Part III of Four part symposium: “Diagnostic Cytopathology of Serous Effusion” on April 19, 2007 at Neenah, WI, USA (2008 Wisconsin Society of Cytology, 40th Anniversary)

1. 1 Diagnostic Cytopathology of Serous Effusions Session III ( 10.00-10.45 ) To view this session on web copy-paste the following URL into your browser: http://www.slideshare.net/vshidham/03-presentations-iii-vs-847mb-32708bpps-3-27-08-b/ Vinod B. Shidham , MD, FRCPath, FIAC Professor Executive editor & coeditor-in-chief, CytoJournal ( www.cytojournal.com ) Department of Pathology Medical College of Wisconsin 9200 W Wisconsin Av, Milwaukee, WI 53226, USA [email_address] 2008 Wisconsin Society of Cytology SPRING MEETING, 40TH ANNIVERSARY Holiday Inn – Riverwalk, Neenah, WI Saturday, April 19, 2008 (7.30 to 3.30)

2. 2 Outline Session I (40 minutes): Anatomy, histology, cytology, and effusions Collection, transportation, and processing of effusion fluids Factors leading to potential diagnostic pitfalls Approach to diagnostic cytopathology of effusions The panorama of different face of mesothelial cells Session II (45 minutes): Benign conditions with/without specific cellular patterns Mesothelioma Metastatic carcinoma Metastatic sarcoma and melanoma Hematolymphoid disorders (Lymphomas and leukemias) Session III (45 minutes): Evaluation of unknown primary sites of origin- Where do they come from? Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Flow cytometry, molecular techniques, and other special techniques Session IV (45 minutes): Diagnostic cytopathology of peritoneal washings Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

3. 2 Outline Session I (40 minutes): Anatomy, histology, cytology, and effusions Collection, transportation, and processing Factors leading to potential diagnostic pitfalls Approach to diagnostic cytopathology of effusions The panorama of different face of mesothelial cells Session II (45 minutes): Benign conditions with/without specific cellular patterns Mesothelioma Metastatic carcinoma Metastatic sarcoma and melanoma Hematolymphoid disorders (Lymphomas and leukemias) Session III (45 minutes): Evaluation of unknown primary sites of origin- Where do they come from? Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Flow cytometry, molecular techniques, and other special techniques Session IV (45 minutes): Diagnostic cytopathology of peritoneal washings Diagnostic pitfalls in cytopathology of serous cavity fluids Study cases

4. Immunocytochemistry of effusion fluids The most important issue to be considered when applying immunocytochemistry to effusion fluids is the significant variation in results due to the many variables incurred from the time of collection of the specimen to its final immunostaining . 3 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

5. Intricacies of finding and locating the cells of interest in cell-block sections may adversely affect the final results. ► Orient the serial sections identically on all slides (to identify more precisely the same cell (or small group of cells) in different sections). Confirmation of a ‘second-foreign’ non-inflammatory population of cells other than mesothelial cells in effusions correlate with metastatic cancer with objectivity. 4 UNIQUENESS OF EFFUSION IMMUNOCYTOCHEMISTRY ► Know the sequence of these serial sections (to evaluate their co-ordinate immunoreactivity pattern). It is crucial to : Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin ► Immunocytochemistry does not have significant role in evaluation of peritoneal washings .

6. Formalin-fixed cell-block sections are recommended- Other protocols such as the evaluation of various cytology preparations (direct smears- wet fixed in alcohol or acetone, air-dried fixed with alcohol, air-dried smears rehydrated and post-fixed in formol alcohol, liquid based cytology preparations- SurePath or ThinPrep, cytospin preparations, etc) should be avoided. For reproducible results a standardized protocol with steps comparable to the processing of formalin-fixed paraffin-embedded tissue sections is essential . 5 Cell-blocks are the preferred choice. Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

7. The proteinaceous effusion fluid around suspended cells may contribute to unexpected nonspecific immunoreactivity . Discrepant results between formalin-fixed paraffin-embedded tissue sections of surgical pathology material and effusion fluid cell-block sections are not uncommon. 6 Reasons for variable reports: The variables responsible for such discrepancies include: sample size (tiny cell groups or single cells), selection of fixatives, antigen retrieval methods (i.e., heat-induced epitope retrieval, enzyme digestion, etc.), antibody clones used, antibody titer, and other variations in immunostaining protocols. Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin

8. It is prudent to be conservative and recommend to repeat. Malignant effusions usually re-accumulate quickly . Acquiring a new sample is generally not a problem . 7 If findings are equivocal: However, it is not uncommon to submit only a small fraction of a large volume of effusion fluid collected. To avoid inadequate resubmission , it may be specifically communicated in the recommendation as comment : “ Recommend submission of most of the drained effusion fluid (up to 1000 mL). Larger volume of specimen facilitates retrieval of adequate cellular material in cell-block sections for immunocytochemical evaluation ”. Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin

9. All aspects of individual and complimentary immunomarkers should be considered collectively rather than applying a reflexive positive-negative approach. 8 Interpretation: Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

10. PGM1 (CD68) WT-1 HBME-1 EMA LCA (CD45) Vimentin Cytokeratin* D2-40 Calretinin Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Immunomarker X X X X X X AdCa X AdCa X X X X X meso X meso

11. Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Immunomarker MOC-31 BerEP4 CA19.9 Ttf-1 Cadherins CD44S Mesothelin CD15 (Lue-M1) CK 5/6 B72.3 mCEA X X X X X X X X X X X

12. EMA Immunoreactivity pattern with EMA (epithelioid mesothelioma, pleural fluid). Mesothelioma cells with membranous (arrow) and cytoplasmic immunostaining. Note the microvilli (arrowhead). [Immunostained cell-block section (100XZoomed)]. 11 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin

13. HBME-1 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Mesothelioma cells with membranous (arrow in a) and cytoplasmic immunostaining. Note the microvilli (arrowhead in b). 11 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques a b HBME-1 HBME-1

14. Pan-cytokeratin immunoreactivity pattern (pleural fluid). Reactive mesothelial cells with cytoplasmic immunostaining (arrow in inset). Some reactive mesothelial cells may show a concentric immunostaining pattern around the nucleus better appreciated by adjusting fine focus. 12 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Pan-cytokeratin

15. Cytokeratin 7 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Neoplastic mesothelial cells with cytoplasmic immunostaining. Note the bushy microvilli (arrowhead). 13 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Cytokeratin 7

16. Calretinin immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Mesothelioma cells (arrow in a) show nuclear (arrowhead 1) immunoreactivity usually with cytoplasmic immunostaining (arrowhead 2) imparting the so called ‘fried-egg’ appearance. 14 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques 1 2 a b Calretinin Calretinin

17. Calretinin immunoreactivity pattern (pleural fluid). Reactive mesothelial cells (blue arrows). The effusion also contains metastatic mammary carcinoma cells (red arrow NC). 15 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques NC RM Calretnin

18. RM WT-1 WT-1 immunoreactivity pattern (Metastatic colonic adenocarcinoma, peritoneal fluid). Reactive mesothelial cells (arrow RM) show nuclear immunoreactivity (arrowhead in inset) with some cytoplasmic immunostaining. Rare adenocarcinoma cells demonstrating nuclear immunoreactivity for CDX2 were also seen in other section. 16 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

19. B72.3 immunoreactivity pattern (Metastatic mammary adenocarcinoma, pleural fluid). Metastatic adenocarcinoma cells (red arrow NC) show a cytoplasmic immunoreactivity pattern. 17 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques NC B72.3

20. RM vimentin Vimentin immunoreactivity pattern (peritoneal wash). Reactive mesothelial cells (arrow RM) show cytoplasmic immunoreactivity pattern (arrowhead in inset). 18 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

21. LCA (CD45 ) immunoreactivity pattern (pleural fluid). Reactive mesothelial cells (blue arrow RM) with chronic inflammatory cells (red arrows). The inflammatory cells show a strong cytoplasmic immunoreactivity pattern obscuring the nucleus (arrowhead in inset). 19 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques RM LCA

