key: cord-0042836-9sdbhw2g authors: Liu, Haiyan; Zhang, Jun; Lin, Fan title: Lung and Mediastinum date: 2017-09-02 journal: Handbook of Practical Fine Needle Aspiration and Small Tissue Biopsies DOI: 10.1007/978-3-319-57386-1_6 sha: 4917d72485813b5a596fa47ba7fcbd6724549ce9 doc_id: 42836 cord_uid: 9sdbhw2g Fine needle aspiration and small tissue biopsies have become a primary modality to achieve a definitive diagnosis of a mass-like lesion of the lung and mediastinum. This chapter delineated cytologic and histologic features of common and rare neoplastic and nonneoplastic mass-like lesions of the lung and mediastinum. The utilities and pitfalls of commonly used diagnostic immunohistochemical (IHC) stains, such as TTF1, Napsin A, p40 and CK5/6, and small diagnostic IHC panels, were described. Multiple challenging and yet practical cases at the end of the chapter were used to reemphasize important points illustrated throughout the chapter. • The concept of personalized medicine and the remarkable advances in lung cancer genetics and therapy in the past decade have changed lung pathology practice dramatically. • The 2015 World Health Organization (WHO) classification of tumors of the lung specifies that immunohistochemistry is required for lung cancer diagnosis, not only for small biopsies and fine needle aspiration (FNA) specimens but also for certain resected specimens such as solid adenocarcinoma (ADC), nonkeratinizing squamous cell carcinoma, large cell carcinoma, neuroendocrine tumors, and sarcomatoid carcinomas. • New criteria and terminology for the diagnosis of lung cancer based on small biopsies and cytology are proposed in the 2015 WHO classification and are summarized in Tables 6.1 and 6.2. • The role of cytopathologists has expanded to not only making a specific diagnosis, including histopathological subtyping of tumors, but also to thoughtfully utilizing the limited material for necessary genetic studies to help personalize treatment strategies for advanced lung cancer patients. • Thyroid transcription factor 1 (TTF1) and napsin A are accepted markers for ADC differentiation; p40 is reported to be the most specific and sensitive marker for squamous cell differentiation. A reasonable recommendation is that, when immunohistochemistry is deemed necessary, at least one antibody each for squamous and glandular differentiation, but no more than two antibodies, should be used for an initial workup (e.g., TTF1 and p40 or p63). Thus a limited panel of TTF1 and p40 (or p63) is suggested for subtyping the tumor to preserve tissue for molecular testing. • Molecular testing for epithelial growth factor receptor (EGFR) mutation and anaplastic lymphoma kinase (ALK) rearrangement is recommended in tumors classified as ADC and in cases where an ADC component cannot be excluded. • When evaluating a computed tomography (CT)guided FNA of a lung lesion in the periphery, mesothelial cells are frequently seen on smears; reactive mesothelial cells may mimic carcinoma, and caution should be taken. • Diagnosis of ADC with a lepidic growth pattern can be challenging in FNA specimens. Both quality (degree of atypia) and quantity (groups of atypical cells) need to be considered. In a typical case of ADC with a lepidic growth pattern, the smears tend to be very cellular, containing many groups of atypical epithelial cells. In contrast, in a reactive condition, such as pulmonary infarction, atypical reactive pneumocytes are usually less numerous. • In mucinous ADC with a lepidic growth pattern, the neoplastic epithelial cells may mimic pulmonary macrophages. Features such as a mucinous background, cytoplasmic mucin/vacuoles, eccentrically located nuclei, and more abundant cytoplasm are helpful in reaching a correct diagnosis. • Basaloid squamous cell carcinoma may mimic small cell carcinoma; therefore, cellblock preparation in conjunction with immunostains will be helpful. • Infection such as aspergillosis may result in squamous cells with significant cytological atypia; therefore, infectious etiologies should be excluded before a diagnosis of well-differentiated squamous cell carcinoma is rendered. • A lesion from the mediastinum should be considered if cytological features do not fit the description of a typical "lung lesion." • Thymoma is rare in young patients and children. In lymphocyte-dominant thymoma, the main component of the aspirate may contain cortical thymocytes with expression of terminal deoxynucleotidyl transferase (TdT) and cluster of differentiation (CD)99; therefore, caution should be taken to avoid misdiagnosing it as a lymphoblastic lymphoma. • Thymic neuroendocrine neoplasm may mimic an epithelial-dominant thymoma; therefore, immunohistochemistry should be performed in cases with equivocal features. Tables 6.1 and 6.2 summarize new criteria and terminology for the diagnosis of lung cancer based on small biopsies and cytology from the 2015 WHO classification. Upper Specific viral infections, such as herpes simplex ( Fig. 6 .6) and cytomegalovirus ( Fig. 6.7) , can cause significant cyto- Severe acute respiratory syndrome (SARS) caused by a novel coronavirus (SARS-CoV) became a worldwide outbreak in 2003, affecting more than 8000 patients, with a fatality rate of 9.2%. SARS-CoV belongs to a family of large, positive, single-stranded ribonucleic acid (RNA) viruses. The key pathologic finding is diffuse alveolar damage (DAD). Depending on different phases in the disease progression, the composition of inflammatory cells may vary; however, macrophages (including multinucleated forms) and lymphocytes usually predominate. Other pathologic findings, such as fibrosis, prominent vascular injury, hemophagocytosis, squamous metaplasia, apoptosis, and atypical pneumocytes, including multinucleated giant pneumocytes with irregularly distributed nuclei or pneumocytes with large atypical nuclei, prominent eosinophilic nucleoli, and granular amphophilic cytoplasm, were reported. Ancillary tests, such as in situ hybridization, immunohistochemistry, viral isolation, or reverse transcription polymerase chain reaction (RT-PCR), are necessary to confirm the diagnosis. Representative images are shown in Fig. 6 .8a, b. FNA is a useful means of diagnosing pulmonary fungal infection, which should be suspected whenever there is granulomatous inflammation. Silver or periodic acid-Schiff (PAS) stains are used on cellblock sections. The common fungal infections include (1) Candida