FNA Cytology of Mediastinal Lesions. Presenters: Xiaoqi Lin, M.D., Ph.D. Ritu Nayar, M.D.

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1 Disclosure information The speakers have no relationship that represents a possible conflict of interest with respect to the content of this presentation. FNA Cytology of Mediastinal Lesions Presenters: Xiaoqi Lin, M.D., Ph.D. Ritu Nayar, M.D. Department of Pathology Northwestern Memorial Hospital Feinberg School of Medicine Northwestern University 675 North St. Clair Street Galter Pavilion 7-132F Chicago, IL Tel: xlin@northwestern.edu r-nayar@northwestern.edu November 16, th Annual Scientific Meeting of American Society of Cytopathology 1

2 FNA Cytology of Mediastinal Lesions Xiaoqi Lin, MD, PhD Ritu Nayar, MD Department of Pathology Northwestern Memorial Hospital Feinberg School of Medicine Northwestern University, Chicago, IL November 16, th Annual Scientific Meeting of American Society of Cytopathology

3 Educational Objectives 1. Use a case based approach to highlight the cytomorphology of common and uncommon mediastinal lesions and their differential diagnosis 2. Discuss diagnostic algorithms based on cytologic findings coupled with ancillary studies and clinical i l information 3. Discuss how to use ancillary studies to reach a final diagnosis

4 Mediastinum 1. Anterior mediastinum: Thymus, lymph nodes, soft tissue, vessels 2. Middle mediastinum: Heart, vessels, trachea, main bronchi, lymph nodes 3. Posterior mediastinum: Vessels, esophagus, lymph nodes, nerves

5 Case 1 59Y, M

6 CT Scan A 5.9 cm anterior mediastinal mass involving gpleura and lung

7 FNA and Touch Prep 1. Scattered single / small clusters of polygonal or round epithelial cells 2. Nuclei: Oval and round, fine chromatin, smooth nuclear membrane 3. Cytoplasm: Scant to moderate, delicate 4. Background: polymorphous lymphoid cells

8 Differential Diagnosis 1. Thymoma (B1, B2, B3) 2. Thymic hyperplasia 3. Thymic or metastatic carcinoid 4. Lymphomas 5. Metastatic carcinoma 6. Germ cell tumor 7. Thymic carcinoma

9 1. Tissue architecture is important for precise subclassification of thymic lesions 2. This case shows 1) Lobular growth pattern 2) Epithelial cells in a delicate loose network, and around perivascular spaces and serum lake 3) Lymphoid cells are present in between epithelial cells Core Biopsy

10 Cytology Diagnosis Thymoma, B2

11 Surgical Pathology Diagnosis Invasive thymoma, B2, with invasion into adjacent pleural tissue and lung parenchyma

12 Thymoma 1. WHO classification: A, AB, B1, B2 & B3 Based on: 1) Cell shape, spindle vs. polygonal/round 2) Extent of lymphocytic y infiltrate and degree of atypia 3) histologic architecture 2. Uncommon, 1-5/million 3. Age: 7-89 yrs, peak yrs 4. Sex: no gender predilection

13 Thymoma 5. Role of FNA 1) Accuracy up to 86% and positive predictive value % for diagnosis of thymic lesions 2) Addition of core biopsy helpful 6. Diagnosis by FNA 1) Characteristic dual population epithelial cells (epithelioid or spindle shaped) small lymphocytes 2) Adequate sampling crucial

14 Differential Diagnosis 1. Thymoma: 1) B1: fewer and smaller clusters of epithelial cells with less atypia and more lymphoid cells. Core bx is helpful.

15 2) B3: more and larger clusters of epithelial cells with more atypia, and much less or no lymphoid cells. core bx is helpful.

16 2. Thymic hyperplasia: Much less and smaller clusters of epithelial cells with no atypia, and more lymphoid cells, which is, sometimes, difficult to be distinguished from thymoma, B1. Follicular hyperplasia

17 3. Thymic or metastatic carcinoid: 1) Plasmacytoid cells with granular cytoplasm, abundant naked nuclei. 2) Neuroendocrine markers (CD56, synaptophysin, chromagranin, and NSE) Synaptophysin

18 6. Germ cell tumor: 1) Obvious malignant cells 2) Serum AFP and hcg 3) IHC (PLAP, OCT4, CD30, AFP, and hcg)

19 Differential Diagnosis 4. Lymphoma: IHC and ISH with lymphoid markers, and flow cytometry for clonal phenotype 5. Metastatic carcinoma: Obvious malignant cells, compared with previous cases on morphology, and IHC 7 Thymic carcinoma: Obvious malignant cells.

20 Take Home Messages 1. FNA/core bx can be used to diagnose thymoma, but, sometimes, may be difficult to subclassify the thymoma 2. FNA/core bx cannot diagnose invasion 3. IHC profile 1) Epithelial cells: positive for CKs (AE1/AE3, CAM5.2, CK5/6, CK7, CK14, CK18, CK19), CD117, and p63 negative: for CK20, EMA, CD5, CD20, CD70 2) Cortical T cells positive for CD1a, CD99, TdT, CD4, CD8, and CD5

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22 59y, M Case 2 Left upper/medial neck mass just inferior to the left lobe of the thyroid, and a 1 cm thyroid nodule History of colonic cancer 3 years ago FNA was performed on the thyroid nodule

23 CT Scan 1 cm thyroid nodule

24 FNA 1. 3 dimensional clusters of cohesive polygonal cells 2. Cytoplasm: Scant, indistinct cell borders 3. Nuclei: Round to oval, stippled and fine granular chromatin, nuclear moldings, conspicuous to prominent nucleoli 4. No acinar/gland formation 5. No intra- or extra-cellular mucin

25 Differential Diagnosis 1. Basaloid squamous cell carcinoma 2. Thymic carcinoma 3. Poorly differentiated thyroid carcinoma 4. Neuroendocrine carcinoma 5. Thyroid medullary carcinoma 6. Metastatic colonic adenocarcinoma 7. Germ cell tumor 8. Thymoma, B3

26 1. Sheets/nests of tumor cells surrounded by dense fibrous stroma 2. Tumor cells strongly positive for CK7 and weakly positive for CK20 Core Biopsy H&E CK20 CK7 CEA, mono

27 Core Biopsy Thyroglobulin, 600x Calcitonin, 600x Chromogranin, 600x NSE, 600x

28 Original Cytology Diagnosis Epithelial neoplasm Favor metastatic carcinoma originating g from the colon

29 Left Cervical Upper/Medial Mass, Excision 1. Tumor shows with a pushing, nodular growth pattern 2. IHC 1) CD1a shows absence of immature T cells in the lymphoid component 2) CD5 shows presence of mature T cells in the lymphoid component 3) Tumor cells are positive for CD5 H&E CD1a Mono CEA CD5

