biopsies are frequently used in addition to ultrasound guidance. Table 2.1  Available imaging methods for biopsy guidance: their use and limitations Modality Fluoroscopy CT

M. Geijer (*) Center for Medical Imaging and Physiology, Skåne University Hospital, Lund, Sweden e-mail: [email protected] H. A. Domanski Department of Pathology, Skåne University Hospital, Lund, Sweden e-mail: [email protected]

Ultrasound

MRI Stereotactic biopsy

Objective Bony lesions in extremities, spinal biopsies, lung biopsies Bony and soft-tissue lesions in musculoskeletal system and chest Abdominal biopsies, subcutaneous musculoskeletal soft-tissue biopsies – Breast lesions. Gives direction and depth

© Springer International Publishing AG, part of Springer Nature 2019 H. A. Domanski (ed.), Atlas of Fine Needle Aspiration Cytology, https://doi.org/10.1007/978-3-319-76980-6_2

Disadvantages Radiation Radiation

Operator dependent. Not for bony lesions Magnetic field

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M. Geijer and H. A. Domanski

Anatomical Regions

Thyroid

Breast

Thyroid gland FNAC may be improved by image guidance [17–19]. In one study, 215 patients with palpable thyroid nodules between 10 and 25  mm diameter were biopsied using both palpation-guided FNAC and ultrasound-guided FNAC by the same examiner, and the samples were evaluated by the same cytologist [17]. There were significantly more cases with inadequate material in the palpation-guided group, especially for small nodules (10–15  mm). The increase in cost for the image-guided procedure of about $20 (US) was deemed acceptable, especially considering the improved sampling from small lesions [17]. The specialty of the person doing the ultrasound-guided FNAC seems less important. One study reported similar rates for diagnostic aspirations by radiologists and surgeons but with a significantly quicker turnaround time for a cytological diagnosis for the surgeon [20]. Ultrasound-guided biopsy should be directed to the periphery of the lesion in solid lesions and to solid areas of cystic lesions [19]. Both 23- and 27-gauge needles may be used. A study evaluating the yield from 23to 27-gauge needles in ultrasound-guided FNAC found no significant difference in adequacy of sampling between the two needle sizes [18]. It was recommended, however, to use both sizes because the number of dry taps was lower with the larger needle, but the quality of samples was higher using the smaller needle [18].

Image-guided breast core biopsies have been accepted as an alternative to open surgical biopsies in nonpalpable lesions, detected at mammography [6]. Biopsies may be performed using ultrasound or MRI for image guidance or by stereotactic-­guided biopsies [6]. Of almost 150,000 breast biopsies performed in the USA in 2004, 86% were performed with image guidance [7]. Long-term follow-up has shown that stereotactic-guided biopsies have the same accuracy as open surgical biopsies [8, 9]. Recently published meta-­analyses report that FNAC is an accurate biopsy when using stringent criteria, but that further invasive procedures are necessary if the sampling is unsatisfactory [10]. Compared with core biopsy, both are reported to have good clinical performance, sometimes with a slightly higher sensitivity for core biopsy, and both can be considered as the first choice for evaluation of a suspicious breast lesion [11]. The advantage of ultrasound guidance is that it not only allows for cytological evaluation by FNA and histologic tissue sampling by core biopsy but also for image characterization of breast lesions and locoregional lymph nodes [12], where the ultrasound examination may be at least as important as the image-­guided biopsy for some patients [13]. In a report from a European discussion forum in 2007, it was reported that most participating countries used a triple assessment approach (i.e., a clinical, imaging, and pathological evaluation) to ensure a definitive pretreatment diagnosis [14]. FNAC was used as the first-line pathological investigation, particularly in symptomatic patients and in some screening populations. Core biopsy was used as a first biopsy modality in some screening populations in, for example, the UK.  Another review reported FNAC as the most common method for evaluation of breast lesions in developing countries mainly for reasons of cost, whereas core needle biopsy is the initial method of choice in developed and Western countries [15]. Breast MRI has evolved in the past decades into an imaging modality well suited for breast imaging. Imaging is usually performed in the prone position, with the breasts falling dependently from the patient, and using special coils. Most lesions will enhance after intravenous contrast administration, making imageguided biopsy possible. The limitations are mainly that biopsy is only possible from the lateral side in most systems, that the time duration for biopsy is short due to washout of contrast from the tissues, and that the biopsied sample cannot be imaged by MRI, as living tissue is needed for MRI [16]. The cost of breast MRI and breast MRI-guided biopsy is high.

Salivary Glands FNAC has for many years been used as the initial biopsy in salivary gland lesions [21]. The literature reports highly variable results with a sensitivity for malignancy in some centers reaching 70–80% but with a nondiagnostic yield as high as 56% in some reports [21]. As in most other fields, the diagnostic yield is reported to increase with image guidance [21]. Ultrasound-guided core biopsy has been reported having higher sensitivity and specificity than non-guided FNAC [22] and should be at the forefront of salivary gland evaluation [22, 23].

Lung With the introduction of multidetector CT and CT fluoroscopy, biopsy of lung nodules has become much easier. In many centers, a CT-guided biopsy is preferred for most lesions because CT gives excellent anatomical detail regarding surrounding tissue such as mediastinum, nerves, and vessels. The major complication, pneumothorax, has been

2  Image-Guided Fine-Needle Aspiration Cytology

reported to be minimized by using fine needles, usually 21 or 22 gauge [24]. On the other hand, the incidence of pneumothorax has been reported to be similar for single-needle FNA, coaxial-needle FNA, and core biopsy [25]. Diagnostic success is higher in lesions close to the pleura and in lesions larger than 1 cm in diameter, in which ultrasound guidance can also be used [26]. With deeper lesions, the risk of pneumothorax also increases, especially if a fissure is traversed, which increases the number of pleural passages. CT guidance requires patients to be able to hold their breath for the time necessary for biopsy. This is a concern especially in the lower lung fields because the basilar parts of the lungs move the most with breathing. This is not of such great concern with fluoroscopy-guided biopsies, which may in these cases be preferred instead. For mediastinal masses, CT guidance can in many cases be applied, e.g. by using a posterior paravertebral approach under CT guidance [27] to approach subcarinal lymph nodes. Also, ultrasound-assisted transthoracic fine-needle aspirations with on-site evaluation followed by core biopsies where indicated are safe and have a high diagnostic yield [28]. Although ultrasound is cheaper than CT and is free from ionizing radiation, lesions obscured by aerated lung, small deep-seated lesions, and cavitary lesions cannot be biopsied with the use of ultrasound [29]. The precision of ultrasound guidance and CT guidance was reported similar in a study evaluating all lesions in the lung, thorax, and thoracic wall [29]. Prior pneumonectomy and other instances of a single lung are possibly the only absolute specific contraindication to image-guided lung biopsy [30]. Among relative contraindications to lung biopsy are suspected hydatid cyst or vascular malformation, significant pulmonary arterial hypertension, or severe obstructive lung disease (forced expiratory volume [FEV]1 < 1.1) [30]. Besides pneumothorax, hemoptysis and hemorrhage occur in up to 10% of patients after lung biopsy and are the major complications after biopsy. Needles larger than 18 gauge and cutting needles are associated with an increased risk for hemorrhage [30].

Abdomen For most abdominal FNAC procedures, ultrasound guidance is the method of choice. Usually, a needle guide is used on the transducer. The method is quick, without radiation to the patient or examiner, and the complete mobility of the ultrasound probe will in most cases give clear access to the lesion, and the ultrasound probe can be used to displace the bowels. Thus, the liver [31], pancreas, kidneys, ovaries [32, 33], lymph nodes [34, 35], and spleen [36] are usually biopsied using ultrasound [37]. Deep-seated lymph nodes, both

45

in the thorax and abdomen, may be biopsied under ultrasound guidance [34]. In some cases with hard-to-reach lesions, CT is preferred [38]. CT may also often be the modality of choice in biopsy of the adrenal glands [39] or the spleen [40]. For hard-to-reach masses in or around the pancreatic head, FNAC using a posterior transcaval approach with CT guidance has been described [41]. Serious complications of abdominal image-guided FNAC are comparatively rare, considering the longer access routes and larger amounts of tissue traversed compared with more superficial FNAC [37, 42, 43].

Musculoskeletal Imaging in conjunction with FNAC may in special cases be diagnostic, obviating the need for a surgical biopsy [44]. For soft-tissue lesions, CT is the best guidance modality, giving excellent soft-tissue contrast to locate the target. Depending on the equipment used, the procedure may differ in detail, but the main procedure commonly involves acquiring a scout scan over the body part to be biopsied, after which a diagnostic CT examination is performed to locate the lesion. The section most optimal for biopsy is selected, and the angle and depth of the approach is determined. After antiseptic cleaning and local anesthesia, the lesion is biopsied. Preferably a coaxial technique is used to avoid multiple skin passages and to facilitate and expedite the procedure, especially if the approach is difficult. If a trocar is used for the coaxial approach, a core biopsy may be performed through the same trocar. The downside of using CT is that an oblique approach in the craniocaudal direction is difficult because, in most CT scanners, the CT gantry must remain perpendicular to the long axis of the body of the patient during the procedure. Superficial lesions or deep-seated lesions close to the muscular fascia may be biopsied with ultrasound guidance. Bony lesions are approached in the same way as soft-tissue lesions using CT, with the exception that a trocar with a drill in most cases is necessary to penetrate the cortical bone. Osteolytic lesions are then easily biopsied with FNAC.  Densely sclerotic lesions may be very difficult to biopsy with FNAC [45]. A high rate of accuracy and clinical usefulness has been reported for image-guided needle biopsies of musculoskeletal lesions [46], where the authors emphasized the importance and benefit of referring patients with a possible musculoskeletal tumor to a specialized referral center. Mehrotra et  al. [47] recommend the use of image-guided FNAC as the first diagnostic method for skeletal lesions of unknown origin. However, image-guided core biopsies have been reported to have a slightly higher accuracy than FNAC [48, 49].

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Special Techniques

Safety

To obtain samples from the bone, the cortical bone must be penetrated by drilling unless there is cortical destruction or softening from the lesion in question. There are several alternatives on the market using a cannula with a trocar and/ or a drill [3]. After cortical penetration, FNAC samples may be acquired from the target lesion using a coaxial technique. The technique can even be used to traverse intact bone to biopsy a soft-tissue lesion in an otherwise inaccessible location [1, 2]. Other ways to reach “inaccessible” lesions include a selection of the proper imaging modality and to consider alternate biopsy routes such as transgluteal, transvaginal, and other transorgan approaches [25, 28]. Another approach is to consider an out-of-plane approach in the CT-guided biopsy, whereby the target is reached by a craniad or caudad approach toward the selected slice. The CT gantry may also be angled to provide an alternate path [37]. Special needles, designed to better reflect the sound waves, may be used for ultrasound biopsies. When losing track of the needle in an ultrasound procedure, it is best to keep the transducer locked on the target lesion and redirect the needle. The next best approach is to keep the needle still and locate it by slightly redirecting the transducers. One should never manipulate the transducer and needle simultaneously. Biopsy needles will deviate in harder tissues, with less deviation with thinner needles. The degree of deviation for core biopsy using 2.1 mm needles has been evaluated using butter blocks at different temperatures to simulate tissue of different hardness in the human breast [38].

With deep-seated intrapulmonary, intra-abdominal, or musculoskeletal lesions, post-biopsy bleeding becomes more important because hemorrhages are harder or virtually impossible to control. If the patient is taking acetylic acid, bleeding time should be checked [17]. If prolonged, the procedure may have to be postponed until the bleeding time has been normalized to avoid the risk of bleeding complications. For patients on anticoagulants such as warfarin, the prothrombin time (PT) or the international normalized ratio (INR) should be checked before the procedure and, with help of the treating physician, the PT should be reduced to a manageable level. This level depends on which tissue will be sampled, the depth and accessibility for manual compression, the surrounding anatomical structures, and possible complications. Besides a bleeding diathesis or anticoagulant therapy, inability of the patient to cooperate is also a relative contraindication to needle biopsy [20], also depending on the situation. In general, FNAC is a safe procedure with few complications reported [39].

Results In order to obtain optimal results, it is probably irrelevant whether a radiologist, cytologist, or another physician performs the actual biopsy [20]. The radiologist should have adequate training in aspiration technique. The most important factor achieving a high success rate is probably the assistance of on-site cytopathology to help with smearing the samples and providing a preliminary evaluation of the FNA quality [50, 51]. When performing image-guided FNAC, it is in many cases reasonable to perform a core biopsy simultaneously [5, 52].

2  Image-Guided Fine-Needle Aspiration Cytology

Illustrative Cases Case 1 A 63-year-old woman with a history of breast carcinoma 10  years ago and squamous cell carcinoma in the gingiva 2 months ago. A single suspect metastatic lesion in the sacral bone was detected with positron emission tomography (PET)/CT.  A single focus of hypermetabolism in the right ischial tuberosity is shown on a coronal section (arrow; see Fig. 2.1). Diagnostic CT before biopsy confirmed the lesion

47

shown as a diffusely sclerotic area in the ischial tuberosity (arrows; see Fig. 2.2). In the prone position, the lesion was biopsied through a trocar to obtain multiple fine-needle samples as well as a core biopsy (see Fig. 2.3). The tip of the drill can be seen in the medullary bone, whereas the trocar is anchored in the cortical bone. FNAC of the sacral lesion shows clusters of the atypical epithelial cells consistent with metastatic carcinoma from the breast (see Fig. 2.4a). Positive results of immunostaining with antibodies against estrogen receptor (ER) confirmed the diagnosis (see Fig. 2.4b).

Fig. 2.2  Metastatic carcinoma from the breast. Diagnostic CT before biopsy shows a diffusely sclerotic area in the ischial tuberosity (arrows)

Fig. 2.1  Metastatic carcinoma from the breast. PET/CT: a single focus of hypermetabolism in the right ischial tuberosity on a coronal section (arrow)

Fig. 2.3  Metastatic carcinoma from the breast. In the prone position, the tip of the drill can be seen in the medullary bone, while the trocar is anchored in the cortical bone

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a

M. Geijer and H. A. Domanski

b

Fig. 2.4  Metastatic carcinoma from the breast. (a) FNAC showing clusters of the malignant epithelial cells (May-Grünwald Giemsa [MGG]). (b) Positive results of immunostaining with antibodies against ER confirmed the diagnosis (ThinPrep; Hologic; Bedford, MA, USA)

2  Image-Guided Fine-Needle Aspiration Cytology

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Case 2 A 13-year-old girl with a 3-month history of left hip pain. Radiography showed an irregular bone structure in the left supra-acetabular area. MRI showed an osteolytic tumor in the supra-acetabular area (arrow) with soft-tissue extension through the quadrilateral plate into the pelvis (arrowheads), with suspicion of Ewing sarcoma (see Fig. 2.5). A diagnostic CT before biopsy confirmed the osteolytic destruction (arrows; see Fig.  2.6a). The soft-tissue extension can be appreciated also with CT (arrowheads; see Fig. 2.6b). With the patient in the prone position, the lesion was biopsied through a trocar to obtain multiple fine-needle samples and a core biopsy (see Fig. 2.6b). FNA smears were hypercellular (see Fig. 2.7a). In high-power view, there was a mixture of small dark cells and larger light cells with occasionally moderate to abundant cytoplasm with characteristic vacuolization/empty spaces after glycogen (see Fig. 2.7b). Ancillary tests confirmed the diagnosis of Ewing sarcoma. a

Fig. 2.5  Ewing sarcoma. MRI showing an osteolytic tumor in the supra-acetabular area (arrow) with soft-tissue extension through the quadrilateral plate into the pelvis (arrowheads)

b

Fig. 2.6  Ewing sarcoma. (a) Diagnostic CT before biopsy confirmed the osteolytic destruction (arrows). (b) The soft-tissue extension can be appreciated also with CT (arrowheads). In the prone position, the lesion was biopsied through a trocar

a

b

Fig. 2.7  Ewing sarcoma. (a) Hypercellular FNA smears. (b) In high-power view, a mixture of small dark cells and larger light cells with moderate to abundant cytoplasm and characteristic vacuolization/empty spaces after glycogen (MGG)

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M. Geijer and H. A. Domanski

Case 3 A 72-year-old overweight man with renal failure and chronic heart failure on antithrombotic treatment with warfarin for previous left ventricle thrombus. Chest radiography had revealed a suspicious lesion in the left lung, which was ­confirmed with PET/CT (see Fig. 2.8a). The same PET/CT also revealed a single suspect metastasis in the right acetabular medial wall (see Fig. 2.8b). Because the patient’s coagulation status could not be restored to normal, it was decided to first biopsy the suspect metastasis instead of the presumed primary lung tumor. The patient’s general poor health made a biopsy from posterior using a prone position unsuitable. From anterior, the direct approach to the tumor (arrow) was obstructed by the femoral vessels (star) and the funicle (rhombus; see Fig. 2.9). By using an indirect lateral oblique Fig. 2.8 Metastatic adenocarcinoma from the lung. (a) PET/CT showing a suspicious lesion in the left lung. (b) A single suspect metastasis in the right acetabular medial wall

a

Fig. 2.9  Metastatic adenocarcinoma from the lung. Diagnostic CT before biopsy. The direct approach to the metastasis (arrow) was obstructed by the femoral vessels (star) and the funicle (rhombus)

approach, a 0.9  mm needle was anchored in the pectineus muscle between the funicle and the vessels (see Fig. 2.10a). The needle was then redirected toward the lesion in the acetabular wall and advanced to its full length (9  cm; see Fig. 2.10b). Coaxial fine-needle aspirations were performed using 15  cm long 0.7  mm needles (arrow indicates outer needle, arrowhead indicates biopsy needle; see Fig. 2.10c). Core biopsy was not performed due to the poor coagulant status. FNA smears were hypercellular showing multiple cohesive sheets and papillary clusters (see Fig.  2.11a) of slight-to-moderate pleomorphic tumor cells with abundant cytoplasm, round nuclei, and inconspicuous nucleoli (see Fig.  2.11b). Positive results of immunostaining with antibody to TTF-1 confirmed the diagnosis of metastatic adenocarcinoma from the lung. b

2  Image-Guided Fine-Needle Aspiration Cytology

a

51

a

b b

c

c

Fig. 2.10  Metastatic adenocarcinoma from the lung. (a) By using an indirect lateral oblique approach, a 0.9 mm needle was anchored in the pectineus muscle between the funicle and the vessels. (b) The needle was then redirected toward the lesion in the acetabular wall and advanced to its full length. (c) Coaxial FNA was performed using 15-cm-long 0.7  mm needles (arrow outer needle, arrowhead biopsy needle) Fig. 2.11  Metastatic adenocarcinoma from the lungs. (a) Hypercellular FNA smears showing multiple cohesive sheets and papillary clusters of (b) slight-to-moderate pleomorphic tumor cells with abundant cytoplasm, round nuclei, and inconspicuous nucleoli (H&E). (c) Positive results of immunostaining with antibody to TTF-1 confirmed the diagnosis of metastatic adenocarcinoma from the lung (ThinPrep; Hologic; Bedford, MA, USA)

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Case 4 A 20-year-old male with an almost 1-year history of low back pain and loss of the left patellar reflex. MRI (see Fig. 2.12a) showed a large tumor destroying a large part of the S1 and S2 vertebral bodies and most of the left lateral mass, with extension into the anterior and posterior soft ­tissues, as well as the spinal canal (see Fig. 2.12b). CT-guided biopsy in the prone position allowed for exact needle placement, thus avoiding the presumed location of the spinal canal and sacral nerve roots (see Fig.  2.13). FNA smears were

M. Geijer and H. A. Domanski

hypercellular and showed clusters of uniform spindle cells embedded within a collagenous and slightly myxoid matrix (see Fig.  2.14a). Nuclear palisading and cell-poor areas in the cell clusters consisted of Verocay bodies (see Fig. 2.14b). Alcohol-fixed smears showed cohesive clusters of relatively uniformed spindle cells with indistinct cytoplasm and elongated nuclei, some with pointed ends (see Fig. 2.14c). Tumor cells expressed diffuse S-100 positivity (see Fig.  2.14d). FNA diagnosis was benign schwannoma, confirmed by examination of a surgical specimen from the removed mass.

a

b

Fig. 2.12  Schwannoma. (a) MRI showing a large tumor destroying a large part of the S1 and S2 vertebral bodies and most of the left lateral mass. (b) Tumor extension into the anterior and posterior soft tissues, as well as the spinal canal

Fig. 2.13  Schwannoma. CT-guided biopsy in the prone position allowing for exact needle placement, thus avoiding the presumed location of the spinal canal and sacral nerve roots

2  Image-Guided Fine-Needle Aspiration Cytology

53

a

b

c

d

Fig. 2.14  Schwannoma. (a) Hypercellular FNA smears showing clusters of uniform spindle cells embedded within a collagenous and slightly myxoid matrix. (b) Nuclear palisading and cell-poor areas in the cell clusters consisted of Verocay bodies (MGG). (c) Alcohol-fixed

smears showing cohesive clusters of relatively uniformed spindle cells with indistinct cytoplasm and elongated nuclei, some with pointed ends (H&E). (d) Diffuse S-100 positivity confirms a diagnosis of schwannoma (ThinPrep; Hologic; Bedford, MA, USA)

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References

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The diagnostic yield and safety of ultrasound-­ WP 3rd, Hunt R, et  al. Stereotactic core-needle breast assisted transthoracic fine-needle aspiration of drowned lung. biopsy: a multi-­ institutional prospective trial. Radiology. Respiration. 2011;81(1):26–31. 2001;218(3):866–72. 29. Kalhan S, Sharma P, Sharma S, Dudani S, Ramakrishnan T, 10. Yu Y-H, Wei W, Liu J-L.  Diagnostic value of fine-needle aspira- Chowdhry A.  Evaluation of precision of guidance techniques in tion biopsy for breast mass: a systematic review and meta-analysis. image guided fine needle aspiration cytology of thoracic mass BMC Cancer. 2012;12(1):41. lesions. J Cytol. 2012;29(1):6–10. 11. Wang M, He X, Chang Y, Sun G, Thabane L.  A sensitivity and specificity comparison of fine needle aspiration cytology and core 30. Murphy JM, Gleeson FV, Flower CD. Percutaneous needle biopsy of the lung and its impact on patient management. World J Surg. needle biopsy in evaluation of suspicious breast lesions: a system2001;25(3):373–80. atic review and meta-analysis. Breast. 2017;31:157–66. 12. Ojeda-Fournier H, Nguyen JQ. Ultrasound evaluation of regional 31. Kaçar Özkara S, Ozöver Tuneli I.  Fine needle aspiration cytopathology of liver masses: 101 cases with cyto-/histopathological breast lymph nodes. Semin Roentgenol. 2011;46(1):51–9. analysis. Acta Cytol. 2013;57(4):332–6. 13. Gipponi M, Fregatti P, Garlaschi A, Murelli F, Margarino C, Depaoli F, et al. Axillary ultrasound and fine-needle aspiration cytology in 32. Mehdi G, Maheshwari V, Afzal S, Ansari HA, Ansari M.  Image-­ guided fine-needle aspiration cytology of ovarian tumors: an assessthe preoperative staging of axillary node metastasis in breast cancer ment of diagnostic efficacy. J Cytol. 2010;27(3):91–5. patients. Breast. 2016;30:146–50. 33. Ray S, Gangopadhyay M, Bandyopadhyay A, Majumdar K, 14. Kocjan G, Bourgain C, Fassina A, Hagmar B, Herbert A, Kapila K, Chaudhury N. USG guided FNAC of ovarian mass lesions: a cyto-­ et al. The role of breast FNAC in diagnosis and clinical management: histopathological correlation, with emphasis on its role in pre-­ a survey of current practice. Cytopathology. 2008;19(5):271–8. operative management guidelines. J Turkish Ger Gynecol Assoc. 15. Nassar A. Core needle biopsy versus fine needle aspiration biopsy 2014;15(1):6–12. in breast – a historical perspective and opportunities in the modern 34. Nahar Saikia U, Khirdwadkar N, Saikia B, Sood B, Goldsmith R, era. Diagn Cytopathol. 2011;39(5):380–8. Dey P, et al. Image-guided fine-needle aspiration cytology of deep-­ 16. Hazard HW, Hansen NM.  Image-guided procedures for breast seated enlarged lymph nodes. Acta Radiol. 2002;43(2):230–4. masses. Adv Surg. 2007;41:257–72. 35. Veerapand P, Chotimanvijit R, Laohasrisakul N, Muennooch 17. Cesur M, Corapcioglu D, Bulut S, Gursoy A, Yilmaz AE, Erdogan W.  Percutaneous ultrasound-guided fine needle aspiration of N, et  al. Comparison of palpation-guided fine-needle aspiration abdominal lymphadenopathy in AIDS patients. J Med Assoc Thail. biopsy to ultrasound-guided fine-needle aspiration biopsy in the 2004;87(4):400–4. evaluation of thyroid nodules. Thyroid. 2006;16(6):555–61. 18. Hanbidge AE, Arenson AM, Shaw PA, Szalai JP, Hamilton PA, 36. Cavanna L, Lazzaro A, Vallisa D, Civardi G, Artioli F.  Role of image-guided fine-needle aspiration biopsy in the management of Leonhardt C.  Needle size and sample adequacy in ultrasound-­ patients with splenic metastasis. World J Surg Oncol. 2007;5:13. guided biopsy of thyroid nodules. Can Assoc Radiol J. 37. Ojalehto M, Tikkakoski T, Rissanen T, Apaja-Sarkkinen 1995;46(3):199–201. M.  Ultrasound-guided percutaneous thoracoabdominal biopsy. 19. Court-Payen M, Nygaard B, Horn T, Krag Jacobsen G, Braendstrup Acta Radiol. 2002;43(2):152–8. O, Narvestad E, et al. US-guided fine-needle aspiration biopsy of 38. Arellano RS, Maher M, Gervais DA, Hahn PF, Mueller PR.  The thyroid nodules. Acta Radiol. 2002;43(2):131–40. difficult biopsy: let’s make it easier. Curr Probl Diagn Radiol. 20. Gu WX, Tan CS, Ho TWT.  Surgeon-performed ultrasound-­ 2003;32(5):218–26. guided fine-needle aspiration cytology (SP-US-FNAC) shortens time for diagnosis of thyroid nodules. Ann Acad Med Singap. 39. Arellano RS, Harisinghani MG, Gervais DA, Hahn PF, Mueller PR. Image-guided percutaneous biopsy of the adrenal gland: review 2014;43(6):320–4.

2  Image-Guided Fine-Needle Aspiration Cytology of indications, technique, and complications. Curr Probl Diagn Radiol. 2003;32(1):3–10. 40. Lieberman S, Libson E, Sella T, Lebensart P, Sosna J. Percutaneous image-guided splenic procedures: update on indications, technique, complications, and outcomes. Semin Ultrasound CT MR. 2007;28(1):57–63. 41. Gupta S, Ahrar K, Morello FA Jr, Wallace MJ, Hicks ME. Masses in or around the pancreatic head: CT-guided coaxial fine-needle aspiration biopsy with a posterior transcaval approach. Radiology. 2002;222(1):63–9. 42. Fornari F, Civardi G, Cavanna L, Di Stasi M, Rossi S, Sbolli G, et al. Complications of ultrasonically guided fine-needle abdominal biopsy. Results of a multicenter Italian study and review of the literature. The cooperative Italian study group. Scand J Gastroenterol. 1989;24(8):949–55. 43. Livraghi T, Damascelli B, Lombardi C, Spagnoli I.  Risk in fine-­ needle abdominal biopsy. J Clin Ultrasound. 1983;11(2):77–81. 44. Domanski HA, Carlen B, Sloth M, Rydholm A. Elastofibroma dorsi has distinct cytomorphologic features, making diagnostic surgical biopsy unnecessary: cytomorphologic study with clinical, radiologic, and electron microscopic correlations. Diagn Cytopathol. 2003;29(6):327–33. 45. Leffler SG, Chew FS. CT-guided percutaneous biopsy of sclerotic bone lesions: diagnostic yield and accuracy. AJR Am J Roentgenol. 1999;172:1389–92.

55 46. Yang YJ, Damron TA.  Comparison of needle core biopsy and fine-needle aspiration for diagnostic accuracy in musculoskeletal lesions. Arch Pathol Lab Med. 2004;128(7):759–64. 4 7. Mehrotra R, Singh M, Singh PA, Mannan R, Ojha VK, Singh P.  Should fine needle aspiration biopsy be the first pathological investigation in the diagnosis of a bone lesion? An algorithmic approach with review of literature. Cytojournal. 2007;4:9. 48. Chojniak R, Isberner RK, Viana LM, Yu LS, Aita AA, Soares FA. Computed tomography guided needle biopsy: experience from 1,300 procedures. Sao Paulo Med J. 2006;124(1):10–4. 49. Hau A, Kim I, Kattapuram S, Hornicek FJ, Rosenberg AE, Gebhardt MC, et  al. Accuracy of CT-guided biopsies in 359 patients with musculoskeletal lesions. Skelet Radiol. 2002;31(6):349–53. 50. Sharma SD, Kumar G, Horsburgh A, Huq M, Alkilani R, Chawda S, et al. Do immediate cytology and specialist radiologists improve the adequacy of ultrasound-guided fine-needle aspiration cytology? Otolaryngol Head Neck Surg. 2015;152(2):292–6. 51. Witt RL, Sukumar VR, Gerges F. Surgeon-performed ultrasound-­ guided FNAC with on-site cytopathology improves adequacy and accuracy. Laryngoscope. 2015;125(7):1633–6. 52. Domanski HA, Åkerman M, Carlén B, Engellau J, Gustafson P, Jonsson K, et al. Core-needle biopsy performed by the cytopathologist: a technique to complement fine-needle aspiration of soft tissue and bone lesions. Cancer. 2005;105(4):229–39.

3

Breast Fernando Schmitt, Rene Gerhard, Donald E. Stanley, and Henryk A. Domanski

 rinciples of Evaluation and Reporting P of Breast FNAC Fine-needle aspiration cytology (FNAC) has been extensively used for many years in the diagnosis of breast lesions because it provides a rapid, accurate, and cost-effective evaluation. However, its use has gradually decreased because of the controversial rates of specimen inadequacy and suboptimal accuracy of diagnoses in inexperienced hands. In many countries, FNAC has been replaced by core needle biopsy (CNB), although often for the wrong reasons. In many places in the world, FNAC is still used as the first-line diagnosis of breast lesions in screening and symptomatic populations. In addition, FNAC is also used alongside with CNB in cases where malignancy is not suspected. Pathologists who specialize in cytopathology are best qualified to collect, prepare, and interpret FNAC samples, but this is not always possible or practical. Radiologists involved in breast imaging diagnosis should ensure that they have the necessary skills to perform FNAC under all forms of image guidance. The best results are achieved by a

F. Schmitt (*) Department of Pathology and Oncology, Medical Faculty of Porto University and IPATIMUP, Porto, Portugal Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal e-mail: [email protected]; [email protected] R. Gerhard Laboratorie national de santé, Dudelange, Luxembourg D. E. Stanley Department of Pathology, University Maine Medical Center, Portland, ME, USA e-mail: [email protected] H. A. Domanski Department of Pathology, Skåne University Hospital, Lund, Sweden e-mail: [email protected]

combination of both disciplines, as shown in the imageguided FNAC with the participation of a cytopathologist. The majority of European countries use the following reporting terminologies according to European guidelines for quality assurance in breast cancer screening and diagnosis: • • • • •

C1 unsatisfactory C2 benign C3 suspicious, probably benign C4 suspicious, probably malignant C5 malignant

In the USA, a similar approach is used, according to the recommendations of the National Cancer Institute (NCI) conference: • • • • •

Benign Atypical/undetermined Suspicious/probably malignant Malignant Unsatisfactory

Recently, the International Academy of Cytology proposed a global uniformization for a structure report for breast FNAC [1] using a five-stage system: • • • •

Code 1 – Insufficient material Code 2 – Benign Code 3 – Suspicious/probably malignant Code 4  – Suspicious, probably in situ or invasive carcinoma • Code 5 – Malignant Despite subtle differences, these systems are similar and claim that the use of a standardized reporting system for breast cytology is important for uniform clinical management of different types of lesions.

© Springer International Publishing AG, part of Springer Nature 2019 H. A. Domanski (ed.), Atlas of Fine Needle Aspiration Cytology, https://doi.org/10.1007/978-3-319-76980-6_3

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The diagnosis section should have a description of the site of lesion, location, and microscopic findings. This section should be followed by FNAC biopsy code and then the ­opinion of the pathologist about the nature of the lesion. Although a degree of flexibility is possible, the diagnostic categories should be followed by the pathological diagnosis. The FNAC report should be used in conjunction with the clinical and imaging findings in the “triple test” approach, which yields very high positive and negative predictive values and provides a solid basis for management decisions in breast lesions. An additional space for comments is present and a section for recommendations to correlate the cytologic with the clinical and imaging findings and the need for clinical follow-up or biopsy of the lesion for a histologic diagnosis. A final area contains the name and signature of the pathologist and the date that the report was given.

F. Schmitt et al.

Accuracy of Diagnosis The sensitivity and specificity of breast FNAC is around 90% in specialized centers [2, 3], combining cases of palpable and nonpalpable (ultrasound-guided) FNAC.  These rates are somewhat lower for stereotactic FNA.  The percentage of false-negative diagnoses should be less (it is suggested that quality assurance studies should be aimed at) than 10%, but in specialized centers, it is usually less than 5%. This can be explained by sampling misses, sampling of benign calcification adjacent to a mammographically undetected carcinoma, and microscopic misinterpretation by the cytopathologist. Concerning false-positive results, most published guidelines point out or suggest that their percentage should be less than 1%. They are always interpretative errors. This occurs in the process of evaluation of rare lesions, diagnostic pitfalls, and look-alikes, such as some fibroadenomas with loss of cellular cohesion that mimic carcinoma, complex sclerosing lesions, and sclerosing adenosis.