22. CD 68 (PGM1 ) immunoreactivity pattern (Metastatic mammary adenocarcinoma with proliferation spheres (red arrow NC), pleural fluid). Histiocytes show CD68 immunoreactivity (blue arrows H). In our experience, PGM1 does not show non-specific immunostaining usually associated with KP1. Inset- Histiocytes (blue arrow H) with cytoplasmic immunoreactivity pattern around the nucleus. 20 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques H NC H H H CD68

23. BerEP4 immunoreactivity pattern (Metastatic mammary adenocarcinoma, pleural fluid). a. The neoplastic cells in proliferation spheres (red arrow NC)- membranous immunostaining with a honey comb-like pattern . b. Solitary adenocarcinoma cells (red arrow NC)- membranous immunostaining pattern along the cell membrane (arrowhead in inset). 21 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques a NC NC b BerEP4 BerEP4 NC

24. Comparison of immunoreactivity with BerEP4 and B72.3 (Metastatic mammary adenocarcinoma, pleural fluid). As Compared to B72.3, most of the adenocarcinoma cells (red arrows NC) show strong membranous BerEP4 immunoreactivity. 22 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques BerEP4 c a b d BerEP4 B72.3 B72.3 NC NC NC NC NC NC

25. mCEA c m Monoclonal CEA (mCEA) immunoreactivity pattern (Metastatic ovarian carcinoma, peritoneal fluid). Metastatic adenocarcinoma cells show cytoplasmic (c) and membranous (m) immunostaining. 23 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

26. CDX2 immunoreactivity pattern (Metastatic colonic adenocarcinoma, peritoneal fluid). The adenocarcinoma cells show nuclear immunoreactivity (arrow NC). Compare with non-immunoreactive nuclei with blue hematoxylin counterstain (arrowhead). 24 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques CDX2 NC

27. TTF-1 immunoreactivity pattern (Metastatic pulmonary carcinoma, pleural fluid). The solitary adenocarcinoma cells as the predominant population (arrows NC) show nuclear immunoreactivity (arrowheads in inset). 25 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques TTF-1 NC NC

28. MOC-31 immunoreactivity pattern (Metastatic mammary carcinoma, pleural fluid). The adenocarcinoma cells show predominantly membranous (m) with cytoplasmic (c) immunoreactivity. 26 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques MOC-31 c m

29. Diffuse malignant mesothelioma of epithelial type, (pleural fluid). Neoplastic cells are immunoreactive for EMA (a & b) and HBME-1 (c, d, & e) with a membranous immunostaining pattern (arrows) highlighting long, slender, microvilli (arrowheads). 27 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques EMA a b c d e HBME-1

30. Adenocarcinoma, peritoneal fluid. Cytoplasmic immunostaining pattern (arrows) with focal blotchy immunostaining for EMA (a) and HBME-1 (b) along the cell membrane . 28 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques a b EMA HBME-1

31. For reproducible results, it is important to select any immunopanel which will fundamentally identify most of the mesothelial and inflammatory cells to create the basic map for confirmation of a ‘second-foreign’ population by ‘Subtractive Coordinate Immunoreactivity Pattern’ (SCIP) approach 29 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

32. Evaluation of ‘ Subtractive Coordinate Immunoreactivity Pattern’ (SCIP) 30 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

33. SCIP approach 31 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques 2 3 1 6 5 4 8 7 Mesothelial & inflammatory cells 2 3 1 6 5 4 8 7 2 3 1 6 5 4 8 7 2 3 1 6 5 4 8 7 2 3 1 6 5 4 8 7 X Metastasis (non-carcinoma) 2 3 1 6 5 4 7 2 3 1 6 5 4 7 2 3 1 6 5 4 7 3 1 6 5 4 7 2 3 1 6 5 4 7 Z Metastasis (carcinoma) 2 1 5 4 3 7 6 2 1 5 4 3 7 6 2 1 5 4 3 7 6 2 1 5 4 3 7 6 2 1 5 4 3 7 6 Y vimentin Pan CK (Mixture of AE1/AE3 & CAM5.2) Calretinin WT-1 LCA (CD45) [or PGM1(CD68) or mixture of LCA & PGM1] A B C D E