30 Surgical Pathology Diagnosis Left Cervical Upper/Medial Mass, Excision Tumor cells are positive for CD5 Consistent with thymic carcinoma

31 Thymic Carcinoma 1. Rare 2. The WHO classification: 10 subtypes 1) Squamous cell carcinoma, most frequent 2) Basaloid carcinoma 3) Mucoepidermoid carcinoma 4) Lymphoepithelioma-like p carcinoma 5) Sarcomatoid carcinoma 6) Clear cell carcinoma 7) Papillary adenocarcinoma 8) Non-papillary adenocarcinoma 9) Carcinoma with t(15;19) 10) Undifferentiated carcinoma

32 Thymic Carcinoma 3. FNA / touch prep: obvious malignant tumor cells with marked atypia, pleomorphism, prominent nucleoli, numerous and/or bizarre mitotic figures, lack of dual cell populations except for the lymphoepithelioma-like p carcinoma 4. Cytomorphology of tumors varies among the subtypes and is similar to their counterpart in other organs

33 Differential Diagnosis 1. Basaloid squamous cell carcinoma: Peripheral palisading 6. Germ cell tumors: 1) Less cohesive 2) Increase serum AFP or hcg 3) Positive for PLAP, OCT4, CD20, AFP, and/or hcg 7. Thymoma, B3: Minimal atypia

34 2. Poorly differentiated thyroid carcinoma: 1) Nuclear inclusions and grooves 2) Positive for thyroglobulin and TTF-1

35 3. Neuroendocrine carcinoma: 1) More discohesive cells with more cytoplasm, some with granules 2) Positive for neuroendocrine markers

36 4. Thyroid medullary carcinoma: 1) More pleomorphic and discohesive 2) Amyloid 3) Positive for calcitonin, neuroendocrine markers, and TTF-1. Calcitonin

37 5 Metastatic colonic adenocarcinoma: 1) Columnar or cuboidal cells forming glandular architectures and palisading arrangement, intra- and extra-cellular mucin 2) Positive for CK20 and CDX-2. CDX-2

38 Take Home Messages 1. FNA / core biopsy useful in diagnosis of thymic carcinoma 2. A metastatic carcinoma should be excluded by comparison with the primary malignancy, clinical presentations, imaging studies, IHC and molecular studies 3. A second and even 3 rd or 4 th primary should be considered for unusual clinical presentations 4. IHC: positivity for CD5, CD70, and CD117 (c-kit) are useful for diagnosis of thymic carcinoma

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40 87y, F Case 3 5 cm pleural based mass, mediastinal mass, and pleural effusion Hx of uterine and colonic carcinoma

41 FNA Single, nests or anatomosing flat sheets of polygonal epithelioid cells Cytoplasm: scant to moderate, dense, welldefined cell borders, intercellular window Nuclei: round or oval, and prominent nucleoli

42 Differential Diagnosis 1. Mesothelioma 2. Metastatic adenocarcinoma 3. Pulmonary adenocarcinoma 4. Non-keratinizing squamous cell carcinoma 5. Thymic carcinoma 6. Benign mesothelial cells from needle track

43 Calretinin Core Biopsy Throbomodulin CK5/6 Negative for CK7, CK20, TTF-1, BRST-2, ER, monoclonal CEA, and mucicarmine

44 Cytology Diagnosis Mesothelioma

45 Mesothelioma 1. Usually in Adults, M/F > 9:1 2. Strongly associated with asbestos exposure and less strongly associated with SV40 3. Pleural l effusions in four fifth 4. Subtypes: 1) Diffuse malignant mesothelioma (epithelioid, sarcomatoid, desmoplastic, and biphasic mesothelioma) 2) Well differentiated papillary mesothelioma 3) Localized malignant mesothelioma 4) Adenomatoid tumor

46 Differential Diagnosis 2. Metastatic adenocarcinoma: 1) Columnar and cuboidal cells with glandular formation, delicate or vacuolated cytoplasm and with no clear cytoplasmic borders 2) Colonic adenocarcinoma is positive for CK20 and CDX-2, and uterine endometrioid adenocarcinoma is positive for ER and Vimentin ER Vimentin

47 3. Pulmonary adenocarcinoma: 1) Glandular formation, more delicate cytoplasm 2) Positive for TTF-1. Negative for thrombomodulin, CK5/6 and calretinin. TTF-1

48 4. Non-keratinizing squamous cell carcinoma: 1) 3 dimensional clusters, and no intercellular window 2) Positive for p63 and CK5/6 Negative for calretinin, WT-1, and D2-40 p63

49 Differential Diagnosis 5. Thymic carcinoma: 1) 3 dimensional clusters, and no intercellular windows 2) Positive for p63, and CD5 Negative for calretinin and WT Benign mesothelial cells from needle track: 1) Flat cohesive nests/sheets. t Similar il morphology with reactive atypia. Sometimes, es, impossible.

50 Take Home Messages 1. Recognize the cytomorphology of subtypes of mesothelioma 2. IHC profiles of mesothelioma: positive for CK7, CK19, CK5/6, WT-1, calretinin, thrombomoculin, D Electron microscopy

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52 33y, M Case 4 A 5 cm anterior mediastinal mass with necrosis and multiple bilateral lung nodules on CT

53 FNA Discohesive polygonal or spindle cells Cytoplasm: dense, orange or green color Nuclei: round or oval, irregular nuclear contour, prominent nucleoli Extensive necrosis

54 Differential Diagnosis 1. Thymic carcinoma 2. Squamous cell carcinoma 3. Mesothelioma 4. Undifferentiated carcinoma/nut midline carcinoma 5. High grade adenocarcinoma 6. Choriocarcinoma/germ cell tumor

55 Core biopsy p63

56 FNA Diagnosis High grade carcinoma favor thymic carcinoma

57 Lung biopsy Sheets of cytotrophoblasts admixed with multinucleated syncytiotrophoblasts Necrosis and hemorrhage Positive for AE1/AE3 and hcg Negative for CD30 and glypican-3 hcg

58 Surgical Diagnosis Metastatic Choriocarcinoma

59 Choriocarcinoma i 1. Highly malignant neoplasm with trophoblastic differentiation (syncytiotrophoblast, cytotrophoblast and variably intermediate trophoblast cells) 2. Rare, 2.5 5% of mediastinal germ cell tumors to 63y, most commonly the 3 rd decade

60 Choriocarcinoma 4. Both anterior and posterior o mediastinum, and early hematogeneous metastasis 5. FNA/needle core biopsy can be used for diagnosis 6. IHC: positive for hcg, p63, cytokeratins, PLAP, CEA, EMA, hpl, EGFR, inhibin, PLAP, and pregnancy-specific betta-1- glycoprotein

61 Differential Diagnosis 1. Thymic carcinoma: 1) Squamous differentiation 2) positive for CD5, and negative for hcg. 3. Mesothelioma: 1) No squamous differentiation 2) Negative for p63 and CD5