species; (2) Aspergillus species, Table 6 .3. Pulmonary strongyloidiasis affects immunocompetent and, more commonly, immunosuppressed persons presenting with pneumonitis with hemoptysis. The etiological agent is the nematode. Strongyloides stercoralis in sputum is shown in Fig. 6 .15. Infection by Mycobacterium tuberculosis is often a granulomatous inflammation containing clusters of epithelioid histiocytes, lymphocytes, and Langhans giant cells, with or without necrosis. In immunocompromised patients there may be abundant acid-fast organisms without obvious granulomatous inflammation. There are pulmonary macrophages and neutrophil-predominant mixed inflammatory cells in the background Acquired immunodeficiency syndrome (AIDS) patients are especially susceptible to Mycobacterium aviumintracellulare, an acid-fast organism producing negative images on Romanowsky stain. Special stains on cellblock section are particularly helpful. Nocardia, a weakly acid-fast filamentous organism, often infects immunocompromised patients, producing cavitary nodules on radiographs, which may mimic a neoplastic process. • Sarcoidosis is characterized by non-caseating granulomas in many organs, most commonly the lung. • Chest x-ray: bilateral hilar adenopathy is a classic finding; variable lung parenchyma changes, from normal, diffuse reticular, or ground glass opacities, nodular consolidation or cystic scarring. • CT and positron emission tomography (PET) scan can also be used. • Aggregates of epithelioid histiocytes, with or without Schaumann and asteroid bodies • Multinucleated giant cells and lymphocytes An essential part of the diagnosis of sarcoidosis is the exclusion of alternative possibilities: • Granulomas caused by infectious agents, such as mycobacterial infection and fungal infection • Drug-induced, hypersensitivity pneumonitis, or foreign body granulomatosis • Wegener granulomatosis is characterized by necrotizing vasculitis and may present as a lung mass with or without involvement of nasal passages and kidneys. • Multiple pulmonary nodules on imaging • Neutrophils, giant cells, necrotic collagen, and epithelioid histiocytes • The findings are nonspecific; therefore, serologic studies are necessary. (c) GMS stain reveals small oval or cup-shaped yeast forms, cellblock The 2015 WHO Classification of Tumors of the Lung, Pleura, Thymus and Heart has been published recently. There are numerous important changes in the classification of lung tumors, as summarized in Table 6 .4. Fig. 6 .16a, b. • Round to multilobulated, well-circumscribed tumor nodules composed of mesenchymal tissues, including chondroid or chondromyxoid tissue, fat, connective tissue, smooth muscle, and bone in various proportions, intermixed with clefts of respiratory epithelial cells • Chondromyxoid tissue usually predominates. • Representative images are shown in Fig. 6 .16c, d. • Immunohistochemistry is usually not necessary for diagnosis. • Pulmonary hamartomas have a high frequency of translocation t(3;12)(q27-28;q14-15) leading to a gene fusion of the high mobility group protein gene AT-hook 2 (HMGA2) and the lipoma preferred partner (LPP) gene. • Most often young patients, under the age of 40 • Male = female • The most common endobronchial mesenchymal lesion in childhood • Chest x-ray: a peripheral, discrete, solitary nodule. If endobronchial location, post-obstructive pneumonia and atelectasis may be evident. Fig. 6 .17a, b. • Positive for vimentin, smooth muscle actin (SMA), and rarely to desmin • Negative for myogenin, myoglobin, CD117, and S100 protein • Focal cytokeratin (CK) positivity was reported in 1/3 of cases, likely due to alveolar entrapment. • Anaplastic lymphoma kinase 1 (ALK1) expression was noted in 40% of cases. • p53 is negative; however, positivity is associated with recurrence and malignant transformation. • Representative images are illustrated in Fig. 6 .17c, d. Squamous cell carcinomas are malignant tumors that either morphologically show squamous cell differentiation (keratinization and/or intercellular bridges) or are morphologically undifferentiated non-small cell carcinomas but show squamous cell differentiation immunohistochemically. The 2015 WHO classifications of tumors of the lung reclassified squamous cell carcinomas into keratinizing, nonkeratinizing, and basaloid subtypes. The nonkeratinizing tumors require immunohistochemical proof of squamous differentiation. • About 20% of all pulmonary malignancies • Clinical presentation is similar to other non-small cell carcinomas. • Usually a central mass with cavitation and post-obstructive pneumonia Keratinizing squamous cell carcinoma: • Abundant dyshesive cells with dense cytoplasm, may be orangeophilic • Polygonal, rounded, or elongated cells • Tadpole or fiber-like cells • Pleomorphic, pyknotic nuclei with obscured nucleoli and chromatin detail • Anucleated cells and twisted keratin strands (Herxheimer spirals) • Representative images are shown in Fig. 6 .18a-d. Nonkeratinizing or basaloid squamous cell carcinoma: • Cohesive groups of cells with larger nuclei and coarsely granular chromatin • Cyanophilic cytoplasm on Pap stain • Rare or no keratinization • Representative images are shown in Fig. 6 .19a-f. WHO classification of tumors of the lung: keratinizing, nonkeratinizing, and basaloid subtypes: 1. Keratinizing squamous cell carcinomas exhibit recognizable keratinization, keratin pearls, and/or intercellular bridges, as illustrated in Fig. 6 .20a, b. 2. Nonkeratinizing squamous cell carcinomas are without recognizable keratinization, keratin pearls, or intercellular bridges, as illustrated in Fig. 6 .20c. 3. Basaloid squamous cell carcinomas are tumors with a basaloid component in greater than 50% of the tumor, regardless of the presence of any keratinization, as illustrated in Fig. 6 .20d. • Immunohistochemistry is required for the diagnosis of nonkeratinizing squamous cell carcinomas. • Squamous cell carcinomas express p40, p63, and CK5/6. • TTF1 is usually negative in keratinizing squamous cell carcinomas, may be weakly focally positive in nonkeratinizing squamous cell carcinomas. • Representative images are illustrated in Fig. 6 .21a-d. • Squamous cell carcinomas of lung are characterized by complex genomic alterations, frequently involving the following pathways: Deletion of chromosome 9p (CDKN2A) was observed in 72% of cases. • The most common primary pulmonary malignancy, accounting for about 40% of cases. • Mortality and incidence rates have generally been highest in high-income countries but are now declining, especially in younger males and females. • Has been more common in men than in women, but has begun to converge. histologic subtype other than a lepidic pattern; tumor cells infiltrating myofibroblastic stroma. 