34. 32 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

35. SCIP approach 33 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques a b

36. 35 Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population Qualitative & quantitative features of mesothelioma Negative for malignancy Absent Present CK-,vim+ Lymphoma Melanoma/ Sarcoma Sarcoma LCA+ LCA – Carcinoma S-100 protein & Melanoma markers Lymphoma panel Cytogenetics Gene rearrangement Immunoreactive for ‘negative’ mesothelial markers such as- BerEP4, B72.3, CEA, MOC-31. Proceed with: Immunopanel for unknown primary OR Restricted panel for known primary Melanoma + – Immunopanel for sarcoma OR Restricted panel for known primary Malignant mesothelioma EMA/HBME-1: Microvillous pattern B72.3 – , BerEP4– CK+,vim –/+ Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin

37. SCIP approach Metastatic colonic adenocarcinoma, (peritoneal fluid). 35 F. CDX2 Immunoreactive nuclear HE stained cell block section 40X B. Pan-cytokeratin Immunoreactive C. LCA (CD45) Non-immunoreactive A. Vimentin Non-immunoreactive D. Calretinin Non-immunoreactive (Inset {2}- Mesothelial cell immunoreactive nuclear-cytoplasmic) E. WT-1 Non-immunoreactive (Arrow 2 with inset: Mesothelial cell- immunoreactive nuclear-cytoplasmic) RM RM ‘ Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections NC 10X 10X 10X 10X 10X 10X 40X 40X 40X 40X 40X 100X 40X NC NC NC NC NC NC NC NC NC NC NC RM RM NC

38. SCIP approach (continued) Metastatic ovarian carcinoma, (peritoneal fluid). 36 HE stained cell block section 10X B. Pan-cytokeratin Immunoreactive C. Calretinin Non-immunoreactive [Mesothelial cells (RM) immunoreactive nuclear-cytoplasmic] A. Vimentin Non-immunoreactive E. Cytokeratin 7 Immunoreactive F. Cytokeratin 20 Non-immunoreactive ‘ Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections 10X 10X 10X 10X 10X D. BerEP4 Immunoreactive 10X Zoomed Zoomed NC RM NC RM Zoomed NC RM NC RM Zoomed

39. SCIP approach Metastatic mammary adenocarcinoma, (pleural effusion). 37 B. CD68 (PGM1) Non-immunoreactive C. Calretinin Non-immunoreactive Mesothelial cell (RM) immunoreactive nuclear-cytoplasmic) A. Vimentin Non-immunoreactive ‘ Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections E. BerEP4 Immunoreactive HE stained cell block section 40X 40X 40X 40X 40X D. Cytokeratin 7 Immunoreactive 40X NC RM NC RM NC RM

40. Metastatic mammary adenocarcinoma, (pleural effusion). SCIP approach (continued) 38 C. Calretinin Non-immunoreactive (Rare mesothelial cell [blue arrow] is immunoreactive nuclear-cytoplasmic) D. BerEP4 Immunoreactive E. Estrogen receptors Immunoreactive B. CD68 (PGM1) Non-immunoreactive (inflammatory cells are immunoreactive) A. Vimentin Non-immunoreactive (Mesothelial & inflammatory cells are immunoreactive) ‘ Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections 20X 20X 20X 20X 20X 40X 40X 40X 40X 40X NC RM NC NC

41. Metastatic small cell carcinoma, (pleural fluid). SCIP approach (continued) 39 B. Cytokeratin 20 Non-immunoreactive C. TTF-1 Immunoreactive Nuclear D. Chromogranin Immunoreactive cytoplasmic F. CD56 Immunoreactive cytoplasmic 40X 100X 40X 100X E. Synaptophysin Weak immunoreactive cytoplasmic 100X 40X 40X 100X 40X 100X 40X 100X A. Cytokeratin 7 Immunoreactive cytoplasmic ‘ Subtractive coordinate immunoreactivity pattern’ (SCIP) in cell block sections NC NC NC NC