62 2 Squamous cell carcinoma: 1) Squamous differentiation 2) Negative for CD5 and hcg Squamous cell carcinoma arising from bronchogenic cyst

63 Differential Diagnosis 5. Undifferentiated carcinoma/nut midline carcinoma: 1) Similar cytomorphology 2) Possibly positive for p63. Negative for hcg 3) Possibly NUT gene rearrangement 6. High grade adenocarcinoma: 1) Cuboidal or columnar cells with possible glandular formation 2) Negative for p63

64 Take home messages 1. Syncytiotrophoblasts may not be seen in FNA/core biopsy of choriocarcinoma 2. If a high grade carcinoma positive for p63, choriocarcinoma should be included in the differential diagnosis, although low incidence 3. p63 positivity is not only seen in squamous cell carcinoma, but also seen in thymoma/thymic carcinoma, poorly differentiated / undifferentiated carcinoma, NUT midline carcinoma, urothelial carcinoma, choriocarcinoma and some trophoblastic neoplasms

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66 42y, M Case 5 Large right pleural effusion, extensive bulky mediastinal lymphadenopathy and a 9.2 x 6.1 cm subcarinal mass Laboratory tests: Elevated AFP (545.4u increased to u 5 days later) and HCG (3.3u)

67 Pleural Fluid Non-cohesive monotonous round cells, approximately 4X the size of a mature lymphocyte Cytoplasm: Scant to moderate, delicate or vacuolated Nuclei: Round, 1 or 2 prominent nucleoli, granular or vesicular chromatin, and minimal nuclear membrane irregularity Frequent mitoses and apoptoses

68 FNA of Subcarinal Mass

69 Differential Diagnosis 1. Germ cell tumor 2. Undifferentiated carcinoma / poorly differentiate carcinoma 3. Thymic carcinoma 4. Sinonasal undifferentiated carcinoma / sinonasal carcinoma 5. Lymphoma 6. PNET 7. Melanoma 8. Mesothelial cells 9. NUT midline carcinoma

70 Flow Cytometry Few lymphoid cells, negative for T or B cell clonality

71 AE1/AE3 CD138 p63 CD99 Vimentin Negative for T and B cell markers

72 Preliminary Pleural Fluid Diagnosis Undifferentiated i d carcinoma

73 IHC NUT FISH BRD3-NUT

74 Final Cytology Diagnosis NUT midline carcinoma with BRD3-NUT rearrangement

75 NUT midline carcinoma 1. High grade carcinoma with t(15;19)(q14;p13.1) - BRD4-NUT and t(9;15) - BRD3-NUT 2. Rare, mostly occurs in young patients ranging from newborn to 78 years old (mean 23 years) 3. Many organs, more frequent involvement of the midline organs 4. Show an undifferentiated carcinoma with occasional abrupt keratinizing squamous, glandular and mesenchymal chondroid differentiation 5. Early metastasis, generally refractory to chemotherapy and radiation, and poor prognosis with survivals measured in months

76 Differential Diagnosis 1. Germ cell tumor: 1) Elevated AFP and hcg 2) May show similar cytomorphology 3) Positive for AFP, hcg, PLAP, CD30, hpl or CD117 Seminoma focally, weakly positive for NUT. 3. Thymic carcinoma: 1) More cohesive clusters, more squamous differentiation 2) Positive for CD5 and CD117 besides p63 5. Lymphoma: 1) May show similar monotonous non-cohesive cells with scant cytoplasm 2) Positive for T or B cell markers 9. Mesothelial cells: 1) More dense cytoplasm, intercellular windows 2) Positive for WT-1, calretinin, CK7, thrombomodulin, CK5/6

77 2. Undifferentiated carcinoma / poorly differentiate t carcinoma: 1) May show similar cytomorphology 2) May be positive for p63 3) Negative for NUT expression and NUT gene rearrangement Negative for NUT expression and NUT gene rearrangement p63

78 4. Sinonasal undifferentiated carcinoma / sinonasal carcinoma: 1) May show similar cytomorphology 2) Location: More restricted area 3) Positive for EBV besides focal or no p63

79 6. PNET: 1) May show similar cytomorphology 2) Positive for neuroendocrine markers besides CD99 Vimentin CD56 Synaptophysin

80 7. Melanoma: 1) May show similar cytomorphology 2) Positive for S100, HMB-45 and Melan A HMB-45 Melan A

81 Take Home Messages 1. NUT midline carcinoma is an undifferentiated carcinoma with NUT gene rearrangement, common involvement of midline organs, generally resistant to chemotherapy and radiation therapy, and very poor prognosis. 2. We advocate performing IHC for NUT and FISH for NUT gene rearrangement for any poorly differentiated or undifferentiated carcinoma when evaluating body cavity fluid on cytology, especially if the tumor involves midline structures, shows little pleomorphism, and expresses p63 and/or CD34

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83 28y, M Case 6 A 10 cm anterior mediastinal mass

84 FNA/TP Single or loosely cohesive clusters of round or oval cells Cytoplasm: Scant to moderate, light blue Nuclei: Round or oval, irregular nuclear contours, prominent nucleoli Lymphoglandular bodies

85 Differential Diagnosis 1. Large cell lymphoma 2. Anaplastic large T-cell lymphoma 3. Precursor lymphoblastic lymphoma/leukemia 4. Thymoma 5. Germ cell tumor 6. Poorly differentiated carcinoma 7. Thymic hyperplasia

86 Flow Cytometry 1. Predominant T cell (CD3+, CD5+) population 2. Too few CD19+ B cells for immunophenotypic / clonality analysis

87 C AS th 58 al nu An c ifi nt ie Sc CD20 CD3 BCL6 tin g ee M

88 Cytology Diagnosis Mediastinal large B-cell lymphoma

89 Primary mediastinal large B-cell lymphoma 1. Young adult (3 rd and 4th), a slight female predominance 2. Commonly yproduces superior vena cava syndrome 3. FNA cytology is useful to make diagnosis with a sensitivity of 42% 4. Positive for CD19, CD20, CD22, CD79a and BCL6 (56-60%), and infrequently for CD10 (20-25%) and immunoglobulin. CD30 stain is weak and focal

90 Differential Diagnosis 2. Anaplastic large T-cell lymphoma: 1) Discohesive large pleomorphic cells with round or indented hyperchromatic nuclei, prominent nucleoli, vacuolated cytoplasm 2) Positive for CD30, ALK-1, CD2, CD4 and CD3 4. Thymoma: cohesive and single epithelial l cells, and lymphoid cells. Positive for cytokeratins.