5. MIA diagnosis is excluded if the tumor invades lymphatics, blood vessels, air spaces, or pleura, contains tumor necrosis, and spreads through air spaces. 6. The cell type is mostly nonmucinous, but rarely may be mucinous. • The 2015 WHO classification of tumors of lung classifies ADC as lepidic, acinar, papillary, micropapillary, or solid according to the predominant pattern after a comprehensive histologic subtyping to identify all of the different histologic patterns in 5% increments. • Other variants of ADCs include invasive mucinous ADC, colloid ADC, fetal ADC, and enteric ADC. • Representative images are shown in Fig. 6 .23a-d. • Positive for TTF1 and napsin A: commonly used markers for pneumocytes, with comparable sensitivity, about 75%; much lower TTF1 expression in solid ADCs and mucinous ADCs • Positive for CK7 • Negative for squamous cell markers; however, p63 positivity was reported in up to 30% of lung ADCs. • EGFR, Kirsten rat sarcoma viral oncogene (KRAS), and ALK mutations are specific for lung ADCs. The ADCs with EGFR or ALK alteration are usually located in the periphery; in contrast, the ADCs with KRAS mutation are frequently located in hilar region. The prevalence for KRAS and EGFR mutations is 10-30%; the transforming fusion gene echinoderm microtubule-associated proteinlike 4 (EML4)-ALK is found in 5% of lung ADCs. The characteristics of patients with EGFR mutation are Asians, never smokers, with nonmucinous tumors; for ALK mutation, patients are younger age, male gender, and never or light smokers; for KRAS mutation patients are non-Asians, smokers, with invasive mucinous ADCs usually negative for TTF1, and positive for mucin (MUC) 2,5,6 immunophenotypes. This group of tumor represents a morphologic and biologic spectrum of tumors that is classified by the 2015 WHO classification of tumors of the lung into four types: preinvasive lesion (including diffuse idiopathic pulmonary neuroendocrine cell hyperplasia), carcinoid tumors (including typical and atypical carcinoid tumors), large cell neuroendocrine carcinoma (LCNEC), including combined LCNEC, and small cell carcinoma (including combined small cell carcinoma). Carcinoid tumors are neuroendocrine epithelial malignancies, including typical carcinoids, defined as those with <2 mitoses/per 2 mm 2 , lacking necrosis, ≥0.5 cm in size, and atypical carcinoids, which are those with 2-10 mitoses/per 2 mm 2 and/or foci of necrosis. • Account for <1% of all lung cancers. • Majority (70-90%) are typical carcinoids. • More common in female, white, and <60 years old. • Clinical syndromes are uncommon, including carcinoid syndrome, Cushing syndrome, and acromegaly. • Chest x-ray: usually a centrally located, lobulated mass with a prominent endobronchial component; one third of tumors in periphery. • CT using intravenous contrast medium shows considerable enhancement; calcification, especially in centrally located tumors; atelectasis; bronchiectasis; and hyperlucency for tumors with bronchial involvement. • Loosely cohesive groups and single uniform cells with granular nuclei and ample eosinophilic cytoplasm and naked nuclei. • Round, columnar, or plasmacytoid cells, forming acinar or rosette-like structures. • Branching capillaries. • Clean background. • Usually uniform polygonal or spindled cells arranged in organoid or trabecular patterns. • Other growth patterns also seen: rosette formation, papillary, pseudoglandular, or follicular patterns. • Significant pleomorphism and prominent nucleoli may be seen in typical carcinoids. • The differential features for atypical carcinoids are the presence of 2-10 mitoses per 2 mm 2 and/or necrosis. These changes may be focal. • Representative images are shown in Fig. 6 .24c-f. • Immunohistochemistry is recommended for the diagnosis of neuroendocrine tumors, not only in small biopsy or cytology cases but also in resected specimens. • The typical phenotype expresses neuroendocrine markers (CD56, synaptophysin, chromogranin); most are positive for panCK and negative for high molecular weight cytokeratins (HMWCKs) and TTF1. However, TTF-1 expression was reported in 43-53% of cases. • The proliferative index (ki-67) is valuable in distinguishing carcinoids from high-grade neuroendocrine carcinomas, especially in small biopsies or cytology specimens with significant crush artifact. Small cell carcinomas have high proliferative index (>50%) in contrast to <10-20% in carcinoids. • Ki-67 is not recommended for the distinction of typical from atypical carcinoids due to the lack of cutoff value. • Representative images are shown in Fig. 6 .25a-d. • Heavy smokers in >90% of cases. • Paraneoplastic syndrome is uncommon. • Usually a periphery mass with irregular margin, with or without intratumoral calcification • The tumors are usually large, showing central inhomogeneous enhancement on CT with contrast. • Cavitation uncommon • Overlapping with other neuroendocrine tumors and ADCs • Loosely cohesive or single, monotonous tumor cells with a hyperchromatic nuclear chromatin pattern but easily appreciated nucleoli, nuclear membrane irregularity, and preserved moderate to abundant, delicate cytoplasm • Necrosis or apoptotic debris may be seen. • Representative images are shown in Fig. 6.26a • The proliferative index (ki-67) is usually in the range of 40-80%. • Necrosis is common. • Representative images are shown in Fig. 6 .26c, d. • Immunohistochemical markers such as synaptophysin, chromogranin, and CD56 are required to confirm neuroendocrine differentiation of the tumor. • CD56 was reported in 92-100% of cases, chromogranin in 80-85%, and synaptophysin in 50-60%. • About 50% of cases are positive for TTF1. • PanCK, low molecular weight cytokeratin (LMWCK), and CK7 are expressed in either dot-like or diffuse cytoplasmic patterns. • CD117 positivity was reported in >70% of cases. • Thirteen percent of all newly diagnosed lung cancers are small cell carcinomas. • Virtually all are heavy smokers, male predominant. • Usually a centrally located lobulated mass, with occasional endobrochial involvement; 5% peripherally located • A large hilar mass with