3 Breast

Normal Breast FNAC The sampling of a normal breast occurs when there are interpretative radiologic variations of normal findings or sampling problems (see Fig. 3.1):

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• Adipose tissue is the most common tissue present in the smears. • Balloon-like fat cells in clusters of variable sizes. • Strands of fibrotic tissue. • Occasional spindled naked nuclei of fibroblasts.

• Variable yield of ductal epithelial cell groups, usually higher in younger age groups.

a

b

Fig. 3.1  Normal ductal epithelial cell groups. (a, b) Flat sheets of ductal cells with a “honeycomb” architecture (MGG and hematoxylin and eosin [H&E])

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 onneoplastic Conditions: Inflammatory N Lesions Inflammatory breast conditions include mastitis, abscess, fat necrosis, and radiation-induced changes.

Acute Mastitis Acute mastitis usually manifests as a palpable lesion associated with local pain and redness. It is commonly observed during lactation in the postpartum period. The lesion may become localized, leading to the formation of an abscess (see Fig.  3.2). Clinically, both abscesses and acute mastitis can

Fig. 3.2  Acute mastitis. Many neutrophils and some histiocytes in a dirty background (Papanicolaou)

F. Schmitt et al.

mimic breast carcinoma. The collected material may be submitted to a culture of infectious agents. Bacteria of the genera Staphylococcus and Streptococcus are the major etiologic agents of mastitis. Cytologic features • Thick, whitish, or yellowish aspirate. • Variable cellularity. • Inflammatory cells, mainly neutrophils, histiocytes, and cellular debris. • With the progression to chronic mastitis, neutrophils are replaced by lymphocytes, plasma cells, fibroblasts, and granulation tissue.

3 Breast

Granulomatous Mastitis Granulomatous mastitis can be idiopathic or secondary to infectious diseases (e.g., tuberculosis, atypical mycobacteriosis, or fungal diseases), reactions to foreign bodies (sutures, silicone), or sarcoidosis (see Fig.  3.3). The presence of necrosis and giant cells of Langerhans type suggests tuberculosis. Specific staining for mycobacteria (Ziehl-Neelsen) and fungi (Grocott) and the culture of the aspirated material may aid in the identification of the etiologic agent. However, PCR-based DNA amplification is the best method for diagnosing mycobacterial infection [4]. Silicone is characterized by globules of liquid material surrounded by vacuolated histiocytes and multinucleated giant cells. Idiopathic granulomatous mastitis is a diagnosis

a

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of exclusion, which occurs in women between the fourth and sixth decades of life or sometimes in association with breastfeeding and the postpartum period. In these cases, numerous epithelioid histiocytes are seen, sometimes with the formation of granulomas, numerous neutrophils, and the absence of necrosis [5]. Cytologic features: • Aggregates of epithelioid histiocytes with kidney-shaped or elongated nuclei. • Multinucleated giant cells of foreign body type. • Chronic inflammatory infiltrate predominantly of lymphocytes and plasma cells. • The presence of necrosis and giant cells of Langerhans type suggests tuberculosis.

b

c

Fig. 3.3  Silicone granuloma. (a, b) Liquid material surrounded by vacuolated histiocytes and multinucleated giant cells (H&E) and granuloma (c) consisting of epithelioid histiocytes arranged in a loose and syncytial aggregate (MGG)

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Recurrent Subareolar Abscess Recurrent subareolar abscess is a chronic inflammatory disease characterized by abscess formation at the base of the nipple. The injury can progress with the formation of fistulous tracts and partial healing. It is believed that squamous metaplasia of columnar epithelial cells of the lactiferous duct may be the cause of subareolar abscess. Keratin plugs could obstruct the duct, leading to its dilatation, rupture, and inflammation in the adjacent stroma. Cytologic features • Richly cellular smears • Mixture of neutrophils, lymphocytes, histiocytes, anucleate squamous cells, parakeratotic cells, granulation tissue, and cholesterol crystals • Commonly multinucleated giant cells of foreign body type involving squamous cells Differential diagnosis • Ruptured epidermoid cyst (often located peripherally in the breast, unrelated to the nipple)

Fat Necrosis Fat necrosis of the subcutaneous tissue of the breast (see Fig. 3.4) is usually the result of trauma. Clinically, fat necrosis may present as a firm, fixed, and painful mass, sometimes

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with skin retraction. Because of calcifications secondary to necrosis, the lesion may mimic carcinoma on mammography. Cytologic features • Hypocellular smears • Fragments of degenerative changed fatty tissue associated with foamy histiocytes • Multinucleated giant cells • Dirty and granular background • Histiocytes with hemosiderin indicating previous bleeding Differential diagnosis and problems in diagnosis • Breast carcinoma • Granulomatous mastitis Tissue changes secondary to radiation (e.g., in patients with breast cancer undergoing adjuvant radiotherapy) can also reveal inflammation. Sometimes, radiation-induced changes are accompanied by fat necrosis. Usually, the cytologic smears are hypocellular and exhibit a lymphocytic infiltrate. Bizarre and atypical epithelial cells with karyomegaly, hyperchromasia, prominent nucleoli, and abundant cytoplasm with coarse vacuoles are also present (see Fig. 3.5). Some of these cells may show bi- or multinucleation, but the nuclearcytoplasmic ratio is low. In contrast to radiation-­ induced changes, the cytologic smears of carcinoma are usually highly cellular with many isolated atypical epithelial cells.

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Fig. 3.4  Fat necrosis. (a–c) Small fragments of degenerated fatty tissue associated with foamy histiocytes and multinucleated giant cells in a dirty and granular background. Histiocytes with hemosiderin indicate previous bleeding, and (d) shows a sheet of benign ductal cells (H&E)

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Fig. 3.5  Changes secondary to radiation. (a) Clusters of duct epithelial cells with karyomegaly and hyperchromasia and abundant cytoplasm (b) with coarse vacuoles (MGG). (Image courtesy of Janne Malina, M.D. Department of Pathology, Skåne University Hospital, Malmö, Sweden)

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Fibrocystic Changes and Breast Cysts Fibrocystic breast changes are common and usually occur in women between the third and fifth decades of life. Such changes result in the formation of multifocal and/or bilateral breast lesions. Patients may refer to palpable and painful nodules, the symptoms of which vary with the menstrual cycle. Fibrocystic changes include nonproliferative lesions (simple cysts, apocrine cysts, apocrine metaplasia, stromal fibrosis, and chronic inflammation) and proliferative lesions (sclerosing adenosis, ductal epithelial hyperplasia, atypical ductal hyperplasia). The proliferative lesions are discussed under the heading of Epithelial Hyperplasia.

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• Ductal epithelial cells, usually arranged in monolayer clusters with a “honeycomb” pattern • Myoepithelial cells appearing as naked, small, round to oval bipolar nuclei with homogeneous chromatin, i­ solated or overlapping epithelial clusters • Apocrine cells usually arranged in cohesive monolayer sheets or as isolated, single cells • Apocrine cells showing abundant, well-defined, finely granular cytoplasm and round nuclei, sometimes with prominent nucleoli • Karyomegaly and irregularity of nuclear contours—can be seen in apocrine cells • Foamy histiocytes • Stromal fragments

Cytologic features of nonproliferative lesions (see Fig. 3.6):

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c

Fig. 3.6  Nonproliferative lesion. (a) Ductal epithelial cells in monolayer cluster and a sheet of apocrine cells. (b) Apocrine cells arranged in cohesive monolayer sheets and apocrine cells (c) with anisokaryosis (H&E)

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Cysts Cysts are the most common lesions of the breast. The aspirated fluid from a breast cyst may be clear or turbid with variable color (yellowish, greenish, brownish). Such cysts have no epithelial lining, and the cytologic smears are char-

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acterized by an amorphous, proteinaceous material and variable numbers of foamy histiocytes. Some cystic lesions of the breast may be lined by epithelium with apocrine metaplasia (apocrine cysts). These cysts generally exhibit a thick content and variable numbers of apocrine cells isolated or arranged in groups of varying sizes (see Fig. 3.7).

b

Fig. 3.7  Cyst. (a) Amorphous, proteinaceous material and apocrine cells. (b) Numerous foamy histiocytes (H&E)

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Epithelial Hyperplasia This group includes epithelial proliferative lesions without atypia (sclerosing adenosis and florid ductal epithelial hyperplasia) and epithelial proliferative lesions with atypia (atypical ductal hyperplasia). The latter subgroup has an increased risk for breast cancer. A study of cytohistologic correlation revealed that 1.7% of epithelial proliferative lesions without atypia were malignant on histologic examination. On the other hand, 36.5% of mammary epithelial lesions with atypia were diagnosed histologically as malignant [6]. According to the authors, the distinction between epithelial proliferative lesions with and without atypia is clinically useful, because patients with atypical lesions may be referred for surgical biopsy for a definitive diagnosis, whereas those with

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lesions without atypia may be managed more conservatively [6]. Cytologic features (see Fig. 3.8) • Moderate to abundant cellularity • Epithelial groups in a monolayer arrangement or some degree of disorganization, nuclear overlapping, and the formation of irregular spacing or secondary lumina • Branched clusters in pseudopapillary arrangement • Myoepithelial cells • In atypical ductal hyperplasia, clusters of monomorphic epithelial cells with slightly hyperchromatic nuclei and myoepithelial cells • A mixture of epithelial groups in monolayer and groups with nuclear crowding and overlapping • Some isolated epithelial cells

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Fig. 3.8  Epithelial hyperplasia. (a) Without atypia. (b) Atypical ductal hyperplasia: cluster of epithelial cells with nuclear crowding and slightly hyperchromatic nuclei (H&E). (c) Sclerosing adenosis: epithe-

lial clusters with myoepithelial cells (MGG) and some degree of disorganization (d) (H&E)

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Differential diagnosis and problems in diagnosis • Low-grade ductal carcinoma in situ (DCIS) The cytologic distinction between atypical ductal epithelial hyperplasia and low-grade ductal carcinoma in situ is virtually impossible (see Fig. 3.9), so it is worth classifying these lesions as “epithelial proliferative lesions with atypia” and suggesting a breast biopsy specimen for histologic diagnosis [7]. The cytologic smears of certain epithelial proliferative lesions (sclerosing adenosis, radial scar/complex sclerosing lesion, columnar cell change, and collagenous spherulosis)

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may simulate malignant epithelial tumors of the breast. Smears from sclerosing adenosis and radial scar may present microglandular structures with some degree of nuclear overlapping and rare myoepithelial cells. These structures resemble those seen in tubular carcinoma of the breast [7]. According to Field and associates [8], the smears of radial scar/complex sclerosing lesion show moderate to abundant cellularity, large and small epithelial groups, tubular epithelial structures, apocrine cells, and many bipolar naked nuclei (see Fig. 3.10). Most of the epithelial groups exhibit myoepithelial cells [8].

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Fig. 3.9  Epithelial hyperplasia. (a, b) Cohesive cluster of epithelial cells with nuclear crowding and formation of bridges and secondary lumina (Papanicolaou stain)

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Fig. 3.10  Radial scar. (a) Cohesive epithelial groups, apocrine metaplasia, naked bipolar nuclei, rare histiocytes, and fragments of stroma in an amorphous background. (b) Collagenous stromal fragments on high-power amplification (MGG)

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Columnar Cell Change The columnar cell change is characterized by terminal duct or lobular units lined by columnar epithelial cells with apical secretion. These lesions may or may not display nuclear atypia, often exhibit luminal microcalcifications, and may be associated with atypical epithelial hyperplasia or ductal carcinoma in situ. The cytologic smears of columnar cell change show epithelial columnar cells with nuclear overlapping, but it is possible to identify myoepithelial cells and peripheral columnar cells with maintenance of polarity [7].

Collagenous Spherulosis

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Cytologic features • Moderate to abundant cellularity • Ductal epithelial cell clusters • Hyaline globules (metachromatically stained by Giemsa) in association with epithelial cell clusters (see Fig. 3.11) Differential diagnosis and problems in diagnosis • Adenoid cystic carcinoma Cytologic smears from an adenoid cystic carcinoma are characterized by small cells with oval or angulated hyperchromatic nuclei. Such cells are isolated or arranged in loose, “mosaic clusters,” sometimes with hyaline globules in between (see Fig. 3.12).

Collagenous spherulosis is a benign lesion usually associated with another breast lesion (papilloma, sclerosing adenosis, radial scar, epithelial hyperplasia).

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Fig. 3.11  Collagenous spherulosis. (a, b) Hyaline globules attached to sheets and clusters of benign epithelial cells. (c, d) Some globules surrounded by epithelial and myoepithelial cells (MGG). (Image courtesy

of Janne Malina, M.D.  Department of Pathology, Skåne University Hospital, Malmö, Sweden)

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Fig. 3.12  Adenoid cystic carcinoma. (a–d) Small cells with oval or angulated hyperchromatic nuclei and scanty cytoplasm in loose, cribriform “mosaic” clusters with myxoid and hyaline basement membrane-like material and globules (a, b H&E; c, d MGG)

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Benign Neoplasms Fibroadenoma Fibroadenomas are benign breast tumors characterized by the proliferation of epithelial and stromal elements of the breast. These tumors occur commonly in young women. They usually present as circumscribed and mobile nodules with fibroelastic consistency. Multiple fibroadenomas can be found in up to one quarter of patients. Smears are usually hypercellular and composed of three elements: epithelial cells, myoepithelial cells, and stromal fragments. The epithelial clusters are, in most cases, cohesive and branched and tend to show an arrangement in monolayer (honeycomb pattern). Some epithelial clusters may show fingerlike or antler-­ horn projections. The epithelial cells may also exhibit some degree of nuclear overlapping in the presence of epithelial hyperplasia. Apocrine metaplasia can be seen. Numerous myoepithelial cells with small and oval nuclei, homogeneous chromatin, and scarce or absent cytoplasm are present in smears (see Fig. 3.13). Cytologic features • Often abundant cellularity • Large sheets of epithelial cells in monolayer • Epithelial three-dimensional, fingerlike, or antler-horn clusters • Numerous naked bipolar nuclei of myoepithelial cells scattered throughout the smear • Fragments of fibromyxoid stroma

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Differential diagnosis and problems in diagnosis • Phyllodes tumor • Ductal proliferations • Breast carcinoma Atypical epithelial cells can be found in some cases of fibroadenomas (see Fig. 3.14). The presence of benign cytologic findings (benign epithelial cells, myoepithelial cells, naked bipolar nuclei) is a clue to benignancy. Although it is rare, the prevalence of atypical cells in smears from a fibroadenoma does not exclude the possibility of a ductal carcinoma in situ or invasive carcinoma associated with fibroadenoma [7]. Another diagnostic problem is the distinction between cases of fibroadenoma with cellular stroma and low-grade phyllodes tumor. The stromal cells of phyllodes tumors exhibit elongated nuclei and delicate cytoplasm, resembling fibroblasts. In the smears of a fibroadenoma, stromal cells appear as small spindle cells immersed in fragments of fibromyxoid stroma and dispersed as bipolar naked nuclei. According to Krishnamurthy and associates [9], a diagnosis of phyllodes tumor is favored over fibroadenoma if more than 30% of the population of single stromal cells in a cytologic smear is made up of long spindle cells with elongated nuclei.

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e

Fig. 3.13  Fibroadenoma. (a–c) Epithelial three-dimensional, fingerlike, or antler-horn clusters and fragments of fibromyxoid stroma, (d) fingerlike clusters, and (e) monolayer sheets of epithelial cells and

numerous naked bipolar nuclei of myoepithelial cells scattered throughout the smears (a, b, d, e H&E; c MGG)

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Fig. 3.14  Atypia in fibroadenoma. (a) Epithelial cells may exhibit some degree of nuclear overlapping (H&E) and (b, c) nuclear pleomorphism/ dissociation (MGG)

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Adenoma of the Nipple Nipple adenoma, also known as papillary adenoma, florid papillomatosis, or erosive adenomatosis, is a rare and benign tumor involving the terminal portion of the lactiferous ducts of the nipple. Nipple adenoma consists of a dual cell population (epithelial and myoepithelial cells) arranged in papillary clusters associated with dense stroma. The tumor occurs commonly in women in the fourth and fifth decades of life and includes a series of clinical manifestations, such as small, circumscribed, unilateral nodules; itching, redness, erosions, and crusts in the papillae; and serous or bloody nipple discharge. The main clinical differential diagnosis is Paget disease [10]. Cytologic features of single cases have been reported [10, 11]. Cytologic features • Cellular smears • Various epithelial cells in monolayer groups or in tight papillary clusters, with or without fibrovascular stroma • Isolated epithelial cells without atypia • Naked bipolar nuclei and histiocytes with hemosiderin granules • Myoepithelial cells overlapping epithelial groups Differential diagnosis and problems in diagnosis • Fibroadenoma • Papilloma • Fibrocystic changes

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Differential diagnoses include lesions with cohesive and branched epithelial groups, sometimes in papillary arrangement. Although nipple adenoma may clinically simulate Paget disease, the latter condition is characterized by isolated and not cohesive pleomorphic epithelial cells.

Myoepithelial Lesions Breast neoplasms consisting of purely myoepithelial or epithelial-­myoepithelial cells are rare and include benign and malignant myoepitheliomas, adenomyoepitheliomas, and tumors similar to those found in the salivary glands, such as pleomorphic adenoma and adenoid cystic carcinoma [12]. Of these, pleomorphic adenoma and adenomyoepithelioma are more commonly reported in the cytologic literature.

Pleomorphic Adenoma Pleomorphic adenoma of the breast (benign mixed tumor) is a very rare tumor that occurs in women from the second to ninth decades of life. About one third of cases occur in the subareolar region. Occasionally, the tumor is multifocal. Clinical and mammographic findings may raise the suspicion of a breast carcinoma. Two elements are commonly present in smears: myoepithelial cells and stroma (see Fig. 3.15).

b

Fig. 3.15  Pleomorphic adenoma. (a) Myoepithelial cells in loose clusters or as single cells in a background of fibromyxoid stroma. (b) Myoepithelial cells with a plasmacytoid morphology embedded in a fibrillar and metachromatic fibromyxoid stroma (MGG)

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Cytologic features • Myoepithelial cells with a plasmacytoid appearance (eccentric round nuclei and homogeneous basophilic cytoplasm) • Loose clusters and isolated myoepithelial cells admixed with fibromyxoid stroma • Intense violet hue and a fibrillar appearance of the stroma (May–Grünwald–Giemsa [MGG] stain) • Karyomegaly and binucleation can be present in isolated myoepithelial cells • Epithelial cells with round nuclei and scant cytoplasm forming tubular structures Differential diagnosis and problems in diagnosis • Metaplastic carcinoma, a rare subtype of breast carcinoma • Mucinous carcinoma Metaplastic carcinoma may display abundant extracellular matrix, including the presence of bone and cartilaginous tissue. Unlike pleomorphic adenoma, these elements are often atypical or malignant in metaplastic carcinoma. The abundant myxoid matrix of mucinous carcinoma may be similar to the mucus matrix of pleomorphic adenoma, especially in smears stained by the Papanicolaou technique [13].

Adenomyoepithelioma Adenomyoepithelioma is a rare benign breast tumor characterized by proliferation of ductal epithelial and myoepithelial cells in different architectural patterns (lobular, papillary, and tubular). The tumor is solitary, unilateral, and usually larger than 1  cm and occurs more commonly in women between the fifth and sixth decades of life. Tumors are usually benign but have a propensity for recurrence and malignant transformation [14].

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Cytologic features (see Fig. 3.16) • Moderately to highly cellular smears. • Cohesive, three-dimensional, branched clusters of epithelial and myoepithelial cells. • Relatively uniform epithelial cells with oval, regular nuclei and occasional nuclear grooves. • Variable spindle, epithelioid, and plasmacytoid appearance of myoepithelial cells. • Tubular structures occur in about one quarter of cases. • Fibromyxoid and metachromatic stroma. • Occasionally stroma with hyaline globules surrounded by myoepithelial cells (similar to collagenous spherulosis) is seen. • Occasional apocrine cells and histiocytes. Differential diagnosis • Fibroadenoma • Adenoid cystic carcinoma Myoepithelial cells are found overlapping the epithelial clusters or as isolated cells with preserved, clear, and vacuolated cytoplasm (“soap bubble” appearance) or in the form of bipolar naked nuclei. Apocrine cells and histiocytes are occasionally present in cytologic smears [15]. Adenomyoepithelioma may be confused with fibroadenoma. In general, the epithelial clusters in adenomyoepithelioma are more three-dimensional and not monolayers. In addition to intranuclear inclusions, found in about one third of cases, myoepithelial cells may show karyomegaly and prominent nucleoli [7]. The presence of mitotic figures and necrosis raises the possibility of a malignant adenomyoepithelioma. The metachromatic hyaline globules may suggest the diagnosis of an adenoid cystic carcinoma. However, in general, these globules are more frequent and in greater numbers in adenoid cystic carcinoma than in adenomyoepithelioma [15].

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Fig. 3.16  Adenomyoepithelioma. (a, b) Clusters of uniform epithelial cells and epithelioid myoepithelial cells with preserved, clear, and vacuolated cytoplasm (MGG). (c, d) Occasional fragments of fibromyxoid stroma and histiocytes in the background (H&E)

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Papillary Tumors

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Some breast lesions showing pseudopapillary clusters may be confused with papillomas. In general, fibroadenoPapillomas are solitary or multiple benign breast lesions that mas exhibit epithelial monolayers that are sometimes folded occur in perimenopausal women. The solitary papilloma is over on themselves, while the epithelial groups of papillousually located in the retroareolar region of the breast. mas are three-dimensional. Fibroadenomas also have Patients may be seen with a palpable nodule or a serous or numerous bipolar naked nuclei scattered throughout the bloody nipple discharge. Field and Mak [8] studied the smear. Fibrocystic changes can simulate a papilloma, pre­cytologic findings of papillary lesions in detail. According to senting cytologic smears with pseudopapillary epithelial these authors, the most characteristic findings of a breast pap- clusters, apocrine cells, and a cystic or hemorrhagic backilloma are the presence of stellate tissue fragments (branched ground. The angulated epithelial clusters of tubular carcifragments of stroma with attached epithelial and myoepithe- noma may have a pseudopapillary appearance, resembling a lial cells in the periphery) (see Figs. 3.17, 3.18, and 3.19) and papillary lesion [16]. meshwork tissue fragments (thin crisscrossing stroma surPapillary carcinoma of the breast affects older women as rounding tubular and acinar epithelial clusters). The authors opposed to patients with benign papillomas. The distinction also observed that the presence of papillary tissue fragments between a papilloma and an intracystic papillary carcinoma or (with fibrovascular stroma) is uncommon and is not neces- a papillary ductal carcinoma in situ is almost impossible based sarily required for the cytologic diagnosis of a papilloma [8]. on cytologic findings alone, and it is advisable to perform a biopsy for histopathologic examination of the lesion [7, 17]. Cytologic features In general, lower cellularity and predominantly cohesive • Moderate to abundant cellularity groups, sometimes with a monolayer arrangement, suggest a • Cuboidal or columnar epithelial cells arranged in three-­ papilloma, whereas the presence of numerous isolated cells dimensional, cohesive, branched groups and papillary (tall columnar cells) favors the diagnosis of a papillary carcifragments noma. According to Tse and coworkers [17], the papillary • Fibrovascular stroma within fragments of epithelial cells fragments of a papilloma have rounded contours and broad• Small groups or isolated columnar cells based projections, while those of papillary carcinoma exhibit • No myoepithelial cells overlapping the epithelial groups [16] ramifying borders and long and slender papillae. Other find• Apocrine cells and histiocytes, occasionally ings that support the cytologic diagnosis of a papilloma are hemosiderin-loaded the presence of apocrine cells, histiocytes, inflammatory cells, and bipolar naked nuclei. Papillary carcinomas do not show Differential diagnosis and problems in diagnosis apocrine metaplasia and usually have a monotonous cellular • Fibroadenoma pattern and a greater number of isolated and atypical epithe• Fibrocystic changes lial cells. However, it should be remembered that cellular • Tubular carcinoma pleomorphism and nuclear atypia can also be found in papil• Papillary carcinoma lomas [17].

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Fig. 3.17  Intraductal papilloma. (a–d) Branched groups and papillary fragments of rounded cuboidal or columnar epithelial cells with some degree of atypia and with fibrovascular stroma (H&E). (Image courtesy

a

of Elwira Bakuła-Zalewska, MD, PhD, Institute of Oncology, Warsaw, Poland)

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Fig. 3.18  Intraductal papilloma. (a) Columnar cells in small and loose clusters and in the periphery (b) of a large three-dimensional cluster (MGG)

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Fig. 3.19  Additional case of intraductal papilloma. (a–d) Smears with fibrovascular stroma with attached fragments of epithelial cells, occasional apocrine cells, and few myoepithelial cells (H&E)

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Granular Cell Tumor Granular cell tumor is a rare lesion that, histogenetically, is related to Schwann cells. Granular cell tumor occurs in any age group, arising most commonly in the skin and subcutaneous tissue, head and neck, and especially in the tongue but only occasionally in the mammary glands in middle-aged women. This neoplasm usually shows a benign clinical behavior (less than 1% are malignant). A stellate appearance on mammography may mimic a breast carcinoma. Cytologic features (see Fig. 3.20) • Polygonal cells with abundant fragile granular cytoplasm • A mixture of loose clusters, isolated cells, and often stripped nuclei due to fragile cytoplasm • Cytoplasmic granular background • Small, round to oval nuclei with finely granular chromatin and small nucleoli

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• Occasional moderately pleomorphic cells with nuclei showing coarse chromatin and macronucleoli • Tumor cells are positive for S-100 and contain periodic acid-Schiff (PAS)-positive cytoplasmic material Differential diagnosis and problems in diagnosis • Ductal carcinoma • Apocrine carcinoma • Secretory carcinoma • Metastatic renal cell carcinoma • Duct ectasia • Fat necrosis The main differential diagnoses include epithelial neoplasms with oncocytic cytoplasm or apocrine differentiation and histiocytic lesions [18].

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Fig. 3.20  Granular cell tumor. (a–d) Loose clusters and some isolated cells with abundant granular cytoplasm and cytoplasmic granular background. Note occasional moderately pleomorphic cells (a H&E; b–d MGG)

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Benign Lesions During Pregnancy The hormonal changes during pregnancy and the postpartum period may promote the formation of nonneoplastic breast lesions or highlight the presence of preexisting breast lumps. Such lesions are commonly referred to as lactating adenomas or lactating nodules. These lesions show a benign course and often regress after cessation of hormonal stimuli present during pregnancy and lactation. Rarely, the nodules may have an ectopic location, such as the axillary region or vulva. Because these lesions are not restricted to the lactational period, some authors propose that they should be named secretory hyperplastic nodules [19]. Cytologic smears show numerous epithelial cells in loose clusters or isolated in a “dirty background” of numerous fat droplets and cellular debris. The cells have abundant, foamy or vacuolated cytoplasm (see Fig.  3.21). Because of the cytoplasmic fragility, most of the cells are

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present as naked nuclei, devoid of cytoplasm. The nuclei exhibit discrete karyomegaly and prominent nucleoli but regular contours and delicate chromatin. A scarce number of bipolar naked nuclei can also be found in these smears. Preexisting breast lumps (such as a fibroadenoma) can also show secretory changes during pregnancy and lactation. In this context, in addition to the epithelial cell groups with secretory changes described previously, the presence of numerous bipolar naked nuclei and fragments of stroma suggests a fibroadenoma with secondary changes due to pregnancy or lactation. Because of the presence of cells with nucleoli and karyomegaly occurring as isolated cells or arranged in loose clusters, the cytologic findings can be interpreted as malignant. In this sense, it is important to recognize secretory changes compatible with the pregnancy or lactational status, such as cells with marked cytoplasmic vacuolization and numerous fat droplets and cellular debris in the background [7].

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Fig. 3.21  Lactation adenoma. (a–d) Loose clusters or isolated cells with abundant, foamy, or vacuolated cytoplasm and nuclei with discrete karyomegaly and prominent nucleoli but regular contours and delicate

chromatin. Note numerous fat droplets in the background (a and b, MGG; c and d, H&E)

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Malignant Neoplasms Breast Carcinoma, Including Special Subtypes Invasive carcinoma of no special type (IC-NST) is the most common malignant neoplasm of the breast. In fact, IC-NST is a heterogeneous entity, with several histologic subtypes, and the recent studies of molecular profiling have demonstrated that this entity is quite heterogeneous [20]. Despite this, the following general criteria of malignancy in breast cytology can be applied to most tumors: • Moderate to abundant cellularity, with some cellular dissociation. • Lack of cell-to-cell adhesion; isolated cells with intact cytoplasm. • Lack of myoepithelial in most invasive carcinomas. • Smears from DCIS, tubular carcinomas, and low-grade ductal carcinomas may contain myoepithelial cells. • Nuclear pleomorphism, nuclear membrane irregularity, and the presence of nucleoli and mitotic figures.

Fig. 3.22  Invasion of stromal or fatty tissue by neoplastic cells is an important criterion suggestive of invasion (H&E)

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• Intracytoplasmic lumina are more common in malignant lesions. • Background littered with cell debris, necrosis, and inflammatory cells. The cytologic diagnosis of DCIS and its distinction from invasive carcinoma have been the subjects of much discussion and controversy [21]. There are some criteria suggestive but not diagnostic of invasion, such as (see Fig. 3.22): • • • •

Presence of elastoid stromal fragments Invasion of stromal or fatty tissue by neoplastic cells Presence of intracytoplasmic vacuoles Presence of tubular structures

None of these criteria is definitive. Despite this limitation, as discussed previously, this is not a reason to reject the use of FNA in the investigation of breast lesions.

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Histologic Subtypes I nvasive Carcinoma of No Special Type (IC-NST) A diagnosis of carcinoma can be made when the breast aspirates display the following cytologic characteristics: Cytologic features • Cellular smear. • Monomorphic cell population with variable cell pattern. • Loss of cellular cohesion, numerous isolated single epithelial cells. • Anisokaryosis. • Irregular three-dimensional clusters, syncytial groupings, or a glandular-like pattern; tumor cells pleomorphic and larger than normal ductal cells. • Nuclei may be eccentric; plasmacytoid appearance of the cells. • Nuclei with prominent nucleoli. • Well-defined, dense to granular, and vacuolated cytoplasm.

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• The background can be bloody, with occasional necrotic debris (rarely clean background). • Myoepithelial cells and stromal cells are absent. High-grade carcinomas are characterized by highly pleomorphic cells with readily identified mitotic figures (see Fig. 3.23), whereas aspirates of low-grade ductal carcinoma display monomorphic cell populations with features similar to those of lobular carcinoma, although the cellularity is usually greater [22]. High-grade ductal carcinomas, with prominent nucleoli, necrosis, and neutrophils, in general, are associated with a triple-negative phenotype [23]. IC-NSTC should be distinguished from atypical proliferative intraductal lesions. Adherence to the strict cytologic characteristics of ductal carcinoma, absence of myoepithelial and stromal cells (see Fig.  3.24), and correlation with imaging findings (triple test) are the keys to making the correct diagnosis [24]. Cases suspected of being low-grade carcinoma should be confirmed by cutting needle biopsy prior to definitive surgery [25].

b

Fig. 3.23  Some features of high-grade ductal carcinoma. (a) Nuclear pleomorphism, nuclear membrane irregularity, nucleoli, and mitotic figures. (b) Background with cell debris, necrosis, and inflammatory cells (H&E)

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Fig. 3.24  Intraductal carcinoma. (a) Tight, three-dimensional, cribriform cluster of atypical cells and cell block section (b) showing cribriform fragment of uniformed, atypical ductal cells suggestive of intraductal carcinoma (H&E). (c) Papillary and cribriform cluster of

moderately pleomorphic ductal cells and few histiocytes (MGG). (d) Loosely cohesive sheets of somewhat pleomorphic cells and calcifications (H&E) are cytologic features suggestive of intraductal carcinoma

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I nfiltrating Lobular Carcinoma (ILC) Aspirates of lobular carcinoma tend to yield poorly cellular specimens composed of small uniform cells that are often dissociated single cells but that occasionally are arranged in loose clusters (≤10 cells), in single files, or with nuclear molding (see Fig. 3.25). Nuclei are often eccentric, round, or oval with finely dispersed chromatin and small, distinct nucleoli. The cytoplasm is clear or vacuolated or may contain a mucin droplet that gives it a target-like appearance. Occasional signet ring cells may be present. A very valuable clue in the diagnosis is the tendency of the cells to form small chains. A pleomorphic variant includes larger cell size with prominent nuclear atypia and may be misclassified as ductal in up to 25% of FNA smears [26].

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• Loose clusters (≤10 cells) and dissociated single cells • Occasional single files of cells with nuclear molding • Often eccentric, round to oval nuclei with finely dispersed chromatin and small, distinct nucleoli • The tendency of the cells to form small chains • Occasional signet ring cells Underdiagnoses of ILC are a major source of false-­ negative findings in breast aspirates. This is because of the low cell yield and the small cell size. A correct diagnosis in the pleomorphic variant is significantly more frequent than in classic types. Bland cytologic findings that are discordant with clinical and imaging findings are the key to avoiding a false-negative diagnosis.