91 3. Precursor lymphoblastic lymphoma/leukemia: 1) Intermediate size lymphoid cells with round or convoluted nuclei with central nucleoli and fine chromatin 2) Positive for TdT. TdT

92 5. Germ cell tumor: 1) Cohesive groups or single cells, prominent nucleoli, tigeroid background 2) Positive for PLAP, CD117, CD34, AFP, hcg, or CD30 6. Poorly differentiated/undifferentiated carcinoma: 1) Large, pleomorphic epithelial l cells with scant or moderate cytoplasm, enlarge, pleomorphic and hyperchromatic nuclei with prominent nucleoli and irregular nuclear membrane 2) Positive for cytokeratins 7. Thymic hyperplasia: 1) Polymorphous lymphoid cells and lymphohistocytic aggregates (germinal centers), tingible macrophages, and immunoblasts.

93 Tk Take Home Messages 1. Flow cytometry may not be useful to detect monoclonal proliferation for mediastinal large cell lymphoma 2. Immunohistochemistry h i t should be used for diagnosis 3. May be positive for CD30, a marker for anaplastic T cell lymphoma

94 Differential Diagnosis for Mediastinal Epithelial Neoplasms Epithelioid id Epithelial l Cells Without Lymphoid cells With Lymphoid Cells Atypia No/Mild Atypia Atypia No/Mild Atypia Thymic carcinoma Thymoma B3 Lymphoepithelial- Thymoma B1, B2 Squamous C Ca Basaloid Ca NET Typical carcinoid like Ca Seminoma Clear Ca Papillary adenoca Non-papillary adenoca Undifferentiated Ca NUT midline carcinoma NET Large C NEC Small C Ca GCT Embryonal Ca Yolk sac tumor Choriocarcinoma Mesothelioma Atypical carcinoid

95 Differential Diagnosis for Mediastinal Epithelial Neoplasms Atypia Spindle Epithelial Cells Sarcomatoid carcinoma Squamous cell carcinoma Sarcomatoid mesothelioma No/Mild Atypia Thymoma A, AB NET Typical carcinoid Atypical carcinoid Desmoplastic mesothelioma

96 Thank You For Your Attention

97 Educational Objective: 1. Use a case based approach to highlight the cytomorphology of common and uncommon mediastinal lesions and their differential diagnosis 2. Discuss the diagnostic algorithms based on cytologic findings coupled with ancillary studies and clinical information 3. Discuss how to use ancillary studies to reach a final diagnosis Abstract We will present 6 diagnostically challenging cases with clinical findings, cytomorphology, ancillary studies and surgical pathology/clinical follow up. The cases will cover thymoma, thymic carcinoma, mesothelioma, lymphoma, germ cell tumors, recently described rare NUT midline carcinoma, and their differential diagnoses. The diagnostic considerations based on FNA and touch prep cytomorphology will be discussed. Current ancillary studies useful in reaching a final diagnosis will also be presented. We will use the following materials in a case based PowerPoint presentation: clinical information, FNA smears/touch preparations (Diff. Quik and Pap stained), H&E stained cell blocks /core biopsies, ancillary studies, and pertinent literature reviews as applicable. Novel Aspects of the Symposium and Assessment of Need: 1. Bring to attention rare or known entities with unusual presentations. 2. Discuss the challenges in making a diagnosis of Nut midline carcinoma. 3. Discuss up to date ancillary studies. 2

98 Introduction Mediastinum is divided into anterior, middle and posterior mediastinum. The anterior mediastinum is composed of thymus, lymph nodes, soft tissue, and vessels. Middle mediastinum is composed of heart, vessels, trachea, main bronchi, and lymph nodes. Posterior mediastinum is composed of vessels, esophagus, lymph nodes, and nerves. Corresponding neoplasms can arise from these normal tissues, e.g., thymic neoplasms (thymoma, thymic carcinoma, thymic neuroendocrine tumors, combined thymic epithelial tumors, etc.), primary germ cell tumors of the mediastinum, primary lymphomas of the mediastinum, myeloid sarcoma, extramedullary acute myeloid leukemia, soft tissue tumors of the mediastinum, ectopic thyroid tumors, ectopic parathyroid tumors, etc. Metastatic malignancies to the mediastinum and neoplasms extending directly from adjacent organs or tissues, especially from lung, are also frequently seen. Case 1 Clinical history: A 59 year-old male presented with a 5.9 x 4.3 cm anterior mediastinal soft tissue mass with a small focus of calcification and involvement of pleura and lung as identified by CT scan. FNA findings: Single or small clusters of polygonal epithelial cells with round or oval nuclei, conspicuous nucleoli, moderate pale cytoplasm, and indistinct cell borders in a background of numerous polymorphous lymphocytes (predominately small lymphocytes). Cytology differential diagnosis: Thymoma (B1, B2 and B3), thymic hyperplasia, thymic carcinoid, thymic carcinoma, lymphomas (small lymphocytic, follicular, MALT, lymphoblastic and Hodgkin s lymphoma), metastatic carcinoma, and germ cell tumor. Core findings: Lobular growth pattern of polygonal epithelial cells with delicate loose network, perivascular space with prominent palisading of large tumor cells, and predominately small lymphocytes. Cytology diagnosis: Thymoma, B2. Surgical pathology diagnosis: b n Invasive thymoma, B2, with invasion into adjacent pleural tissue and lung parenchyma. Discussion: Differential diagnosis for anterior mediastinal mass includes thymoma, thymic carcinoma, thymic hyperplasia, lymphoma, germ cell tumor, endocrine neoplasm, metastatic carcinoma, and so on. 3