bulky mediastinal lymph nodes is characteristic; invasion of hilar vessels and vena cava is common; cavitation is rare. • Small cells with hyperchromatic nuclei, powdery chromatin texture, indistinct nucleoli, nuclear molding, and scant cytoplasm • Marked mitosis and single cell necrosis • Nuclear debris and crush artifact in the background • Representative images are shown in Fig. 6 .27a-d. • Reserve cell hyperplasia • Carcinoids • Small blue cell tumors, such as lymphoma, Ewing sarcoma, and rhabdomyosarcoma • Non-small cell carcinoma • Pulmonary blastoma • Merkel cell carcinoma • The tumor usually presents in a sheetlike pattern. • The tumor cells are small, containing round, oval, or spindled nuclei, with a fine granular chromatin pattern, inconspicuous nucleoli, and scant cytoplasm, without defined cell borders. • Nuclear molding is frequent. • High mitotic rate is seen, at least 10 mitoses/per 2 mm 2 , with an average of 60 mitoses/per 2 mm 2 . The proliferative index (ki-67) is >50%, with an average of >80%. • May show extensive necrosis • Representative image is shown in Fig. 6 .27e. • Immunohistochemical studies may be required to confirm neuroendocrine and epithelial differentiation of the tumor. • CK AE1/3, CAM5.2, and MNF116 are positive in nearly 100% of cases, with either dot-like, paranuclear, or diffuse cytoplasmic staining patterns. • Neuroendocrine markers (CD56, synaptophysin, and chromogranin) are reactive in the majority of cases; CD56 and synaptophysin are usually diffuse and strong, but chromogranin is often focal and weak. • <10% of small cell carcinomas may lack or have only very focal expression of neuroendocrine markers. • TTF1 is positive in up to 90-95% of cases. • CD117 positivity was reported in 60% of cases. • Representative images are shown in Fig. 6 .27f-j. The features of differential diagnosis for neuroendocrine tumors are summarized in Table 6 .5. • About 2.3% of all pulmonary malignancies. • Usually in the sixth decade, male predominant. • Most patients are smokers. • Chest x-ray: usually a peripheral mass, rarely may be cavitated • Single cells or loose clusters • Vesicular, pleomorphic nuclei with irregular nuclear membrane, multiple nucleoli and high nuclear-tocytoplasmic ratio • Ill-defined, feathery cytoplasm types. Therefore, molecular testing is recommended for large cell carcinomas. Sarcomatoid carcinoma is a general term that includes pleomorphic carcinoma, carcinosarcoma, and pulmonary blastoma, which are individual entities in the 2015 WHO classification. This group of tumors accounts for 2-3% of all cancer cases in a surgical series and <1% of all lung cancers. Definitive diagnosis of this group of tumors is very difficult or impossible in small biopsies and cytology specimens. The characteristic features of individual tumors in this group are summarized in Table 6 .6. Representative images are illustrated in Fig. 6 .28a-h. This is a new entity in the 2015 WHO classification of tumors of lung. The group of carcinomas associated with • Affects people of all ages, although, it was originally reported in children and younger adults • Male equals female • Fewer than 100 cases reported • Usually presents at an advanced stage, with pleural effusion, chest pain, weight loss, and respiratory symptoms • Chest x-ray: extremely rapid-growing tumor, with complete opacification of the thorax within 2-8 weeks • CT: a hypoattenuating, heterogeneously enhancing, often extensively necrotic mass with poorly defined, infiltrative borders. High fluorodeoxyglucose (FDG) uptake is characteristic. • Usually cellular smears • Discohesive clusters and single of small to intermediate size, monomorphic cells with irregular nuclear membrane, granular to coarse chromatin pattern, and discrete nucleoli • Mitoses, necrotic debris, and crush artifact are common. • Representative image is shown in Fig. 6 Fig. 6 .29b. • Speckled nuclear positivity in more than 50% of tumor cells with NUT antibody is a constant finding and is diagnostic. • Broad-spectrum cytokeratins are positive in majority of cases. • Other epithelial markers, such as epithelial membrane antigen (EMA), epithelial cell adhesion molecule (BerEP4), and carcinoembryonic (CEA) antigen showed variable results. • Most of cases are positive for p63/p40 and CD34. • Occasional reactivity to synaptophysin, chromogranin, and even TTF1 was observed. • NUT carcinomas are defined by the presence of NUT gene rearrangement, which is a chromosomal translocation between the NUT gene (NUTM1) on chromosome 15q14 and other genes: Bromodomain containing 4 (BRD4) on chromosome 19p13.1 (70%), BRD3 on chromosome 9q34.2 (6%), or an unknown partner gene (24%). The most common metastatic tumors are carcinomas, especially from the gastrointestinal tract, gynecological tract, breast, urothelial, head and neck, prostate, and other sites, followed by sarcomas, melanomas, and germ cell tumors. Sex and age distribution depend on tumor types, such as colorectal cancers in elderly patients of both sexes, breast cancer and melanoma in younger adults, and germ cell tumors and sarcomas in young adults or children. Metastases can be single or multiple, usually involving the lung parenchyma and the pleura. However, bilateral, multiple, peripherally located round, variable-size nodules (hematogenous metastases), or diffuse thickening of the interstitium (lymphangitic carcinomatosis) are typical. Cytological features, histological findings, and immunoprofile are the same as the primary tumors. The differential immunophenotypes for the most common metastatic carcinomas are summarized in Table 6 .7. The WHO classifications of tumors of the pleura, 2015, are summarized in Table 6 .8. • Usually occurs in older adults, ≥60 years • More often in men • A strong association with asbestos exposure • Pleural effusion is very common. • Variety of presentations, classically exhibiting a diffuse circumferential ring of nodular pleura associated with ipsilateral effusion • Less commonly presents with pleural effusion without obvious pleural nodularity • Pleural plaques, especially associated with calcifications, are suggestive of asbestos exposure. • Hypercellular specimen with mixed epithelial and spindle cells. • Can be epithelial or spindle cell dominate. • Resemble ordinary mesothelial cells. • Tumor giant cells, granulomas, and psammoma can be seen. • Representative images are shown in Fig. 6 .30a-c. • Pulmonary ADC (Table 