Cytologic features • Poorly cellular smears • Small monomorphic cell population

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Fig. 3.25  Infiltrating lobular carcinoma. (a, b) Small and loose clusters of neoplastic cells with eccentric nuclei and intracytoplasmic vacuoles. (c, d) Some cells arranged in a single-file pattern (H&E)

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Mucinous (Colloid) Carcinoma Mucinous carcinoma is needle-soft due to production of mucin. The aspirated material is gelatinous, and the smears are cellular with abundant extracellular mucin (see Fig. 3.26). Generally, nuclear pleomorphism in mucinous carcinoma is minimal. Branching, thin-walled blood vessels may be prominent [27]. Cytologic features • Usually cellular smears • Bland-looking or slightly pleomorphic cells • Tumor cells in groups and as single cells • Tight cell balls, flat sheets, and loosely cohesive cell clusters • Moderate to numerous small- to medium-sized single cells with round, often eccentric nuclei

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• Pale green to clear (on Papanicolaou stain) or pink to blue (on Diff-Quik stain) cytoplasm • Branching, thin-walled blood vessels

Differential diagnosis and problems in diagnosis • Mucocele-like lesions • Fibroadenoma The aspirates from mucocele-like lesions show abundant extracellular mucin indistinguishable from that seen in mucinous carcinoma [28]; however, the epithelial cells are present in only a few flat sheets with few or no single cells. Myoepithelial cells are also present. A mucoid background may be seen in aspirates from fibroadenomas. The concomitant presence of naked bipolar nuclei and normal ductal epithelium serves to distinguish the mass from a mucinous carcinoma.

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Fig. 3.26  Mucinous carcinoma. (a–c) Smears with abundant extracellular mucin, capillaries, and small- to medium-sized single cells with round, often eccentric nuclei (H&E)

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Tubular Carcinoma Because of the minimal cytologic atypia and of the cohesiveness of the cells, tubular carcinoma may be mistaken for fibroadenoma or fibrocystic change. Especially at low magnification, a pattern somewhat similar to that of fibroadenoma may be visible, but detailed examination shows the tubular structures to be three-dimensional with a central lumen. The characteristic angular tubular structures (see Fig. 3.27), coupled with clinical and mammographic findings, are clues to the correct diagnosis. Aspirates of radial scars may reveal a monomorphic cell population with a tubular arrangement that mimics tubular carcinoma [22]. Cases suspected of representing a tubular carcinoma or radial scar should be followed by core needle biopsy for a definitive diagnosis [27].

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Cytologic features • Moderately cellular smears • Cohesive clusters of uniform, bland epithelial cells • Tubular structures with an angular or comma-like appearance • Tumor cells with loss of polarity and no associated myoepithelial cells • Occasional cribriform fragments of uniform epithelial cells • Rarely present bare nuclei or bipolar cells Differential diagnosis • Radial scar

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Fig. 3.27  Tubular carcinoma. (a–d) Tubular and cribriform structures with an angular or comma-like appearance of bland-looking slightly atypical cells (H&E)

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Medullary Carcinoma A definitive diagnosis of medullary carcinoma requires evaluation of tissue sections to determine margin circumscription and other parameters. Therefore, a diagnosis of medullary carcinoma on FNA can only be suggested when the clinical and imaging findings—well-circumscribed, mobile mass—coupled with the cytologic appearance are suggestive of this carcinoma. However, “medullary features” can be recognized in aspirates, and this can be clinically useful because these tumors are associated with the BRCA 1 mutation and basal-like phenotype. Medullary carcinoma presenting at the tail of the breast must be distinguished from metastatic carcinoma to the lymph node. In such cases, correlation with clinical findings and/or core needle biopsy is necessary for establishing the correct diagnosis. Aspirates from medullary carcinoma are usually cellular with large pleomorphic tumor cells in a background of lymphocytes and plasma cells (see Fig. 3.28).

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Cytologic features • Moderately cellular smears. • Large tumor cells (range from 15 to 20 μm in diameter). • Cells in clusters, syncytial groups, or scattered individual cells. • Irregular nuclei with clumped chromatin and macronucleoli. • Bare nuclei or tumor cells with only a thin rim of cytoplasm. • Homogeneous or granular, poorly demarcated cytoplasm. • Lymphocytes in the background. Usually abundant. • Occasional smears may show predominantly lymphoid tissue and a few small groups of malignant cells. Differential diagnosis • Lymph node metastasis

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Fig. 3.28  Medullary carcinoma. (a–c) Small clusters, syncytial groups, or scattered individual cells with irregular nuclei, clumped chromatin, macronucleoli, and lymphocytes in the background (H&E)

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Papillary Carcinoma Papillary carcinoma of the breast is a variant with an excellent prognosis. Because of the possibility of a papillary carcinoma’s being entirely in situ, this is one of the variants of breast carcinoma in which definitive diagnosis is not possible on cytologic examination. Additionally, breast papillary proliferations constitute a group of lesions that show a broad spectrum of morphologic changes, ranging from benign to malignant and posing challenges at all diagnostic levels. In FNA material, the presence of a papillary architecture, spherical papillae (cell balls), and columnar cell coating or disso-

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ciated from the papillae are signs of a papillary tumor that could be benign or malignant. Samples of papillary carcinomas are characterized by an abundance of material, including three-dimensional papillary clusters, small papillae arranged in cell balls, isolated columnar cells, and the absence of bipolar naked nuclei (see Fig. 3.29). The aspirates may be associated with much dark blood. The presence of cell balls and the absence of bipolar naked nuclei are two of the most distinctive findings in papillary carcinoma. Ancillary techniques can be useful in the distinction of benign and malignant papillary tumors. A potential adjunct in papillary lesions of the breast is p63, being positive in benign papillary tumors [29].

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Fig. 3.29  Papillary carcinoma. (a–d) Three-dimensional papillary clusters of slightly to moderately pleomorphic ductal round to oval and columnar cells and absence of bipolar naked nuclei suggestive of papillary carcinoma (a, b, d H&E; c MGG)

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Invasive Micropapillary Carcinoma

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architectural arrangement (see Fig. 3.30). The nuclear atypia is usually high grade.

Aspirates from micropapillary carcinoma are cellular and display small cell clusters of tumor cells with rare individual cells. Tumor morules and staghorn structures are the main

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Fig. 3.30  Invasive micropapillary carcinoma. (a, b) Cellular smears showing small pseudopapillary and morule-like clusters and rare individual tumor cells with moderate or prominent nuclear atypia (H&E)

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Apocrine Carcinoma

tiple nucleoli; mitotic figures may be present. Pleomorphic nuclei, poorly defined cell borders, and cell discohesion are criteria that favor malignancy when compared with benign apocrine lesions. Also, the presence of a polymorphous cell population should be the clue to the correct diagnosis of benign lesions [30]. Apocrine metaplasia with atypia may be difficult to distinguish from well-differentiated apocrine carcinoma.

Aspirates from apocrine carcinoma are characterized by high tumor cellularity. The tumor cells are arranged singly and in syncytial tissue fragments. Both the cells and the nuclei are enlarged and pleomorphic (see Fig. 3.31). The cytoplasm is abundant, finely granular, and amphophilic. The cell outline is polygonal. The nuclei are oval to round and contain mul-

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Fig. 3.31  Apocrine carcinoma. (a–d) Predominantly round and polygonal single cells with enlarged pleomorphic nuclei, prominent nucleoli, and abundant, finely granular, and amphophilic cytoplasm (a, b H&E; c, d MGG)

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 reast Carcinoma with Osteoclast-Like B Giant Cells This is a variant of breast carcinoma, in general a ductal type, characterized by the presence of osteoclast-like stromal giant cells. The aspirates are cellular and composed of cohesive groups of epithelial cells, with low-grade atypia. Frequently, there are associated groups of plump spindle cells, but the

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main characteristic is the presence of osteoclast-like multinucleated cells at the periphery of the epithelial cells or in the background of the smears (see Fig. 3.32). The mild atypia of the epithelial cells and the presence of spindle cells can raise the differential diagnosis of fibroepithelial tumors. However, because of the presence of giant cells, some entities must be ruled out: granulomatous inflammation, myeloid metaplasia, and metaplastic carcinoma.

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Fig. 3.32  Breast carcinoma with osteoclast-like giant cells. (a) Smears showing poorly cohesive sheets and dispersed cancer cells with admixture of few osteoclast-like giant cells (MGG). (b) Cell block section with mixture of cancer cells and osteoclast-like giant cells (H&E).

(c) Immunostains on cell block section and liquid-based preparation (ThinPrep). (d) Groups of malignant cells with positive keratin staining, while giant cells are negative

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Secretory Carcinoma Aspirates of secretory carcinoma are characterized by the presence of globular structures consisting of small, centrally located, mucoid material with covering epithelia, usually composed of two or three and occasionally more cells. The globular structures are generally uniform in size. The mucoid

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substance within these structures can be round, crescentic, dented, irregular, or tadpole-shaped. The nuclei are crescentic or ovoid with no atypia. Mitotic figures are not seen. Other findings of secretory carcinoma include prominent intracytoplasmic vacuolization and occasional signet ring cells (see Fig. 3.33).

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Fig. 3.33  Secretory carcinoma. (a, b) Loosely cohesive clusters of slightly atypical cells with abundant cytoplasm and some cells with intracytoplasmic mucoid material and small eosinophilic globular

structures clearly visible also in liquid-based preparation (c, d) (ThinPrep). Note some signet ring cells (H&E)

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Acinic Cell Carcinoma

Uncommon Breast Neoplasms

This is a variant of breast carcinoma, similar in morphology to the acinic cell carcinoma of the salivary glands and pancreas. There are few descriptions of the cytologic aspects of this tumor. It is characterized by cellular smears composed of isolated and grouped tumor cells, sometimes with papillary arrangements, and cells showing moderate atypia with a clear and granular cytoplasm. The definitive diagnosis is possible only with the use of immunocytochemistry or ultrastructural studies. All types of breast cancer with clear cells must enter into the differential diagnosis.

Phyllodes Tumor

Glycogen-Rich Carcinoma This tumor is characterized by the presence of clear cells that are filled with glycogen. Aspirates are cellular with tumor cells in groups or clusters or as single dissociated forms. The cytoplasm is large, clear, and fragile and with a central nucleus with moderate to marked pleomorphism. Metastatic clear cell carcinoma of the kidney should be differentiated from this variant.

Lipid-Rich Carcinoma In the few reported cases, aspirates of lipid-secreting carcinoma were moderately cellular with loosely cohesive tumor cells. The cells displayed well-demarcated cytoplasm containing many large and small vacuoles. The number of vacuoles varied from a single one, occupying most of the cytoplasm, to so many that they gave the cytoplasm a foamy appearance. The nuclei were slightly pleomorphic, with distinct nuclear membranes, coarse or fine chromatin, and small nucleoli. Some tumor cells showed deeply indented nuclei as well as nuclear vacuoles.

Phyllodes tumor is a rare type of breast neoplasm occurring in women in their 30s and 40s, but they may be diagnosed at any age. Phyllodes tumors tend to grow quickly, within a period of weeks or months. Like fibroadenoma, phyllodes tumor contains stromal and glandular components, but the main difference from fibroadenoma is an overgrowth of stromal tissue. It is usually difficult to distinguish benign or borderline phyllodes tumor from fibroadenoma on FNA smears. The predominance of the mesenchymal component and occasional cellular and nuclear pleomorphism in the mesenchymal component favor phyllodes tumor (see Fig.  3.34). Smears from malignant phyllodes tumors usually contain sparse epithelial components but predominant stromal components with obvious malignant sarcomatous features.

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Fig. 3.34  Phyllodes tumor. (a, b) Moderately to highly cellular fragments of myxoid stroma, sometimes in a fingerlike pattern. (c) Cellular stromal fragment with atypical spindle cells (a MGG; b, c H&E)

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Metaplastic Carcinoma Metaplastic carcinoma (matrix-producing carcinoma) is a rare malignant neoplasm of the breast, comprising 1% of all malignant breast tumors. It is defined as a carcinoma with a nonepithelial component or with epithelial squamous differ-

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entiation. Diagnosis of metaplastic carcinoma in FNA smears may be difficult. Poorly differentiated carcinoma cells mixed with atypical spindle cells, osteoclastic giant cells, and collagenous-­myxoid or chondroid matrix are the cytologic features of metaplastic carcinoma seen in FNA smears (see Figs. 3.35 and 3.36).

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Fig. 3.35  Metaplastic carcinoma. (a–c) The presence of poorly differentiated carcinoma cells mixed with malignant spindle cells (sarcomatous component) and osteoclastic giant cells (d) (MGG; H&E)

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Fig. 3.36  Metaplastic carcinoma. (a) Atypical cells embedded in a chondromyxoid stroma (MGG). (b) Atypical squamous cells with dense, homogeneous, and polygonal cytoplasm (Papanicolaou)

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Mesenchymal Neoplasms Nonepithelial neoplasms include a spectrum of benign and malignant (sarcomas) lesions with morphology similar to soft tissue tumors in other body locations. Angiosarcoma is the most common sarcoma of the breast.

Malignant Lymphomas

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other settings (see Fig. 3.37). Ancillary techniques such as flow cytometry and immunocytochemical and molecular examination are required to arrive at a correct cytologic diagnosis of NHL in the breast. Other lymphoproliferative neoplasms such as myeloma (see Fig. 3.38) and leukemia may also rarely involve the breast. a

Secondary involvement of the breast in patients with disseminated non-Hodgkin lymphoma (NHL) is not uncommon. A primary NHL of the breast is rare accounting for 0.1–0.5% of all malignant breast tumors. Most patients with primary breast lymphoma will also develop lymphoma in the lymph nodes and other regions of the body. The most common breast lymphoma is the B-cell NHL. It can sometimes be difficult to distinguish NHL from breast cancer in FNA smears. The FNA smears from breast NHL are usually cellular and show morphology comparable to that of NHL in a

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Fig. 3.37  Non-Hodgkin lymphoma. (a, b) Monomorphic medium to large lymphoid cells with slightly irregular nuclei and scanty cytoplasm (MGG)

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Fig. 3.38  Myeloma manifestation in the breast. (a–c) Smears from breast nodule show scattered atypical plasma cells (MGG)

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Male Breast FNAC Gynecomastia Gynecomastia is the most common disease in the male breast; it can affect 30–40% of teenagers and young adults. Both breasts are usually affected, but in general one side is

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more obvious. Gynecomastia is a benign condition with no increased risk of carcinoma. The cytologic aspects are similar to those of fibroadenoma, with a variable cellularity showing a bimodal pattern (epithelial and stromal). Epithelial sheets are often large and cohesive and may be hyperplastic and three-dimensional. In the background, small to moderate numbers of bipolar cells can be present (see Fig. 3.39).

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Fig. 3.39  Gynecomastia. (a–d) Large, cohesive, three-dimensional clusters and flat sheets of bland ductal cells with admixture of bipolar nuclei (MGG)

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Male Breast Carcinoma

Metastatic Neoplasms

Male breast carcinoma accounts only for 1% of all breast carcinomas. Family history, hormonal changes, age, radiation exposure, and obesity are risk factors. About 4–14% of the cases are related to gene mutation in the BRCA 2 gene and 3–8% to Klinefelter syndrome. There is no evidence of a causal relationship with gynecomastia. In general, the tumor presents as nodules, most often in the elderly, and the presence of axillary metastases is common at diagnosis. Nipple discharge and Paget disease are relatively common forms of clinical presentation. The cytologic aspects are similar to those in carcinomas in females as previously described, especially ductal carcinomas, although all types described for the female breast may also be found. Most of the tumors in males are high grade. Often, male breast cancer is positive for estrogen receptors. Prostate cancer can metastasize to the male breast, simulating primary tumors. As in the female breast, axillary metastases, tumor size, and histologic grade are the most important prognostic factors.

Metastases to the breast are rare. According to large studies, metastatic lesions from extramammary neoplasms constitute 0.4–2.0% of all breast malignancies. The most common extramammary malignancies in the breast are hematologic neoplasms, followed by malignant melanoma and lung carcinoma. Breast metastases usually appear as solitary, superficial masses, without skin ulceration and nipple discharge. In mammography breast metastases are difficult to distinguish from primary carcinoma, and occasionally it may not be possible to distinguish them from benign lesions such as cysts and fibroadenomas. Correct diagnosis is very important, since treatment options for primary and metastatic neoplasm to the breast are different. Some metastatic malignancies attempt to recapitulate morphology of the original tumor; in others, the use of ancillary techniques may be necessary to establish the origin of the metastasis (see Fig. 3.40).

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Fig. 3.40  Metastatic neoplasm to the breast. (a, b) FNA of breast nodule in patient with history of ovarian serous papillary carcinoma. Note papillary clusters of the tumor cells and psammoma bodies, features suggestive of metastatic ovarian carcinoma (H&E). (c) Breast metastasis

of malignant melanoma showing loosely cohesive sheets of malignant cells with pleomorphic nuclei and prominent nucleoli. Note intranuclear inclusion (H&E). (d) Strong positivity for HMB 45 in the tumor cells confirms diagnosis of metastatic melanoma (ThinPrep, HMB 45)

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Ancillary Techniques in Breast FNAC  valuation of Estrogen Receptor, Progesterone E Receptor, and the Human Epidermal Growth Factor in FNAC (See Fig. 3.41) Specific receptors such as hormonal receptors—estrogen receptor (ER) and progesterone receptor (PR)—and the human epidermal growth factor receptor-2 (HER2) are therapeutic targets in breast cancer. The assessment of ER, PR, and HER2 is mandatory in all cases of invasive breast carcinoma, and detailed guidelines have been published by the American Society of Clinical Oncology (ASCO) and the College of American Pathologists (CAP) for their evaluation [31, 32]. Nuclear expression of ER and PR is assessed by immunohistochemistry (IHC) and around 80% of invasive breast carcinomas are ER positive and 60–70% are PR positive. Positivity for ER and/or PR is related to response to hormonal therapies, and patients with positive tumors have a better outcome compared to those with negative ones [30]. The HER2 status in breast cancer is assessed by IHC and in situ hybridization (ISH) techniques such as fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH), or silver in situ hybridization (SISH). The expression of HER2 in breast cancer is evaluated by a semiquantitative approach and classified as negative (scores 0 or 1+), equivocal (score 2+), or positive (score 3+) according to the extension and intensity of membrane immunostaining of the tumor cells. Tumors graded as 2+ are further analyzed by the ISH technique. The HER2 gene is amplified in 15% of inva-

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sive breast carcinomas, and patients whose tumors are HER2 positive (3+ by IHC immunohistochemistry or amplified by ISH) are eligible for treatment with targeted therapies against the HER2 protein [33]. The ASCO/CAP guidelines for hormonal receptors and HER2 detection were established in formalin-fixed and paraffin-­ embedded (FFPE) tissue specimens obtained by core needle biopsies or surgical resection specimens [31, 32]. Despite the ASCO/CAP recommendations, many studies compared the immunohistochemical expression of ER, PR, and HER2 between cytological samples and FFPE tissue specimens. Air-dried or alcohol-fixed cytologic smears as well as cell block preparations can be used for the analysis of hormonal receptors and HER2 [34]. In general, studies have been shown that the detection of hormonal receptors (ER, PR) in cytological samples is reliable, with a good agreement when compared to the FFPE tissue specimens [35–37]. Domanski et al. [35], for example, compared the expression of hormonal receptors in 267 breast FNA aspirates (cell blocks and monolayer preparations) with the corresponding FFPE tissue specimens (core needle biopsies or excised tumours). The concordance rates with the FFPE specimens were very high either for the monolayer (98% and 96% for ER and PR, respectively) or for the cell block (92% and 96% for ER and PR, respectively) preparations [35]. Ferguson et al. [38] analyzed the ER and PR expression on alcohol-­ fixed smears from 47 FNA breast samples, using the cell transfer technique. The authors found a good correlation between the cell transfer smears and cell blocks or concurrent biopsies. The sensitivity rate for ER and PR was 95%

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Fig. 3.41 (a) Positive immunostaining for estrogen receptor and (b) HER 2 on liquid-based preparation of smears from breast carcinoma (ThinPrep, ER, HER-2)

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and 90%, respectively, with a specificity of 100% for both hormonal receptors [38]. In one study, the concordance of ER IHC expression between cell block preparations and the corresponding tissue specimens was very high (96.2%). This was in contrast with the PR IHC expression which showed only a moderate agreement (77.5%) [39]. In another study, a moderate correlation of ER expression, with a kappa value of 0.446, was observed between 97 paired cytological and histological breast specimens [40]. Indeed, a multinational study in Europe that analyzed the detection of ER and PR in FNACs of breast carcinomas showed a great variability of results because of the different types of specimen preparations that were used by the laboratory participants, including fixation and immunostaining methods. This study also concluded that the best results were achieved when antigen retrieval as well as cytospins or monolayer preparations were used for ER and PR detection [41]. Rather controversial evidence exists regarding HER2 IHC in cytologic samples. A poor to moderate kappa agreement between FNAC and tissue specimens tested for HER2 was obtained by Beatty and coworkers [42]. One study showed that a moderate agreement for the detection of HER2 by IHC was observed between cell block specimens that were initially fixed in ethanol (followed by formalin fixation) and FFPE tissue samples. The authors also found an increase in contradictory results and in the false-positive rate for HER2 expression [43]. Another study revealed a discrepancy rate of 26% for detection of HER2 protein between cell blocks fixed in formalin and FFPE core needle and resection specimens [44]. Analyzing 542 breast cancer FNAs using the monolayer preparations, Zhang et al. [45] demonstrated a moderate concordance (80%, with a kappa value of 0.62) of HER2 IHC expression between the cytological samples and the matched FFPE tissue specimens. In spite of the high specificity rate (97.3%), the sensitivity rate was modest (67.1%), and the authors concluded that the HER2 IHC assessment in cyto-

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logical specimens could not be reliable for clinical use [45]. A similar result was found in another study where a moderate correlation of HER2 IHC expression (kappa value of 0.541) between 77 paired cytological and histological breast specimens was demonstrated [40]. On the other hand, Shabaik and associates [46] revealed a 100% correlation for HER2 IHC results between FFPE cell blocks of FNAC and serous effusions from primary and metastatic breast carcinomas and tissue sections. Regarding the type of fixation, it was recently demonstrated that either formalin-fixed or methanol-fixed cell blocks showed similar results for ER and HER2 IHC expression [47]. Other studies also demonstrated a satisfactory concordance between cytology and tissue specimens for the detection of HER2 IHC expression. Ferguson et  al. [38], for example, found a concordance of 88% between cell transfer smears and cell blocks or concurrent biopsies [38]. Durgapal et al. [48] detected a 99% diagnostic accuracy of HER2 IHC expression in 100 breast FNAs when compared with the corresponding tissue sections [48]. In a recent study, the HER2 IHC expression in breast FNA cell blocks demonstrated a high positive and negative concordance rate (≥98%) with the FISH test as the reference method [39]. Several studies have demonstrated a good correlation between cytologic samples and FFPE tissue specimens for the detection of HER2 gene amplification by the FISH technique, with concordance rates higher than 90% [40, 42, 48, 49]. Nishimura et al. [50] evaluated the HER2 status using the FISH technique in 100 FNAs samples obtained from surgically resect breast cancer specimens. From 98 cases with successful cytological FISH results, an agreement with the paired histological sections was achieved in 97 cases, providing an accuracy rate of 99% [50]. In another study, the concordance of HER2 FISH testing between cell block preparations and the corresponding tissue breast specimens was 96.7% [39].

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Molecular Genetic Examination

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3. Dong J, Ly A, Arpin R, Ahmed Q, Brachtel E. Breast fine needle aspiration continues to be relevant in a large academic medical center: experience from Massachusetts General Hospital. Breast Molecular testing of breast carcinomas is emerging as benCancer Res Treat. 2016;158:297–305. eficial for supplementing the information obtained by con- 4. Canberk S, Longatto-Filho A, Schmitt F.  Molecular diagnosis of infectious diseases using cytological specimens. Diagn Cytopathol. ventional morphologic tools. DNA and RNA of good quality 2016;44:156–64. can be extracted from breast FNAC for using in molecular 5. Tse GM, Poon CS, Law BK, Pang LM, Chu WC, Ma TK.  Fine techniques such as microarray analysis and next-generation needle aspiration cytology of granulomatous mastitis. J Clin Pathol. 2003;56:519–21. sequencing [51–53]. Performing genomic studies in small amounts of material minimizes the need for invasive proce- 6. Zhao C, Raza A, Martin SE, Pan J, Greaves TS, Cobb CJ. Breast fine-needle aspiration samples reported as “proliferative breast dures while simultaneously allowing more frequent re-­ lesion”: clinical utility of the subcategory “proliferative breast biopsy and therefore enabling longitudinal monitoring lesion with atypia”. Cancer. 2009;117:137–47. tumors and metastases. The phenotypic and genotypic 7. Orell SR, Miliauskas J.  Fine needle biopsy cytology of breast lesions: a review of interpretative difficulties. Adv Anat Pathol. changes observed in metastatic breast cancer make funda2005;12:233–45. mental to obtain cells from metastatic sites to study bio- 8. Field A, Mak A. The fine needle aspiration biopsy diagnostic critemarkers of therapeutic response [53]. The molecular ria of proliferative breast lesions: a retrospective statistical analysis of criteria for papillomas and radial scar lesions. Diagn Cytopathol. techniques classically used in cytology are polymerase 2007;35:386–97. chain reaction (PCR) and in situ hybridization (ISH). 9. Krishnamurthy S, Ashfaq R, Shin HJ, Sneige N.  Distinction of However sequencing techniques with the introduction of phyllodes tumor from fibroadenoma: a reappraisal of an old probmassive parallel sequencing or next-generation sequencing lem. Cancer. 2000;90:342–9. (NGS) using target panels have been gaining significant ter- 10. Kijima Y, Matsukita S, Yoshinaka H, Owaki T, Aikou T. Adenoma of the nipple: report of a case. Breast Cancer. 2006;13:95–9. rain. The main indication to properly select the molecular 11. Gupta RK, Dowle CS, Naran S, Lallu S.  Fine-needle aspiration technique to use in cytology will largely depend on the cytodiagnosis of nipple adenoma (papillomatosis) in a man and specificity of the target(s) to be identified, the type of alterawoman. Diagn Cytopathol. 2004;31:432–3. tion, and if there is a need to perform an in situ analysis 12. Pia-Foschini M, Reis-Filho JS, Eusebi V, Lakhani SR.  Salivary gland-like tumours of the breast: surgical and molecular pathology. rather than bulk tissue/tumor analysis. Differently than J Clin Pathol. 2003;56:497–506. these more specific approaches, sequencing techniques have 13. Iyengar P, Cody HS 3rd, Brogi E.  Pleomorphic adenoma of the the ability to investigate more genes or sequences of interest breast: case report and review of the literature. Diagn Cytopathol. 2005;33:416–20. simultaneously. Sequencing techniques have been being 1 4. Hayes MM. Adenomyoepithelioma of the breast: a review stressapplied in cytology samples for some time, but the applicaing its propensity for malignant transformation. J Clin Pathol. tion of sequencing techniques has gained considerable trac2011;64:477–84. tion with the use of NGS [54]. NGS is highly scalable and 15. Iyengar P, Ali SZ, Brogi E.  Fine-needle aspiration cytology of mammary adenomyoepithelioma: a study of 12 patients. Cancer. allows to tune the level of resolution to meet specific needs, 2006;108:250–6. making possible to obtain clinically significant resolution 16. Nayar R, De Frias DV, Bourtsos EP, Sutton V, Bedrossian with minimal amounts of material. Additionally, despite C. Cytologic differential diagnosis of papillary pattern in breast aspipresently with limited clinical interest, NGS also allows rates: correlation with histology. Ann Diagn Pathol. 2001;5:34–42. easier genome-wide methylation or DNA-­protein interac- 17. Tse GM, Ma TK, Lui PC, Ng DC, Yu AM, Vong JS, et  al. Fine needle aspiration cytology of papillary lesions of the breast: how tion profiling. The application of NGS in FNAC samples accurate is the diagnosis? J Clin Pathol. 2008;61:945–9. obtained from metastatic breast cancer to study mutations in 18. Pieterse AS, Mahar A, Orell S.  Granular cell tumour: a pitfall in the ER gene related to resistance to the treatment is one of FNA cytology of breast lesions. Pathology. 2004;36:58–62. 19. Niveditha SR, Kusuma V, Krishnamurthy, Bhagawan the possible applications of these techniques in the clinical BC. Cytomorphology of secretory hyperplastic (lactational) nodule practice [55]. breast. Cytopathol: Off J Br Soc Clin Cytol. 2006;17:156–8. 20. Ellis IO, Collins L, Ischihara S, et al. Invasive carcinoma of no special type. In: Lakhani SR, Ellis IO, Schnitt SJ, et al., editors. WHO classification of tumors of the breast. Lyon: IARC; 2012. p. 34–8. References 21. Sneige N, Staerkel GA. Fine-needle aspiration cytology of ductal hyperplasia with and without atypia and ductal carcinoma in situ. Hum Pathol. 1994;25:485–92. 1. Field AS, Schmitt F, Vielh P.  IAC standardized reporting of breast fine-needle aspiration biopsy cytology. Acta Cytol. 2017; 22. de la Torre M, Lindholm K, Lindgren A.  Fine needle aspiration cytology of tubular breast carcinoma and radial scar. Acta Cytol. 61:3–6. 1994;38:884–90. 2. Kocjan G, Bourgain C, Fassina A, Hagmar B, Herbert A, Kapila K, et al. The role of breast FNAC in diagnosis and clinical manage- 23. Dufloth RM, Alves JM, Martins D, Vieira DS, Chikota H, Zeferino LC, et al. Cytological criteria to predict basal phenotype of breast ment: a survey of current practice. Cytopathol: Off J Br Soc Clin carcinomas. Diagn Cytopathol. 2009;37:809–14. Cytol. 2008;19:271–8.

3 Breast 24. Morris KT, Pommier RF, Morris A, Schmidt WA, Beagle G, Alexander PW, et  al. Usefulness of the triple test score for palpable breast masses; discussion 1012-3. Arch Surg. 2001;136: 1008–12. 25. Tse GM, Tan PH. Diagnosing breast lesions by fine needle aspiration cytology or core biopsy: which is better? Breast Cancer Res Treat. 2010;123:1–8. 26. Rajesh L, Dey P, Joshi K. Fine needle aspiration cytology of lobular breast carcinoma. Comparison with other breast lesions. Acta Cytol. 2003;47:177–82. 27. Schmitt F, Sneige N, Lee A. Classification using needle-core biopsy and fine needle aspiration. In: Lakhani SR, Ellis IO, Schnitt SJ, et  al., editors. WHO classification of tumors of the breast. Lyon: IARC; 2012. p. 26–7. 28. Stanley MW, Tani EM, Skoog L. Mucinous breast carcinoma and mixed mucinous-infiltrating ductal carcinoma: a comparative cytologic study. Diagn Cytopathol. 1989;5:134–8. 29. Reis-Filho JS, Milanezi F, Amendoeira I, et  al. Distribution of p63, a novel myoepithelial marker, in fine-needle aspiration biopsies of the breast: an analysis of 82 samples. Cancer Cytopathol. 2003;99:172–9. 30. Gerhard R, Costa JL, Schmitt F.  Benign and malignant apo crine lesions of the breast. Expert Rev Anticancer Ther. 2012;12: 215–21. 31. Hammond ME, Hayes DF, Dowsett M, Allred DC, Hagerty KL, Badve S, et  al. American Society of Clinical Oncology/College of American Pathologists guideline recommendations for immunohistochemical testing of estrogen and progesterone receptors in breast cancer (unabridged version). Arch Pathol Lab Med. 2010;134:e48–72. 32. Wolff AC, Hammond ME, Hicks DG, Dowsett M, LM MS, Allison KH, et  al. Recommendations for human epidermal growth factor receptor 2 testing in breast cancer: American Society of Clinical Oncology/College of American Pathologists clinical practice guideline update. Arch Pathol Lab Med. 2014;138:241–56. 33. Allred C, Miller K, Viale G. Molecular testing for estrogen receptor, progesterone receptor, and HER2. In: Lakhani SR, Ellis IO, Schnitt SJ, et al., editors. WHO classification of tumors of the breast. Lyon: IARC; 2012. p. 22–3. 34. Skoog L, Tani E.  Immunocytochemistry: an indispensable technique in routine cytology. Cytopathol: Off J Br Soc Clin Cytol. 2011;22:215–29. 35. Domanski AM, Monsef N, Domanski HA, Grabau D, Ferno M.  Comparison of the oestrogen and progesterone receptor status in primary breast carcinomas as evaluated by immunohistochemistry and immunocytochemistry: a consecutive series of 267 patients. Cytopathol: Off J Br Soc Clin Cytol. 2013;24:21–5. 36. Schmitt FC, Bento MJ, Amendoeira I.  Estimation of estrogen receptor content in fine-needle aspirates from breast cancer using the monoclonal antibody 1D5 and microwave oven processing: correlation with paraffin embedded and frozen sections determinations. Diagn Cytopathol. 1995;13:347–51. 37. Cano G, Milanezi F, Leitao D, Ricardo S, Brito MJ, Schmitt FC. Estimation of hormone receptor status in fine-needle aspirates and paraffin-embedded sections from breast cancer using the novel rabbit monoclonal antibodies SP1 and SP2. Diagn Cytopathol. 2003;29:207–11. 38. Ferguson J, Chamberlain P, Cramer HM, Wu HH.  ER, PR, and Her2 immunocytochemistry on cell-transferred cytologic smears of primary and metastatic breast carcinomas: a comparison study with formalin-fixed cell blocks and surgical biopsies. Diagn Cytopathol. 2013;41:575–81.