99 Thymoma and thymic carcinoma are uncommon with an annual incidence of 1 5/million, age ranges from 7 89 years, peak age of years, and no sex predilection 1. Thymoma accounts for < 1% of human neoplasms 2. WHO classifies thymoma into type A, AB, B1, B2 and B3 based on cell shape (spindle versus polygonal/round epithelial cells), extent of lymphocytic infiltrate, degree of atypia of epithelial cells, and histologic architecture. FNA has variable accuracy rates in diagnosing thymoma (86% in one report) 3-4 and variable positive predictive value (from 69% to 100%) 3, 5. The characteristic feature is presence of dual population, epithelial cells (epithelioid or spindle in shape) and small lymphocytes 3-4. FNA can be used in diagnosis of thymic neoplasms 2-4. FNA of the type B2 thymoma shows small discohesive clusters of single large polygonal epithelial cells surrounded by small lymphoid cells. The cytoplasm varies from scant to moderate in amount, and from delicate to dense, usually with indistinct cell borders 2, 6. The nuclei are round or oval, and bland with smooth or slightly irregular contour, fine and evenly dispersed granular chromatin, and frequently small nucleoli 2, 7. Mitoses are rare. FNA cytology of the type B1 thymoma shows scattered small discohesive clusters or single polygonal epithelial cells surrounded by numerous small lymphoid cells on the slides. The epithelial cell number is much less than type B2 thymoma. Thymoma B1 can be extremely difficult to distinguish from thymic hyperplasia based on cytology findings, and separation of thymoma B1 from thymic hyperplasia may need histologic architecture. FNA cytology of thymoma B3 shows much less lymphocytes and consists of predominately epithelial cells, especially large clusters of epithelial cells. The epithelial cells of thymoma B3 are more atypical than B1 and B2. Although WHO classification claims that there is a definite histologic difference between thymoma B1 and B2 with the former having smaller epithelial cells with smaller nuclei and smaller nucleoli 1, 8-9, there is not a distinction that anyone has reported in FNA preparations 2. In addition, it is not infrequent that mixture of thymoma subtypes may be present in the same mass. 10 Therefore, an author suggested it would be unwise to attempt to subclassify thymoma on FNA biopsies that sample only a small fraction of the tumor. However, by using needle core biopsy, we subclassified a few cases of thymomas that correlated with the subclassification on the excised surgical specimens. In our cases, needle core biopsy did not help in determining invasion. Identification of epithelial cells in the FNA smears is the key to distinguish thymoma, especially type B1 and B2, from lymphoproliferative lesions, such as reactive lymph nodes, thymic lymphoid hyperplasia, non-hodgkin lymphoma, and Hodgkin lymphoma. Sometimes, the dendritic cells in the germinal centers and epithelioid cells in the granuloma are easy to misinterpret as epithelial cells that appear in the thymoma. Cytokeratin stains (CAM5.2, CK19 and AE1/3) performed on cell block, FNA smear and needle core are helpful to identify the epithelial cells in thymoma. CD21 stain is helpful to identify dendritic cells in the germinal centers of reactive lymph node and lymphoid follicles in thymic hyperplasia. CD68 and CD163 stains are helpful to identify epithelioid histiocytes in the granuloma. 4

100 The lymphoid cells in thymoma are polymorphous and predominately small lymphocytes, which may be misinterpreted as monotonous population. If suspicious for a non-hodgkin lymphoma, a portion of sample should be sent to flow cytometry and immunohistochemical stains should be performed on cell block or needle cores. In addition Reed-Sternberg cells and Reed-Sternberg variants should be carefully searched for to exclude Hodgkin lymphoma. Thymic hyperplasia is divided into two subtypes, true thymic hyperplasia and follicular hyperplasia. True thymic hyperplasia consists of thymic tissue with normal architecture, but increases in the weight and size of thymic tissue including size of individual cortex and medullar. Follicular hyperplasia consists of proliferation of lymphoid cells with formation of germinal centers. FNA cytology of true thymic hyperplasia shows much less and smaller clusters of epithelial cells with no atypia, and more lymphoid cells, which is difficult to be separated from B1 thymoma on FNA cytology only and often needs histologic architecture to separate it from B1 thymoma. FNA cytology of thymic or metastatic carcinoid may show single or clusters of plasmacytoid cells with acinar, core or glandular formation, granular cytoplasm, and abundant naked nuclei. Carcinoid is positive for neuroendocrine markers (CD56, synaptophysin, chromagranin, and NSE) and negative for thymoma markers (CD117 and p63). FNA cytology of metastatic carcinoma shows obvious malignant cells. Comparison with morphology of patient s previous cases as well as IHC studies is helpful to distinguish it from thymoma. FNA cytology of germ cell tumors shows obvious malignant cells with cytomorphology depending on subtypes. Increased serum AFP and hcg levels are seen in germ cell tumors, and germ cell rumors are variably positive for PLAP, OCT4, CD30, AFP, and hcg depending on subtypes and negative for thymoma markers. FNA cytology of thymic carcinoma shows obvious malignant cells. Therefore, by recognition of cytomorphology, comparison with the patient s previous pathology morphology, and ancillary studies, thymoma can be diagnosed by FNA and/or core biopsy. Take home messages: 1. FNA and core biopsy can accurately diagnose thymoma, but it is, sometimes, difficult to subclassify the thymoma, especially if sampling is not adequate and core biopsy is not available. 2. IHC: epithelial cells are positive for CK (AE1/AE3, CAM5.2, CK5/6, CK7, CK14, CK18, CK19), CD117 and p63, while are negative for CK20, EMA, CD5, CD20, and CD70. Cortical T cells are positive for CD1a, CD4, CD8, CD5, CD99 and TdT. 3. FNA and needle core biopsy are not ideal tools to diagnose invasion (invasive thymoma). Case 2 Clinical history: A 59 years old male presented with a left upper/medial neck mass located just inferior to the left lobe of the thyroid, and a 1 cm thyroid nodule. He had a history of colon cancer diagnosed and treated 3 years ago. FNA biopsy was performed on the thyroid nodule. FNA findings: Three dimensional clusters of cohesive polygonal tumor cells with scant delicate cytoplasm, indistinct cytoplasmic borders, and round to oval nuclei (varying in size) with stippled and fine 5

101 granular chromatin. Nuclear molding was seen. Some cells had conspicuous nucleoli. No evidences of intracellular and extracellular mucin and glandular formation was seen. Cytology differential diagnosis: Neuroendocrine carcinoma, thymic carcinoma, thymoma, basaloid squamous cell carcinoma, metastatic colonic adenocarcinoma per patient s history of colonic cancer 3 years ago, poorly differentiated thyroid carcinoma, thyroid medullary carcinoma, and germ cell tumor. Core biopsy findings: Sheets of tumor cells with round nuclei, scant delicate cytoplasm, and indistinct cell borders. Tumor cells are surrounded by dense fibroconnective tissue. Foci of lymphoid cell aggregates. Tumor cells are focally positive for CK7 and CK20, and negative for monoclonal CEA, thyroglobulin, Calcitonin, NSE, and chromagranin. Cytology diagnosis: Consistent with an epithelial neoplasm, favor metastatic carcinoma from the colon. Surgical pathology findings: Excision of the mass showed histologic features that were different from previous colonic carcinoma. Precious colonic carcinoma was a moderately differentiated mucinous adenocarcinoma. The current tumor is heterogeneous, consisting of nests and trabeculae of tumor cells associated with a mononuclear cell infiltrate and separated by dense collagenous tissue. The tumor overall had a pushing, nodular growth pattern. The tumor cells grow in a syncytial pattern with multifocal squamous metaplasia and focal microcyst formation, and spindle cell morphology. Individual tumor cells have vesicular nuclear chromatin with prominent nucleoli and extensive apoptosis. IHC stains to Calcitonin and thyroglobulin are negative, ruling out a medullary carcinoma or an unusual primary thyroid carcinoma, respectively. A stain to CK7 shows strong staining of many tumor cells at the periphery of the tumor nests and a stain to CK20 shows significant staining of many tumor cells. Tumor cells are also positive for CEA. The results would be unusual for lung (expected CK7 positive and CK20 negative), colon (expected CK7 negative and CK20 positive) metastasis, or thymic carcinoma (expected CK7 positive and CK20 negative). IHC stain to CD5 shows staining of many tumor cells and most of the associated mononuclear cells. A stain to CD1a shows staining of many of the tumor associated mononuclear cells with dendritic processes. The majority of lymphoid cells are small lymphocytes, most of which are positive for CD5 and negative for CD1a. In summary, considering the location of the tumor, the overall growth pattern, heterogeneous morphological features, and the IHC profile (particularly the CD5 immunopositivity), a diagnosis of the primary thymic carcinoma is favored. Surgical pathology diagnosis: Consistent with thymic carcinoma. Discussion: Thymic carcinoma is rare. The WHO classifies it into 10 subtypes, squamous cell carcinoma, basaloid carcinoma, mucoepidermoid carcinoma, lymphoepithelioma-like carcinoma, sarcomatoid carcinoma, clear cell carcinoma, papillary adenocarcinoma, non-papillary 6