6 .9) • Metastatic carcinoma • Reactive mesothelial cells (Table 6 .10) • Account for 60-80% of malignant mesotheliomas • Variety of histological patterns. Most common patterns: solid, tubulopapillary, and trabecular; less common patterns: micropapillary, adenomatoid (microcystic), clear cell, transitional, deciduoid, and small cell showing varying degree of cellularity from hyalinized acellular to highly cellular stroma. Myxoid changes may be seen in 5-10% of cases, with nests of bland-looking, vacuolated epithelioid cells floating in the matrix. • Representative images are shown in Fig. 6 .30d-f. • Characterized by a proliferation of spindle cells arranged in fascicles or with a haphazard pattern and involves the adipose tissue of the parietal pleura or the adjacent lung parenchyma. • The tumor cells show variety of morphology, from plump to thin, elongated cells with scant cytoplasm. • The degree of nuclear atypia, mitotic activity, and necrosis vary from minimal to moderate to marked. • Characterized by areas of atypical spindle cells arranged in a so-called patternless pattern within a dense, hyalinized, fibrous stroma constituting at least 50% of the tumor • Invasion of adipose tissue is the most reliable criterion to distinguish desmoplastic mesothelioma from organizing pleuritis. • Show any combination of the patterns described above constituting at least 10% of the tumor • Positive for CK AE1/3, calretinin, CK5/6, WT1, and D2-40. • Negative for BerEP4, MOC-31, B72.3, and CEA. • See Tables 6.8 and 6.9 for differential diagnosis. • Multiple chromosomal alterations, more common with chromosomal losses, especially on chromosomal arms 1p, 3p, 4q, 6q, 9p, 13q, 14q, and 22q; alterations in several tumor suppressor genes, including neurofibromin 2 (NF2), CDKN2A (p16INK4a), CDKN2B (p15INK4b), and BRCA1-associated protein 1 (BAP1). • Representative images are shown in Fig. 6 .31a-f. • Most common in sixth to seventh decades • No sex predisposition • Accounting for <5% of primary pleural tumors • Unknown etiology • Slow-growing, relatively benign tumors, up to 10% malignant • Usually asymptomatic, incidental finding • Usually solitary, well-circumscribed mass arising from visceral pleura • Can be multiple and distributed throughout the pleural cavity • Cellular smear with bland-appearing, small, oval to spindled cells • No mitosis or necrosis • Representative images are shown in Fig. 6.32a Fig. 6 .32c, d. • Positive for signal transducer and activator of transcription 6 (STAT6): > 95% of cases, specific. • Positive for CD34, B-cell CLL/lymphoma 2 (Bcl2), and CD99: nonspecific. • Occasionally may be positive for SMA, epithelial membrane antigen (EMA), keratin, S100, or desmin. • Representative images are shown in Fig. 6 .32e, f. • The tumor harbors characteristic gene fusion NGFI-A binding protein 2 (NAB2)-STAT6. The mediastinum can be divided into four hypothetical compartments: superior, anterior, middle, and posterior. The distribution of organs and tumors of the mediastinum is summarized in Table 6 .11. In adults, the distribution of tumors is as follows: 46% thymic epithelial tumors, 23% lymphomas, 16% endocrine tumors, and 15% germ cell tumors. In children, the frequency of tumors is 47% neurogenic neoplasms, 19% germ cell tumors, 12% lymphomas, 10% thymic epithelial tumors, 6% cysts, and 6% mesenchymal tumors. The 2015 WHO classification of tumors of the thymus is summarized in Table 6 .12. Selected entities will be discussed in the section below. • Bronchogenic cyst -any part of the mediastinum • Enteric cyst -posterior mediastinum • Thymic cyst -anterior mediastinum • Pericardial cyst -middle mediastinum • Hypocellular smear • Dependent upon the type of the cyst Thymoma • Rare malignancy overall, but the most common mediastinal tumors in adults • Median age of 50 years, rare in children • May present as a mass lesion or an incidental finding • May be associated with myasthenia gravis, pure red cell aplasia, hypogammaglobulinemia (Good syndrome), and/ or other autoimmune disorders • Usually a mass lesion, well-circumscribed or invasive borders, depending on the subtypes of thymoma • Usually moderate to hypercellular smear • Two populations of relatively bland neoplastic epithelial cells and lymphocytes • The proportion of epithelial cells and lymphocytes is dependent upon subtype. • In epithelial subtype, epithelial cells are dominant; in lymphocyte-predominant subtype, small mature lymphocytes are dominant; and in spindle cell type, spindle epithelial cells are predominant. • Neoplastic epithelial cells tend to be cohesive and have a delicate nuclear membrane, fine nuclear chromatin, and small nucleoli. • Few or no mitoses are present. • Representative images are shown in Fig. 6 .33a-f. • Type A thymoma: 1. Complete or incomplete fibrous capsule may display coarse lobulation with thick fibrous bands. 2. Microcystic pattern is most common, more prominent in subcapsular areas. 3. Other patterns: rosettes (with or without a central lumen), glandular or glomeruloid structures, Masson's hemangioma-like papillary projections in cystic spaces, meningioma-like whorls, fascicular growth, and storiform growth. 4. Hassall corpuscles are absent. 5. The tumor cells are spindled and/or oval-shaped with bland nuclei, finely dispersed powdery chromatin, and inconspicuous nucleoli. 6. Low mitotic activity, usually <4 mitoses/per 2 mm 2 . 7. No or very few immature lymphocytes. 8. When hypercellularity, increased mitotic counts, and focal necrosis are present, designate as atypical type A thymoma variant. 