103 39. Vohra P, Buelow B, Chen YY, Serrano M, Vohra MS, Berry A, et al. Estrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 expression in breast cancer FNA cell blocks and paired histologic specimens: a large retrospective study. Cancer Cytopathol. 2016;124:828–35. 40. Acs B, Szekely N, Szasz AM, Lotz G, Szekely T, Istok R, et  al. Reliability of immunocytochemistry and fluorescence in situ hybridization on fine-needle aspiration cytology samples of breast cancers: a comparative study. Diagn Cytopathol. 2016;44:466–71. 41. Marinsek ZP, Nolde N, Kardum-Skelin I, Nizzoli R, Onal B, Rezanko T, et al. Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates. Cytopathol: Off J Br Soc Clin Cytol. 2013;24:7–20. 42. Beatty BG, Bryant R, Wang W, Ashikaga T, Gibson PC, Leiman G, et al. HER-2/neu detection in fine-needle aspirates of breast cancer: fluorescence in situ hybridization and immunocytochemical analysis. Am J Clin Pathol. 2004;122:246–55. 43. Williams SL, Birdsong GG, Cohen C, Siddiqui MT. Immunohistochemical detection of estrogen and progesterone receptor and HER2 expression in breast carcinomas: comparison of cell block and tissue block preparations. Int J Clin Exp Pathol. 2009;2:476–80. 44. Kinsella MD, Birdsong GG, Siddiqui MT, Cohen C, Hanley KZ. Immunohistochemical detection of estrogen receptor, progesterone receptor and human epidermal growth factor receptor 2 in formalin-fixed breast carcinoma cell block preparations: correlation of results to corresponding tissue block (needle core and excision) samples. Diagn Cytopathol. 2013;41:192–8. 45. Zhang Z, Yuan P, Guo H, Zhao L, Ying J, Wang M, et al. Assessment of hormone receptor and human epidermal growth factor receptor 2 status in breast carcinoma using thin-prep cytology fine needle aspiration cytology FISH experience from China. Medicine (Baltimore). 2015;94:e981. 46. Shabaik A, Lin G, Peterson M, Hasteh F, Tipps A, Datnow B, et al. Reliability of Her2/neu, estrogen receptor, and progesterone receptor testing by immunohistochemistry on cell block of FNA and serous effusions from patients with primary and metastatic breast carcinoma. Diagn Cytopathol. 2011;39:328–32. 47. Gorman BK, Kosarac O, Chakraborty S, Schwartz MR, Mody DR.  Comparison of breast carcinoma prognostic/predictive biomarkers on cell blocks obtained by various methods: Cellient, formalin and thrombin. Acta Cytol. 2012;56:289–96. 48. Durgapal P, Mathur SR, Kalamuddin M, Datta Gupta S, Parshad R, Julka PK, et al. Assessment of Her-2/neu status using immunocytochemistry and fluorescence in situ hybridization on fine-needle aspiration cytology smears: experience from a tertiary care centre in India. Diagn Cytopathol. 2014;42:726–31. 49. Bozzetti C, Nizzoli R, Guazzi A, Flora M, Bassano C, Crafa P, et al. HER-2/neu amplification detected by fluorescence in situ hybridization in fine needle aspirates from primary breast cancer. Ann Oncol: Off J Eur Soc Med Oncol/ESMO. 2002;13:1398–403. 50. Nishimura R, Kagawa A, Tamogami S, Kojima K, Satou M, Yamashita N, et  al. Correlation of HER2 gene status assessment by fluorescence in situ hybridization between histological sections and cytological specimens of breast cancer. Breast Cancer. 2016;23:211–5. 51. Schmitt F, Barroca H.  Role of ancillary studies in fine-needle aspiration from selected tumors. Cancer Cytopathol. 2012;120:145–60. 52. Annaratone L, Marchio C, Renzulli T, Castellano I, Cantarella D, Isella C, et  al. High-throughput molecular analysis from leftover of fine needle aspiration cytology of mammographically detected breast cancer. Transl Oncol. 2012;5:180–9.

104 53. Beca F, Schmitt F. Growing indication for FNA to study and analyze tumor heterogeneity at metastatic sites. Cancer Cytopathol. 2014;122:504–11. 54. Malapelle U, Mayo-de-Las-Casas C, Molina-Vila MA, Rosell R, Savic S, Bihl M, Bubendorf L, Salto-Tellez M, de Biase D, Tallini G, Hwang DH, Sholl LM, Luthra R, Weynand B, Vander Borght S, Missiaglia E, Bongiovanni M, Stieber D, Vielh P, Schmitt F, Rappa A, Barberis M, Pepe F, Pisapia P, Serra N, Vigliar E, Bellevicine C, Fassan M, Rugge M, de Andrea CE, Lozano MD, Basolo F, Fontanini G, Nikiforov YE, Kamel-Reid S, da

F. Schmitt et al. Cunha Santos G, Nikiforova MN, Roy-Chowdhuri S, Troncone G, Molecular Cytopathology Meeting G.  Consistency and reproducibility of next-generation sequencing and other multigene mutational assays: a worldwide ring trial study on quantitative cytological molecular reference specimens. Cancer Cytopathol. 2017;125:615–626. 55. Yanagawa T, Kagara N, Miyake T, Tanei T, Naoi Y, Shimoda M, et  al. Detection of ESR1 mutations in plasma and tumors from metastatic breast cancer patients using next-generation sequencing. Breast Cancer Res Treat. 2017;163:231–40.

4

Salivary Glands and Head and Neck Elwira Bakuła-Zalewska, Henryk A. Domanski, and Gabrijela Kocjan

 he Role and Diagnostic Accuracy of FNA T in the Management of Salivary Glands Cytological examination aims to determine if a process is of salivary or non-salivary origin, if it is inflammatory and/or reactive, and if it is neoplastic and either benign or malignant and, if possible, to give a specific diagnosis [1, 2]. Almost all neoplastic salivary gland lesions undergo surgical excision. In patients undergoing radical surgery, preoperative FNA determines the degree of urgency and helps plan the surgical approach, particularly the decision to preserve or sacrifice the facial nerve. Importantly, a preoperative diagnosis allows for presurgical counseling of the patient. In patients not undergoing surgery, FNA can confirm both benign and malignant disease, the latter in those unsuitable for attempted curative surgery or with recurrent disease before palliative treatment. With experience and good team work, sensitivity and specificity of FNA are 81–100% and 94–100%, respectively [3–11]. Accuracy in diagnosis of pleomorphic adenoma is invariably high, which is important since this is by far the most common FNA finding in routine practice. The diagnostic accuracy of ThinPrep LBC (Cytyc; Marlborough, MA, USA)-processed salivary gland FNA approaches that of the direct smears [12]. Causes of false-negative and false-positive errors and diagnostic problems have been reviewed in several papers E. Bakuła-Zalewska (*) Department of Pathology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Warsaw, Poland e-mail: [email protected] H. A. Domanski Department of Pathology, Skåne University Hospital, Lund, Sweden e-mail: [email protected] G. Kocjan Department of Cellular Pathology, University College London, London, UK e-mail: [email protected]

[13–20]. False-negative results are generally due to unrepresentative sampling, especially from cystic tumors [21–25], and false-positive reports from failure to appreciate the difficulties associated with pleomorphic adenoma and basal cell adenoma [26–31]. Successful FNAC of the salivary glands depends on the recognition of the problem lesions. In many cases, giving a list of differential possibilities is better than making a definite, specific diagnosis.

 ilan System for Reporting Salivary Gland M Cytopathology Salivary gland FNA reporting has always generated some confusion for both pathologists and clinicians due to lack of uniform and generally accepted classification scheme for reporting of salivary gland FNA. Currently there are different reporting schemes with diversity of diagnostic categories as well as descriptive reports. Because of the need for a defined set of diagnostic categories for salivary gland FNA, a group of international pathologists (over 40 participants from 14 countries) proposed recently a classification scheme for reporting salivary gland FNA specimens, known as the “Milan System for Reporting Salivary Gland Cytopathology.” Similar to the Bethesda system for reporting thyroid cytopathology, this scheme consists of six main categories: nondiagnostic; nonneoplastic; atypia of undetermined significance; neoplasm-benign; salivary gland neoplasm of undetermined malignant potential, suspicious for malignancy; and malignant. The objective of developing the Milan System was to create a practical classification system that is evidence-­ based, user-friendly, and internationally accepted. The Milan System is an important improvement in the field of salivary gland FNA and thus provides a uniform diagnostic terminology and optimizes communication between pathologist and clinician, data evaluation, and a cytologic and histologic correlation of cases [32–34].

© Springer International Publishing AG, part of Springer Nature 2019 H. A. Domanski (ed.), Atlas of Fine Needle Aspiration Cytology, https://doi.org/10.1007/978-3-319-76980-6_4

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 spiration and Specimen Preparation A Techniques Depending on the clinical situation, the cytopathologist, surgeon, or radiologist may perform the FNA. Identification of the nodule is essential to successful aspiration. Deep lesions smaller than 1 cm in diameter are best aspirated under ultrasonographic (US) or computed tomography (CT) scanning guidance [14, 35, 36]. Cystic masses are best aspirated using a syringe attached to the needle, with or without a syringe holder. Material can be sent to microbiology. The fluid is processed in a routine manner. US-guided FNA, with immediate assessment of the material by a pathologist or cytotechnologist (ROSE), can be more accurate than specimens obtained in other ways [37, 38]. In general, US cannot reliably distinguish benign from malignant lesions. However, benign lesions tend to have well-defined margins with no infiltration, whereas malignant lesions tend to have ill-defined, infiltrating margins into the normal parotid parenchyma [39]. Preoperative histological biopsies using a core biopsy needle [40] or intraoperative frozen sections have their own hazards and organizational problems. As such, they are recommended in cases of soft-tissue lesions, and some lymphoid infiltrates in which FNAC does not yield enough cells for diagnosis.

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Normal Salivary Gland Components Acinar cells are large, with abundant cytoplasm and small, round, uniform nuclei (see Fig. 4.1). The cytoplasm is finely granular in serous glands and clear or lightly vacuolated in mucous glands. Either type is fragile and easily disrupted by smearing, so that numerous dispersed, bare nuclei are often found in the background. When complete acini are aspirated, the cells are arranged in compact spheres. Intact groups of acini can also be aspirated and have been likened to grapes in a cluster. Ductal cells. The larger ducts are lined by columnar and the smaller ones by cuboidal cells. The cells are usually arranged in flat sheets displaying good cohesion and uniform morphology. Slender pointed nuclei arising from myoepithelial cells may occasionally be identified between the epithelium and the basement membrane. Intraparotid lymph nodes. Due to late encapsulation in fetal life, small lymph nodes are commonly enclosed within the parotid. It is not possible to distinguish between intraparotid lymphadenopathy and true salivary gland pathology reliably by palpation or imaging. FNA of a hyperplastic lymph node yields a mixed population of lymphocytes, follicle center cells, and “tingible body” macrophages.

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Fig. 4.1  Normal salivary gland components. (a) A complete acini with central ducts and intact groups of acini remaining grapes in cluster. (b) Similar pattern is obvious in the cell block section; hematoxylin and eosin (H&E). (c–f) Scanning and high-power view of acinar cell groups showing a “bunch of grapes” arrangement. Note the very regular nuclei

and abundant bubbly cytoplasm (H&E and May–Grünwald–Giemsa [MGG]). (g, h) Ductal epithelial fragments with darker nuclei arranged in sheets. In the periphery of the fragments, some spindled cells can represent myoepithelial cells (H&E and MGG)

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Fig. 4.1 (continued)

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Cysts Careful attention should be directed at identifying the extracellular fluid component (mucoid vs. watery proteinaceous) and the predominant cellular component (e.g., lymphocytes,

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histiocytes, epithelial cells, and oncocytes). Occasionally, epithelial cells may not be obtained on FNA of cystic salivary gland lesions. Some cysts may contain crystals of varying sizes (see Fig. 4.2).

b

Fig. 4.2  Crystals. (a, b) Smears with crystals and inflammation (MGG and H&E)

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Mucous Cysts (Mucocele) Mucocele commonly arise in minor salivary glands of the lip and oral cavity, as well as in the sublingual gland (ranula). When tense and deep seated, these may lead to clinical suspicion of neoplasia. Aspirates contain mucous and macrophages (see Fig.  4.3), as well as lymphocytes and/or granulocytes if the process is inflamed. Even cytologically, it may be difficult to rule out the rare mucinous cystadenoma of salivary glands.

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• Leucocytes and macrophages in variable numbers • Few epithelial cells Differential diagnosis and problems in diagnosis: • Warthin tumor • Pleomorphic adenoma • Mucoepidermoid carcinoma • Acinic cell carcinoma • High-grade carcinoma • Low-grade mucoepidermoid carcinoma

Cytologic features: • Watery or viscous fluid • Scanty cellularity

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Fig. 4.3  Mucocele. (a-d) Thick, viscous fluid contains macrophages and a few leukocytes against a mucinous background (MGG; Papanicolaou (PAP) and H&E)

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Lymphoepithelial Cysts

• Lymphocytes including follicle center cells • A few squamous epithelial cells • Occasional multinucleated giant cells and crystalloids

Lymphoepithelial cysts can represent an early manifestation of HIV infection [41] and are rarely found in patients with advanced disease. They are the most common presentation of HIV infection in children. Bilateral and multiple lymphoepithelial cysts are uncommon. Aspirates contain serous fluid and scattered lymphocytes with an ordinary appearance (see Fig. 4.4).

Differential diagnosis and problems in diagnosis: • Non-Hodgkin lymphoma • Intraparotid lymph node • Chronic sialoadenitis • Warthin tumor • Branchial cleft cyst

Cytologic features: • Cystic aspirates • Foamy macrophages a

b

c

Fig. 4.4  Lymphoepithelial cyst. (a) Bilateral and multiple lymphoepithelial cysts of major salivary glands, in particular of parotid glands, are quite rare and have been reported in human immunodeficiency virus

(HIV) infected patients. (b, c) The aspirates contain foamy macrophages, lymphoid cells, epithelial (squamous) cells, and, occasionally, multinucleated giant cells (MGG)

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Branchial Cleft Cysts Branchial cleft cysts are usually asymptomatic but one-third present acutely due to inflammation [42]. Aspirates show degenerated squamous epithelium (see Fig.  4.5) [43]. In cases in which inflammation is active, there may be marked atypia in the epithelium, and a diagnosis of squamous cancer metastasis may be suspected. Cytologic features: • Abundant viscous fluid • Mature squamous cells and keratinous debris • Macrophages, cholesterol crystals • Acute inflammatory cells if inflamed

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Differential diagnosis and problems in diagnosis: • Cystic metastasis of a squamous cell carcinoma • Ruptured epidermoid cyst

Midline Cysts The most common midline cyst is thyroglossal cyst. In majority of cases, FNA smears of thyroglossal cyst are indistinguishable from that of branchial cleft cyst.

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d

Fig. 4.5  Branchial cleft cyst. (a–c) Cyst is usually lined by squamous and occasionally by columnar epithelium. It may contain variable degree of and keratinous debris and inflammatory cells but usually does

not contain necrotic background (H&E, PAP, and MGG). (d) The aspirates contain occasionally cholesterol crystals and calcifications (MGG)

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Inflammation

cess termed Kuttner tumor or chronic sclerosing sialoadenitis is a tumor-like condition of the salivary glands, which is now often considered an IgG4-related process [45, 46]. Aspirates can contain ductal epithelium with reactive atypia, which is so severe that a diagnosis of neoplasm can be considered (see Fig. 4.6).

Acute Sialoadenitis Acute sialoadenitis is due to specific bacterial or viral infection such as mumps or cytomegalovirus (CMV) parotitis [44]. FNA is not indicated. The presence of acute inflammatory cells in a salivary gland FNA does not always indicate acute sialoadenitis; some salivary gland tumors may contain acute inflammation, either due to necrosis or as a result of a previous FNA.

Cytologic features: • Scanty aspirate • Ductal epithelium, usually a few tightly cohesive clusters • Possible metaplasia or atypia • Scanty or absent acinar cells due to atrophy and fibrosis of acinar tissue • Inflammatory cells and background debris • Sometimes cystic change with a variety of crystalloids

Chronic Sialoadenitis Chronic sialoadenitis is very frequently caused by calculi (sialoliths), which form in the salivary ducts as the result of mineralization of debris. The submandibular gland is the most common site. Clinically, calculi are associated with pain and swelling, and retrograde infection results in acute or chronic sialoadenitis with fibrosis. A particular fibrosing pro-

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Differential diagnosis and problems in diagnosis: • Warthin tumor • Lymphoepithelial cyst • Metastatic squamous cell carcinoma • Low-grade mucoepidermoid carcinoma

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Fig. 4.6  Inflammation. (a) Ductal epithelial fragment with disorder and slight dissociation. (b) Ductal fragments with hyperchromasia. Note inflammation in background. (c) Ductal epithelium with swollen cytoplasm simulating acinar cells. (d) Acinar group and a granuloma (MGG)

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Granulomatous Sialoadenitis Granulomatous sialoadenitis reflects involvement of salivary glands in either a local or generalized granulomatous process, such as sarcoidosis, tuberculosis, fungal infection, cat scratch disease, brucellosis, and toxoplasmosis [47]. Granulomatous reaction to nontyrosine crystalloids can be associated with both neoplastic and nonneoplastic salivary gland disease, and they may be a product of oncocytic cell secretion (see Fig. 4.6). Cytologic features: • Clusters of epithelioid cells • Multinucleated histiocytes • Lymphoid cells

Lymphoepithelial Sialoadenitis Lymphoepithelial sialoadenitis (LESA) is commonest in middle-aged or elderly women and is usually bilateral and symmetrical, although sometimes initially unilateral [48]. In cases of Sjögren’s syndrome, other manifestations such as dryness of the eyes and mouth, rheumatoid arthritis, and hypergammaglobulinemia are present. Malignant lymphomas can develop from these lesions [49].

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Cytologic features: • Reactive lymphoid cells, plasma cells, and histiocytes • Clusters of myoepithelial cells sometimes present Differential diagnosis and problem in diagnosis: • Chronic sialoadenitis • Warthin tumor • Lymphoepithelial cyst • Mucoepidermoid carcinoma • Acinic cell carcinoma • Non-Hodgkin lymphoma • Perisalivary gland lymph nodes

Other Conditions Foreign Material Foreign material, particularly gels injected as part of cosmetic surgery, can be problematic. Patients can present with lumps in the cheeks and near the parotid gland long after the cosmetic procedure has been performed. Awareness of the possibility of foreign material and reactive inflammation can enable a correct evaluation of the aspirate (see Fig. 4.7).

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Fig. 4.7  Foreign material. (a) Structureless, blue, hyaline material (not to be confused with mucin). Air-dried smear (MGG). (b, c) Blue, hyaline material, inflammatory cells, and multinucleated giant cells of for-

eign body type. Alcohol-fixed smear (H&E). (d) Some inflammatory cells can appear atypical (MGG)

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Sialadenosis Sialadenosis is a noninflammatory, often bilateral, swelling of the salivary glands, particularly of the parotid, and shows a hypertrophy of the acinar cells. It is associated with hormonal, nutritional, or neurogenic disorders and with ­systemic

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diseases such as diabetes. A deficiency in myoepithelial cells may play a pathogenetic role [50]. Characteristically, numerous large, benign acinar cells are present, and ductal cells are sparse [51]. A similar picture may be seen, however, in welldifferentiated acinic cell carcinoma, and a definite diagnosis can be hard to establish (see Fig. 4.8).

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Fig. 4.8  Sialadenosis. (a, b) Large acinar cluster with some dissociation. (c, d) Acinar cells with overabundant cytoplasm and poor cohesion (H&E and MGG)

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Lipomatosis Lipomatosis is a diffuse excess of interstitial fat, which may occur in obesity and diabetes, and occasionally presents as a swelling [52]. Fatty replacement of atrophic parenchyma,

a

sialolipoma, periparotid lipomata, and lipometaplasia in pleomorphic adenomas are other causes of fat cells in an aspirate (see Fig. 4.9).

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Fig. 4.9  Lipomatosis. (a) Acinar formations embedded in fat. (b) Widely spaced acinar cells closely associated with fat cells (MGG)

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Sclerosing Polycystic Sialadenosis

Neoplastic Lesions

This infrequently described lesion has been likened to fibrocystic disease of the breast. There is some evidence that it is of neoplastic nature, however, and a progression into malignant disease has been reported (see Fig. 4.10) [53–55].

Almost all neoplastic lesions will undergo surgical excision. Cytology can help in determining the degree of urgency of surgery and also its extent, particularly with regard to the facial nerve. A preoperative diagnosis allows for counseling of the patient prior to surgery or other therapy. If a cytological diagnosis is unclear or potentially malignant, the case should be discussed in a multidisciplinary conference where an appropriate treatment plan can be formulated [1, 2, 56–58].

a

 umors Composed of Both Epithelial T and Myoepithelial Cells

b

Fig. 4.10  Polycystic sclerosing sialadenopathy. (a) Smears are characterized by the tightly cohesive, sharply outlined clusters of hyperplastic ductal epithelium. Low-power view does not show any inflammation or acinar cells in the background. (b) Ductal epithelium is associated with smaller, darker, myoepithelial cells lying in a different plane. Some sheets of ductal epithelium show mild architectural atypia, such as loss of polarity, and occasional mitoses (MGG)

It has been postulated that many salivary gland tumors derive from a primitive cell that has the ability to differentiate into both myoepithelial cells (sometimes termed the basal cell) and ductal epithelial cells [59]. The relative proportion of these two cell types varies considerably in different tumor types and even in different areas of an individual tumor. This heterogeneity is reflected in the appearance of the aspirate. Epithelial cells can be of simple ductal type or have a squamous, mucinous, or oncocytic appearance. To further complicate matters, the myoepithelial cell can also present with several different morphological phenotypes: plasmacytoid, spindled, epithelioid, and clear. These aspects must be kept in mind when considering tumor types discussed below.

Pleomorphic Adenoma This benign tumor is the most common neoplasm of salivary gland origin and usually presents in the parotid gland [60]. It can be found in any salivary gland, however, including minor glands [61, 62]. The diagnosis can be made with some confidence if the characteristic fibrillary ground substance is abundant, if there are numerous plasmacytoid myoepithelial cells, and if some ductal epithelial elements are seen. This picture, in the absence of any atypical cells, is almost pathognomonic of pleomorphic adenoma (PA). However, just as the histological appearance is very variable, the cytological findings can vary [26, 28, 29, 63–68]. Epithelial tissue that shows metaplasia of squamous, mucinous, oncocytic, and clear cell type can complicate the cytological evaluation of PA. Myoepithelial cells are typically plasmacytoid but can be epithelioid, spindled, or clear. In rare cases, cell fragments with an appearance typical for fat, cartilage, or osteoid can be aspirated. It is not unusual that the aspiration diagnosis is uncertain and coupled with a list of differential possibilities (see Figs. 4.11 and 4.12).

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Cytologic features: • Fibrillary, metachromatic extracellular material, often abundant. • Moderately sized, uniform myoepithelial cells, often plasmacytoid, in groups or as dissociated cells, usually dominate. • Epithelial cells may be difficult to see in the thick metachromatic substance. • Some nuclear atypia can be seen (see Fig. 4.13). a

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Differential diagnosis and problems with diagnosis: • Basal cell adenoma • Myoepithelioma • Carcinoma ex pleomorphic adenoma • Adenoid cystic carcinoma • Epithelial–myoepithelial carcinoma • Polymorphous low-grade adenocarcinoma

b

c d

e

f

Fig. 4.11  Pleomorphic adenoma. (a) Abundant, dark, fibrillary matrix. (b) Matrix material can be hyaline and not fibrillary. (c) Plasmacytoid myoepithelial cells characteristic of the diagnosis (MGG). (d, e) Spindled myoepithelial cells in a smear and (f) a cell block (H&E)

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Fig. 4.12  Pleomorphic adenoma. (a, b) Matrix is pale in H&E and PAP. (c, d) Characteristic plasmacytoid myoepithelial cells in H&E and PAP

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a

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d

Fig. 4.13  Pleomorphic adenoma with atypia. (a–d) Occasional, clearly atypical cells in otherwise ordinary pleomorphic adenomas (MGG and H&E)

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 asal Cell Adenoma B An unusual tumor, basal cell adenoma (BCA) presents most often in the major salivary glands [69]. Basaloid cells dominate the aspirate completely, although the sparse presence of a metachromatic ground substance, often hyaline, gives witness to an occasional participation of myoepithelial cells even in this tumor type [30, 63, 70–72]. Trabecular, tubular, membranous, and solid variants have been described (see Fig. 4.14) [31]. a

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Cytologic features: • Numerous, regular epithelioid cells in groups or dissociated. • Sparse amounts of hyaline and even faintly fibrillary metachromatic, extracellular material can be present. Differential diagnosis and problems with diagnosis: • Basal cell adenocarcinoma • Adenoid cystic carcinoma • Pleomorphic adenoma b

c d

e

f

Fig. 4.14  Basal cell adenoma. (a–f) Tightly cohesive cell groups. Very regular cells and nuclei. Some fibrillary and/or hyaline matrix substance (MGG and H&E)

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Canalicular Adenoma This type of benign adenoma occurs almost exclusively on the upper lip [73–76]. Aspirates show a distinctive narrow pattern of single- and double-cell rows of epithelial cells resembling a band of pearls. The presence of a metachromatic ground substance is often sparse in canalicular adenomas (see Fig. 4.15). Cytologic features: • Upper lip is the typical location. • Cell yield can be abundant. • Cells are usually small and very regular. • Tissue fragments can be trabecular or tubular. • Little or no stroma component of any kind. Differential diagnosis and problems with diagnosis: • Basal cell carcinoma of the skin (of the lip) • Stroma poor (cellular) pleomorphic adenoma • Basal cell adenoma

a

b

Fig. 4.15  Canalicular adenoma. (a, b) Some dissociation but regular cells and nuclei. Obvious hyaline matrix in globules (MGG)

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 olymorphous Adenocarcinoma (PAC) P These malignant tumors often arise from minor glands and have a predilection for the hard palate. The histological diagnosis is based on heterogeneity of growth patterns (solid, trabecular, tubular, and cribriform) couples with a cytological homogeneity [66]. The tumors are not encapsulated and often show neural invasion. The cytological diagnosis can be suggested if a high index of suspicion is maintained in the appropriate clinical setting (see Fig. 4.16) [77–80]. Cytologic features: • Cellular aspirate with uniform cells—no atypia • Cells are loosely cohesive and show pale cytoplasm • Sparse extracellular metachromatic material Differential diagnosis and problems with diagnosis: • Pleomorphic adenoma • Adenoid cystic carcinoma

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b

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d

Fig. 4.16  Polymorphous adenocarcinoma. (a, b) Large-cell fragments, some of which show slight dissociation. Cells somewhat larger than adenomas and hyperchromatic (MGG). (c) Large crowded cluster of

slightly pleomorphic cells with small nucleoli (H&E). (d) The tumor cells surround metachromatic matrix reminiscent of adenoid cystic carcinoma (MGG)

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 pithelial/Myoepithelial Carcinoma (EMC) E This tumor variant shows a very specific relationship between epithelial and myoepithelial cells in tissue sections [81]. If tumor microbiopsies are present in aspirates, the diagnosis can be suggested, but other tumors in this group of salivary gland neoplasms can also show the same growth pattern in small foci, making a firm diagnosis difficult (see Fig. 4.17) [82–86]. Cytologic features: • Variable cell yield • Cells are moderately large and show little atypia.

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• Cell groups can show a biphasic appearance with smaller ductal cells and larger, often pale, peripherally situated cells. • A metachromatic extracellular substance is usually present. Differential diagnosis and problems with diagnosis: • Pleomorphic adenoma • Adenoid cystic carcinoma • Basal cell adenoma

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d

Fig. 4.17  Epithelial–myoepithelial carcinoma. (a–d) Large cell clusters in which a core of smaller, darker cells can sometimes be appreciated. The outer cell layer, myoepithelium, can be spindled or vacuolated (MGG)

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 denoid Cystic Carcinoma (ACC) A The specter of this cancer makes the importance of adequate cytological diagnosis in the whole group of salivary gland tumors obvious [87]. Insufficient first surgery in a patient with this diagnosis may have serious prognostic implications. Therefore, in aspirates from this family of salivary gland tumors, the possibility of ACC should be raised in cases in which another tumor diagnosis is not obvious. The histological spectrum of ACC is reflected in the aspiration cytology (see Figs. 4.18 and 4.19) [65, 88, 89].

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• Hyaline “balls” closely associated with epithelial clusters. • Usually abundant cell yield. • Nuclei are dark and can be angular. • Cytoplasm is relatively sparse. Differential diagnosis and problems with diagnosis: • Pleomorphic adenoma • Basal cell adenoma/adenocarcinoma • Myoepithelioma

Cytologic features: • Cases with broad cylinders of metachromatic hyaline material are very characteristic for the “cylindroma” type of ACC. a

b

c

d

Fig. 4.18  Adenoid cystic carcinoma. (a–d) Cohesive cell groups with small amounts of hyaline matrix. Cells have scant pale-staining cytoplasm, and nuclei can be dark and angular (H&E and PAP)

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a

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c

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Fig. 4.19  Adenoid cystic carcinoma. (a–c) Large and smaller balls and cylinders of a metachromatic, hyaline matrix substance, an appearance that speaks strongly for ACC.  Cells with sparse cytoplasm and dark,

oval nuclei showing some dissociation (MGG). (d–f) Basal dark cells and tubular structures—irregular hyaline matrix, characteristic pattern (PAP and H&E)

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 arcinoma Ex Pleomorphic Adenoma (CEPA) C The cell picture of a malignant tumor must be obvious to make this diagnosis [90]. CEPA usually shows the picture of an adenocarcinoma UNS but can have the appearance of adenoid cystic cancer, salivary duct cancer, or ­mucoepidermoid cancer [91–96]. In addition, aspects that are typical for PA must also be found. Carcinoma in situ ex PA and microinvasive carcinoma ex PA are entities that also must be kept in mind (see Fig. 4.20).

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• Presence of epithelial cells with poor cohesion and obvious nuclear irregularity Differential diagnosis and problems with diagnosis: • Pleomorphic adenoma with atypia • Adenocarcinoma UNS • Adenoid cystic carcinoma • High-grade mucoepidermoid carcinoma • Salivary duct carcinoma

Cytologic features: • Presence of the chondromyxoid stroma, which is typical for pleomorphic adenoma

a

b

c

d

Fig. 4.20  Carcinoma ex pleomorphic adenoma. (a–d) Cell clusters with a matrix suggestive of pleomorphic adenoma but with a cellular disorder and nuclear atypia greater than what is usually tolerated in the benign tumor (MGG and PAP)

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Myoepithelioma/Myoepithelial Carcinoma This group of tumors is defined by exclusive composition of myoepithelial cells [97]. Pleomorphic adenomas with a very small component of epithelial cells, which may not be represented in an aspirate, pose an obvious differential diagnostic difficulty [98]. Basal cell adenomas can also be difficult to differentiate due to the variability of the appearance of both basal and myoepithelial cells. A confident diagnosis of myoepithelial tumors of the salivary glands should be rare [82, 99]. In most cases, a differential diagnosis must be rendered (see Fig. 4.21). An unusual variant of myoepithelioma is the myxoid type (see Fig. 4.22).

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• Plasmacytoid, epithelioid, and clear cell variants common. • Some intercellular hyaline ground substance can be present. • Atypia is absent or minimal. • Cells are positive in caldesmon and actin stains. Differential diagnosis and problems with diagnosis: • Pleomorphic adenoma • Basal cell adenoma • Myoepithelial carcinoma

Cytologic features: • Variable cell yield, often very cellular. • Spindled cells in fragments with limited dissociation.

a

b

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d

Fig. 4.21  Myoepithelioma. (a, b) Dissociated oval nuclei with sparse, pale cytosol. Note the clumps of hyaline matrix (MGG). (c, d) More spindled nuclei in alcohol-fixed smears. Obvious eosinophilic matrix, similar to adenoid cystic cancer but with paler, spindled cells (H&E)

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c

d

Fig. 4.22  Myoepithelioma. Myxoid variant. (a) Histological appearance of this unusual variant. (b, c) Smears show an abundant yield with loosely cohesive fragments of pale spindled cells and a very pale matrix

(H&E). (d) Abundant matrix substance with metachromatic appearance (MGG)

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 asal Cell Adenocarcinoma B This malignant counterpart to basal cell adenomas may comprise up to 3% of salivary gland malignancies and occurs largely in the major glands [100]. Histologically, it must be differentiated from adenoid cystic cancer, basaloid squamous cell cancer, cellular pleomorphic adenoma, and basal cell adenoma. It shows growth patterns that reflect the various patterns seen in the benign counterpart. Very few cases detailing the cytological features have been reported (see Fig. 4.23) [101–106].

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Cytologic features: • Very similar to basal cell adenoma • Morphological signs of malignancy can be subtle Differential diagnosis and problems with diagnosis • Basal cell adenoma • Pleomorphic adenoma

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d

Fig. 4.23  Basal cell adenocarcinoma. (a, b) Low-power views showing an abundant, dissociated cell yield. (c, d) Very regular nuclei and sparse, pale cytoplasm. No background matrix (MGG and H&E)

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Tumors of Acinar Cells This category is comprised essentially of acinic cell carcinoma (AcCC). There are, however, almost as many cytological variations in its appearance as there are histological subtypes (see Figs. 4.24 and 4.25) [107]. Well-differentiated forms are often easily recognized and need to be separated from normal salivary gland epithelium, from adenomatoid hyperplasia (see Fig.  4.26), and from sialadenosis (see Fig.  4.8). AcCC with ductal differentiation can have mucinous aspects. It is common for AcCC to be associated with lymphoid cells [108–111]. The unusual cases of dedifferentiated AcCC may be recognized as malignant but may be difficult to diagnose as being derived from acinar cells [112, 113].