102 adenocarcinoma, carcinoma with t(15;19) translocation, and undifferentiated carcinoma 11. Squamous cell carcinoma is the most frequent subtype 12. FNA cytology of thymic carcinoma shows obvious malignant tumor cells with markedly atypia, pleomorphism, prominent nucleoli, and numerous or bizarre mitotic figures. Except for the lymphoepithelioma-like carcinoma, thymic carcinomas usually lack the dual cell population, a characteristic of other types of thymomas. Cytomorphology of tumors varies among the subtypes of thymic carcinomas and is similar to their counterpart in other organs. Necrosis may be seen. Cytology correctly diagnosed the thyroid nodule as a metastatic carcinoma, but misinterpreted the origin. A second and even 3 rd primary is not uncommonly seen clinically. There were three main causes resulting in the misinterpretation of the origin of the malignancy in cytology. First, the morphology of primary colonic adenocarcinoma is not compared. The primary colonic adenocarcinoma was a moderately differentiated mucinous adenocarcinoma. In contrast, the FNA smears and core biopsy show 3 dimensional clusters or sheets of cohesive polygonal tumor cells with scant delicate cytoplasm, indistinct cytoplasmic borders and conspicuous nucleoli, no evidence of any intracellular and extracellular mucin and glandular formation, which are different from the primary colonic mucinous adenocarcinoma and also not typical for a colonic adenocarcinoma. Therefore, the cytomorphology of the colonic adenocarcinoma and thyroid nodule are different. Secondly, the patient also had a large left cervical upper/medial cervical mass (7.0 x 4.5 x 4.0 cm), a second primary from thyroid, parathyroid, thymus or lung should have been considered. We excluded thyroid and lung primary in cytology by immunostaining. The cytomorphology and IHC profile also exclude the possibility of a parathyroid neoplasm, in which FNA would show follicular, papillary, sheets/nests, acinar, and single cell patterns, clear, vacuolated or granular cytoplasm, and positivity for chromagranin. Thirdly, interpretation of IHC stains should have been done more critically. The thyroid nodule is positive for both CK7 and CK20, which is not a common IHC profile for colonic adenocarcinoma, pulmonary carcinoma and thymic carcinoma. A panel of IHC stains for p63, CD5, and CK5/6 plus CK7 and CK20 are helpful in distinguish thymic carcinoma from metastatic colonic adenocarcinoma, pulmonary adenocarcinoma and thyroid carcinoma. Peripheral palisading pattern of basaloid squamous cell carcinoma is not present. FNA cytology of poorly differentiated thyroid carcinoma may show nuclear inclusions, grooves and powdering chromatin. Thyroid carcinoma is positive for thyroglobulin and TTF-1. FNA cytology of neuroendocrine carcinoma shows more discohesive cells with more cytoplasm than our case, some with granules. Neuroendocrine carcinoma is positive for neuroendocrine markers, while negative for thymic carcinoma markers (p63, CD117 and CD5). FNA cytology of thyroid medullary carcinoma shows more pleomorphic and amyloid. It is positive for calcitonin, neuroendocrine markers and TTF-1, while negative for thymic carcinoma markers. FNA cytology of metastatic colonic adenocarcinoma shows columnar or cuboidal cells forming glandular architectures and palisading arrangement, intra- and extra-cellular mucin and extensive necrosis. It is positive for CK20 and CDX-2, while negative for thymic carcinoma markers. Increased AFP and hcg levels are seen in germ cell tumors. Germ cell tumors are variably positive for PLAP, OCT4, CD20, AFP, or hcg depending on subtypes. FNA cytology of thymoma B3 shows minimal atypia. These cytology features and IHC profile as well as comparison with available patient s previous pathology examination are helpful to distinguish them from thymic carcinoma. 7

103 Take home messages: 1. FNA and core biopsy can be used in diagnosis of thymic carcinoma. 2. A metastatic carcinoma from other organs must be excluded by comparison of cytomorphology and histology with the primary malignancy, clinical presentations, imaging studies, IHC and molecular studies. 3. A secondary and even 3 rd or 4 th primary should be considered for an unusual clinical presentation. 4. IHC: positivity for CD5, CD70, and CD117 (c-kit) are useful for diagnosis of thymic carcinoma Case 3 Clinical history: An 87 years old female presented with a 5 cm pleural based mass and mediastinal mass and pleural effusion. She had a history of uterine and colonic carcinoma, FNA findings: Single, nests or anatomosing flat sheets of polygonal epithelioid cells with scant to moderate slightly dense cytoplasm, well-defined cell borders, intercellular window, round or oval nuclei, and prominent nucleoli. Cytology differential diagnosis: Mesothelioma, metastatic adenocarcinoma, pulmonary carcinoma, non-keratinizing squamous cell carcinoma, and thymic carcinoma. Core biopsy findings: Infiltrating epithelioid cells with the same cells as described in FNA findings. The tumor cells are positive for thrombomodulin, calretinin, CK5/6, while are negative for CK7, CK20, TTF-1, BRST-2, ER, monoclonal CEA, and mucicarmine. Cytology diagnosis: Mesothelioma. Discussion Mesothelioma is a malignant tumor arising from mesothelial cells 13. It is subclassified into diffuse malignant mesothelioma (epithelioid, sarcomatoid, desmoplastic, and biphasic mesothelioma), well differentiated papillary mesothelioma, localized malignant mesothelioma and adenomatoid tumor. Mesothelioma usually happens in adults, but occasionally in children or even infants. Male/female ratio is > 9:1 13. Mesothelioma is strongly associated with asbestos exposure and less strongly associated with SV40. Pleural effusions are found in four fifth 14. FNA cytology of diffuse mesothelioma depends on the subtypes. Epithelioid mesothelioma 8