9. Representative images are shown in Fig. 6.34a (a) predominance of polygonal epithelial cells forming solid sheets, resulting in a pink appearance on H&E; (b) paucity of admixed nonneoplastic immature T cells. • Epithelial cells: usually positive for AE1/3, CK7, CK19, p63, PAX8, forkhead box N1 (FOXN1), CD57, and CD205; negative for EMA, CD117, and CD5. • Small lymphocytes (thymocytes) are mature T-cells, but positive for CD3, CD5, TdT, CD99, with a high MIB1 (Ki-67) proliferative index. • Representative images are shown in Fig. 6 .34e-h. Thymic carcinomas include of a variety of histological types of tumors, accounting for approximately 22% of all thymic epithelial neoplasms. The squamous cell carcinomas are the most common type, accounting for approximately 70% of all thymic carcinoma cases. Lymphoepithelioma-like carcinomas are rare, accounting for 6-32% of all thymic carcinomas; sarcomatoid carcinomas account for 5-10%; and basaloid carcinomas account for <5%. Other types, such as mucoepidermoid carcinomas, clear cell carcinomas, ADCs, NUT carcinomas, and undifferentiated carcinomas, are very few. The characteristic features of the three most common types are summarized in Table 6 .13. Thymic neuroendocrine tumors are categorized into two major groups: (1) low-grade typical carcinoids and intermediategrade atypical carcinoids, which always show characteristic morphological and immunohistochemical neuroendocrine features, and (2) high-grade LCNECs and small cell carcinomas, which may lack some neuroendocrine features. • No established association with smoking. • More aggressive clinical course: propensity for recurrence, lymph node or distant metastases, and tumorassociated death. • Atypical carcinoids account for the majority of the tumors. • Only carcinoids reported in setting of multiple endocrine neoplasia type 1 (MEN1). • Average age at presentation: 49 years. • Carcinoids show a strong male predominance. • Typically a lobulated, heterogeneous mass in the anterior mediastinum • Same as pulmonary neuroendocrine tumors; please refer that section in this chapter. • Representative images are shown in Fig. 6 .36a-i. • Pulmonary neuroendocrine tumors: careful clinical and radiological correlation is the primary way to distinguish pulmonary from thymic neuroendocrine tumors. • Thymoma, especially type A, to distinguish from carcinoids. • Thymic carcinoma usually shows focal and/or weak expression of neuroendocrine markers. The same as pulmonary neuroendocrine tumors, respectively. Same as the pulmonary neuroendocrine tumors, respectively. 1. Mature teratoma: cytology diagnosis is difficult; may be paucicellular with few anucleated squamous cells and macrophages in a proteinaceous background; ciliated bronchial cells, smooth muscle, and cartilage are present; a mucoid background with bland-looking signet ring cell-like mucus cells may be seen. 2. Immature teratoma: may be cellular, composed of aggregates or individual small, round, hyperchromatic cells with high nuclear-to-cytoplasmic ratio, and inconspicuous nucleoli; rare rosettes with neuropils, rhabdomyoblasts, immature cartilage, and blastemalike stromal cells may be identified. 2. Immature teratoma: the immature teratomatous elements are mostly cellular spindle mesenchymal components, but immature neural and epithelial elements can be seen; frequent mitoses; embryonic rhabdomyoblastic tissue, blastomatous tissue resembling embryonic kidney or lung, primitive neuroectodermal tumor (PNET), and other epithelial or mesenchymal malignant transformations can be seen. The immunophenotypes of mediastinal germ cell tumors are summarized in Table 6 .14. sex determining region Y box 10 (SOX10) and cytoplasmic staining for vimentin and human melanoma black 45 (HMB45), focally for melanoma antigen recognized by T cells (MART1) and S100 (both nuclear and cytoplasmic), while CK AE1/3 and neuroendocrine markers (synaptophysin, chromogranin and CD56) were nonreactive. Representative images are illustrated in Fig. 6 .39e-h. The overall findings are those of a metastatic spindle cell melanoma. The diagnosis of metastatic spindle cell melanoma can be challenging on FNA specimens. The spindled tumor cells can display a wide range of morphologies, from deceptively bland-appearing reactive fibroblastlike cells to highly pleomorphic, high-grade sarcomatous spindle cells. The diagnostic cytological features of conventional melanoma, such as a dispersed single cell pattern, eccentric nuclei, cytoplasmic melanin pigments, intranuclear pseudoinclusions, prominent macronucleoli, and bi-or multinucleations, are often subtle or even lacking. For the index case, the presence of melanin pigments is a valuable clue to raise the suspicion for melanoma which, in conjunction with the immunophenotype, justified the diagnosis of spindle cell melanoma. The presence of cytoplasmic melanin (continued) Immunohistochemistry plays a critical role in the diagnosis of spindle cell melanoma, especially in the clinical scenario of metastasis. Metastatic spindle cell melanomas tend to lose expression of some melanoma markers. Piao and colleagues reported that spindle cell melanomas express S100, HMB45, and MART1 in 67%, 50%, and 18% of cases, respectively. Although the sensitivity of S100 for melanoma in general is very high (~93-100%), its specificity is low. The expression of S100 can be seen in a variety of tumors. HMB45 is expressed in melanocytic tumors, also in clear cell sarcomas, perivascular epithelial cell tumors (PEComas), melanocytic schwannomas, meningeal melanocytomas, some ovarian steroid tumors, and renal cell carcinomas with the t(6;11)(p29;q12) translocation. The sensitivity of HMB45 for melanoma in general is in the range of 70-90%, but only 0-30% for desmoplastic melanomas. MART1 has a sensitivity of ~85-97% for primary and ~57-92% for metastatic melanomas, but only 0-33% for desmoplastic melanomas; its specificity for melanoma is 95-100%. MART1 expression is also seen in PEComas and clear cell sarcomas. SOX10 is a transcription factor that is essential for the survival of neural crest-derived cells and for the maintenance of the multipotency of neural crest cells. The cells derived from neural crest multipotential cells include neurons and glial cells in the peripheral nervous system, melanocytes of the skin, C cells of the thyroid, catecholaminergic cells of the adrenal gland, and cartilage and bone of the face. Studies have shown that, among tumors, SOX10 is commonly expressed in melanomas, including desmoplastic melanomas, tumors with Schwann cell differentiation, myoepithelial cell tumors of the soft tissue and some salivary gland neoplasms, particularly those with myoepithelial differentiation, and acinic cell carcinomas. When compared with other melanocytic-associated markers, SOX10 is highly sensitive and specific and can assist in the differential diagnosis of melanomas. Even in desmoplastic melanomas, SOX10 expression was reported in 100% of cases. Studies of SOX10 expression in tumors of the nervous system reported that SOX10 is commonly expressed in schwannomas (100%), neurofibromas (98-100%), and, less frequently, MPNSTs (~50-55%). The differential diagnosis of this case is summarized in Table 6 .15. Learning Objectives 1. To describe the cytological features of this entity 