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Cytologic features: • Abundant cell yield. • Uniform, pale, dissociated cells. • Abundant pale, granular, or finely vacuolated cytoplasm. • Lymphocytes often present. • Clear cells, oncocytes, and mucinous cells can be seen. Differential diagnosis and problems with diagnosis: • Normal salivary gland cytology • Sialadenosis • Mucoepidermoid carcinoma • Warthin tumor

b

d

Fig. 4.24  Acinic cell carcinoma. (a–d) Acinic cell groups showing variation in cytoplasmic appearance and nuclei. Note abundant lymphoid cells in (a) (MGG)

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d

e

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Fig. 4.25  Acinic cell carcinoma. (a–c) In alcohol-fixed smears, the nuclei appear very regular (H&E and Pap) as they do in liquid based preparation (d) (H&E). Microscopic findings in smears (e) are very similar in this section from a cell block (f) (H&E)

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Fig. 4.26  Adenomatoid hyperplasia. (a) Acinar fragments without usual grape cluster-like architecture. (b) Sheets of acinar cells with bland nuclei are difficult to differentiate from acinic cell carcinoma. (c)

Acinar cells lacking normal organization and showing some hyperchromasia. (d) Histological section showing sheets of bland acini (MGG, PAP, H&E)

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Mucinous Tumors Mucoepidermoid Carcinoma Mucoepidermoid cancer (MEC) is the most common of mucinous tumors and is defined by the presence of ­mucous-­producing cells. Intermediate cells and squamoid cells also need to be present to make a confident diagnosis [114]. Several other tumor types can contain mucinous cells including mucinous adenocarcinoma, cystadenocarcinoma, AcCC, adenocarcinoma not otherwise specified (NOS), Warthin tumor, and rarely PA. MEC can be well, moderately, or poorly differentiated and the cytology correspondingly very variable (see Fig. 4.27) [115–121].

a

Fig. 4.27  Mucoepidermoid carcinoma. (a, b) A mucoid background is a common and helpful finding. Cells often have well-defined cytoplasmic margins (MGG). (c–e) The relatively abundant cytoplasm cohesive cell groups are usual. Intracellular mucous production should be seen to suggest the diagnosis (H&E, MGG). (f) The mucous cells contain

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Cytologic features: • Commonly cystic with a mucinous background. • Heterogeneous cell population; mucinous, squamous, ductal. • Lymphocytes often present. • Clear cells and oncocytes can be present and even dominant. Differential diagnosis and problems with diagnosis: • Mucocele • Mucinous cystadenoma/adenocarcinoma • Warthin tumor • Acinic cell carcinoma • Squamous cell carcinoma (poorly differentiated MEC)

b

faintly eosinophilic mucin vacuoles on Papanicolaou stain. The remaining cells represent intermediate cells, and a few platelike squamous cells are present at the periphery. This epithelial heterogeneity is characteristic of mucoepidermoid carcinoma (PAP)

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d

e

f

Fig. 4.27 (continued)

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Secretory Carcinoma (Mammary Analogue Secretory Carcinoma) Secretory carcinoma is a low-grade salivary gland carcinoma with morphological resemblance to mammary secretory carcinoma. Secretory carcinoma usually occurs in adults and most often involves the parotid gland followed by the oral cavity and submandibular gland. Secretory carcinoma harbors a recurrent translocation t(12;15)(p13;q25), which results in the ETV6-NTRK3 gene fusion. Tumor cells are positive for S100 protein and mammaglobin. Secretory material stains for PAS/PASD and Alcian blue. FNA smears are usually cellular and show loosely cohesive clusters or isolated cells. A mucinous material and histiocytes, occasionally contained hemosiderin pigments, are often seen in the background of the smears or within epithelial clusters. The tumor cells show epithelioid morphology with abundant, eosinophilic, granular or vacuolated cytoplasm, and small uniform nuclei (see Fig. 4.28) [122–124].

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Cytologic features: • Usually abundant cell yield • Loosely cohesive occasionally papillary and follicular clusters or isolated cells • Intermediate to large cuboidal, epithelial, bland tumor cells • Small to medium-sized round to oval nuclei with small nucleoli • Soap bubble-like microvacuolated or finely eosinophilic, variable granular cytoplasm • A mucinous material and hemosiderin-laden histiocytes in the background of the smears Differential diagnosis and problems with diagnosis: • Acinic cell carcinoma • Adenocarcinoma NOS • Mucoepidermoid carcinoma

b

c

Fig. 4.28  Secretory carcinoma. (a, b) Loosely cohesive clusters or isolated intermediate to large cuboidal cells with round to oval nuclei with small nucleoli, microvacuolated cytoplasm, and hemosiderin-laden

histiocytes in the background of the smears (MGG, Pap). (c) Corresponding cell block section (H&E)

138

Tumors with Oncocytes Warthin Tumor (WT) Warthin tumor dominates this group. A combination of oncocytic cell sheets and lymphocytes in an aspirate with cystic aspects can reliably diagnose WT (see Fig.  4.29) [125]. Some tumors are almost entirely necrotic, and cellular degeneration and inflammation can mask the more typical elements. Oncocytomas and oncocytic hyperplasia are less common than WT but must be considered when lymphocytes and cystic elements are lacking (see Fig.  4.31). Several tumors can contain epithelium with oncocytic metaplasia, for example, PA, WT, AcCC, and, rarely, MEC.  Another diagnostic pitfall in smears is that oncocytes, clear cells, and acinic cells can be difficult to differentiate reliably from each other. Squamous metaplasia can be seen and is sometimes atypical (see Fig. 4.30) [17]. A prominent lymphocytic com-

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ponent can be seen, raising the possibility of another differential diagnostic group [126–128]. Cytologic features: • Often a cystic aspirate and “machine-oil” appearance. • Degenerated cells and numerous lymphocytes. • Fragments and sheets of uniform oncocytic cells. • Presence of mast cells in the epithelium is common. Differential diagnosis and problems with diagnosis: • Oncocytoma • Mucoepidermoid carcinoma • Squamous cell cancer (metastasis) • Chronic inflammation, lymphoma, LESA • Branchial cleft cyst • Mucinous cyst or cystadenoma

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b

c

d

Fig. 4.29  Warthin tumor. (a) Cohesive oncocytic cell sheet and lymphocytes in background. (b) Regular oncocytic cells with occasional mast cells (MGG). (c–e) Nuclear details seen better in alcohol-fixed smears (H&E, PAP) and (f) typical tumor architecture in cell block sections (H&E)

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e

139

f

Fig. 4.29 (continued)

a

b

Fig. 4.30  Warthin tumor with atypia. (a, b) Degenerated cells with squamous epithelial aspects can be very worrisome (H&E, MGG)

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Oncocytoma Oncocytoma is an uncommon benign tumor that can be multifocal. It does not display a lymphocytic component and is rarely cystic [126, 127]. Malignant variants are very unusual (see Fig. 4.31). Oncocytic hyperplasia, often multifocal, can be tumor-forming and can only be diagnosed histologically [128, 129]. Cytologic features: • Abundant cell yield. • Cells have uniform nuclei and often a small, obvious nucleolus.

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• Large amount of cytoplasm, which is eosinophilic and granular. • Cell fragments can be loosely cohesive and dissociated cells are common. • No signs of cystic degeneration and no lymphocytes. Differential diagnosis and problems with diagnosis: • Oncocytic hyperplasia • Acinic cell carcinoma with oncocytic metaplasia • Mucoepidermoid carcinoma with oncocytic metaplasia

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a

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d

Fig. 4.31  Oncocytoma. (a–c) Benign tumor with regular cell picture. Cells have abundant evenly granular cytoplasm without lymphoid or pseudoneurotic background (MGG, H&E). (d) A rare malignant tumor showing obvious atypia (MGG)

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Tumors with Obvious Signs of Malignancy  alivary Duct Carcinoma S Salivary duct cancer (SDC) is an especially interesting entity in this group because its cytology is similar to that of mammary cancer [130–132]. The type of SDC first described was similar to the comedo cancer of the breast with large, pleomorphic cells and necrosis [133]. Variants with less cellular atypia and termed well-differentiated SDC have been reported but probably should be classified as low-grade cribriform adenocarcinomas. They have a better prognosis and show some similarity to other types of breast cancer (see Fig. 4.32) [134, 135].

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Cytologic features: • Abundant cell yield with numerous, often pleomorphic malignant cells with large nucleoli. • Necrosis and degeneration common. • Few specific cell or tissue fragment findings; sievelike pattern in sheets can be seen (cribriform appearance). • Squamous differentiation can be seen. Differential diagnosis and problems with diagnosis: • Poorly differentiated mucoepidermoid carcinoma • Salivary gland adenocarcinoma NOS • CEPA • Metastatic adenocarcinoma

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Fig. 4.32  Salivary duct carcinoma. (a–c) Cohesive cell groups can be large and show variable dissociation and clearly malignant features (H&E). (d, e) Large cells with moderate to abundant cytoplasm and

nuclei with distinctive nucleoli (MGG). (f) Corresponding cell block section (Cell block; H&E)

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 oorly Differentiated Mucoepidermoid P Carcinoma Poorly differentiated mucoepidermoid carcinoma can be dominated by pleomorphic squamous cells and mimic primary or secondary squamous epithelial carcinoma (see Fig. 4.33) [136, 137].

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• Squamous cell differentiation prominent. • Some mucinous/signet ring cells must be identified. Differential diagnosis and problems with diagnosis: • Squamous cell carcinoma (primary or metastatic) • Salivary duct carcinoma

Cytologic features: • Often cellular yield with cells, which are obviously malignant.

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Fig. 4.33  Poorly differentiated mucoepidermoid carcinoma. (a, b) Cells with squamoid features and large atypical nuclei and vacuolated cytoplasm (MGG, H&E)

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 rimary Poorly Differentiated Small-Cell P (Neuroendocrine) Carcinoma Primary poorly differentiated small-cell carcinoma has similar morphology to those seen in aspirates of small-cell carcinomas from other sites including the lung. Smears are composed of small cells with scant cytoplasm, irregular and hyperchromatic nuclei with nuclear molding, and often smearing artifact and necrotic background (see Fig.  4.34) [138].

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Cytologic features: • Often cellular yield with small, obviously malignant cells • Scant cytoplasm • Irregular hyperchromatic nuclei • Molding • Background necrosis Differential diagnosis and problems with diagnosis: • Metastatic small-cell carcinoma

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Fig. 4.34  Primary poorly differentiated small-cell (neuroendocrine) carcinoma. (a–d) Cellular smears with dissociated small cells showing clearly malignant features. Scant cytoplasm, irregular hyperchromatic nuclei with molding, and apoptosis/background necrosis (MGG, PAP)

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 rimary Squamous Cell Carcinoma (SCC) P Primary SCC has similar morphology to those seen in aspirates of squamous cell carcinomas from other sites (see

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Fig. 4.35). Primary salivary gland SCC is very rare, and the diagnosis can be made after the exclusion of prior cutaneous SCC or metastatic lesion [139].

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c

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Fig. 4.35  Primary squamous cell carcinoma. (a) This degree of keratinization is a feature of squamous cell carcinoma and not mucoepidermoid carcinoma (PAP). (b, c) Tight clusters of basaloid cells and a

fragment of metachromatic matrix remaining primary salivary gland tumor of basaloid cells (MGG) and (d) corresponding histology of primary salivary gland SCC (H&E)

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Adenocarcinoma NOS Adenocarcinoma NOS primary in the salivary glands is not an uncommon tumor and must be included in the diagnostic differential [140].

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• No specific aspects of differentiation, squamous, mucinous, or oncocytic (see Fig. 4.36) Differential diagnosis and problems with diagnosis: • Metastatic adenocarcinoma

Cytologic features: • Cellular yield with cytologic finding consistent with malignancy

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Fig. 4.36  Adenocarcinoma NOS. (a, b) Malignant cells without any specific aspects of differentiation (MGG)

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Metastatic Tumors Metastatic tumors comprise about 5% of all malignant tumors of salivary gland; the majority of cases are squamous cell carcinoma with melanoma second in frequency. Primary sites are frequently the upper and middle parts of the facial

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region (including the skin, mucous membrane, deep soft tissues, eyes, and ears) [141]. About 10% originate from distant sites such as the lung (especially small-cell carcinoma), kidney (see Fig. 4.37), and breast. About 10% of tumors remain undefined as to their origin.

b

Fig. 4.37  Clear cell carcinoma of the kidney metastasizing to the salivary gland. (a, b) Clusters of malignant cells with abundant, fragile, vacuolated cytoplasm, slightly pleomorphic nuclei and capillaries (MGG)

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Tongue and Oral Cavity Intraoral lesions are not frequently aspirated, and it can be difficult to obtain adequate material. In certain cases, however, FNA can be of help in the diagnosis of lesions even in this area [142–148]. Incorrect diagnosis have most often been found to be due to inadequate sampling but even to incorrect interpretation, especially in cases of salivary gland lesions. Again, it can be prudent to provide a differential diagnosis in such cases, instead of a categorical one, given the difficulties outlined above.

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in the skin, and criteria of malignancy are nearly identical (see Chap. 15).

Salivary Gland-Type Tumors The widespread presence of minor salivary glands implies that tumors of this type can be expected in the oral cavity (see entities above) (see Fig. 4.38).

Squamous Cell Carcinoma The cytomorphology of squamous cell carcinoma in mucosa of the ear–nose–throat (ENT) area is similar to what is seen

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Fig. 4.38  Minor salivary gland-type tumor in the oral cavity. (a) A small mass in the palate and (b) FNAC smears of the mass showing cytologic pattern of adenoid cystic carcinoma (MGG)

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Mesenchymal Tumors

Nasal Cavity and Pharynx

Any type of soft-tissue tumor can arise in this area. Aspirates show findings similar to those from other anatomic sites (see Chap. 14). The problem of differentiating spindle cell lesions from salivary gland tumors with a prominent myoepithelial component can be difficult, and sometimes only a differential diagnosis can be given.

Lesions in these areas are very seldom the object of aspiration cytology due to difficulties in obtaining adequate specimens. A wide variety of lesions can present including carcinomas, melanomas, and sarcomas. The cytological appearance of these processes is discussed elsewhere in this chapter and in chapters dealing with soft tissue, bone tumors, and skin tumors.

Metastases It is often assumed that smears showing findings of an adenocarcinoma in the oral cavity indicate a primary tumor. The possibility of carcinoma spread from elsewhere must be born in mind, however. Certain cancer entities present a characteristic picture, renal cell carcinoma, prostate carcinoma, and carcinomas from the gastrointestinal tract, for example. Immunocytochemistry can be helpful in making a correct diagnosis.

Lymphoma Lymphomas often present in the ENT area. Benign lymph node enlargement is even more common and can be difficult to differentiate from neoplastic processes in the microscope. Flow cytometry with lymphocytic phenotyping can be important in the diagnosis of such lesions [149]. Polymerase chain reaction (PCR) analysis can be a useful diagnostic tool when there is a suspicion of T-cell lymphoma [150]. Specific and nonspecific infections and salivary gland lesions with a considerable lymphocytic component can also cause diagnostic problems in aspirates. The cytology of lymphoid lesions is described in Chap. 9.

Neck Lesions Metastases Squamous cell carcinoma metastases in the upper and middle neck often come from primary tumors in the ENT area, whereas those in the lower neck are more likely to arise from intrathoracic primaries [151]. Metastases of adenocarcinoma type can come from the GI tract, the ENT area, or even the female genital tract. Some metastases have a particular cytological picture (renal, prostate) and should be diagnosed specifically. A smear showing clear cells not thought to be characteristic for renal carcinoma may be a metastasis from a salivary gland tumor. Aspirates showing very poorly differentiated epithelial cells can be obtained from metastases from an undifferentiated carcinoma (lymphoepithelioma-­ like) primary in the nasopharynx (see Fig. 4.39) or an olfactory neuroblastoma (see Fig. 4.40).

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a

b

c

d

Fig. 4.39  Undifferentiated (lymphoepithelioma-like) carcinoma. (a–d) Very large tumor cells with obvious nucleoli. Fragile, pale cytoplasm. Numerous lymphocytes in the background (MGG and H&E)

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b

c

d

Fig. 4.40  Olfactory neuroblastoma. (a–d) Moderately enlarged, angular nuclei with irregular nucleoli. Abundant cytoplasm and visible cell membranes. Some lymphocytes in the background indicating lymph

node. (a, b) MGG and H&E. (c) Cell block (Cellient; Hologic; Bedford, MA, USA) and H&E. (d) Cell block stained for chromogranin

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Cysts and Cystic Metastases A wide variety of cysts can present in the neck. Most of these are lined by a respiratory and/or squamous epithelium, and many are complicated by inflammation. The epithelium which is aspirated can thus show reactive changes with

a

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atypia, raising the possibility of neoplasm. Differentiating between a benign, inflamed cyst and a necrotic carcinoma metastasis is a major problem, and much care must be taken in interpretation and reporting (see Fig.  4.41). Carcinomas primarily in the Waldeyer ring have a tendency to be associated with cystic metastases [152].

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Fig. 4.41  Cystic metastasis of well-differentiated squamous cell carcinoma in the neck lymph node. (a, b) Dispersed slightly atypical tumor cells with obvious squamous differentiation (H&E, MGG). (c) Cell block section confirms diagnosis (H&E)

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Carotid Body Paraganglioma These unusual tumors present at the bifurcation of the carotid artery which can make puncture and aspiration hazardous [153]. In addition, they are often richly vascular. The cell yield varies but can be abundant and shows moderately large cells with regular nuclei and abundant granulated cytoplasm [154–156]. When these features are recognized and the tumor is in a typical location, the diagnosis can be made with some confidence. There is often little nuclear pleomorphism, but some tumors can show obvious anisokaryosis (see Fig. 4.42).

Mesenchymal Tumors Benign and malignant soft-tissue tumors of all kinds can present in the neck and show the same appearance as those from other locales. They are discussed in Chap. 14.

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Nonneoplastic soft-tissue lesions, particularly nodular fasciitis, can be diagnostic dilemmas. If spindle-cell lesions (e.g., schwannoma or solitary fibrous tumor) present close to the parotid gland, the differential diagnosis of pleomorphic adenoma or myoepithelioma may be considered.

Cervical Rib This anomaly, associated with thoracic outlet syndrome, can be found in about 1% of the population, somewhat more frequently in women. Elongated transversal processes are present in about 2% and, again, are more usual in women [157]. These structures can be palpated in the neck, particularly in thin individuals and are occasionally referred for FNA biopsy. Locating and finding a hard, fixed mass when punctured is often diagnostic, even before the slides are ­examined. Connective tissue, cartilage, and bone elements can be aspirated.

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b

c

d

Fig. 4.42  Carotid body tumor. (a–d) Small tumor cells with regular nuclei, although with occasional nuclei, which appear twice as large. Sparse, fragile, pale cytoplasm that can be fibrillary (MGG and H&E)

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References

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E. Bakuła-Zalewska et al. ration cytology and clinicopathologic study of a case. Acta Cytol. 1989;33:195–200. 138. Klijanienko J, Lagace R, Servois V, Lussier C, El-Naggar AK, Vielh P. Fine-needle sampling of primary neuroendocrine carcinomas of salivary glands: cytohistological correlations and clinical analysis. Diagn Cytopathol. 2001;24(3):163–6. 139. Klijanienko J, Vielh P.  Fine-needle sampling of salivary gland lesions. VI.  Cytological review of 44 cases of primary salivary squamous-cell carcinoma with histological correlation. Diagn Cytopathol. 1998;18(3):174–8. 140. Bjørndal K, Krogdahl A, Therkildsen MH, Overgaard J, Johansen J, Kristensen CA, et  al. Salivary gland carcinoma in Denmark 1990–2005: outcome and prognostic factors. Results of the Danish Head and Neck Cancer Group (DAHANCA). Oral Oncol. 2012;48:179–85. 141. Lussier C, Klijanienko J, Vielh P. Fine-needle aspiration of metastatic nonlymphomatous tumors to the major salivary glands: a clinicopathologic study of 40 cases cytologically diagnosed and histologically correlated. Cancer. 2000;90(6):350–6. 142. Gupta N, Banik T, Rajwanshi A, Radotra BD, Panda N, Dey P, et al. Fine needle aspiration cytology of oral and oropharyngeal lesions with an emphasis on the diagnostic utility and pitfalls. J Cancer Res Ther. 2012;8:626–9. 143. Shah SB, Singer MI, Liberman E, Ljung BM.  Transmucosal fine-needle aspiration diagnosis of intraoral and intrapharyngeal lesions. Laryngoscope. 1999;109:1232–7. 144. Daskalopoulou D, Rapidis AD, Maounis N, Markidou S.  Fine-­ needle aspiration cytology in tumors and tumor-like conditions of the oral and maxillofacial region: diagnostic reliability and limitations. Cancer. 1997;81:238–52. 145. Scher RL, Oostingh PE, Levine PA, Cantrell RW, Feldman PS. Role of fine needle aspiration in the diagnosis of lesions of the oral cavity, oropharynx, and nasopharynx. Cancer. 1988;62:2602–6. 146. Bardales RH, Baker SJ, Mukunyadzi P.  Fine-needle aspiration cytology findings in 214 cases of nonparotid lesions of the head. Diagn Cytopathol. 2000;22:211–7. 147. Domanski HA, Akerman M.  Fine-needle aspiration cytology of tongue swellings: a study of 75 cases. Diagn Cytopathol. 1998;18:387–92. 148. Persson PG, Domanski HA.  Fine needle aspiration cytology of uterine leiomyosarcoma metastatic to the tongue. Acta Cytol. 1998;42:1066–7. 149. Schmid S, Tinguely M, Cione P, Moch H, Bode B. Flow cytometry as an accurate tool to complement fine needle aspiration cytology in the diagnosis of low grade malignant lymphomas. Cytopathology. 2011;22:397–406. 150. Dictor M, Skogvall I, Warenholt J, Rambech E. Multiplex polymerase chain reaction on FTA cards vs. flow cytometry for B-lymphocyte clonality. Clin Chem Lab Med. 2007;45:339–45. 151. Jereczek-Fossa BA, Jassem J, Orecchia R. Cervical lymph node metastases of squamous cell carcinoma from an unknown primary. Cancer Treat Rev. 2004;30:153–64. 152. Goldenberg D, Sciubba J, Koch WM. Cystic metastasis from head and neck squamous cell cancer: a distinct disease variant? Head Neck. 2006;28:633–8. 153. Rosa M, Sahoo S. Bilateral carotid body tumor: the role of fine-­ needle aspiration biopsy in the preoperative diagnosis. Diagn Cytopathol. 2008;36:178–80. 154. Masilamani S, Duvuru P, Sundaram S.  Fine needle aspiration cytology diagnosis of a case of carotid body tumour. Singap Med J. 2012;53:e35–7. 155. Monabati A, Hodjati H, Kumar PV. Cytologic findings in carotid body tumors. Acta Cytol. 2002;46:1101–4. 156. Rana RS, Dey P, Das A. Fine needle aspiration (FNA) cytology of extra-adrenal paragangliomas. Cytopathology. 1997;8:108–13. 157. Brewin J, Hill M, Ellis H.  The prevalence of cervical ribs in a London population. Clin Anat. 2009;22:331–6.

5

Head and Neck: Thyroid Paul A. VanderLaan and Jeffrey F. Krane

 rinciples of Evaluation and Reporting P of Thyroid Fine-Needle Aspiration Thyroid nodules are a frequent finding in the general population, with palpable nodules found in 4–7% of adults and subclinical nodules identified in up to 70% of adults, although the vast majority (90–95%) of these nodules are benign [1]. Since its introduction in the early 1950s [2], fine-needle aspiration (FNA) of the thyroid has proven to be an integral and reliable method of evaluating patients presenting with a thyroid mass. The addition of thyroid FNA to clinical evaluation of a thyroid nodule has dramatically increased triage efficiency: not only determining which nodules are at greatest risk of malignancy and should undergo surgery but also indicating which nodules are benign, thus sparing the patient unnecessary surgery [3]. As such, thyroid FNA acts as both a diagnostic test and a screening test. FNA of a palpable thyroid mass may be performed by any clinician with appropriate experience; however, thyroid FNA is being increasingly performed using ultrasound guidance for clinically occult nodules (although this practice is generally discouraged for nodules less than 1  cm in size [4]). Repeat aspirates of nondiagnostic specimens should be performed with ultrasound guidance to ensure proper needle placement. On-site adequacy evaluation can be helpful in reducing nondiagnostic findings and is also recommended in the setting of a repeat aspirate of a nondiagnostic specimen [4]. Largely determined by local practice patterns, both air-­dried and alcohol-fixed smears as well as liquid-based preparations have been used in evaluating thyroid FNAs. Whereas both methods have been shown to provide roughly P. A. VanderLaan Department of Pathology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA e-mail: [email protected] J. F. Krane (*) Department of Pathology, Brigham & Women’s Hospital, Harvard Medical School, Boston, MA, USA e-mail: [email protected]

equivalent results, each technique has been shown to have characteristic strengths and limitations [5, 6]. Adjunct use of cell block preparations and/or core biopsies may also be helpful in certain situations [7, 8]. In the past, thyroid FNA reporting generated much confusion for both clinicians and pathologists due to multiple different reporting schemes and descriptive reports that did not clearly convey malignancy risk [9]. In the past decade, more uniform and evidence-based reporting schemes have been instituted, including the six-tiered Bethesda System for Reporting Thyroid Cytopathology (TBSRTC), arising from the 2007 National Cancer Institute Thyroid FNA State of the Science Conference in Bethesda, Maryland, USA [10, 11], as well as the virtually identical updated Royal College of Pathologists Thy1–5 reporting system [12]. As a result, these systems provide clarity of communication, facilitating exchange of data across institutions, as well as providing an implicit cancer risk associated with each category to guide appropriate clinical management.

Benign Components Normal components of a thyroid aspirate are primarily comprised of follicular cells and colloid. Intact follicles exhibit a spherical configuration with centrally located colloid (see Fig.  5.1). Architecturally, macrofollicles predominate in benign aspirates, while microfollicles (composed of 15 or fewer follicular cells in a tight spherical configuration with scant centrally located colloid) are absent or a minor component. Most commonly, macrofollicles rupture when aspirated, releasing their luminal colloid and thereby appearing as flat sheets of uniformly spaced follicular cells with small round nuclei and condensed chromatin (see Fig. 5.2). Nuclear size variation (so-called endocrine atypia) may be observed intermittently and is of no consequence (see Fig. 5.2c). As follicular cells undergo oncocytic (Hürthle cell) metaplasia, the cells exhibit more abundant granular cytoplasm with enlarged, round, eccentrically placed nuclei with prominent nucleoli

© Springer International Publishing AG, part of Springer Nature 2019 H. A. Domanski (ed.), Atlas of Fine Needle Aspiration Cytology, https://doi.org/10.1007/978-3-319-76980-6_5

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Fig. 5.1  Intact benign follicles. Intact follicles maintain a three-­ dimensional spherical shape with regular spacing of follicular cells around a central core of colloid. A predominance of variably sized macrofollicles is present; however, a single microfollicle is present (lower left). The even spacing of the round follicular cell nuclei is apparent (Papanicolaou [Pap] stain)

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Fig. 5.2  Benign macrofollicular fragments. (a, b) Macrofollicles are usually disrupted when aspirated, presenting as flat or twisted sheets of follicular cells with regular spacing of round nuclei (a, ThinPrep; b, SurePath). (c) On higher power, modest variation in nuclear size and spacing is evident. Nuclei have granular chromatin with occasional

small, indistinct nucleoli (ThinPrep). (d) On air-dried smears, nuclear detail is not as crisp, but the nuclei are uniform and round with smooth nuclear membranes. Mild crowding may be seen, most likely an artifact of smearing (Diff-Quik [DQ])

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(see Fig. 5.3). Colloid has a variable appearance commonly seen as rounded or irregular aggregates with jagged “cracking” artifact (see Fig. 5.4a, b). “Bubbles” may also be apparent (see Fig.  5.4c). Liquid colloid has a coating or filmy quality present in the aspirate background. In liquid-­based cytologic preparations, “tissue paper” colloid may be noted with fine wrinkles and folds that may represent the counterpart to the liquid colloid of smears (see Fig. 5.4d) [13]. Cystic thyroid nodules are commonly encountered and characteristically exhibit hemosiderin-laden macrophages in the setting of intracystic hemorrhage (see Fig. 5.5a, b). Follicular cells lining distended thyroid cysts (known as cyst-lining cells) have elongated, drawn-out cytoplasm and can exhibit nuclear pallor and longitudinal grooves that can be mistaken for evidence of papillary carcinoma if not appropriately recognized (see Fig. 5.5c, d) [14]. Follicular cells also may have cytoplasmic hemosiderin in this setting (see Fig. 5.5e). Multinucleated giant cells are often encountered in thyroid aspirates, although are not specific for either benignity or malignancy (see Fig. 5.5f). Extensive pigment deposition in follicular cells also occurs in “black thyroid” seen in patients with long-term use of antibiotics related to tetracycline (see Fig. 5.6). Nonthyroidal elements including the skin, skeletal muscle, adipose tissue, and cartilage may be obtained through transit of the needle to the target lesion (see Fig. 5.7a). Ciliated columnar cells (see Fig. 5.7b) inadvertently sampled from the trachea can elicit a cough response in the patient but are of no other concern, although if seen in conjunction with anucleated squamous cells, sampling of a branchial cleft cyst or thyroglossal duct cyst should be considered if clinically suggested (see Fig. 5.7c). Of greater consequence are lymph nodes and parathyroid nodules that may mimic thyroid lesions. In addition to lymph node sampling, when numerous lymphocytes are seen in a supposed thyroid aspirate, there may be concern for Hashimoto thyroiditis or lymphoma, with flow cytometry extremely useful for diagnosing the latter. Parathyroid tissue exhibits small uniform cells resembling thyroid follicular epithelium. These cells present in crowded aggregates and cords that suggest a follicular neoplasm (see Fig. 5.8). The absence of colloid is a clue to their proper recognition. Immunocytochemical stains and fluid parathyroid hormone levels are valuable if the diagnosis is suspected [15]. Adequacy in thyroid aspirates generally requires identification of six or more groups of follicular cells comprised of at least ten cells per group. Any aspirate with a diagnostic abnormality is adequate regardless of overall cellularity. Diagnoses of specific conditions like Hashimoto thyroiditis or subacute thyroiditis also obviate the need for a minimum number of follicular cells. A colloid nodule (see Fig. 5.9) is also considered benign as the presence of abundant colloid is deemed evidence of an underlying macrofollicular process even in the absence of any follicular cells. Classification of cystic lesions lacking adequate follicular cells is controversial, but such aspirates are generally classified as nondiagnostic with explanatory text [11].

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Fig. 5.3  Hürthle cell change. (a) A spectrum of oncocytic (Hürthle) cell change can be seen in benign reactive conditions, characterized by abundant granular cytoplasm. The extent of Hürthle cell change is most pronounced in the group at upper left and least developed in the cells at the lower right (ThinPrep). (b) Significant size heterogeneity can be seen in a reactive Hürthle cell population, typifying benign “endocrine atypia” (DQ). (c) On high power, the granular nature of the polygonal cytoplasm is appreciated, as well as the prominent nucleoli and occasional binucleation (ThinPrep)

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Fig. 5.4  Colloid. (a) On air-dried smears, colloid often appears as sheets of metachromatic translucent material with characteristic “cracking” artifact, likened to a broken stained glass window (DQ). (b, c) Dense colloid fragments can have smooth or jagged edges and may have entrapped “bubbles,” although cracks in dense colloid fragments

are seen more often than not (ThinPrep). (d) The thin or watery colloid seen on liquid-based preparations has a delicate tissue paper-like quality and can be red-pink to blue-green in color. Clumps of fibrin can have a somewhat similar appearance, although fibrin is more granular and stranded without the delicate folds of colloid (ThinPrep)

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Fig. 5.5  Cystic degeneration. (a) Hemosiderin-laden macrophages are common findings in aspirates from nodules with either cystic degeneration or prior aspiration. These cells have ample bubbly cytoplasm and round to kidney bean-shaped nuclei with variably prominent nucleoli. Golden refractile hemosiderin pigment or red cell fragments fill the cytoplasm (ThinPrep). (b) On air-dried preparations, the cells appear larger with more prominent cytoplasmic vacuolization and dark blue hemosiderin granules (DQ). (c) Benign follicular cells lining cysts can adopt an elongated, spindled, or streaming conformation with nuclear enlargement and more vesicular chromatin (ThinPrep). (d) Cyst-lining

cells on air-dried smear can appear quite bizarre, although the cohesive cytoplasmic streaming, preserved N/C ratio, and background macrophages help identify them as benign (DQ). (e) Hemosiderin can also be deposited in the cytoplasm of follicular cells in cystic lesions (ThinPrep). (f) Multinucleated histiocyte giant cells are often encountered in thyroid aspirates, although are not specific for either benignity or malignancy. Foreign body giant cells can be observed with engulfed colloid or a fragment of suture material (as illustrated here), suggesting prior procedure (DQ)

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b Fig. 5.6  Black thyroid. Long-term use of the tetracycline class of antibiotics (minocycline in this example) can lead to dark brown pigment deposition in follicular cells. This pigment is melanin-like, staining with a Fontana-Masson stain, with a less refractile nature than hemosiderin (ThinPrep)

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Fig. 5.7  Nonthyroidal elements. (a) Skeletal muscle and adipose tissue are occasionally seen in thyroid aspirates, quite easy to recognize on cell block preparations shown here. On smears or liquid-based preparations, the striations seen in skeletal muscle differentiate it from colloid (hematoxylin and eosin [H&E]). (b) Benign columnar cells with terminal bars and cilia can be encountered in thyroid aspirates when the operator inadvertently punctures the trachea, as well as when sampling a thyroglossal duct cyst (ThinPrep). (c) Squamous cells and anucleated squames can be seen when aspirating branchial cleft cysts or thyroglossal duct cysts, rarely due to cutaneous contamination (ThinPrep)

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Fig. 5.8  Parathyroid. (a) Parathyroid aspirate showing a vaguely follicular architectural pattern; notice the lack of colloid in the background (Pap stain). (b) Parathyroid tissue aspirates can very closely resemble follicular neoplasms of the thyroid: both are highly cellular and colloid

Fig. 5.9  Colloid nodule. Aspiration of a benign colloid nodule will yield abundant colloid, either thick colloid as illustrated here or thin watery colloid (see Fig.  5.4d), and a paucity or absence of follicular cells. These findings constitute an adequate aspirate, warranting a benign diagnosis (ThinPrep)

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poor and have cells in flat sheets or crowded clusters, with relatively uniform round nuclei, although focal variation in nuclear size (endocrine atypia) can sometimes be seen. Immunohistochemistry for parathyroid hormone can be useful in difficult cases (ThinPrep)

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Nonneoplastic Conditions Chronic Lymphocytic (Hashimoto) Thyroiditis Chronic lymphocytic (Hashimoto) thyroiditis is characterized on FNA by a prominent lymphocytic infiltrate in an aspirate also demonstrating the presence of benign-­appearing follicular cells and Hürthle cells. The lymphoid component is polymorphous, and lymphohistiocytic aggregates with tingible body macrophages are an especially helpful finding in recognizing Hashimoto thyroiditis (see Fig. 5.10). Hashimoto thyroiditis can pose significant challenges in thyroid FNA interpretation including difficulty recognizing the inflammatory component of the process, atrophic follicles or prominent Hürthle cell proliferations that mimic a follicular neoplasm, and reactive nuclear changes that suggest the possibility of a papillary carcinoma [16].