42. Large B-cell lymphoma, (peritoneal fluid). SCIP approach (continued) 40 HE stained cell block Section (d) A. Cytokeratin 7 Non-immunoreactive [Mesothelial cell Immunoreactive (red arrow) Cytoplasmic] B. Calretinin Non-immunoreactive [Mesothelial cell Immunoreactive (red arrow) nuclear-cytoplasmic] D. Bcl2 Immunoreactive Cytoplasmic (red arrow) E. CD3 Non-immunoreactive ‘ Subtractive coordinate immunoreactivity Pattern’ (SCIP) in cell block sections C. CD 20 Immunoreactive Cytoplasmic (red arrow) PAP stained Cytospin preparation (a-c) 40X 40X 40X 40X 40X a b c d 10X 40X NC NC RM NC RM NC NC 40X

43. SCIP with dual staining method a b d c Vimentin (Brown)-Cytokeratin 7 (Red) Vimentin (Brown)-Cytokeratin 7 (Red) Vimentin (Brown)-Cytokeratin 7 (Red) Vimentin (Brown)-Cytokeratin 7 (Red) Mammary carcinoma, (effusion fluid). 41 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

44. SCIP with dual staining method a b c Calretinin (Brown)-BerEP4 (Red) Vimentin (Brown)-Cytokeratin 7 (Red) Metastatic mammary adenocarcinoma, pleural fluid. 42 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

45. SCIP with dual staining method a b c Calretinin (Brown)-BerEP4 (Red) Vimentin (Brown)-Cytokeratin 7 (Rred) Metastatic gastric adenocarcinoma, peritoneal fluid. 43 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

46. 45 Effusion cytology 1 Evaluation of unknown primary sites of origin- Where do they come from? Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Unequivocal for malignant cells 4 Equivocal for malignant cells 2 Negative for malignant cells 3

47. 46 Cell-block Evaluation of unknown primary sites of origin- Where do they come from? (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Equivocal for malignant cells 2 5 Positive for malignant cells, depending on results of immunocytochemistry, comment about the primary site Report 18 Negative for malignant cells Report 17 Immunocytochemical characterization on cell-block sections to confirm presence of second population by SCIP with characterization of this second population for possible primary site 12 Not available Or insufficient 9 Suspicious for malignant cells with recommendation to submit additional specimen for confirmation with additional cytopathological evaluation with cell-block preparation if effusion reaccumilates b . Report 13 Available 8

48. 47 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Evaluation of unknown primary sites of origin- Where do they come from? (continued) Negative for malignant cells 3 Negative for malignant cells Report 6

49. Clinical correlation c 7 Not possible 11 Possible 10 Comparative review of primary lesion 14 48 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Evaluation of unknown primary sites of origin- Where do they come from? (continued) Unequivocal for malignant cells 4

50. Cytomorphological features suggestive of a primary site 49 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Cytomorphological patterns Possible primary carcinoma 1 Three dimensional round cell groups- proliferation spheres or ‘cannonballs’ Breast adenocarcinoma Ovarian adenocarcinoma Mesothelioma of epithelioid type Reactive mesothelial proliferations 2 Acini / glands Adenocarcinomas of Breast, Lung, Colorectum, Stomach, Ovary, Endometrium, etc. Mesothelioma of epithelioid type 3 Predominantly scattered isolated malignant cells Gastric adenocarcinoma Non-cohesive variant of lung adenocarcinoma Breast lobular carcinoma Adrenocortical carcinoma (Also Lymphoma, Melanoma , & Sarcoma)

51. Cytomorphological features suggestive of a primary site (continued) 50 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Cytomorphological patterns Possible primary carcinoma 4 Carcinoma cells in chains and rows (‘Indian file’ pattern) Breast- Lobular and ductal carcinoma Poorly differentiated small cell carcinoma Gastric adenocarcinoma Ovarian adenocarcinoma 5 Extensive cytoplasmic vacuolization Renal cell adenocarcinoma (glycogen, fat) Adrenocortical carcinoma (fat) Benign mesothelial cells Pancreatic adenocarcinoma (mucin) Ovarian adenocarcinoma (mucin) Lung adenocarcinoma Clear cell carcinoma endometrium 6 Signet ring cells Gastric adenocarcinoma Colorectal adenocarcinoma 7 Intracytoptasmic lumina Breast adenocarcinoma