104 1. Characteristically presence of single, loosely cohesive to cohesive sheets, papillae, balls, or glandular clusters of bland to malignant-appearing epithelioid cells often with mesothelial features. The cells are round or polygonal. The cytoplasm varies from scant to abundant and from dense and squamoid to delicate and vacuolated, and has relatively well-defined cell borders. Windows are often seen. The nuclei are centrally or eccentrically located, and round to oval, and have fine to coarse chromatin and single or multiple, small to prominent nucleoli. Binucleation or multinucleation is common. 2. Psammoma bodies and asbestos bodies can be seen Differential diagnoses include reactive mesothelial cell hyperplasia, adenocarcinoma, large cell carcinoma, and melanoma. 4. Mesothelioma is positive for CK5/6, WT-1, calretinin, thrombomodulin, D2-40, and N- cadherin. Electron microscopy shows long, slender microvilli. Mesothelioma is mucicarmine negative. Sarcomatoid Mesothelioma 1. Characteristically presence of single or loose clusters of spindle cells. The spindle cells have a moderate amount of delicate cytoplasm. The nuclei are relatively large, oval to spindle, and variably pleomorphic, and often have coarse, hyperchromatic chromatin with small nucleoli. Naked nuclei may be conspicuous 15. Mitoses and necrosis are commonly seen. 2. The differential diagnoses include chronic pleuritis with reactive inflammatory fibrosis, primary and metastatic sarcomas, spindle cell melanoma or squamous cell carcinoma, and sarcomatoid carcinoma of the lung. 3. Sarcomatoid mesothelioma cells are positive for cytokeratins, vimentin, actin, desmin, S- 100 and calretinin. Desmoplastic mesothelioma 1. Often presence of low cellularity of atypical spindle cells, which may be inadequate for evaluation. 2. The differential diagnoses include those as sarcomatoid mesothelioma, as well as pleural plaque. 3. Clinical correlation and image studies as well as IHC stains will be helpful in differential diagnosis. Biphasic malignant mesothelioma 1. Contain both epithelioid and sarcomatoid patterns. Each component is present at least 10%. Account for 30% of malignant mesotheliomas. 2. IHC stains and electron microscopy may be helpful in differential diagnoses. FNA cytology of metastatic uterine endometrioid adenocarcinoma and colonic adenocarcinoma characteristically shows columnar and cuboidal cells with glandular formation, and delicate or vacuolated cytoplasm. Tumor cells are positive for ER and vimentin, or CK20 and CDX-2. FNA cytology of pulmonary adenocarcinoma should show more glandular formation, and more delicate or vacuolated cytoplasm with no clear cytoplasmic border. The tumor cells are positive for TTF-1, while are negative for thrombomodulin, CK5/6 and calretinin. FNA cytology of nonkeratinizing squamous cell carcinoma shows more 3 dimensional clusters, and no intercellular 9

105 windows. Tumor cells are positive for p63, and negative for calretinin, WT-1, and D2-40. FNA cytology of thymic carcinoma should not show intercellular windows. Tumor cells are positive for p63 and CD5, and negative for calretinin and WT-1. The FNA cytology of benign mesothelial cells from needle track are flat cohesive nests or sheets. Reactive atypia may be seen and misinterpreted. Sometimes, It is extremely challenging to separate benign mesothelial cells from mesothelioma. Take home messages: 1. Recognize the cytomorphology of subtypes of mesothelioma. 2. IHC profile of mesothelioma: positive for CK7, CK19, CK5/6, WT-1, calretinin, thrombomoculin, and D Electromicroscopy. Case 4 Clinical history A 33 years old male presented with cough, shortness of breath, and a 5 cm anterior mediastinal mass with necrosis on CT and multiple bilateral lung nodules. Patient had no medical history. FNA findings: Discohesive polygonal or spindle cells with dense cytoplasm that is stained orange or green color with Pap stain, round or oval nuclei, irregular nuclear contour, prominent nucleoli, and extensive necrosis. FNA cytology differential diagnosis Thymic carcinoma, squamous cell carcinoma, mesothelioma, undifferentiated carcinoma, high grade adenocarcinoma, and choriocarcinoma/germ cell tumor. Core biopsy findings: Sheets of pleomorphic malignant polygonal tumor cells with round or oval, hyperchromatic nuclei, moderate to abundant eosinophilic cytoplasm, necrosis and surrounding fibrous tissue. Tumor cells are positive for p63. Because there are only 2 levels with tumor cells, no more IHC stains were performed. FNA/core biopsy diagnosis High grade carcinoma, favoring thymic carcinoma Transbronchial lung biopsy findings Sheets of cytotrophoblasts admixed with multinucleated syncytiotrophoblasts, necrosis and hemorrhage. Tumor cells are positive for AE1/AE3 and hcg, and were negative for CD30, glypican-3. Surgical pathology diagnosis of lung biopsy Metastatic choriocarcinoma 10

106 Discussion Choriocarcinoma is a highly malignant neoplasm displaying trophoblastic differentiation. It is composed of syncytiotrophoblast, cytotrophoblast and variably intermediate trophoblast cells 16. Pure mediastinal choriocarcinomas are exceedingly rare, only accounting for 2.5 5% of Mediastinal germ cell tumors Patients age ranges from 17 to 63 years old and is most commonly the 3 rd decade. It often presents as large mass in both anterior and posterior mediastinum 19, and early hematogeneous metastasis 16. There are only a few case reports regarding FNA cytology of choriocarcinoma of lung primary 20, metastatic to breast or spleen 23, of testicular primary 24, and of vulvar primary 25. Choriocarcinoma consists of syncytiotrophoblasts and cytotrophoblats 26. FNA smears show a cellular specimen with single or clusters of malignant cells 20. Syncytiotrophoblasts are very large cells with eosinophilic/dark pink cytoplasm, one to several smudgy nuclei and distinct nucleoli 22, 26, that is not seen in FNA and core of our case. The cytotrophoblasts are mediumsized cells with moderate vacuolated, basophilic cytoplasm and pleomorphic, irregular, eccentric, hyperchromatic nuclei with prominent nucleoli 20, 22, 26. Occasional atypical mitotic figures can be seen 20. Extensive necrosis is present 20, 23. One case was misinterpreted as adenocarcinoma of lung primary 20. Choriocarcinoma is positive for hcg, p63, cytokeratins, PLAP, CEA, EMA, hpl, EGFR, inhibin, and pregnancy-specific betta-1-glycoprotein. FNA cytology of thymic carcinoma and squamous cell carcinoma shows squamous differentiation. Thymic carcinoma is also positive for CD5, while squamous cell carcinoma is negative for CD5. FNA cytology of mesothelioma shows intercellular windows and no squamous differentiation. Tumor cells are negative for CD5 and p63. FNA cytology of undifferentiated carcinoma/nut midline carcinoma may show similar cytomorphology and tumor cells may be positive for p63. NUT midline carcinoma is positive for NUT expression and NUT gene rearrangement. FNA cytology of high grade adenocarcinoma shows cuboidal or columnar cells with possible glandular formation. Tumor cells are negative for p63. Therefore, based on cytomorphology only, it is challenging to distinguish these malignancies from choriocarcinoma without presence of syncytiotrophoblasts. IHC stains for hcg, PLAP, hpl, inhibin and pregnancy-specific beta-1-glycoprotein should be performed if diagnostic material available. Choriocarcinoma is treated by cisplatin-based chemotherapy followed by resection of tumor remnants 16. Therefore, correct diagnosis is very critical to guide the clinician in treating the patient effectively. Take home messages 1. Syncytiotrophoblasts may not be seen in FNA/core biopsy of choriocarcinoma. 2. If a high grade carcinoma positive for p63, choriocarcinoma should be included in the differential diagnosis, although its incidence is very low. 11