Other Inflammatory and Reactive Conditions Other inflammatory and reactive conditions involving the thyroid are usually recognized clinically and do not require FNA for diagnosis; however, these conditions can sometimes present with discrete nodules that require evaluation. The cytologic findings in the autoimmune-mediated hyperplastic condition of Graves’ disease can mimic those seen in papillary thyroid carcinoma [17]. As with other hyperfunctioning a

Fig. 5.10  Chronic lymphocytic (Hashimoto) thyroiditis. (a) This low-­ power image highlights the two key findings: numerous lymphocytes and lymphohistiocytic aggregates, as well as oncocytic (Hürthle) cell change, in follicular cells (Pap stain). (b–d) Higher-power views highlight the small lymphocytes and histiocytes admixed with oncocytic

P. A. VanderLaan and J. F. Krane

thyroid nodules, the follicular cells in Graves’ disease possess the so-called flame cell morphology with peripherally located cytoplasmic vacuoles and red irregular edges to the cytoplasm best appreciable on Romanowsky stains (see Fig.  5.11). Subacute (granulomatous) thyroiditis resembles acute thyroiditis in early stages with an acute inflammatory infiltrate but more characteristically has epithelioid granulomas and numerous multinucleated giant cells as the disease progresses (see Fig.  5.12) [18]. The dense and infiltrative fibrosis seen in Riedel thyroiditis translates to a paucicellular aspirate with scant spindled cells, fibrous tissue, and sparse chronic inflammation without follicular cells. Such aspirates require clinical correlation to recognize the disease and to prevent overdiagnosis as sarcoma or paucicellular anaplastic thyroid carcinoma [19]. As in other organs, chemotherapy or radiation can induce cellular changes in follicular cells that may be confused for malignancy. Typical cellular changes include macrocytosis with preservation of the nucleus to cytoplasmic ratio, prominent nucleoli, and cytoplasmic vacuolization (see Fig. 5.13). Extensive amyloid deposition with amyloid goiter is often a manifestation of systemic amyloidosis. The amyloid may bear a strong resemblance to colloid but often has distorted spindled fibroblasts intercalated among the amyloid deposits (see Fig.  5.14). Apple-green birefringence with polarized light using the Congo red stain confirms the diagnosis. Distinction from amyloid-rich medullary carcinoma is essential. b

follicular cells. A spectrum of granular eosinophilic cytoplasm is most evident in (c) (Pap stain). (e) Lymphohistiocytic aggregate with tingible body macrophages as seen on a liquid-based preparation (ThinPrep). (f) Tingible body macrophages, with cytoplasmic phagocytized basophilic cellular debris, are indicative of germinal center sampling (Pap stain)

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Fig. 5.10 (continued)

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Fig. 5.11  Graves’ disease. The hyperfunctioning follicular cells in Graves’ disease can possess the so-called flame cell morphology with peripherally located cytoplasmic vacuoles and irregular edges to the

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cytoplasm. Although these features can be seen in Pap-stained material (a) (ThinPrep), they are more readily appreciated on Romanowsky stains (b) (DQ)

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Fig. 5.12  Subacute (de Quervain or granulomatous) thyroiditis. (a) On low power from this air-dried smear, the immediately apparent feature is that of a highly cellular aspirate with numerous multinucleated giant cells (May-Grünwald-Giemsa [MGG]). (b, c) Higher power reveals granulomas composed of spindled-to-epithelioid histiocytes in addition

to large multinucleated giant cells (MGG). (d–f) Alcohol-fixed smears from a different case showing similar findings: multinucleated giant cells with round to oval nuclei and vesicular chromatin, as well as aggregates of elongated epithelioid histiocytes (Pap stain)

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b Fig. 5.14  Amyloid goiter. Amyloid often has distorted spindled fibroblasts interdigitating among the amorphous deposits. When stained with Congo red, the amyloid demonstrates diagnostic apple-green birefringence when viewed under polarized light (Pap stain)

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Fig. 5.13  Treatment effects. (a) Follicular cells from a hyperthyroid patient treated with methimazole. Note the striking anisonucleosis between adjacent cells, although the chromatin texture and nuclear membrane contours resemble that of benign follicular cells (ThinPrep). (b) Treated Graves’ disease, showing cells with similar nuclear enlargement and slightly irregular nuclear membranes, although with benign-­ appearing chromatin (ThinPrep). (c) Radiation therapy (either external beam or radioactive iodine) can induce striking cellular atypia and macrocytosis. The common theme in these examples is the benign reactive-­ appearing nature of the nuclear chromatin (ThinPrep)

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Primary Thyroid Neoplasms  ollicular Neoplasm (Follicular Adenoma F and Follicular Carcinoma) Histologically, follicular carcinoma is distinguished from follicular adenoma by the presence of either capsular penetration or lymphovascular invasion. Since neither of these parameters can be routinely assessed on cytologic material, follicular carcinoma and follicular adenoma (or some adenomatous/hyperplastic nodules in multinodular goiters for that matter) cannot be reliably distinguished on cytologic grounds alone.

P. A. VanderLaan and J. F. Krane

For this class of lesions, cytology instead serves as a screening test relying on the consistently observed architectural abnormalities of follicular carcinoma to identify those lesions most likely to harbor a malignancy on resection. Although most benign thyroid nodules are characterized by a predominance of follicular cells with macrofollicular architecture and abundant colloid, follicular carcinoma is consistently characterized by cellular aspirates exhibiting follicular cells with architectural abnormalities. These follicular cells present predominantly as hypercellular aspirates in crowded, microfollicular, and/or trabecular arrangements, often with scant colloid (see Figs. 5.15 and 5.16) [20]. Based on these cytologic patterns, the large majority of FNAs can

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Fig. 5.15  Follicular neoplasm. (a, b) On low power, a microfollicular architectural pattern without background colloid is present (ThinPrep and MGG). (c, d) On higher power, the tight spherical arrangement of

microfollicles with nuclear crowding is more apparent. Nuclei are round with coarse chromatin (MGG and ThinPrep)

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be characterized as “benign” with those harboring architectural abnormalities classified as “suspicious for a follicular neoplasm” (or alternatively simply “follicular neoplasm”). It is important to recognize that while this approach will identify almost all follicular carcinomas, malignancy in this category still represents a minority of the nodules resected (25–40% overall) with most lesions proving to be follicular adenomas or hyperplastic nodules with prominent microfollicular architecture. Cytologic features: • Hypercellular aspirate • Follicular cells in crowded, microfollicular, and/or trabecular arrangements • Scant colloid Differential diagnosis and problems in diagnosis: • Hyperplastic nodule • Noninvasive follicular thyroid neoplasm with papillary-­ like nuclear features (NIFTP) • Invasive follicular variant of papillary carcinoma • Poorly differentiated carcinoma

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Fig. 5.16  Follicular neoplasm. (a) In addition to microfollicles, follicular neoplasms also demonstrate cells with a trabecular or cord-like arrangement (MGG). (b) The crowded nature of the nuclei is evident here, with nuclear overlap and molding (MGG). (c) Microfollicle with a central droplet of dense colloid (MGG)

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 ollicular Neoplasm, Oncocytic F (Hürthle Cell) Type As is the case with follicular carcinoma, specific distinction of Hürthle cell adenoma or hyperplastic Hürthle cell nodules from Hürthle cell carcinoma cannot be made on cytologic grounds alone, with the same histologic criteria of lymphovascular or capsular invasion needed to make this determination. Cytologic evaluation of Hürthle cell lesions is often quite challenging but relies on the identification of a cellular aspirate comprised almost exclusively of Hürthle cells (see Fig. 5.17) and an absence of lymphocytes, which if present would favor reactive Hürthle cell change in the context of chronic lymphocytic thyroiditis. A useful point to reiterate though is that Hürthle cell carcinomas are almost always characterized cytologically by the presence of one or more of the following features: dyshesion (numerous single cells), crowding (adjacent nuclei touching one another), small cell dysplasia (nucleus accounting for >50% of cell volume), or large cell dysplasia (greater than twofold variation in nuclear size) [21]. Paucity of colloid and the presence of transgressing blood vessels are also common features of Hürthle cell neoplasms [22, 23]. Like the follicular neoplasm category, a cytologic diagnosis of “suspicious for a follicular neoplasm, Hürthle cell type,” results in malignancy at resection in 25–40% of cases.

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Cytologic features: • Hypercellular aspirate • Aspirate comprised almost exclusively of Hürthle cells • Dyshesion, crowding, increased nuclear to cytoplasmic ratio, and anisonucleosis more common in carcinoma • Scant colloid • Lack lymphocytes Differential diagnosis and problems in diagnosis: • Hyperplastic Hürthle cell nodule • Hashimoto thyroiditis • Medullary carcinoma • Oncocytic variant of papillary carcinoma • Poorly differentiated carcinoma

Papillary Carcinoma Papillary carcinoma accounts for 80% of all thyroid cancers and has readily recognizable cytologic features that make thyroid FNA the preferred diagnostic test for this entity. Several histologic variants of papillary carcinoma have been described; however, the cytologic features of most of these overlap to an extent that specific recognition of subtypes is usually neither possible nor is it clinically necessary in most instances.

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Fig. 5.17  Follicular neoplasm of Hürthle cell type. (a–d) Hürthle cell neoplasms are characterized by an essentially pure population of Hürthle cells. These images from Hürthle cell carcinomas additionally demonstrate features more commonly encountered in malignant Hürthle cell neoplasms: a dispersed population of oncocytic follicular

cells (a, b), marked nuclear size variation (b, c), and nuclear crowding and overlap (d) (ThinPrep). (e) Low-power view of an air-dried smear demonstrating hypercellularity, lack of colloid, and a mixture of single dyshesive oncocytic cells as well as cell clusters (DQ). (f) High-power view reveals the oncocytic cells clinging to transgressing vessels (DQ)

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Classical Type The conventional variant of papillary carcinoma has papillary architecture that may be appreciable cytologically, with the malignant cells lining true papillae with fibrovascular cores (see Fig.  5.18) [24]. Laminated psammomatous ­calcifications may also be appreciated (see Fig. 5.19) [25]. The diagnosis of papillary carcinoma most often lies in recognizing the characteristic nuclear features (see Figs. 5.20, 5.21, and 5.22), which include enlarged, crowded, and often molded nuclei with pale, fine nuclear chromatin and a small but distinct nucleolus. Nuclear membrane contour irregularities are evident and may manifest as longitudinal nuclear grooves or nuclear pseudoinclusions. Aspirates from papillary carcinoma may demonstrate one or more of these features, with no single cytologic feature being truly pathognomonic [26–29]. Intranuclear pseudoinclusions appear to be a highly specific finding [30, 31], but even these can be encountered in other tumors (especially medullary carcinoma and hyalinizing trabecular tumor) as well as nonneoplastic conditions. The introduction of noninvasive follicular thyroid neoplasm with papillary-like nuclear features (NIFTP) terminology [32] (see section “Follicular Variant of Papillary Carcinoma and Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features (NIFTP)”) has increased concern about falsely “malignant” cytologic diagnoses of papillary carcinoma potentially leading to more aggressive surgical management than is needed (subtotal thyroidectomy rather than lobectomy) [33, 34]. To minimize this risk, it has been proposed that a “malignant” for papillary carcinoma diagnosis be reserved for cases with nuclear features of papillary carcinoma and containing at least one of the following: (1) psammomatous calcifications, (2) true papillae, or (3) frequent (three or more) nuclear pseudoinclusions [11, 35, 36]. These features are all typically absent in NIFTP [35, 37, 38].

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• Hürthle cell neoplasms • Hyalinizing trabecular tumor a

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Cytologic features: • Hypercellular aspirate • Papillary architecture • Psammomatous calcifications • Enlarged, crowded, and often molded nuclei • Pale, fine nuclear chromatin • Nuclear membrane contour irregularities • Nuclear grooves • Nuclear pseudoinclusions • Small but distinct nucleolus, usually solitary but may be multiple • “Bubble gum” colloid Differential diagnosis and problems in diagnosis: • Papillary hyperplasia • Other variants of papillary carcinoma and NIFTP • Hashimoto thyroiditis

Fig. 5.18  Papillary carcinoma, classical type. (a) Intact papillary structures with fibrovascular cores may be seen in classical type papillary carcinoma (Pap stain). (b) Another example with papillary projections (upper right) and cells with nuclear crowding and pale chromatin (Pap stain). (c) At low power, this hypercellular air-dried preparation also shows numerous irregular papillary groups (MGG)

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Fig. 5.19  Psammoma bodies. (a) These concentrically lamellated calcified structures derived from the infarcted tips of papillary structures are commonly seen in papillary carcinoma. On air-dried smears, they often appear as poorly staining, variably sized nodules, although the concentric rings are easily appreciated (DQ). (b) Psammoma bodies

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appear aqua to purple on ThinPrep slides. Isolated psammoma bodies should raise concern for papillary carcinoma even in the absence of lesional cells (ThinPrep)

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Fig. 5.20  Nuclear features of papillary carcinoma. (a, b) Nuclear enlargement, crowding, and pale chromatin are appreciable even at low power (MGG and ThinPrep). (c) At higher power, chromatin pallor and nuclear membrane irregularities are pronounced. Single, small, but

prominent nucleoli are commonly seen (Thin Prep). (d) Another manifestation of the nuclear membrane irregularities is the presence of nuclear grooves, a very sensitive finding in papillary carcinoma (Pap stain)

176 Fig. 5.21  Nuclear features of papillary carcinoma. (a) Perhaps the most specific nuclear finding of papillary carcinoma (although not sufficient for the diagnosis by itself) is that of intranuclear pseudoinclusions. This air-dried smear juxtaposes two true nuclear pseudoinclusions (at 12 and 4 o’clock) with air bubbles that may be mistaken for inclusions if they overlie the nucleus (MGG). (b, c) Macrofollicular fragments with extensive nuclear changes of papillary carcinoma: intranuclear pseudoinclusions, grooves, nuclear membrane abnormalities, powdery chromatin, crowding, and nuclear enlargement (ThinPrep)

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Fig. 5.22  Squamoid cytoplasm in papillary carcinoma. (a–d) The malignant cells of papillary carcinoma often have a squamoid appearance, with dense, almost waxy cytoplasm as demonstrated by these images, which also exhibit the common nuclear features of papillary

carcinoma: nuclear enlargement, pale and powdery chromatin, irregular nuclear membranes with grooves, and intranuclear pseudoinclusions (MGG, DQ, ThinPrep, and Pap stain)

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 ollicular Variant of Papillary Carcinoma F and Noninvasive Follicular Thyroid Neoplasm with Papillary-Like Nuclear Features (NIFTP) The follicular variant of papillary carcinoma is a problematic diagnostic entity both histologically and cytologically, as lesions with progressively subtler and less well-developed cytologic features have been accepted for this diagnosis. As a result, the follicular variant of papillary carcinoma became the most commonly diagnosed variant of papillary carcinoma. Since circumscribed/encapsulated follicular variants of papillary carcinoma behave in an indolent fashion, the term noninvasive follicular thyroid neoplasm with papillary-­like nuclear features (NIFTP) has been proposed for such tumors [32]. The NIFTP terminology removes the stigma of a cancer diagnosis and is intended to promote more conservative surgical management (i.e., lobectomy rather than subtotal thyroidectomy). Aspirates from NIFTP and invasive follicular variant of papillary carcinoma are most frequently diagnosed in an “indeterminate” category: “atypia of undetermined significance/follicular lesion of undetermined significance,” “suspicious for a follicular neoplasm” (if the architectural features are more prominent than the cytologic ones), or “suspicious for malignancy” (if the cytologic features are more prominent than the architectural ones) (see Fig. 5.23) [33, 34, 39, 40]. Aspirates tend to be cellular with a microfollicular architecture and some thick or thin colloid in the background [37–40]. The typical nuclear features of papillary carcinoma tend to be either focal or less well developed than the classical variant (see

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Fig. 5.23 NIFTP and follicular variant of papillary carcinoma. Aspirates from nodules diagnosed as NIFTP or the follicular variant of papillary carcinoma on surgical resection overlap one another and typically show only mild nuclear changes including pallor and grooves in conjunction with a microfollicular or crowded architectural pattern. Diagnostic thresholds for classifying such lesions inevitably vary. (a) If architectural features are more prominent than nuclear features, these

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section “Nonneoplastic Conditions”) [37–40]. In particular, NIFTP can be reliably distinguished from classical papillary carcinoma in most instances by noting the absence of papillary architecture, psammomatous calcifications, and frequent (three or more) intranuclear pseudoinclusions in NIFTP [35, 37]. Distinction of NIFTP from invasive follicular variant of papillary carcinoma cannot be made reliably on cytology although the latter is more likely to be called “malignant” [41]; surgical pathology evaluation is needed for definitive classification. If NIFTP or the follicular variant of papillary carcinoma is suspected on FNA, an explanatory note may help promote more limited surgical management, particularly for those aspirates classified as “suspicious for malignancy” [36]. Cytologic features: • Hypercellular aspirate • Follicular cells in crowded, microfollicular, and/or trabecular arrangements • Variably developed nuclear features of papillary carcinoma, typically less pronounced than in classical variant, and usually lacking nuclear pseudoinclusions • Lacks papillary architecture • Lacks psammomatous calcifications Differential diagnosis and problems in diagnosis: • Hyperplastic nodule • Follicular neoplasm • Other variants of papillary carcinoma

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lesions may be diagnosed as “atypia of undetermined significance/follicular lesion of undetermined significance” or “suspicious for a follicular neoplasm” on FNA (ThinPrep). (b) When nuclear features suggestive of papillary carcinoma are more pronounced, “suspicious for malignancy” may be preferred. An explanatory note indicating the possibility of NIFTP helps to encourage conservative surgical management (ThinPrep)

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Macrofollicular Variant This uncommon variant of papillary carcinoma is characterized architecturally by a tumor composed of at least 50% enlarged macrofollicles. Aspirates of this variant possess abundant colloid, are moderately cellular with follicular cells in a predominantly macrofollicular pattern, and demonstrate focal or subtle nuclear changes as seen in the more common follicular variant [42, 43]. Cytologic features: • Moderately cellular aspirate • Follicular cells in macrofollicular arrangements • Variably developed nuclear features of papillary carcinoma • Abundant colloid • Lacks papillary architecture • Lacks psammomatous calcifications

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and infrequent grooves or pseudoinclusions (see Fig. 5.24) [47]. Cytologic features: • Sparsely cellular aspirate • Prominent cyst contents with cyst-lining cells and hemosiderin-­laden macrophages • Few lesional follicular cells • Variably developed cytologic features of papillary carcinoma, typically less pronounced than in classical variant • Histiocytoid cells with abundant vacuolated cytoplasm, grainy chromatin, and occasional nucleoli with infrequent nuclear pseudoinclusions or grooves Differential diagnosis and problems in diagnosis: • Nonneoplastic cyst • Benign cyst-lining cells • Histiocytes

Differential diagnosis and problems in diagnosis: • Hyperplastic nodule • Follicular neoplasm • Other variants of papillary carcinoma • Noninvasive follicular thyroid neoplasm with papillary-­ like nuclear features (NIFTP)

Cystic Variant Papillary carcinoma often has a cystic component, but up to 10% of cases may be mostly cystic. Cystic papillary carcinoma is often cytologically challenging because of the paucity of malignant cells in an aspirate dominated by nonspecific cyst contents including cyst-lining cells and hemosiderin-­ laden macrophages [44]. This variant may be a significant source of false-negative diagnoses by FNA, and even when malignant cells are seen, their paucity may favor a “suspicious for malignancy” diagnosis [45]. An unusual presentation of papillary carcinoma has been described with malignant cells demonstrating an atypical histiocytoid appearance, mimicking benign histiocytes seen in cystic nodules [46]. The malignant follicular cells tend to have abundant vacuolated cytoplasm and nuclei with grainy chromatin, occasional nucleoli,

Fig. 5.24  Histiocytoid cells in papillary carcinoma. Especially in cystic papillary carcinomas, lesional cells may have a striking resemblance to histiocytes, with abundant bubbly cytoplasm, round to oval nuclei, mildly vesicular chromatin, and only minor nuclear membrane irregularities. These cells represent a second population amid the other cyst content cells, including histiocytes and hemosiderin-laden macrophages, and are a useful clue to the diagnosis of papillary carcinoma (ThinPrep)

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Tall Cell Variant The tall cell variant is an aggressive variant of papillary carcinoma characterized by follicular cells that are approximately three times as tall as they are wide, with more oncocytic cytoplasm and distinct cell borders [48, 49]. Cytologically, this variant has papillary architecture and prominent intranuclear pseudoinclusions, including forms with a “soap bubble-like” appearance (see Fig.  5.25) [50, 51]. Although it may not be possible to specifically render a cytologic diagnosis of this variant in all instances, they are usually readily recognizable as malignant. Due to the aggressive nature of this variant, some surgeons will opt for a central compartment lymph node dissection at the time of thyroidectomy in patients with this diagnosis. Therefore, a a

Fig. 5.25  Tall cell variant of papillary carcinoma. (a) Although cells tend to round up in liquid-based preparations, tall cell variant cells do maintain a somewhat rectangular shape, with a height of at least three times the cell width (ThinPrep). (b–d) This variant is easy to recognize as papillary carcinoma, as nuclear inclusions are frequent and nuclear

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comment suggesting the possibility of a tall cell variant when these features are present on FNA may be considered [42, 52]. Cytologic features: • Cellular aspirate • Papillary architecture • Oncocytic cytoplasm with distinct cell borders • Tall cells (at least three times as tall as they are wide) • Prominent nuclear pseudoinclusions, including “soap bubble” pseudoinclusions Differential diagnosis and problems in diagnosis: • Other variants of papillary carcinoma

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membrane abnormalities are not subtle. Recognition of a population of elongated cells is needed to suggest the tall cell variant (ThinPrep). (e) The intranuclear pseudoinclusions seen in the tall cell variant are often multiple in the same nucleus imparting a “soap bubble” appearance (ThinPrep)

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Fig. 5.25 (continued)

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Oncocytic Variant The oncocytic variant of papillary thyroid carcinoma is uncommon and is characterized by diffuse cellular oncocytic changes coupled with the nuclear features of papillary carcinoma (see Fig. 5.26) [53]. The greatest difficulty is distinguishing such cells from a Hürthle cell neoplasm or from Hürthle cell changes associated with chronic lymphocytic thyroiditis, which in both cases requires careful attention to the nuclear cytomorphology [54]. Oncocytic cytoplasm can also be seen in the tall cell variant, which is frequently seen in conjunction with the oncocytic variant [55]. Cytologically, the rounded cell shape of the oncocytic variant can help differentiate it from the more narrow rectangular shape of the tall cell variant. Cytologic features: • Diffuse oncocytic cytoplasmic changes • Typical nuclear features of papillary carcinoma Differential diagnosis and problems in diagnosis: • Hürthle cell neoplasm • Other variants of papillary carcinoma, especially the tall cell variant • Hürthle cell changes associated with chronic lymphocytic thyroiditis

Fig. 5.26  Oncocytic variant of papillary carcinoma. As with traditional Hürthle cells, this variant demonstrates cells with abundant round to polygonal granular cytoplasm; however, the nuclear features (including inclusions, nuclear membrane irregularities, and nuclear pallor) are the diagnostic clue to papillary carcinoma. Although any follicular cell with Hürthle cell change may show some mild degree of nuclear membrane irregularity, the severity of the nuclear changes in the oncocytic variant identifies these cells as papillary carcinoma (ThinPrep)

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Warthin-Like Variant This rare variant is subclassified under the oncocytic variant of papillary carcinoma in the World Health Organization terminology. As its name implies, it is characterized by oncocytic cells with papillary architecture and the nuclear features of papillary carcinoma along with a prominent lymphoid component with reactive germinal centers within the papillary cores (much like the salivary gland Warthin tumor) (see Fig. 5.27) [56]. Distinction must be made from Hashimoto thyroiditis and rests on identifying the papillary architecture and nuclear cytomorphology of papillary carcinoma [57]. Cytologic features: • Papillary architecture • Prominent lymphoid cells with reactive germinal centers within papillary cores • Oncocytic epithelial cells with typical nuclear features of papillary carcinoma Differential diagnosis and problems in diagnosis: • Other variants of papillary carcinoma • Hashimoto thyroiditis

5  Head and Neck: Thyroid Fig. 5.27  Warthin-like variant of papillary carcinoma. (a, b) On low power, a dense lymphoid aggregate forms a core that is surrounded by a population of epithelial cells with oncocytic cytoplasm. At high power, the follicular cells lining these lymphohistiocytic aggregates have abundant granular oncocytic cytoplasm, and the nuclei demonstrate the traditional nuclear features of papillary carcinoma (Pap stain). (c) Another example shows lymphohistiocytic aggregates forming a central core and nearly obscuring the surrounding lesional epithelial cells (ThinPrep)

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Columnar Cell Variant This is a rare, aggressive variant of papillary carcinoma resembling colonic or endometrioid adenocarcinoma [58]. As the name implies, the cells are columnar and elongated in shape with occasional clear cytoplasm (see Fig. 5.28). The pseudostratified, elongated nuclei are typically hyperchromatic in contrast to the fine powdery chromatin of most papillary carcinomas. Furthermore, the characteristic nuclear contour irregularities of papillary carcinoma, including pseudoinclusions and grooves, are generally absent [59]. Clinical history and immunostains can help distinguish this variant from metastatic disease to the thyroid.

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Fig. 5.28  Columnar variant of papillary carcinoma. (a) Nuclei are somewhat elongate, crowded, and polarized to one side of the cytoplasm. The chromatin is more hyperchromatic than is typical in most

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Cytologic features: • Columnar cells with occasional clear cytoplasm • Elongated, pseudostratified nuclei • Hyperchromatic chromatin • Lacks typical nuclear features of papillary carcinoma Differential diagnosis and problems in diagnosis: • Metastatic carcinoma

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papillary carcinomas (ThinPrep). (b) At higher power, crowding and pseudostratification can be appreciated with only limited nuclear contour irregularities present (ThinPrep)

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Cribriform-Morular Variant This papillary carcinoma variant occurs sporadically (usually solitary) but may also be associated with adenomatous polyposis coli (APC) gene germline mutations in the autosomal dominant disorder familial adenomatous polyposis (FAP), particularly when present as multiple nodules. This variant has not been well characterized cytologically, but aspirates usually are highly cellular with crowded clusters of ovoid to columnar cells with cribriform architecture and focal squamous morules (see Fig. 5.29) [60]. Typically, cytologic features of papillary carcinoma are also present, but the nuclei tend to be more hyperchromatic. Of note, both the cribriform structures and background are devoid of colloid. Beta-catenin immunohistochemistry characteristically shows strong nuclear and cytoplasmic staining with this entity [61]. Since the thyroid tumor(s) may represent the initial manifestation of FAP, recognition of this variant can be an invaluable clue to the clinical syndrome. Cytologic features: • Highly cellular • Crowded cluster of ovoid to columnar cells • Cribriform architecture devoid of colloid • Focal squamous morules • Nuclear features of papillary carcinoma but somewhat more hyperchromatic nuclei Differential diagnosis and problems in diagnosis: • Other variants of papillary carcinoma

Fig. 5.29  Cribriform-morular variant of papillary carcinoma. In this uncommon variant, the aspirate shows crowded cell clusters with a cribriform architectural arrangement. The nuclei are more hyperchromatic than traditional papillary carcinoma, though intranuclear inclusions and membrane abnormalities are readily appreciated (ThinPrep)

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Diffuse Sclerosing Variant This uncommon variant is seen in younger patients, and the diagnosis is suggested by the clinical and radiologic presentation of a mass encompassing an entire lobe or the entire thyroid. Cytologically, these tumors have numerous psammoma bodies, well-developed cytologic and architectural features of papillary carcinoma, and prominent squamous morules and/or squamoid cytologic features (see Fig. 5.30). Lymphoid cells are often an appreciable component of the aspirate as well [62, 63]. Cytologic features: • Cellular aspirate • Prominent nuclear features of papillary carcinoma • Numerous psammoma bodies • Squamoid cells or squamous morules • May have lymphoid cells Differential diagnosis and problems in diagnosis: • Other variants of papillary carcinoma

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Hyalinizing Trabecular Tumor Although this tumor almost invariably behaves in a benign fashion, it is thought by some to represent a variant of papillary carcinoma. Aspirates typically have a bloody background and are variably cellular with cohesive clusters of round to oval cells with a low nuclear to cytoplasmic (N/C) ratio radiating from a hyaline stromal core (see Fig.  5.31) [64]. These tumors have frequent intranuclear pseudoinclusions and grooves; psammoma bodies may also be present. Hyalinizing trabecular tumors are most often diagnosed as papillary carcinoma or suspicious for papillary carcinoma on cytologic aspirates [65]. The hyaline material can mimic amyloid, potentially leading to a misdiagnosis as medullary carcinoma. Paraganglioma is also often in the differential diagnosis. By immunohistochemistry, the cells are positive for TTF-1 and thyroglobulin, are negative for calcitonin, and demonstrate aberrant peripheral cytoplasmic staining for MIB-1 [66]. Cytologic features: • Variably cellular • Cohesive clusters of round to oval cells • Cells radiate from a hyaline stromal core • Low N/C ratio • May have frequent intranuclear pseudoinclusions and grooves • May have psammoma bodies

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Fig. 5.30 Diffuse sclerosing variant of papillary carcinoma. (a) Numerous psammoma bodies are seen in aspirates from this variant, occasionally accompanied by fragments of fibrous stromal tissue (DQ). (b, c) The psammoma bodies illustrated here are closely approximated to follicular cells that demonstrate a squamoid morphology (ThinPrep and Pap stain)

Differential diagnosis and problems in diagnosis: • Papillary carcinoma • Medullary carcinoma • Paraganglioma

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Fig. 5.31  Hyalinizing trabecular tumor. (a, b) On air-dried smears, cells with round to oval nuclei are seen to be emanating from a central hyaline stromal core (MGG). (c) Higher power highlights the pale staining hyaline material, as well as the prominent nuclear membrane irregularities (MGG). (d, e) Alcohol-fixed smears highlight the pow-

dery chromatin and nuclear grooving of rectangular-shaped cells protruding from the central avascular hyaline core (H&E). (f) Cell block preparation shows the trabecular arrangement of rectangular-shaped cells with granular oncocytic cytoplasm projecting from the central hyaline core (H&E)

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Secretory Carcinoma Primary secretory carcinoma (previously termed mammary analogue secretory carcinoma) of the thyroid is a rare malignancy resembling its counterparts in the salivary gland and breast and harboring the same translocation resulting in an ETV6-NTRK3 fusion product [67–70]. Aspirates are cellular with cells present singly and in clusters often with associated dense secretory colloid-like material. Cytoplasm is dense with occasional cells having a single prominent cytoplasmic vacuole conferring a signet ring cell-like morphology (see Fig. 5.32) [71–73]. Nuclei are round to oval with pale chromatin and some nuclear contour irregularities but typically do not have the elongate grooves or nuclear pseudoinclusions of papillary carcinoma. Nucleoli are more prominent and centrally located than the small, distinct, and peripherally situated nucleoli of papillary carcinoma. The differential diagnosis is primarily with papillary carcinoma. The signet ring-like cytoplasmic vacuoles and lack of prominent nuclear grooves and pseudoinclusions are