2. To become aware of the subtle differences in cytology and to raise the question of a metastasis 3. To become familiar with the potential utility of immunohistochemical markers in the diagnosis of this tumor A 72-year-old female with a past medical history of noninvasive low-grade papillary urothelial carcinoma a year ago presented with coughing and chest pain for month. CT imaging of the chest revealed multiple lung nodules and lymphadenopathy. An ultrasound-guided FNA and biopsy of the hilar lymph node were performed. The smears were highly cellular, composed of loosely cohesive clusters and single spindle to plump cells with hyperchromatic nuclei, coarse chromatin, occasional discernible to prominent nucleoli, and abundant delicate cytoplasm without defined borders. Some of the cells appeared spindled, columnar or racket-shaped, with eccentrically located nuclei and cytoplasmic tails, also called cercariform cells. Abundant necrosis was noted in the background. Representative images of the FNA smears and biopsy are shown in Fig. 6 .40a-f. The cytological features raise a differential diagnosis that includes a non-small cell carcinoma of lung, especially squamous cell carcinoma, a sarcomatoid carcinoma, and metastatic carcinoma, especially metastatic urothelial carcinoma. A panel of immunoassays was performed, revealing that the tumor cells were positive for CK7, CK20 (focal), CK5/6, uroplakin II, p40, and placental S100 (S100P) while negative for TTF1, napsin A, synaptophysin, and chromogranin. Representative images are illustrated in Fig. 6 .40g-j. The final diagnosis is metastatic carcinoma with urothelial differentiation There are limited reports in literature regarding the cytology of metastatic urothelial carcinomas. The following features were reported: (1) loosely cohesive cells occurring singly and in syncytial clusters; (2) large hyperchromatic nuclei with irregularly distrib-uted granular chromatin and abundant granular or fibrillar cytoplasm; (3) distinct cell borders; (4) multilayered papillary fragments; (5) cells with eccentric nuclei, multiple nucleoli, and intracytoplasmic vacuoles; and (6) cercariform cells, "bipolar" cells, and spindled-shaped cells. Among those, multilayered papillary fragments and cercariform cells are the most helpful features in the distinction of urothelial carcinoma from others. The cercariform cells, first described in 1993 by Johnson and Kini and further defined in 1995 by Powers and Elbadawi, are fusiform, pyramidal, or racket-like cells with eccentric nuclei that form non-tapering, flattened, bulbous, or fishtail-like cytoplasmic extensions in varying lengths. The presence of a small vacuole in the bulbous tail was also a helpful criterion. These cells, which are encountered in 57-100% of metastatic urothelial carcinomas, are interpreted in favor of urothelial carcinoma, particularly when they are observed in large numbers. However, they are not specific and must be considered alongside other clinical and morphological characteristics and immunohistochemical phenotypes. The cytological features of metastatic urothelial carcinomas are very difficult to differentiate from mesenchymal tumors and squamous cell carcinomas. Immunohistochemical analyses play a crucial role. Urothelial cell carcinomas express both CK7 and CK20. In addition, they express HMWCKs (CK5/6, CK903), p63, GATA binding protein 3 (GATA3), uroplakin II, and S100P. GATA3 is a zinc finger transcription factor with a diverse range of biologic roles. GATA3 is a newer generation of urothelial specific marker. GATA3 expression was reported in approximately 80% of urothelial carcinomas, over 90% of breast carcinomas, 100% of parathyroid gland tissue or tumors, salivary gland tumors, especially the salivary ductal carcinomas (~90%), and transitional proliferations of the gynecological tract. In addition, 0-12% of pulmonary squamous cell carcinomas and approximately 10% of pancreatic carcinomas were also reported to be positive for GATA3, although weakly and focally in the majority of positive cases. The immunohistochemical differential diagnosis of metastatic urothelial carcinomas is summarized in Table 6 .16. Met UCA metastatic urothelial carcinoma, SqCC squamous cell carcinoma, CA carcinoma, CK cytokeratin, TTF1 thyroid transcription factor 1, GATA3 GATA binding protein 3, S100P placental S100 a GATA3 expression in squamous cell carcinoma of lung was reported in 0-12% of cases; when positive, it is usually weak and focal. Comprehensive cytopathology Cytology: diagnostic principles and clinical correlates The art & science of cytopathology Fine needle aspiration cytology of metastatic urothelial carcinoma: study of seven cases with review of literature Transthoracic fine-needle aspiration cytology of non-invasive, low-grade urothelial carcinoma with lung metastasis: a case report with review of the literature Comprehensive genomic characterization of squamous cell lung cancers Lung pathology of severe acute respiratory syndrome (SARS): a study of 8 autopsy cases from Singapore NUT midline carcinomas in the thymic region Pathology and pathogenesis of severe acute respiratory syndrome Lung pathology of fatal severe acute respiratory syndrome Diagnostic histopathology of tumors WHO classification of tumours of the lung, pleura, thymus and heart. Lyon: IARC WHO panel. The 2015 World Health Organization classification of lung tumors: impact of genetic, clinical and radiologic advances since the 2004 classification Diagnosis of lung cancer in small biopsies and cytology: implications of the 2011 International Association for the Study of Lung Cancer Diagnosis of lung adenocarcinoma in resected specimens: implications of the 2011 International Association for the Study of Lung Cancer Pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (SARS) Ancillary Tests Utility of GATA3 immunohistochemistry in differentiating urothelial carcinoma from prostate adenocarcinoma and squamous cell carcinomas of the uterine cervix, anus, and lung Neural crest progenitors and stem cells Expression of the urothelial differentiation markers GATA3 and placental S100 (S100P) in female genital tract transitional cell proliferations Temporally regulated neural crest transcription factors distinguish neuroectodermal tumors of varying malignancy and differentiation Selective immunohistochemical markers to distinguish between metastatic high-grade urothelial carcinoma and primary