52. 51 Cytomorphological features suggestive of a primary site (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Cytomorphological patterns Possible primary carcinoma 8 Giant tumor cells Lung large cell carcinoma- giant cell type Pancreatic adenocarcinoma Thyroid anaplastic carcinoma Squamous cell carcinoma (Also Melanoma & pleomorphic sarcoma) 9 Targetoid intracytoplasmic vacuole containing secretion Breast adenocarcinoma (especially lobular) Thyroid carcinoma (colloid) Ovarian carcinoma Pancreatic carcinoma 10 Three dimensional groups in papillary configurations Bronchioloalveolar carcinoma Colonic adenocarcinoma Endometrial adenocarcinoma Mammary adenocarcinoma

53. 52 Cytomorphological features suggestive of a primary site (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Cytomorphological patterns Possible primary carcinoma 11 Three dimensional papillary groups containing psammoma bodies Ovarian carcinoma- serous papillary Thyroid papillary carcinoma Pancreatic papillary carcinoma 12 Cell groups of tall columnar cells with a picket fence pattern Colonic adenocarcinoma Pancreato-biliary carcinoma 13 Cellular pleomorphism Poorly differentiated carcinomas of lung, pancreas, ovary, thyroid, urothelium 14 Large polyhedral cells Hepatocellular carcinoma Transitional cell carcinoma Large c ell type squamous cell carcinoma 15 Cytoplasmic pigment Hepatocellular carcinoma- Bile, (Melanoma- melanin) 16 Prominent nucleoli Hepatocellular carcinoma Renal cell carcinoma Prostatic adenocarcinoma

54. Clinical correlation c 7 Not possible 11 Possible 10 Cytomorphology not classical for the known primary neoplasm 16 Cytomorphology consistent with primary site 15 Comparative review of primary lesion 14 Available 21 Not available Or insufficient 22 Immunocytochemical characterization 23 48 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Evaluation of unknown primary sites of origin- Where do they come from? (continued) Cell-block 20 Positive for malignant cells with broad cytomorphological characterization (such as non-small cell carcinoma vs small cell carcinoma vs lymphoma ). Recommend additional specimen for cell-block for immunocharacterization of neoplastic cells if effusion reaccumilates. Report 26 Unequivocal for malignant cells 4 Positive for malignant cells, consistent with metastatic cancer from previous neoplasm . Report 19

55. Immunoprofile of the second population is not distinct for primary neoplasm. 25 Immunoprofile of the second population is consistent with primary neoplasm. 24 54 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Immunocytochemical characterization 23 Positive for malignant cells, consistent with XYZ primary. Report 27 Positive for malignant cells, And suggest a differential diagnosis for the primary sites. Report 28

56. Flow cytometry, molecular techniques, and other special techniques A. SURFACE MARKER FLOW CYTOMETRY A1. HEMATOPOIETIC NEOPLASMS a. Use of flow cytometry as a screening tool b. Flow cytometry in selected patient population c. Aberrant or monoclonal lymphoid population- Significance A2. NONHEMATOPOIETIC NEOPLASM B. ELECTRON MICROSCOPY C. FISH AND METASTATIC SEROUS EFFUSION D. MOLECULAR GENETICS D1. HEMATOPOIETIC NEOPLASM D2. SOFT TISSUE TUMORS D3. PCR DIAGNOSIS OF PLEURAL TUBERCULOSIS E. OTHER TECHNIQUES E1. CYTOGENETICS E2. CISH E3. DIGITIZED IMAGING E4. PROTEOMICS; 2-DIMENSIONAL GEL ELECTROPHORESIS; MALDI AND SELDI E5. DNA PLOIDY ANALYSIS 54 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

57. Flow cytometry It is usually difficult to differentiate between reactive lymphocytosis and malignant lymphoma in serous effusions especially in low-grade malignant lymphoma on morphology alone. Flow cytometric analysis can be useful in this situation. The decision to send the specimen for flow cytometric analysis should be based primarily on: cytomorphologic evaluation, clinical context, and patient population. 55 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