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Fig. 5.32  Secretory carcinoma. (a) Tumor cells have dense cytoplasm with occasional cells with a signet ring cell-like cytoplasmic vacuole (top and bottom). Nuclei have a central prominent nucleolus and pale chromatin with some nuclear contour irregularity but lack the

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helpful distinguishing features. By immunohistochemistry, secretory carcinoma is positive for S100 and mammaglobin but may also exhibit weak PAX-8 immunoreactivity that may cause diagnostic confusion. Demonstration of the characteristic ETV6 translocation is diagnostic. Cytologic features: • Cellular aspirate • Sheets, papillary clusters, and single cells • Dense cytoplasm, sometimes with a signet ring-like cytoplasmic vacuole • Extracellular dense secretory material resembling colloid • Enlarged nuclei with pale chromatin, minimal nuclear contour irregularity, and a central distinct nucleolus • May rarely have nuclear pseudoinclusions • May rarely have psammoma bodies Differential diagnosis and problems in diagnosis: • Papillary carcinoma • Metastatic carcinoma

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longitudinal nuclear grooves of papillary carcinoma (ThinPrep). (b) Cell block preparation demonstrates similar nuclear features along with glandular spaces containing dense secretory material (H&E)

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Poorly Differentiated (Insular) Carcinoma

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especially when the poorly differentiated carcinoma arises in the setting of a more well-differentiated tumor. When a dysThis class of thyroid carcinoma is characterized by clinical hesive cell pattern is pronounced, distinction from medullary behavior that falls between the relatively indolent well-­ carcinoma may be difficult (see Fig.  5.34). differentiated follicular and papillary thyroid carcinomas Immunocytochemistry for thyroglobulin and calcitonin is and the extremely aggressive undifferentiated (anaplastic) useful in making this distinction (but not TTF-1 as it is posicarcinoma. Poorly differentiated carcinoma is challenging tive in both tumors). Undifferentiated (anaplastic) carcinoma to recognize by cytology and often requires subsequent his- has more pronounced atypia and pleomorphism, often with tologic evaluation for definitive diagnosis. Poorly differen- squamoid or sarcomatous features, and typically loses thyrotiated carcinoma may be seen in pure form or may arise in globulin and TTF-1 expression. Metastatic disease is also an association with a well-differentiated component of follicu- important consideration; in such cases, immunoreactivity for lar carcinoma or papillary carcinoma, adding further com- TTF-1, PAX-8, and thyroglobulin is diagnostically useful. plexity to the cytologic diagnosis. Aspirates are typically highly cellular with pronounced nuclear crowding [74, 75]. Cytologic features: Solid, trabecular, or insular (nested) architecture may be • Highly cellular aspirate appreciable, and individual cells with high N/C ratios are • Dyshesive pattern or demonstrating solid, insular, or traalso seen (see Fig.  5.33) [76]. While atypia may be probecular growth nounced, often the presentation is of a monotonous popula- • Nuclear crowding tion of relatively uniform cells (see Fig. 5.34). Worrisome • May have pronounced nuclear atypia features that are not associated with well-differentiated car- • May be strikingly monotonous cinomas (such as mitotic activity, apoptosis, and necrosis) • May have necrosis, apoptosis, or increased mitotic suggest the diagnosis when present [77]. In most instances, activity these tumors are diagnosed as “suspicious for a follicular neoplasm” with the diagnosis of poorly differentiated car- Differential diagnosis and problems in diagnosis: cinoma only prospectively offered in about one third of • Well-differentiated follicular neoplasm cases. • Undifferentiated (anaplastic) carcinoma The differential diagnosis of poorly differentiated carci- • Medullary carcinoma noma includes follicular neoplasms and papillary carcinoma, • Metastatic carcinoma

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Fig. 5.33  Poorly differentiated (insular) carcinoma. (a) This example of a poorly differentiated carcinoma shows the crowded nature of this group of relatively monotonous follicular cells. No follicular architectural arrangement is appreciated (ThinPrep). (b, c) Aspirates are hypercellular with scant colloid. Trabecular and microfollicular patterns are

prominent in these examples, similar to aspirates of well-differentiated follicular neoplasms (Pap stain and DQ). (d–f) The nuclei are enlarged, crowded, and overlapping with round to oval contours and coarse chromatin. Although not present here, the presence of mitoses, apoptosis, or necrosis is an important clue to the diagnosis (DQ and Pap stain)

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Fig. 5.34  Poorly differentiated (insular) carcinoma. (a, b) This aspirate is extremely cellular and comprised of a dispersed population of cells. Distinction from medullary carcinoma required immunohisto-

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chemistry to demonstrate the presence of immunoreactivity for thyroglobulin and the absence of calcitonin staining (ThinPrep)

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Medullary Carcinoma Medullary carcinoma is a neuroendocrine carcinoma derived from the calcitonin-producing parafollicular C cells of the thyroid. The appearance of the tumor cells can be notoriously variable but shares the characteristic fine or coarsely granular “salt and pepper” chromatin of other neuroendocrine tumors [78]. Aspirates are typically cellular with loosely cohesive aggregates of cells as well as numerous single cells (see Figs.  5.35 and 5.36). Cell shapes are variable, including small rounded cells, plasmacytoid variants, and spindled forms. Slender cytoplasmic tails are often appreciable and are a helpful diagnostic clue (see Fig.  5.36a). Cytoplasm is often granular such that distinction from a Hürthle cell neoplasm is a frequent consideration. On Romanowsky stains, small red granules may be seen in the cytoplasm (see Fig. 5.35d). The nuclei are rounded and often eccentrically located. Binucleation is common (see Figs. 5.35b, d and 5.36b) with rare giant cells also seen (especially prominent in the giant cell variant). Nucleoli vary but are not usually prominent. Like papillary carcinoma, intranuclear pseudoinclusions may be seen (see Fig.  5.36d). In the majority of cases, waxy amorphous amyloid is present in the background (see Fig. 5.36a) [79, 80]. As previously noted, the differential diagnosis is most often with a Hürthle cell neoplasm. The granules of Hürthle cell neoplasms are blue on Romanowsky stains rather than red, and the chromatin is fine, often having more prominent nucleoli. Pseudoinclusions suggest the possibility of papillary carcinoma or hyalinizing trabecular tumor, but again the presence of coarse rather than fine chromatin is helpful. Particularly when spindled cells are prominent, distinction

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from melanoma and undifferentiated carcinoma may be difficult. As a general principle, confirmatory immunostains are advisable before rendering a definitive diagnosis. In particular, calcitonin versus thyroglobulin expression for distinction from other thyroid epithelial neoplasms is helpful. Although 10–15% of cases may be calcitonin negative by immunohistochemistry, positivity for CEA, as well as chromogranin and/or synaptophysin, is typically retained [81]. Congo red, trichrome, or sulfated Alcian blue stains can be used to highlight amyloid. If suitable cytologic material is not available for special stains, evaluation of serum calcitonin levels is recommended as these will be invariably elevated in patients with medullary carcinoma. Cytologic features: • Cellular aspirate • Dyshesive cells • Highly variable appearance • Epithelioid, plasmacytoid, spindled, small cell, or giant cell morphology • Binucleation common • “Salt and pepper” chromatin with variable nucleoli • May have nuclear pseudoinclusions • Red cytoplasmic granules with Romanowsky stain • Amyloid Differential diagnosis and problems in diagnosis: • Hürthle cell neoplasm • Melanoma • Papillary carcinoma • Hyalinizing trabecular tumor • Poorly differentiated thyroid carcinoma • Undifferentiated (anaplastic) carcinoma

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a

b

c

d

Fig. 5.35  Medullary carcinoma. (a) On low power, one appreciates the hypercellular nature of the aspirate, composed of individual cells and loosely cohesive clusters (DQ). (b) Higher power highlights the plasma-

cytoid cell shape, with binucleation a common finding (MGG). (c, d) The cell nuclei are round to oval and eccentrically placed, with abundant cytoplasm with metachromatic neurosecretory granules apparent (DQ)

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a

b

c

d

Fig. 5.36  Medullary carcinoma. (a) The cellular morphology can be quite varied, including spindled cells as seen here. Additionally, the presence of waxy amorphous amyloid material (upper left) is an important diagnostic clue (ThinPrep). (b) Nuclear size variation is common, as well as the presence of giant tumor cells (MGG). (c) Alcohol fixation

preserves the neuroendocrine quality of the chromatin, characterized by the “salt and pepper” nuclei illustrated here (Pap stain). (d) Intranuclear pseudoinclusions may raise concern for papillary carcinoma, but the other typical nuclear features of medullary carcinoma are evident (Pap stain)

5  Head and Neck: Thyroid

Undifferentiated (Anaplastic) Carcinoma Mostly affecting older patients, undifferentiated (or anaplastic) thyroid carcinoma behaves in an extremely aggressive manner. The diagnosis is often clinically suspected with a hard, rapidly enlarging, infiltrative mass in the thyroid. Tumor often metastasizes to the lymph nodes and/or lungs, but death usually results in a period of months from rapid locoregional spread. Since surgical management is an infrequent occurrence, these tumors are more commonly encountered as diagnostic cytologic aspirates as opposed to surgical resection specimens. Undifferentiated thyroid carcinomas can be variably cellular, depending on the extent of associated tumor fibrosis. The cytologic appearance of the tumor cells is also quite variable, with marked nuclear pleomorphism encountered in cells with spindle, epithelioid, rhabdoid, squamoid, or giant cell morphology (see Figs. 5.37, 5.38, 5.39, 5.40, and 5.41) [82]. Necrosis (see Fig. 5.39)- and abscess-like neutrophilic infiltrates (see Figs. 5.38 and 5.39) may be prominent along with frequent mitotic figures (see Figs. 5.37a, 5.38a, f, and 5.41b). Osteoclast-like giant cells are also frequently seen (see Fig. 5.37). As undifferentiated thyroid carcinoma com-

195

monly is found to arise in the setting of a more well-­ differentiated thyroid carcinoma [83], cells with nuclear features of papillary carcinoma or architectural groups of follicular carcinoma may also be present [84]. The differential diagnosis may include well-differentiated thyroid carcinomas as well as medullary carcinoma, but the pleomorphism of undifferentiated carcinoma exceeds that seen with these tumors. Immunohistochemically, undifferentiated carcinoma is negative for calcitonin as well as most markers of thyroid differentiation (thyroglobulin, TTF-1). Keratin staining may be only focal or altogether absent. Distinction from metastatic disease (especially lung carcinoma since patients often have both lung metastasis and a neck mass at presentation) can be particularly challenging. PAX-8 expression is retained in the majority of undifferentiated thyroid carcinomas and is especially helpful in this context [85]. Otherwise, clinical correlation is needed to confirm the diagnosis. Cytologic features: • Variably cellular • Markedly atypical cells with epithelioid, spindle, squamous, rhabdoid, or giant cell features

a

b

c

d

Fig. 5.37  Undifferentiated (anaplastic) carcinoma, giant cell variant. (a) Aspirates of this entity are quite variable in appearance and cellularity. Here, multinucleated giant cells and mitotic figures are prominent (MGG).

(b) Cell block preparation with multinucleated giant cells and pleomorphic mononuclear tumor cells (H&E). (c, d) The giant cells resemble osteoclast-like giant cells, with dozens of round to oval nuclei (Pap stain)

196

• • • •

Necrosis Abundant mitoses Neutrophilic infiltrate May coexist with a well-differentiated component

P. A. VanderLaan and J. F. Krane

Differential diagnosis and problems in diagnosis: • Metastatic carcinoma • Sarcoma • Medullary carcinoma • Poorly differentiated thyroid carcinoma

a

b

c

d

e

f

Fig. 5.38  Undifferentiated (anaplastic) carcinoma, squamous variant. (a, b) The malignant cells exhibit striking pleomorphism as well as mitotic figures (H&E, MGG). (c–e) Bizarre squamoid morphology is

present in a background of marked acute inflammation (H&E, MGG, and cell block H&E). (f) A different example with pleomorphic squamoid cells, an atypical mitosis, and scattered neutrophils (Pap stain)

5  Head and Neck: Thyroid

a

197

b

Fig. 5.39  Undifferentiated (anaplastic) carcinoma, squamous variant. (a, b) In addition to the bizarre orangeophilic squamoid cells, a background tumor diathesis is present (Pap stain and ThinPrep)

a

Fig. 5.40  Undifferentiated (anaplastic) carcinoma, spindled variant. (a, b) Malignant cells may have a spindled morphology, making medullary carcinoma a diagnostic consideration; immunohistochemistry for

a

b

calcitonin can prove helpful in such cases. Additionally, the extent of the nuclear atypia exceeds that seen in medullary carcinoma (DQ)

b

Fig. 5.41  Undifferentiated (anaplastic) carcinoma. (a, b) Pleomorphic cells with a predominantly epithelioid shape have irregular nuclear membranes, prominent nucleoli, coarse chromatin, and frequent mitotic figures (ThinPrep)

198

Lymphoma Primary non-Hodgkin lymphoma (NHL) of the thyroid gland accounts for an estimated 2–8% of thyroid malignancies [86]. Most patients are older females, with the lymphoma arising in a background of Hashimoto thyroiditis. These patients may present with a thyroid mass or diffuse glandular enlargement, frequently with compressive airway symptoms. Three broad classes of lymphoma are most typically observed: marginal zone B-cell lymphoma of mucosa-­ associated lymphoid tissue (MALT lymphoma) (see Fig.  5.42a), diffuse large B-cell lymphoma (DLBCL) (see Fig. 5.42b), or less frequently follicular lymphoma [87]. The cytologic preparations are typically highly cellular comprised of a dyshesive single-cell population of lymphoid cells. Ancillary studies (i.e., flow cytometry, immunohistochemistry, and molecular studies) are necessary for the diag-

a

Fig. 5.42  Lymphoma. (a) In this example of marginal zone B-cell lymphoma (MALT lymphoma), the aspirate is polymorphous with predominantly small lymphoid cells, making distinction from a reactive process impossible on morphologic grounds alone. Ancillary studies including flow cytometry and immunophenotyping are essential

P. A. VanderLaan and J. F. Krane

nosis and proper classification of lymphoma of the thyroid gland. Cytologic features: • Highly cellular specimen • Prominent lymphoid infiltrate, often devoid of epithelial elements • Polymorphous lymphoid population with prominent component of monocytoid B cells (MALT lymphoma) • Monotonous population of small- to intermediate-sized lymphoid cells with round to slightly irregular nuclei (follicular lymphoma) or of large lymphoid cells (DLBCL) Differential diagnosis and problems in diagnosis: • Reactive lymphoid infiltrate, especially Hashimoto thyroiditis • Inadvertent lymph node sampling

b

(ThinPrep). (b) When larger, more malignant lymphoid cells with scant cytoplasm and coarse nuclear chromatin are a prominent finding in a thyroid aspirate, suspicion for involvement by diffuse large B-cell lymphoma is warranted (ThinPrep)

5  Head and Neck: Thyroid

Metastasis Metastatic disease involving the thyroid is a relatively rare event. Although autopsy studies have shown that up to 10–20% of patients with widespread metastasis have thyroid involvement, these tend to be subclinical with only about 0.1% of thyroid FNAs diagnosed as metastatic disease [88, 89]. Nodules may be single, multifocal, or diffusely present throughout the gland. Tumor involvement, especially by squamous cell carcinoma, may occur by local direct extension. Metastasis to the thyroid is most frequently from the lung (see Fig. 5.43a) and breast (see Fig.  5.43b) (commonest sources identified at autopsy), kidney (see Fig. 5.43c) (most common clinically apparent secondary thyroid malignancy), as well as colon, esophagus, and melanoma [90]. These tumors are often recognized by the clinical history and their lack of resemblance to well-differentiated follicular neoplasms. Metastatic renal cell carcinoma is worth special note since the primary tumor may be occult, the metastasis may occur

a

Fig. 5.43  Metastatic carcinoma to the thyroid. (a) Metastatic lung adenocarcinoma demonstrating tumor cells with enlarged, round to oval nuclei with vesicular chromatin and prominent nucleoli and delicate vacuolated cytoplasm (ThinPrep). (b) Metastatic breast carcinoma demonstrating large cytoplasmic vacuoles and eccentrically placed large nuclei with prominent nucleoli (DQ). (c) Metastatic clear cell renal cell carcinoma. On liquid-based preparations, the nuclear features are pronounced, demonstrating round nuclei with single prominent nucleoli. The vacuolated cytoplasm can resemble that of histiocytes (ThinPrep). (d) Prominent clear cell change can rarely be seen in hyperplastic (as in this example) or neoplastic thyroid follicular cells.

199

many years after the primary tumor, and the typical clear cell morphology overlaps significantly with clear cell change that can be prominent in follicular-derived neoplasms (see Fig  5.43d). Immunohistochemistry is often useful in demonstrating a nonthyroid-derived origin for these malignant cells. Cytologic features: • Variable, but recognized by lack of resemblance to normal thyroid elements or typical well-differentiated follicular neoplasms Differential diagnosis and problems in diagnosis: • Undifferentiated (anaplastic) carcinoma • Poorly differentiated thyroid carcinoma • Papillary carcinoma, columnar variant (especially colon primary) • Clear cell hyperplasia or neoplasia of follicular origin (especially renal cell carcinoma) • Medullary carcinoma (especially melanoma)

b

Immunohistochemistry can be used to exclude renal cell carcinoma in difficult cases (Pap stain). (e) In contrast to the benign follicular cells at left, the metastatic ovarian carcinoma cells present on the right are much larger, with hyperchromatic nuclei and high N/C ratios. The appearance is that of a population of cells foreign to those typically encountered in the thyroid (ThinPrep). (f) Metastatic ovarian carcinoma with a papillary cluster of cells with hyperchromatic nuclei, high N/C ratios, and necrotic debris in the background. The pleomorphism and necrosis should raise the possibility of an anaplastic carcinoma as well as metastasis (ThinPrep)

200

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c

d

e

f

Fig. 5.43 (continued)

5  Head and Neck: Thyroid

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6

Head and Neck: Parathyroid Elwira Bakuła-Zalewska

Introduction The normal parathyroid glands are oval to reniform measuring approximately 2–7 mm, and most of the glands is of size and shape comparable with a grain of rice. In majority of normal individuals, there are four parathyroid glands with most common anatomical distribution of two lower glands in the dorsal extracapsular aspects of the lower thyroid poles and two upper glands in the dorsomedial extracapsular aspect of the upper thyroid poles at the level of isthmus. The number and location of parathyroid glands may be variable, and supernumerary parathyroid glands occur in up to 15% of individuals. As lower parathyroid glands share their embryologic descent with the thymus and upper parathyroid glands with the thyroid gland, abnormal location of the parathyroid glands such as mediastinum, around the carotid sheath, in the aortopulmonary window and less often intrathyroidal occurs in 10–20% of individuals. Histologically, the parathyroid is composed of multiple nodules of parenchymal epithelial cells (chief cells) arranged in nests and cords, a fibrovascular stroma, and fat cells. Chief cells are polygonal in shape and have slightly eosinophilic granular cytoplasm with secretory (PTH) granules and small round nucleus. The chief cells may have morphologic variations such as oxyphilic cells with deeply eosinophilic cytoplasm, clear cells with clear cytoplasm and transitional cells, all those depending of different physiologic states of the parathyroid glands activity. In addition, the parathyroid glands contain varying amounts of stromal fat. Sporadic hyperparathyroidism is the most common etiology of hypercalcemia, and a parathyroid adenoma is found in approximately 85% of patients with hypercalcemia. In addition approximately 14% of primary hyperparathyroidism are caused by primary parathyroid

hyperplasia. Very rare parathyroid carcinoma is cause of hyperparathyroidism in less than 1% of cases. Secondary hyperparathyroidism associated with parathyroid hyperplasia is usually response to hypercalcemia or hyperphosphatemia due to chronical renal failure and rarely to malabsorption or vitamin D deficiency. Localization of abnormal parathyroid gland/glands is crucial as curative treatment of hyperparathyroidism is parathyroidectomy of affected gland/glands.

Indications for Parathyroid FNA FNAC is of limited value in the preoperative assessment of patients with hyperparathyroidism [1, 2]. Normal parathyroid glands are not visible in ultrasound (US) because they are too small and have an echotexture similar to thyroid. Evaluation of enlarged parathyroid glands and determination of parathyroid tissue are not possible by using US alone. Majority FNA of parathyroid lesions is unintended, as intrathyroidal parathyroid adenomas and parathyroid cysts are often mistaken for thyroid lesions [3–5]. Uncommon ectopic neck and mediastinal parathyroid adenomas in patients with persistent hypercalcemia are important differential diagnosis in the FNA of head and neck and mediastinal masses [6–9]. Many investigators have stressed the difficulty in discerning thyroid from parathyroid in FNA smears. However, US-guided FNA of the parathyroid is helpful in patients with persistent hypercalcemia after failed surgery when the neck anatomy is distorted and in patients with atypical or intrathyroidal parathyroid gland location [10]. US-guided FNA may be also useful to localize parathyroid lesions in recurrent disease and to distinguish abnormal parathyroid glands from lymph nodes.

E. Bakuła-Zalewska Department of Pathology, The Maria Sklodowska-Curie Memorial Cancer Centre and Institute of Oncology, Warsaw, Poland e-mail: [email protected] © Springer International Publishing AG, part of Springer Nature 2019 H. A. Domanski (ed.), Atlas of Fine Needle Aspiration Cytology, https://doi.org/10.1007/978-3-319-76980-6_6

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 pecimen Collection, Preparation, S and Ancillary Tests US-guided FNA sampling of parathyroid lesions can be performed by non-aspirating Zaidela technique using preferably 27-gauge needles or by regular FNA techniques using syringe and syringe holder. FNA technique of parathyroid lesions can be mainly limited by the previous surgical procedures in the neck as well as coexisting goiter or other lesions of the thyroid gland. Immunocytochemical examination (IC) of aspirated material helps to determine whether the specimen obtain by FNA represents parathyroid or thyroid lesions [5, 11–13]. In our experience results of IC of specimens obtain from parathyroid lesions can vary depending on specimen preparation and antibody used. Cell blocks are usually guaranteed of the best results of IC, but results depend also

E. Bakuła-Zalewska

on amount of material collected by FNA [14]. All cell type of parathyroid express positive cytoplasmic immunoreactivity against antibodies to parathormone (PTH), keratin, chromogranin, and neuron-specific enolase (NSE). Problem in cases examined in our clinic was variable results of IC for antibodies against parathormone (PTH), while antibodies against keratin, chromogranin, and NSE work well with clear positivity in all cases examined. The measurement of PTH levels in the needle washout fluid (FNA-PTH testing) is yet another technique which increases the diagnostic accuracy of FNAC of the parathyroid lesions [15–20]. The remaining aspirate in the syringe and needles can be immediately rinsed with 0.5 ml of isotonic 0.9% saline and the washouts processed to measure PTH using electrochemiluminescent (ECLIA) assay. A high PTH concentration in FNA specimens supports a parathyroid origin.

6  Head and Neck: Parathyroid

Solid Parathyroid Lesions  arathyroid Hyperplasia/Chief Cell P Hyperplasia Nonneoplastic enlargement of all parathyroid glands and increase in parenchymal cell mass (diffuse or nodular) result in increased secretion of parathyroid hormone (PTH). Parathyroid hyperplasia includes proliferation of parathyroid cells: chief, water-clear, and oxyphilic cells. Secondary and tertiary hyperplasia is microscopically indistinguishable from that seen in primary hyperplasia. a

Fig. 6.1  Normal parathyroid (a) and hyperplastic parathyroid (b). Aspirate showing a vaguely follicular architectural pattern and cells with relatively uniform round nuclei arranged as flat sheets or crowded

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In some of the early paper reporting cytologic features of hyperparathyroidism, the authors indicated the possibility to distinguish between parathyroid adenoma and parathyroid hyperplasia by FNAC, based on smears pattern and cell morphology in FNA smears [21–24]. Later publications and our experiences indicate however that smears of hyperplastic parathyroid glands are indistinguishable from that of adenoma although cells from parathyroid hyperplasia usually are arranged in sheets and syncytia compared to loose sheets and microfollicles in parathyroid adenoma (see Figs. 6.1 and 6.2).

b

clusters closely resembling thyroid aspirates. Notice the lack of colloid in the background (H&E)

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a

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Fig. 6.2  Hyperplastic parathyroid and parathyroid adenoma. Smears of both parathyroid adenoma and hyperplastic parathyroid (a–d) are moderate to highly cellular and show cells arranged either as loose sheets and clusters with microfollicles, single cells, and naked nuclei in the background or cohesive three-dimensional crowded tissue frag-

ments with an organoid-cribriform pattern in a bloody background. (e, f) Both have cells with relatively uniform round nuclei with the neuroendocrine quality of the chromatin, characterized by the stippled “saltand-pepper” nuclei (H&E)

6  Head and Neck: Parathyroid

Parathyroid Adenoma Parathyroid adenomas are more frequent in women than men and occur at any age but are most often common in adults particularly in the fourth decade of life. One of the lower parathyroid gland is mostly involved. Rare double adenomas are described, accounting 1.7–12% of primary parathyroidism. Histologically, adenomas are cellular and are usually composed of chief cells or combination of the other cell types. Microscopic variants of parathyroid adenoma include rare histological subtypes such as lipoadenoma (parathyroid hamartoma), papillary, water-clear, follicular, and oxyphilic variants. Papillary variant shows papillary pattern and follicular variant follicular/acinar pattern that may mimic papillary thyroid carcinoma or thyroid follicular neoplasm, respectively. Rarely intrafollicular deposition of amyloid or more often colloid-like material can be seen in follicular variant of parathyroid adenoma simulating a thyroid follicular neoplasm. Molecular genetic abnormalities of parathyroid adenomas are diverse and vary and are not conclusively distinct from parathyroid hyperplasia. As mentioned above microscopic examination of FNA smears from solid parathyroid lesions do not allow for distinction of hyperplastic glands from adenomas. The cytologic features of parathyroid lesions may vary depending on preparation. Along with the paper by Odronic et al. [25], many of the common features of parathyroid aspirates are lost on ThinPrep preparations which may lead to difficulty in recognizing parathyroid origin on FNAC. Cytologic features of solid lesions: • Moderate to hypercellular smears. • Three-dimensional crowded clusters and sheets with organoid/cribriform/trabecular architecture and nuclear overlap. • Loose clusters and sheets with admixture of single cells and naked nuclei in the background. • Microacini/microfollicles. • Round, papillary, and pseudopapillary clusters. • Occasionally vascular proliferation. • Cells are somewhat smaller and have less cytoplasm than thyroid follicular cells. • Cells with lacy cytoplasm. • Occasionally oncocytic cells with abundant granular cytoplasm. • Rarely cells with cytoplasm vacuoles. • Nuclei small, uniform, round to oval with stippled (saltand-pepper) chromatin. • Rarely tiny nucleoli but usually nucleoli absent. • Occasionally few atypical larger nuclei. • Very rare nuclear pseudoinclusions. • Often clean or bloody background. • Background colloid absent. • Rarely colloid-like material; follicular elements may contain colloid-like substances and may resemble a thyroid neoplasm.

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• Rarely lymphocytes and plasma cells. • Rarely macrophages in the background. • Mitoses absent. Differential diagnosis and problems in diagnosis: • Hyperplastic nodule • Noninvasive follicular thyroid neoplasm with papillarylike nuclear features (NIFTP) • Invasive follicular variant of papillary carcinoma • Poorly differentiated carcinoma • Medullary thyroid carcinoma Architectural pattern in smears helps to distinguish between parathyroid and thyroid aspirates [11, 13, 26, 27]. Overall organoid and cribriform or trabecular architecture of three-dimensional and frequently crowded clusters with overlap of cells as well as loose sheets and clusters with acinar-follicular and/or rounded-pseudopapillary or papillary [5, 11, 27–29] formations are suggestive of a parathyroid lesion [11, 13, 28, 30–32] (see Figs. 6.1, 6.2, 6.3, and 6.4). Cells with small, dark nuclei, somewhat smaller than nuclei in follicular thyroid cells, and with stippled chromatin [5, 13, 27, 29, 30, 32] (see Fig. 6.2e, f) are another sign suggestive of parathyroid origin, as is clean (see Fig. 6.2c– e) or bloody, colloid-free background [33] (see Figs. 6.2f and 5-3a–c). However, colloid-like material occasionally seen in the FNA smears from hyperplastic parathyroid may be confused with true colloid [11, 28] (see Fig. 6.7). True colloid may be also accidentally collected in the FNA of the intrathyroidal parathyroid, when needle crosses thyroid tissue on the way to target. Another distinctive feature that helps to distinguish smears of parathyroid from thyroid is the presence of single cells and bare nuclei [11, 29, 33] (see Figs. 6.2a, b, 6.5a, 6.6c, and 6.7a–d) in addition to cohesive groups of cells as well as prominent capillary network and vascular proliferation appearing occasionally in the parathyroid FNA [13, 27, 29, 31, 33] (see Fig. 6.6). Cytoplasmic tiny fat vacuoles have been reported as frequent and important sign helping distinguishing of parathyroid from thyroid aspirated in two papers [11, 33], but in our experience vacuolated cytoplasm belongs rare findings and appears exclusively in air-dried smears (see Fig. 6.8b). Few, occasionally numerous oxyphilic (oncocytic) cells can be seen in aspirates from parathyroid adenomas, and predominant or exclusive oxyphil component is a feature of oxyphil (oncocytic) parathyroid adenoma [5, 13, 29, 31] (see Fig.  6.5). Differential diagnosis includes oncocytic thyroid neoplasms as oncocytic cells of parathyroid adenoma can be easily misinterpreted as Hürthle cells. A combination of naked nuclei of chief cells together with oncocytic cells in parathyroid smears may be misdiagnosed as Hashimoto thyroiditis. Compared to Hürthle cells in the thyroid, oncocytic cells in the parathyroid are smaller, and the cell borders are poorly defined. The nuclei of parathyroid cells show neuroendocrine quality with stippled chromatin or

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Fig. 6.3  Common features of parathyroid lesions. (a–d) Organoid two- and three-dimensional, crowded clusters of uniform cells with small round nuclei without nucleoli and scant to moderate granular

cytoplasm. (e) Cohesive and (f) somewhat loose cribriform clusters of uniform cells with small, round nuclei and granular/lacy cytoplasm (H&E)

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a

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c

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Fig. 6.4  Common features of parathyroid lesions. Parathyroid adenoma. (a–d) Loose clusters and sheets of uniform and slightly pleomorphic cells with moderate to abundant granular cytoplasm and small

round bare nuclei in the background. Occasional microfollicular pattern can be easily confused with thyroid FNA-follicular lesion (H&E)

salt-and-pepper appearance [32, 34]. Hemosiderin-loaded macrophages indicative of cystic change, commonly seen in the smears from thyroid, are unusual finding in smears from the parathyroid [28] (see Fig. 6.9b). Nuclear enlargement or cellular and nuclear ­pleomorphism occasionally seen in smears of solid parathyroid lesions do not conclusively correspond to the type of the lesions [13]. Rare parathyroid adenoma may show nuclear pseudoinclusions and may be confused with papillary thyroid carcinoma, especially when located within the thyroid gland [6]. Cells of medullary carcinoma may be confused with cells of parathyroid lesions as these cells share some microscopic features of round to oval cells in loose clusters, single cells, and stippled nuclear chromatin. Presence of spindled cells and binucleated cells and absence of bare nuclei as well as immunoreactivity for calcitonin favor medullary carcinoma. Parathyroid hormone measurements in the needle washout fluid are helpful in the differential diagnosis of parathyroid lesions. IC for TTF1, thyroglobulin, parathy-

roid hormone, chromogranin, and GATA-3 all are useful to support parathyroid or thyroid origin.

Parathyroid Carcinoma Parathyroid carcinoma is rare and may be seen as a component of MEN1. Hereditary predisposition for parathyroid carcinoma is seen in patients with the hyperparathyroidismjaw tumor syndrome. Parathyroid carcinoma can be suspected when there is local invasion or nodal metastasis. Patients may have very high calcium and PTH serum levels. Carcinomas composed of chief cells are more common than oxyphilic cell carcinomas. Although smears of carcinoma may display pleomorphic cells and enlarged nuclei, the distinction between carcinoma and benign solid parathyroid lesions is very difficult if possible [35] as both smears of adenomas and hyperplastic parathyroid may occasionally display cellular and nuclear pleomorphism [10].

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Fig. 6.5  Uncommon features of parathyroid lesions. Oncocytic cell pattern in oncocytic parathyroid adenoma. (a, b) Hypercellular smears composed almost exclusively of round to polygonal oncocytic cells with abundant granular cytoplasm and naked nuclei in the background (H&E stain). (c) Cell block section and (d) PTH and (e) chromogranin

positivity confirm parathyroid origin and diagnosis of oncocytic parathyroid adenoma (cell block; H&E stain; chromogranin and PTH). (f) Corresponding histologic section of the removed oncocytic parathyroid adenoma (H&E stain)

6  Head and Neck: Parathyroid

a

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b

c d

e f

Fig. 6.6  Uncommon features of parathyroid lesions. Parathyroid adenoma. (a–f) A prominent vascular network and pseudo papillary pattern (H&E; MGG and Pap stain) (Images c–f courtesy of Anna Maria

Domanski, CTMIAC, Department of Pathology, Skåne University Hospital, Lund, Sweden)

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Fig. 6.7 Uncommon features of parathyroid lesions. Parathyroid hyperplasia (a–c) Colloid-like material was present within the clusters of tumor cells and in the background of the smears. (d) Colloid-like material and a cell cluster with rounded configuration mimicking thy-

roid follicle (H&E stain). (e) Corresponding histologic section of the removed hyperplastic parathyroid and (f) positive immunoreactivity for PTH (H&E; PTH)

6  Head and Neck: Parathyroid

a

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b

Fig. 6.8  Uncommon features of parathyroid lesions. Parathyroid adenoma (a) a mild to moderate nuclear pleomorphism and (b) occasional small intracytoplasmic vacuole (Pap and MGG stains)

a

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Fig. 6.9  Uncommon features of parathyroid lesions. Parathyroid adenoma (a) cell block section with (b) hemosiderin-loaded macrophages. Negative immunoreactivity for thyroglobulin (c) and positive immunoreactivity for PTH (d) confirm parathyroid origin (H&E; PTH)

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Parathyroid Cyst Parathyroid cysts are rare and develop more frequently in women than in men. The majority of cysts are non-functioning and are commonly present as asymptomatic cervical nodules [36]. The cyst wall contains parathyroid tissue and is usually lined with cuboidal or columnar epithelium. The cyst fluid is usually clear and transparent without colloid and may contain variable numbers of macrophages and few or absent epithelial cells [36]. Parathyroid origin can be established by measuring the fluid content of parathyroid hormone [37, 38]. Differential diagnosis includes thymic and thyroid cyst or cystic metastasis of papillary carcinoma.