107 3. p63 positivity is not only seen in squamous cell carcinoma, but also seen in thymoma/thymic carcinoma, poorly differentiated/undifferentiated carcinoma, NUT midline carcinoma, urothelial carcinoma, choriocarcinoma and some trophoblastic neoplasms. Case 5 Clinical history A 42-year-old man developed worsening deep, non-productive cough, breathlessness and chest pressure after sinus infection and surgery for recurrent inguinal hernia. He reported 10-15lb loss intentionally and childhood asthma. Physical examination revealed a large right pleural effusion confirmed by chest X-ray. Pulmonary CT angiography showed extensive bulky mediastinal lymphadenopathy and a 9.2 x 6.1cm subcarinal mass narrowing and encasing the right lower lobe bronchus. Laboratory tests revealed elevated AFP (545.4u increased to u 5 days later) and HCG (3.3u). Scrotal ultrasound was normal. He received four cycles of chemotherapy with VIP (Etoposide, ifosfamide, and cisplatin), a regimen for mediastinal germ cell tumor. The tumor initially responded chemotherapy with normalization of -AFP but then rapidly progressed after the forth cycle. He refused further chemotherapy and was lost to follow-up 4 months later. Cytology findings Cytospins of pleural fluid showed noncohesive monotonous round cells, approximately 4X the size of a mature lymphocyte, with scant to moderate, delicate or vacuolated cytoplasm, round nuclei, 1 or 2 prominent nucleoli, granular or vesicular chromatin, and minimal nuclear membrane irregularity. Mitoses and apoptoses were frequent. BAL showed rare clusters of cells with similar cytologic features. FNA smears showed single, loosely cohesive or cohesive nests or sheets of cells with similar cytologic features. Occasional nuclear grooves, crushing, and extensive necrosis were seen. Cytologic differential diagnosis Differential diagnosis includes germ cell tumor, undifferentiated carcinoma, poorly differentiated carcinoma, nasopharyngeal carcinoma, thymic carcinoma, sinonasal undifferentiated carcinoma, lymphoma, PNET, mesothelial cells, and NUT midline carcinoma. Histology Excisional biopsy showed nests, sheets or pseudopapillary architecture of undifferentiated cells with similar cytologic features. Slight anisonucloesis and anisocytosis, frequent mitoses, apoptotic figures, and extensive necrosis were seen. Immunohistochemistry, ISH/FISH, and FC Tumor cells were diffusely positive for vimentin and NUT, and focally positive for P63, CD99, CD138, and EMA. Further FISH was positive for BRD3-NUT translocation. Flow cytometry and immunohistochemistry showed tumor cells had increased scattered light characteristics and were negative for all lymphoid markers. Cytology diagnosis 12

108 Undifferentiated carcinoma NUT midline carcinoma with BRD3-NUT rearrangement Discussion Nuclear protein in testis (NUT) midline carcinoma (NMC) is a rare, recently described, high grade carcinoma NMC is defined by the demonstration of rearrangement of the NUT gene on chromosome 15q , 30. Two-thirds of cases is involved in a balanced translocation t(15;19)(q14;p13.1), that creates BRD4-NUT fusion gene 27-28, While in the remaining cases, NUT is rearranged with variant fusion partners such as BRD3 gene on chromosome 9 that creates BRD3-NUT fusion gene The average survival of patients with BRD3-NUT carcinoma was longer than that of BRD4-NUT carcinoma 33. BRD-NUT oncogenes are a family of closely related nuclear proteins that block epithelial differentiation and maintain the growth of carcinoma cells 32. NMC mostly occurs in young patients ranging from newborn to 78 years old (mean 23 years) 27-28, 34. Many organs are affected with more frequent involvement of the midline organs, including the orbit, nasal cavity, paranasal sinuses, parotid gland, upper aerodigestive tract, mediastinum, lung, liver, pancreas, iliac bone, or bladder 27-29, NMC shows an undifferentiated carcinoma with occasional abrupt keratinizing squamous, glandular and mesenchymal chondroid differentiation 29, 33-34, 36, 38. The NMC tumor has early metastasis, generally refractory to chemotherapy and radiation, and poor prognosis with survivals measured in months 27, 36. The morphology of NMC is indistinguishable from poorly differentiated or undifferentiated carcinoma Fine needle aspiration (FNA) smears were highly cellular and generally noncohesive with focal cellular cohesion 39. Cells were 2-3 times the diameter of a small lymphocyte with irregular nuclear contours, discrete nucleoli, and fine/granular to vesicular chromatin, scant and delicate cytoplasm with occasional denser or vacuolated cytoplasm 39. Slight to moderate anisonucleosis and anisocytosis, mitotic figures, and extracytoplamic lumina (negative for epithelial mucin) were seen The background contained naked nuclei and karyorrhectic debris, and nuclear crush was noted 39. Experience with the cytological diagnosis of this entity is limited to one report describing FNA cytology of 3 NMC cases 39. We found that the initial diagnosis of NMC in pleural fluid can be very challenging, because the cytomorphology of the tumor mimics both benign mesothelial cells and malignant entities. Our FNA cytological findings differed somewhat from those first reported by Bellizi 39 by the presence of more cellular cohesion, prominent nucleoli, and cytoplasmic vacuoles 39, and absence of cells with dense cytoplasm. Excisional biopsy showed a monomorphic population of undifferentiated small epithelioid cells with similar cytomorphology as previously described 30, 34, 36. Foci of more differentiated components 29, 33, 36 were not identified in our case. Tumor cells expressed keratin, CD138 EMA, vimentin, CD34, and particularly, p63. In this case, morphologic analysis, immunohistochemistry, and flow cytometry excluded melanoma, lymphoma, and tumors of muscle, primitive neuroectodermal, and mesothelial origin as described in other reports 28, 30, 36. A confounding factor in the diagnosis was the elevated serum -AFP and HCG that decreased after initial chemotherapy and strongly suggested the possibility of a mediastinal germ cell neoplasm. However, all germ cell immunomarkers were 13