poorly differentiated invasive squamous cell carcinoma of the lung Placental S100 (S100P) and GATA3: markers for transitional epithelium and urothelial carcinoma discovered by complementary DNA microarray Induction of the neural crest and the opportunities of life on the edge Diagnostic utility of SOX10 to distinguish malignant peripheral nerve sheath tumor from synovial sarcoma, including intraneural synovial sarcoma Sorting out Sox10 functions in neural crest development Unknown primary/undifferentiated neoplasm Immunohistochemical evaluation of GATA-3 expression in ER-negative breast carcinomas Immunohistochemical evaluation of GATA3 expression in tumors and normal tissues: a useful immunomarker for breast and urothelial carcinomas Sox10-a marker for not only schwannian and melanocytic neoplasms but also myoepithelial cell tumors of soft tissue: a systematic analysis of 5134 tumors Sox10: a pan-schwannian and melanocytic marker Value of GATA3 immunostaining in tumor diagnosis: a review Value of melanocytic-associated immunohistochemical markers in the diagnosis of malignant melanoma: a review and update Value of SOX10 immunostaining in tumor diagnosis Survival and glial fate acquisition of neural crest cells are regulated by an interplay between the transcription factor Sox10 and extrinsic combinatorial signaling Urinary bladder and urachus The application of immunohistochemical biomarkers in urologic surgical pathology Rabbit polyclonal anti-SOX10 is a reliable IHC marker for melanoma and its mimics Handbook of practical immunohistochemistry: frequently asked questions Lymphoepithelioma-like carcinoma Sarcomatoid carcinoma Clinical features 1. 70% of all cases 2. Most common in sixth decade 3. Men slightly more than women 4. <5% associated with myasthenia gravis cartilage, spicules of bone, patches of pigmented tissue, or brain tissue may be discernible; an admixture of ectoderm, endoderm, and mesoderm is seen, assembled in either a disorganized or organized pattern. Learning Objectives 1. To describe the cytological features of this entity 2. To become aware of its cytologic atypia, a potential mimic of malignancy 3. To become familiar with the utility of lab tests in the diagnosis of this entity Case A 14-year-old female with a medical history of Rett syndrome presented with respiratory illness, fatigue, and pneumonia for 2-3 months. Respiratory secretion for culture and RT-PCR and bronchial washing for cytology and differential cell count were received. The bronchial washing specimen revealed scattered atypical squamoid cells with large nuclei, high nuclearto-cytoplasmic ratio, and hyperchromatic, smudged chromatin with a suggestion of nuclear inclusions in background of intense acute inflammation, reactive respiratory epithelial cells, and pulmonary macrophages. The cellblock material showed similar findings. Immunohistochemically, the scattered atypical cells were decorated by CK7, but nonreactive to p40; the profile suggested that those cells are pneumocytes. Representative images are shown in Fig. 6 .38a-d.SARS, a new human emergent infectious disease mainly involving the lower respiratory tract, became a worldwide outbreak in 2003, affecting more than 8000 patients, with a fatality rate of 9.2%. A novel coronavirus (SARS-CoV), a member of a family of large, positive, single-stranded RNA viruses, was identified as the etiological agent for this disease. The key pulmonary pathology is that of DAD, featured by pronounced pulmonary edema and hyaline membrane formation. There are interstitial thickening, fibrosis, and intraalveolar exudates with granulation tissue formation, as well as sparsely inflammatory cell infiltrates, including macrophages (often multinucleated forms) and lymphocytes. Depending on different phases in the disease progression, the degree of fibrosis and the composition of inflammatory cells may vary. Other pathologic findings, such as prominent vascular injury, hemophagocytosis, squamous metaplasia, apoptosis, and atypical pneumocytes, including multinucleated giant pneumocytes with irregularly distributed nuclei or pneumocytes with large atypical nuclei, prominent eosinophilic nucleoli, and granular amphophilic cytoplasm, were reported. However, distinct viral inclusions were not apparent. Ancillary tests, such as in situ hybridization, immunohistochemistry, viral isolation, or RT-PCR are necessary to confirm the viral infection.For the index case, serology testing and culture of the respiratory secretion were positive for coronavirus OC43. Electron microscopy (EM) revealed viral-like particles in pneumocytes, but not in macrophages or other cell types of lung. The findings confirmed the viral infection.The atypical pneumocytes, metaplastic squamous cells, and apoptosis may mimic malignancy. Caution should be exercised in the interpretation of specimens from patients with a history of SARS and coronavirus infection. The awareness of clinical history and the limited amount of atypical cells should alert the pathologists to avoid overdiagnosis in this clinical setting. Learning Objectives 1. To describe the cytological features of this entity 2. To become aware of the common differential diagnoses of this tumor 3. To become familiar with the potential utility of the immunohistochemical markers in the diagnosis of this tumor An 81-year-old gentleman with no documented past history of malignancy presented with dizziness and difficulty of breathing. CT imaging of the chest revealed multiple lung nodules and lymphadenopathy. In addition, a possible brain lesion was noted on imaging. An ultrasound-guided FNA of the hilar lymph node was performed. The smears were highly cellular, composed of clusters, aggregated cell groups, and abundant single spindled to plump cells with hyperchromatic nuclei without prominent nucleoli. Some rosette-like structures were seen, as well as scattered pigment-laden macrophages. The pigments were dusky and brown granules on Pap stain and blue-black on Diff-Quik stain. Abundant necrosis was noted. Representative images of the FNA smears and cellblock material were shown in Fig. 6 .39a-d.The cytological features raise a differential diagnosis that includes neuroendocrine tumor, spindle cell tumors, including sarcomatoid carcinoma and metastatic melanoma of the spindle cell type. A limited panel of immunoassays was performed, revealing tumor cells with strong and diffuse nuclear staining for (continued)