58. Flow cytometry is proved to be more valuable tool in selected patient population with- ► A known history of malignant lymphoma or ► Clinical suspicion of malignant lymphoma or ► Cytomorphologic features suggestive of lymphoma Important caveat: The finding of aberrant or monoclonal lymphoid population by flow cytometry in serous effusions is not equal to a diagnosis of malignant lymphoma . 56 Flow cytometry (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

59. Diffuse large B-cell lymphoma , pericardial fluid The two-dimensional histograms show four-color flow cytometric analysis of B lymphocytes which express CD19 and CD20 (D-F). These neoplastic B cells are medium to large as illustrated by intermediate forward scatter (FSC) (C), correlating with the cytomorphological correlation (A&B). They also coexpress CD10 (E&F), are negative for CD5 (H), and demonstrate kappa restriction (G). C D E F G H A B 57 Flow cytometry (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques 0 256 512 768 1024 FSC-Height -> 10 10 10 10 10 0 1 2 3 4 CD20 PerCP -> 10 10 10 10 10 0 1 2 3 4 CD10 APC -> 10 10 10 10 10 0 1 2 3 4 pL FITC -> 10 10 10 10 10 0 1 2 3 4 CD5 APC -> 10 10 10 10 10 0 1 2 3 4 CD10 APC ->

60. Follicular lymphoma , ascitic fluid. C-H: The two-dimensional histograms show four-color flow cytometric analysis of B lymphocytes which express CD19 and CD20 (D-F). These neoplastic B cells are medium to large as illustrated by intermediate forward scatter (FSC) (C), correlating with the cytomorphological correlation (A&B). They also coexpress CD10 (E&F), are negative for CD5 (H), and demonstrate kappa restriction (G). 58 Flow cytometry (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques 0 256 512 768 1024 FSC-Height -> C 10 10 10 10 10 0 1 2 3 4 CD19 PerCP-CY5 -> D 10 10 10 10 10 0 1 2 3 4 CD19 PerCP -> E 10 10 10 10 10 0 1 2 3 4 CD20 FITC -> F 10 10 10 10 10 0 1 2 3 4 pLAMBDA FITC -> G 10 10 10 10 10 0 1 2 3 4 CD5 APC -> H A B

61. Algorithm for evaluation of serous effusion suspicious for lymphoma 59 Flow cytometry (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Cytomorphologic Evaluation of neoplastic cells Small cell* Large cell ¶ Flow cytometry Immuno - phenotyping on cell block If enough tissue, save some for molecular study. If enough specimen, save some for FCM and molecular study. ¶ Large cell : > 2x of resting lymphocyte or histiocyte nuclei Small cell : < 2x of resting lymphocyte or histiocyte nuclei *

62. Flow cytometry immunophenotyping of lymphomas/lymphoid leukemias 60 Flow cytometry (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

63. Other molecular and special techniques It should be highlighted that cytomorphologic examination is still a goal standard for tumor staging and is the only method accepted in the classical AJCC cancer staging . FISH AND METASTATIC SEROUS EFFUSION 61 Prospective studies are needed to demonstrate the clinical benefit of FISH to detect disseminated tumor cells in correlation with clinical outcomes. Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

64. The morphology and immunophenotyping (both flow cytometry and immunocytochemistry) remain the preferred standard for diagnosis and classification . MOLECULAR GENETICS (PCR, Southern blot) 62 However, in difficult cases with inconclusive morphology and immunophenotyping results, these techniques can be important ancillary for reaching an accurate diagnosis. Other molecular and special techniques (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

65. PCR FOR DIAGNOSIS OF PLEURAL TUBERCULOSIS The reported sensitivity of PCR testing for pleural tuberculosis range from 17.5 - 83% (probably due to different PCR methods and diverse clinical diagnostic criteria). The reported specificity of the tests ranged from 78 to 100%, approaching100% in most. The data suggests that PCR is not recommended in a routine clinical practice , though clinical utility of PCR testing for diagnosis of pleural tuberculosis may be improved in the future . 63 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques

66. Major molecular genetic abnormalities in lymphomas CYTOGENETICS & MOLECULAR TESTS 64 Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Lymphomas Cytogenetic Abn Molecular Genetic Abn Prognotic significance CLL/SLL 13q14 abnormalities 13q14 abnormalities Favorable Follicular lymphoma t(14;18)(q32;q21) BCL2-IGH fusion None Mantle cell lymphoma t(11;14)(q13;q32) BCL1-IGH fusion None MALT lymphoma t(11;18)(q21;q21) API2-MALT1 fusion No responses to H. pylori eradication therapy t(14;18)(q32;q21) IGH-MALT1 fusion N/A t(1;14)(p22;q32) BCL10-IGH fusion N/A Burkitt lymphoma t(8;14)(q24;q32) cMYC-IGH fusion N/A ALCL t(2;5)(p23;q35) ALK-NPM fusion Favorable

67. Major molecular genetic abnormalities in acute leukemias 65 CYTOGENETICS & MOLECULAR TESTS (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Leukemias Cytogenetic Abn Molecular Genetic Abn Prognostic significance AML t(8;21)(q22;q22) ETO-AML1 fusion Favorable inv(16)(p13;q22) MYH11-CBFβ fusion Favorable t(16;16)(p13;q22) MYH11-CBFβ fusion Favorable t(15;17)(q22;q21) PML-RARα fusion Favorable t(6;9)(p23;q34) DEK-CAN fusion Intermediate t(11;q23) Fusions involving MLL Unfavorable t(9;11)(p22;q23) MLL-AF9 fusion Unfavorable ALL t(9;22)(q34;q11) BCR-ABL fusion Unfavorable t(12;21)cryptic TEL-AML1 fusion Favorable t(1;19)(q23;p13) E2A-PBX fusion Unfavorable t(11;q23) Fusions involving MLL Unfavorable t(4;11)(q21;q23) MLL-AF4 fusion Unfavorable

68. 66 CYTOGENETICS & MOLECULAR TESTS (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Type of soft tissue tumor Cytogenetic Abn Molecular Genetic Abn 1 Rhabdomyosarcoma Botryoid NA NA Spindle cell NA NA Embryonal Gains of 2, 7, 8, 12, 13; losses of 1, 6, 9, 14, and 17 IGF2, GOK, PTCH TP53 Alveolar t(2;13)(q35;q14); t(1;13)(p36;q14) PAX3-FKHR PAX7-FKHR

69. 67 CYTOGENETICS & MOLECULAR TESTS (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Type of soft tissue tumor Cytogenetic Abn Molecular Genetic Abn 2A. Non Rhabdomyosarcoma-EWS family EWS/PNET t(11;22)(q24;q12) t(21;22)(q22;q12) t(7;22)(p22;q12) EWS-FLI-1 EWS-ERG EWS-ETV1 DSRCT t(11;22)(p13;q12) EWS-WT1 Clear cell sarcoma t(12;22)(q13;q12) EWS-ATF1 Extraskeletal myxoid chondrosarcoma t(9;22)(q22-23;q11-12) t(9;15)(q22;q21) t(9;17)(q22;q11) EWS-TEC (CHN) TCF12-TEC TAF2N-TEC Extraskeletal mesenchymal chondrosarcoma der(13;21)(q10;q10) NA

70. 68 CYTOGENETICS & MOLECULAR TESTS (continued) Immunocytochemistry of effusion fluids Evaluation of unknown primary sites of origin Flow cytometry, molecular techniques, and other special techniques Type of soft tissue tumor Cytogenetic Abn Molecular Genetic Abn 2B. Non Rhabdomyosarcoma - Others Synovial sarcoma t(X;18)(p11;q11) SYT-SSX1 SYT-SSX2 Inflammatory myofibroblastic tumor t(1;2)(q25;p23) t(2;19)(p23;p13) TPM3-ALK TPM4-ALK Myxoid/round cell liposarcoma t(12;16)(q13;p11) t(12;22)(q13;q12) TLS-CHOP EWS-CHOP Alveolar soft part sarcoma t(X;17)(p11;q25) ASPL- TFE3

71. Thank you Milwaukee Art Museum [email_address] End Diagnostic Cytopathology of Serous Effusions III