Secondary Tumors of the Parathyroid The most common sites of origin of tumors involving the parathyroid glands that arise as results of direct extension from contiguous structures (thyroid) or as a result of hematogenous/lymphatic spread include the breast, melanoma, lung, kidney, and soft tissue. However metastases to the parathyroid gland are exceedingly rare [39, 40].

References 1. Erbil Y, Salmaslioglu A, Kabul E, Issever H, Tunaci M, Adalet I, et  al. Use of preoperative parathyroid fine-needle aspiration and parathormone assay in the primary hyperparathyroidism with concomitant thyroid nodules. Am J Surg. 2007;193(6):665–71. 2. Abraham D, Sharma PK, Bentz J, Gault PM, Neumayer L, McClain DA.  Utility of ultrasound-guided fine-needle aspiration of ­parathyroid adenomas for localization before minimally invasive parathyroidectomy. Endocr Pract. 2007;13(4):333–7. 3. Shi C, Guan H, Qi W, Ji J, Wu J, Yan F, et  al. Intrathyroidal parathyroid adenoma: diagnostic pitfalls on fine-needle aspiration: two case reports and literature review. Diagn Cytopathol. 2016;44(11):921–5. 4. Paker I, Yilmazer D, Yandakci K, Arikok AT, Alper M. Intrathyroidal oncocytic parathyroid adenoma: a diagnostic pitfall on fine-needle aspiration. Diagn Cytopathol. 2010;38(11):833–6. 5. Domingo RP, Ogden LL, Been LC, Kennedy GC, Traweek ST. Identification of parathyroid tissue in thyroid fine-needle aspiration: a combined approach using cytology, immunohistochemical, and molecular methods. Diagn Cytopathol. 2017;45(6):526–32. 6. Vaidya A, Gouri M, Sudha HM, Mysorekar V, Balekudura A. Ectopic parathyroid adenoma presenting as a mediastinal mass. J Clin Diagn Res JCDR. 2017;11(5):ED40–ED2. 7. Vu DH, Erickson RA.  Endoscopic ultrasound-guided fine-needle aspiration with aspirate assay to diagnose suspected mediastinal parathyroid adenomas. Endocr Pract. 2010;16(3):437–40. 8. Noussios G, Anagnostis P, Natsis K.  Ectopic parathyroid glands and their anatomical, clinical and surgical implications. Exp Clin Endocrinol Diabetes. 2012;120(10):604–10. 9. Graff-Baker A, Roman SA, Boffa D, Aslanian H, Sosa JA. Diagnosis of ectopic middle mediastinal parathyroid adenoma using endoscopic ultrasonography-guided fine-needle aspiration

E. Bakuła-Zalewska with real-time rapid parathyroid hormone assay. J Am Coll Surg. 2009;209(3):e1–4. 10. Balakrishnan M, George SA, Rajab SH, Francis IM, Kapila K. Cytological challenges in the diagnosis of intrathyroidal parathyroid carcinoma: a case report and review of literature. Diagn Cytopathol. 2018;46(1):47–52. 11. Cho M, Oweity T, Brandler TC, Fried K, Levine P. Distinguishing parathyroid and thyroid lesions on ultrasound-guided fine-needle aspiration: a correlation of clinical data, ancillary studies, and molecular analysis. Cancer Cytopathol. 2017;125(9):674–82. 12. Lieu D.  Cytopathologist-performed ultrasound-guided fineneedle aspiration of parathyroid lesions. Diagn Cytopathol. 2010;38(5):327–32. 13. Abati A, Skarulis MC, Shawker T, Solomon D.  Ultrasound guided fine-needle aspiration of parathyroid lesions: a morphological and immunocytochemical approach. Hum Pathol. 1995;26(3):338–43. 14. Wuertz FG, Kresnik E, Malle P, Hyden M, Lind P, Rogatsch H, et  al. Fine-needle aspiration with immunohistochemistry using a modified scrape cell block technique for the diagnosis of thyroid and parathyroid nodules. Acta Cytol. 2016;60(2):118–30. 15. Trimboli P, D’Aurizio F, Tozzoli R, Giovanella L.  Measurement of thyroglobulin, calcitonin, and PTH in FNA washout fluids. Clin Chem Lab Med. 2017;55(7):914–25. 16. Ozderya A, Temizkan S, Cetin K, Ozugur S, Gul AE, Aydin K. The results of parathyroid hormone assay in parathyroid aspirates in pre-operative localization of parathyroid adenomas for focused parathyroidectomy in patients with negative or suspicious technetium-99m-sestamibi scans. Endocr Pract. 2017;23(9):1101–6. 17. Fustar Preradovic L, Danic D, Dzodic R.  Small nonfunc tional parathyroid cysts: single institution experience. Endocr J. 2017;64(2):151–6. 18. Abdelghani R, Noureldine S, Abbas A, Moroz K, Kandil E.  The diagnostic value of parathyroid hormone washout after fine-needle aspiration of suspicious cervical lesions in patients with hyperparathyroidism. Laryngoscope. 2013;123(5):1310–3. 19. Bancos I, Grant CS, Nadeem S, Stan MN, Reading CC, Sebo TJ, et al. Risks and benefits of parathyroid fine-needle aspiration with parathyroid hormone washout. Endocr Pract. 2012;18(4):441–9. 20. Owens CL, Rekhtman N, Sokoll L, Ali SZ. Parathyroid hormone assay in fine-needle aspirate is useful in differentiating inadvertently sampled parathyroid tissue from thyroid lesions. Diagn Cytopathol. 2008;36(4):227–31. 21. Halbauer M, Crepinko I, Tomc Brzac H, Simonovic I. Fine needle aspiration cytology in the preoperative diagnosis of ultrasonically enlarged parathyroid glands. Acta Cytol. 1991;35(6):728–35. 22. Mincione GP, Borrelli D, Cicchi P, Ipponi PL, Fiorini A. Fine needle aspiration cytology of parathyroid adenoma. A review of seven cases. Acta Cytol. 1986;30(1):65–9. 23. Tseng FY, Hsiao YL, Chang TC.  Ultrasound-guided fine needle aspiration cytology of parathyroid lesions. A review of 72 cases. Acta Cytol. 2002;46(6):1029–36. 24. Liu F, Gnepp DR, Pisharodi LR. Fine needle aspiration of parathyroid lesions. Acta Cytol. 2004;48(2):133–6. 25. Odronic SI, Reynolds JP, Chute DJ.  Cytologic features of parathyroid fine-needle aspiration on ThinPrep preparations. Cancer Cytopathol. 2014;122(9):678–84. 26. Dimashkieh H, Krishnamurthy S.  Ultrasound guided fine needle aspiration biopsy of parathyroid gland and lesions. CytoJournal. 2006;3:6. 27. Agarwal C, Kaushal M. Parathyroid lesions: difficult diagnosis on cytology. Diagn Cytopathol. 2016;44(8):704–9. 28. Absher KJ, Truong LD, Khurana KK, Ramzy I. Parathyroid cytology: avoiding diagnostic pitfalls. Head Neck. 2002;24(2):157–64.

6  Head and Neck: Parathyroid 29. Giorgadze T, Stratton B, Baloch ZW, Livolsi VA.  Oncocytic parathyroid adenoma: problem in cytological diagnosis. Diagn Cytopathol. 2004;31(4):276–80. 30. Papanicolau-Sengos A, Brumund K, Lin G, Hasteh F.  Cytologic findings of a clear cell parathyroid lesion. Diagn Cytopathol. 2013;41(8):725–8. 31. Park GS, Lee SH, Jung SL, Jung CK. Liquid-based cytology in the fine needle aspiration of parathyroid lesions: a comparison study with the conventional smear, ThinPrep, and SurePath. Int J Clin Exp Pathol. 2015;8(10):12160–8. 32. Agarwal AM, Bentz JS, Hungerford R, Abraham D.  Parathyroid fine-needle aspiration cytology in the evaluation of parathyroid adenoma: cytologic findings from 53 patients. Diagn Cytopathol. 2009;37(6):407–10. 33. Kumari N, Mishra D, Pradhan R, Agarwal A, Krishnani N.  Utility of fine-needle aspiration cytology in the identification of parathyroid lesions. J Cytol Indian Acad Cytologists. 2016;33(1):17–21. 34. Sung S, Saqi A, Margolskee EM, Crapanzano JP. Cytomorphologic features distinguishing Bethesda category IV thyroid lesions from parathyroid. CytoJournal. 2017;14:10.

217 35. Wong YP, Sharifah NA, Tan GC, Gill AJ, Ali SZ.  Intrathyroidal oxyphilic parathyroid carcinoma: a potential diagnostic caveat in cytology? Diagn Cytopathol. 2016;44(8):688–92. 36. Rossi ED, Revelli L, Giustozzi E, Straccia P, Stigliano E, Lombardi CP, et al. Large non-functioning parathyroid cysts: our institutional experience of a rare entity and a possible pitfall in thyroid cytology. Cytopathology. 2015;26(2):114–21. 37. Kaplanoglu V, Kaplanoglu H, Ciliz DS, Duran S.  A rare cystic lesion of the neck: parathyroid cyst. BMJ Case Rep. 2013;2013. 38. Ogo A, Komori K, Nishida K, Hiramatsu S, Sakai Y, Matoba Y, et  al. Fluid aspiration identified the primary cyst among multiple cervical cysts in a case of hyperparathyroidism. J Endocrinol Investig. 2010;33(5):360–1. 39. Shifrin A, LiVolsi V, Shifrin-Douglas S, Zheng M, Erler B, Matulewicz T, et al. Primary and metastatic parathyroid malignancies: a rare or underdiagnosed condition? J Clin Endocrinol Metab. 2015;100(3):E478–81. 40. Fulciniti F, Pezzullo L, Chiofalo MG, Butera D, Losito NS, Tommaselli AP, et  al. Metastatic breast carcinoma to parathyroid adenoma on fine needle cytology sample: report of a case. Diagn Cytopathol. 2011;39(9):681–5.

7

Lung Henryk A. Domanski, Nastaran Monsef, and Anna M. Domanski

Introduction Cytological examination of specimens obtained from bronchial tree and lung tissue is a cost-effective, safe, and fast method of diagnosing lung disease. There are many established sampling and preparation techniques, producing cytological specimens for routine microscopic examinations, and a spectrum of ancillary methods such as microbiology, cytochemical, immunocytochemical, and molecular genetic examinations. Procedures such as examination of sputum are focused on lesions affecting the bronchial mucosa [1–4], whereas bronchoalveolar lavage (BAL) focuses on distal airways and alveolar mucosa [4, 5]. Via a bronchoscope, samples can be taken directly from larger- or middle-sized branches by aspiration of bronchial secretions, bronchial washing, and brushing. Lesions located beneath the bronchial surface and mediastinal targets such as mediastinal lymph nodes can be sampled by transbronchial fine-needle aspiration (FNA). Pulmonary masses not accessible through the bronchial tree, often peripherally located in the lungs, may be reached by percutaneous transthoracic FNA.  The main limitations of FNA include size of the sample and lack of architectural/histologic pattern. Core needle biopsy (CNB) is another minimally invasive procedure commonly used in the evaluation of lung masses. Thus, CNB preserves tissue architecture, it is a slightly more invasive procedure than FNA, and samplings obtained by CNB are of limited size. FNA and CNB often yielded similar diagnostic accuracy for malignant tumors and epithelial malignancies [6], whereas CNB is slightly superior in the diagnosis of nonepithelial and

H. A. Domanski (*) · A. M. Domanski Department of Pathology, Skåne University Hospital, Lund, Sweden e-mail: [email protected]; [email protected] N. Monsef Department of Pathology, Linköping University Hospital, Linköping, Sweden e-mail: [email protected]

benign neoplasms [7, 8] and provision of specific histologic diagnosis [9–12].

 ampling Techniques, Diagnostic Accuracy, S and Complications Percutaneous FNA is an outpatient procedure that can be performed transthoracically with the guidance of computed tomography (CT) [13], fluoroscopy, ultrasound (US) [14], or magnetic resonance imaging (MRI). CT allows safer FNA for small and deep-seated lesions, or lesions located near blood vessels, airways, or nerves. During the examination, a needle will be inserted percutaneously through the chest wall and advanced to the site of the lesion to remove samples of tissue. Generally, two needling techniques exist: a single-­ needle technique with a pleural puncture made when every FNA of the lesion is performed and a coaxial system in which multiple FNA samples may be taken through a single pleural puncture [13]. Published studies report the sensitivity of FNA for lung malignancy ranging from 56% to more than 90%. False-­ positive diagnoses are rare, usually under 1%, and positive predictive values approach 100% [6, 15–21]. Thus, overdiagnosing malignancy is less of a problem than underdiagnosing, with most studies reporting a false-negative rate of around 10%. Reasons for false negatives include paucity of tumor cells or nondiagnostic smears due to sampling error [18–21]. Similar to FNA cytology in other organs, rapid on-­ site evaluation (ROSE) of the aspirated material may improve diagnostic accuracy of percutaneous and transbronchial FNA of lung lesions [22–25]. Complications to transthoracic FNA are pneumothorax, hemorrhage, air embolism, hemoptysis and needle tract seeding of tumor cells. The most common complication is pneumothorax, with an average incidence of approximately 25% [13]. Most cases of pneumothorax are minor and do not require any medical intervention (pleural drainage). Needle tract seeding of tumor cells is related to the size of the ­needles

© Springer International Publishing AG, part of Springer Nature 2019 H. A. Domanski (ed.), Atlas of Fine Needle Aspiration Cytology, https://doi.org/10.1007/978-3-319-76980-6_7

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and using regular 22–23 gauge needles, although the incidence of this event is exceedingly low [26–28]. Rare cases of infection have been reported as a complication of endobronchial US-guided transbronchial needle aspiration [29, 30]. FNA can also be performed through the bronchoscope. This technique is usually performed with the aid of endobronchial US, in which a needle is inserted through the bronchial wall and cells are aspirated from the desired range

H. A. Domanski et al.

(endobronchial US-guided transbronchial needle aspiration [EBUS-TBNA]). This type of sampling presents demands on the sampler’s ability to aspirate representative and sufficient material [31–33], but after appropriate training, the technique is suitable for all experienced bronchoscopists [34]. EBUS-TBNA is an important part of lung cancer staging and has increasingly replaced more invasive and risky mediastinoscopy (see Chap. 8).

7 Lung

 NA of Lung Neoplasm and the Significance F of Tumor Subtyping The main purpose of FNAC examination of the lung masses is to determine whether the mass is neoplastic (benign or malignant) and to classify as accurately as possible the histologic type of the neoplasm. The most common malignant lung neoplasms are epithelial, divided into two main groups: small cell and nonsmall cell. This classification has been based on different treatment strategies for each group. Increased understanding of lung tumor pathobiology and new therapies for lung carcinomas have resulted in a new classification of lung neoplasms [35–37]. An important point in the new classification is an introduction of a new concept addressed specifically to the surgical specimen such as adenocarcinoma in situ and minimally invasive adenocarcinoma. With regard to FNAC examination of lung neoplasms, invasive carcinomas, especially those in advanced stages, are to be classified according to specific type (Table  7.1) [35–38]. The broader category of non-small carcinoma should be specifically diagnosed as adenocarcinoma, squamous cell carcinoma, or large cell carcinoma. Tumors classified previously as non-small cell carcinoma, not otherwise specified (NOS), should be further classified using a limited panel of antibodies, such as TTF1 or Napsin A (as markers of adenocarcinoma) and P40 or CK5 (as markers of squamous cell carcinoma), no more than two antibodies in each case [36, 39–42]. Other previously recommended antibodies of squamous cell carcinoma include cytokeratin 5/6 and P63 [43–45]. Identification of genomic alternations, essential for survival of lung tumor cells, has in recent years provided new treatment targets and improved lung cancer therapeutics. The genetic testing with additional immunochemistry can be performed mostly on both cytology specimens and biopsies [46–53]. EGFR mutations and ALK, RET, and ROS1 translocations have been mainly found in adenocarcinoma from nonsmokers and light smokers, while KRAS and BRAF mutations have been identified mostly in heavy smokers. EGFR inhibitors are available for treatment of patients with EGFR mutations. ALK-EML4 translocation is detected with both immunohistochemistry and ALK FISH testing on smears. Lung tumors with this translocation can effectively be treated with ALK/MET/ROS1 inhibitor and other ALK inhibitors. ROS1-driven lung adenocarcinoma and lung tumors with RET translocation appear responsive to ALK and ROS 1 inhibitor and tyrosine kinase c met and VEGFR 2 inhibitor, respectively. Even

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though there are no specific treatments for tumors with KRAS mutation, these tumors have shown lack of response to EGFR-targeted agents, and identification of these tumors is essential in selecting the right therapeutic method for lung cancer patients. Treatment of lung tumors, harboring ERBB2 and BRAF mutations with their inhibitors, is under evaluation. Table 7.1  Comparison of 2004 and 2015 WHO classification of lung carcinoma to the reporting of FNA samplings and small biopsies 2004 WHO classification Adenocarcinoma (mixed subtype, acinar, papillary, solid); bronchioloalveolar carcinoma (nonmucinous); fetal, colloid, signet ring, and clear cell adenocarcinoma Solid adenocarcinoma

Bronchioloalveolar carcinoma (mucinous) Squamous cell carcinoma

Small cell carcinoma Large cell carcinoma

Adenosquamous cell carcinoma

Sarcomatoid carcinoma

2015 WHO classification Adenocarcinoma; describe identifiable patterns including micropapillary pattern (due to its association with poor prognosis) not included in 2004 WHO classification

Non-small cell carcinoma, favor adenocarcinoma if definite adenocarcinoma morphology is not represented and/or presence of mucin, TTF1+/NapsinA+/P40-/CK5– Mucinous adenocarcinoma; describe pattern present Squamous cell carcinoma; morphological squamous cell patterns clearly present Non-small cell carcinoma, favor squamous cell carcinoma Morphological squamous cell patterns not clearly present but supported by immunostains, TTF1−/NapsinA−/p40+/ CK5+ Small cell carcinoma Non-small cell carcinoma, not otherwise specified (NOS); no clear differentiation by morphology or immunohistochemistry, TTF1−/negative staining for squamous cell markers The term non-small cell carcinoma NOS can also be used when morphology and immunocytochemistry results are conflicting Non-small cell carcinoma with neuroendocrine (NE) morphology; supported by NE stains, possible large cell neuroendocrine carcinoma (LCNEC) Non-small cell carcinoma with squamous and adenocarcinoma patterns; morphological squamous cell and adenocarcinoma patterns present in separate sets of tumor cells Poorly differentiated non-small cell lung carcinoma with spindle and/or giant cell carcinoma

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PD-1/PDL-1 immunohistochemistry can be used to identify patients who might have benefit of PD-1/PDL-1 checkpoint inhibitor. These agents make the immune system more effective in their attacks on cancer cells. Cytology specimens as well as biopsies might be used in detection of PD-1/PDL-1 status of tumor cells. Evaluation of lung carcinomas in advanced stages requires minimal invasive examination techniques such as FNAC or core biopsies. In conjunction with ancillary procedures such as liquid-based and cell block preparations for immunocytochemical and molecular genetic examinations, FNAC is an excellent tool to obtain specific diagnosis [38, 54–63] and specimens for gene mutation analysis predicting responsiveness, toxicities, and therapeutic efficacy of new target therapies for lung carcinoma (see Fig. 7.1) [46–53, 64–68].

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Fig. 7.1  EGFR. Fluorescence in situ hybridization (FISH) performed on a cytological specimen. There is amplification with clusters of amplified copies of the EGFR gene (red signal) in all cells. Chromosome 7 shows polysomia (i.e., >2 copies of chromosome 7 in each cell; green signal). (Image courtesy of Leif Johansson, MD, PhD, Department of Pathology, Skåne University Hospital, Lund, Sweden)

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Normal Elements The bronchial mucosa is lined by ciliated cylinder cells intermingled with few goblet cells (see Fig.  7.2) that might increase in number due to inflammatory conditions. Bronchial epithelium in aspiration smears appears as sheets of various size containing benign glandular cells. Cilia may be seen in some of these cells, depending on the quality of smears and sampling/preparation techniques (see Fig. 7.2). Bronchiolar epithelium appears as sheets and clusters of uniform round-to-oval cells and do not contain ciliated cells (see Fig.  7.3). In inflammatory/reactive processes, cells in bronchiolar epithelium may show atypical features such as moderate nuclear enlarging, irregularity, and hyperchromasia (see Fig. 7.4). Reactive changes in bronchiolar epithelium

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might be sometimes pronounced, making it difficult to distinguish these from malignant epithelial cells. Other cells and noncellular elements frequently seen in smears from transbronchial and transthoracic aspirates include cartilage, macrophages, and contaminations such as fat fragments, squamous epithelial cells, bone marrow cells, and mesothelium. Alveolar macrophages are a normal population in the alveolar space but might increase in different conditions such as smoking-associated respiratory bronchiolitis or the rare condition of desquamative interstitial pneumonia. Hemorrhage leads to increased numbers of alveolar macrophages, which contain hemosiderin pigment (see Fig. 7.5). Contamination by squamous cells may be occasionally difficult to differentiate from reactive squamous metaplasia and atypical squamous metaplasia in some inflammatory/reactive conditions of bronchial epithelium from squamous cell carcinoma (see Fig. 7.6).

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a

b

c

d

e

f

Fig. 7.2  Normal respiratory epithelium. (a) Columnar cell with terminal bars and cilia (MGG). (b) Sheet of columnar cells with few goblet cells (H&E). Bronchial epithelium in the FNA smears. (c, d) Loose

sheets of varying size containing benign glandular cells (Pap stain; MGG). (e, f) Cilia may be seen in some of these cells (MGG)

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a

a

b

b

c

c

Fig. 7.3  Bronchiolar epithelium. (a–c) Sheets of uniform, round- to-­ oval cells (H&E; MGG; Diff-Quik)

Fig. 7.4  Reactive atypia in bronchiolar epithelium. (a–c) Loose sheets of bronchiolar cells with moderate nuclear enlargement, irregularity, and hyperchromasia (MGG; Pap stain)

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a

b

c

d

Fig. 7.5  Normal elements. (a) Normal cartilage fragments from transthoracic FNA. (b, c) Pulmonary macrophages with abundant foamy cytoplasm and frequent intracytoplasmic carbon particles (MGG). (d) Macrophages may contain hemosiderin pigment, likely after hemorrhage (H&E)

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a

b

c

d

Fig. 7.6 (a) Mature squamous cells adjacent to respiratory epithelium cells showing likely contamination from mouth epithelium (MGG). (b) Reactive squamous metaplasia in the smears from lung FNA (Pap

stain). (c, d) Smears from cavitating squamous cell carcinoma show atypical squamous cells with poorly preserved morphology and inflammatory background (MGG)

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Inflammatory and Reactive Conditions The main indication of FNAC of the lung is to evaluate lesions suspected of being malignant. Many inflammatory, reactive, and systemic disorders affect the lung, showing clinical symptoms and radiological abnormalities of the bronchial tree and lung tissue that can be suspicious for malignancy. Aspiration of nonneoplastic conditions occasionally yields smears with microscopic features, allowing a specific and precise diagnosis such as tuberculosis, fungal infections, some parasite infestation, and other specific entities [69–72]. In many cases, FNA of nonneoplastic lung conditions can only exclude malignancy and suggest a differential diagnoses and additional diagnostic procedures leading to diagnosis.

Infections Smears from localized pneumonia and lung abscess yield purulent fluid showing, on microscopic examination, polymorphonuclear leukocytes admixed with granular debris

a

Fig. 7.7  FNA from cavitary infection. (a, b) Aspergillus hyphae (H&E)

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and histiocytes. Problems occur when smears contain viable reactive epithelial cells. These cells are enlarged with hyperchromatic nuclei. Occasional multinucleated bizarre cells may be seen. Usually, these atypical reactive cells occur as a few dispersed cells or few small cell clusters in the background of purulent inflammation, which helps to avoid a diagnosis of malignancy. The main differential diagnosis concerning lung abscess is smears from necrotic squamous cell carcinoma showing debris and inflammatory cells with features resembling abscess. In addition, dispersed atypical squamous cells with small pyknotic nuclei can resemble histiocytes or metaplastic squamous cells (see Fig.  7.6). An inflammatory smear consisting of polymorphonuclear leukocytes might indicate fungal infection, as well. Fungal infections are usually diagnosed in specimens obtained from bronchial brushings, washings, and BAL, but fungal hyphae can also appear in aspiration smears from lung abscess and granulomas (see Fig. 7.7) [72, 73].

b

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Granulomatous Inflammation Many granulomatous smears are associated with tuberculosis, atypical mycobacteria (see Fig.  7.8), and other infections. Some noninfectious diseases showing granulomatous lung nodules, occasionally examined by FNA cytology, include sarcoidosis [74, 75], Wegener’s granulomatosis [76– 80], and rheumatoid nodules (see Fig. 7.9) [81].

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• Occasionally multinucleated cells • Necrotic granular or amorphous debris • Polymorphonuclear leukocytes In cases in which specific mycobacterial infection is suspected, polymerase chain reaction (PCR) examination of aspiration smears is the most reliable and rapid technique to provide diagnosis.

Cytologic features • Clusters of epithelioid histiocytes • Admixture of lymphocytes

a

b

c

d

Fig. 7.8  Granulomatous inflammation. (a, b) Tuberculosis. Granular and amorphous debris and poorly preserved granulomas (H&E; MGG). (c, d) Epithelioid cell granulomas and scattered lymphocytes (Pap stain; MGG)

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Fig. 7.9  Granulomas. (a, b) Non-necrotizing epithelioid cell granulomas in sarcoidosis (MGG; H&E)

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Benign Neoplasm Pulmonary Hamartoma Pulmonary hamartoma is a common benign lesion of the lung with characteristic cytomorphological features. Hamartomas show tufts of a chondroid substance, fragments of myxoid connective tissue, mature fat and cartilage, and respiratory epithelium without atypical features (see Fig.  7.10) [82–84]. Rare cases of so-called endobronchial hamartoma arise in or near the bronchial wall, often protruding to the bronchi. The dominance of the epithelial component with reactive atypia in the FNA smears from

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endobronchial hamartomas (see Fig. 7.11) may be a source of false-positive diagnosis. Cytologic features • Cartilage • Bronchioloalveolar cells in small sheets and clusters • Fragments of myxoid connective tissue • Histiocytes • Occasionally fat Possible pitfall • Endobronchial hamartoma

a

b

c

d

Fig. 7.10  Hamartoma. (a, b) Scattered epithelial elements, macrophages, and fragments of cartilaginous tissue in the myxoid background (MGG). (c) Fragment of mesenchymal matrix (MGG). (d) Sheet of

respiratory epithelium and myxoid mesenchymal tissue in the background of the smears (H&E)

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a

b

c

d

e

f

Fig. 7.11  Endobronchial hamartoma. (a, b) Papillary clusters of based preparation (Pap stain; ThinPrep; Hologic; Bedford, MA, USA). tightly packed epithelial cells (MGG; Pap stain). (c, d) Reactive atypia (f) Cell block section with both epithelial and mesenchymal compoof epithelial elements and (e) cartilage fragments appears in liquid-­ nents (H&E; Cellient; Hologic; Bedford, MA, USA)

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Inflammatory Pseudotumor

Malignant Neoplasm

Inflammatory pseudotumor is a rare lesion mostly seen in young adults. The diagnosis of this lesion by aspiration cytology specimen requires usually clinical and radiological correlation since the cytomorphological features are not specific [85–88]. The background consists of plasma cells, lymphocytes, mast cells, and histiocytes. In addition, the cytological specimen usually contains a variable amount of spindle cells with mild nuclear atypia, micronucleoli, and finely granular chromatin.

FNAC has been generally accepted as a diagnostic modality capable of distinguishing carcinoma from other types of lung malignancies and to distinguish two main categories of small cell and non-small cell carcinomas. In recently published studies, it appears clearly that FNA complemented by ancillary techniques can also distinguish squamous cell carcinoma from adenocarcinoma [36, 39–42, 54, 55] and provide precise diagnosis with up to 97% accuracy [6, 15–21]. Summary of cytological features of most common types of lung carcinoma in FNA smears are presented in Table 7.2.

Bland, finely textured

Coarse

Clean or diathesis Easily detected

Diathesis Present

Slight to moderate Salt and pepper

Nuclear molding

Salt and pepper

None

May be present

Scant to moderate, coarsely granular, occasionally dense

Conspicuous

Salt and pepper

Slight to moderate

Not detected Easily detected, may be numerous

Clean

Clean or diathesis, necrosis Present

Crush artifact, Tumor cells attached Tumor cells apoptotic cells to capillaries attached to capillaries

Clean or diathesis Diathesis

Crush artifact

Uniform, round to oval, plasmacytoid, spindled

Small and medium

Atypical carcinoid; cytologic diagnosis: carcinoid, possible atypical Loosely cohesive groups and isolated single cells, rosettes

Eccentrically placed Eccentrically placed

Typical carcinoid; cytologic diagnosis, carcinoid, possible Small cell typical carcinoma Isolated single Loosely cohesive or tight groups and cells, small isolated single cells, dyscohesive rosettes clusters and sheets, single file pattern Small, high Small and medium N/C ratio Round to oval, Uniform, round to spindled oval, plasmacytoid, spindled

Inconspicuous Inconspicuous or small Variable, usually Scant or absent Scant to moderate, coarsely granular, moderate occasionally dense amounts

Prominent

Variable size and shape, nuclear molding Moderate to prominent Variable, often coarsely granular

Large and medium Variable

LCNEC; Cytologic diagnosis: Non- small cell carcinoma,with NE morphology, possible LCNEC Nuclear palisading, rosette formations

BAC bronchioloalveolar carcinoma, MGG May–Grünwald–Giemsa, N/C nuclear/cytoplasmic, Pap Papanicolaou stain, LCNEC large cell neuroendocrine carcinoma

Multinucleated giant cells

Prominent, one or more Variable amounts, often moderate; lacy or vacuolated, ill-defined borders

Irregular

Keratinization rare to absent

Variable amounts, most often dense

Prominent

Coarse

Prominent

Variable, undifferentiated cells, tumor giant cells Variable size and shape

Variable, often polygonal, elongated, and round to oval Large, often centrally placed, dark Variable

Variable, irregular, polygonal, rounded tadpole cells Centrally placed, irregular Prominent

Large or variable

Variable

Large cell Squamous cell carcinoma Cytologic carcinoma; moderately and diagnosis: non Small cell poorly carcinoma NOS differentiated Syncytial sheets Isolated single cells, small and clusters, dyscohesive single cells clusters and sheets

Variable

Squamous cell carcinoma; well differentiated Isolated single cells, small clusters

Small, conspicuos Absent or ill defined Variable amounts, Abundant/ moderate amounts; dense, welldefined borders small vacuoles orangeophilic (Pap), turquoise (MGG) Anucleated cells, Special Goblet and/or keratin structures columnar cell morphology. Small basally oriented nuclei Background Clean or diathesis, Clean or mucin Necrotic diathesis mucin Mitotic figures Present Not detected May be present

Nuclear Variable pleomorphism Chromatin Finely textured, pattern dusty, rarely coarse Nucleoli Prominent, one or more Cytoplasm Variable amounts; translucent, foamy, mucin vacuoles

Eccentrically placed

Nucleus

Eccentrically placed, round to ovoid None or slight

Columnar, round, Uniform, cuboidal polygonal, or columnar

Cell shape

Small/Medium

Variable

Adenocarcinoma Cell sheets and clusters; acinar, papillary, honeycomb-like, single cells

Cell size

Cytological features Architecture/ cell arrangement

Invasive mucinous Adenocarcinoma (including former BAC); Cytologic diagnosis: Mucinous adenocarcinoma Monolayer sheets, isolated single cells and 3D clusters

Table 7.2  Summary of cytological features of the most common types of lung carcinoma in FNA smears

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Adenocarcinoma Adenocarcinoma is the most common primary lung tumor and displays a variety of morphological features [9, 35, 89, 90]. Lung adenocarcinoma is heterogenous neoplasm with various histological subtypes that have been shown correlate with molecular genetic as well as clinicopathological f­ eatures. The current classifications of lung adenocarcinoma by International Association for the Study of Lung Cancer/ American Thoracic Society/European Respiratory Society (IASLC/ATS/ERS classification) and World Health Organization (WHO classification) include new entities such as adenocarcinoma in situ (AIS) for small (=