Shidham- Immunohistochemistry in Serous Fluid Cytopathology

Vinod B. Shidham , MD, FRCPath, FIAC Professor Co-editor-in-chief & Executive editor, CytoJournal (www.cytojournal.com) Vice-chair - AP Director of Cytopathology, Residency training program, Cytotechnology School, Cytopathology fellowship, & GI fellowship Dept of Pathology, Wayne State University Medical School Karmanos Cancer Institute & Detroit Medical Center Detroit, MI 48201, USA [email_address] Scope and limitations of Immunohistochemistry in Serous Fluid Cytopathology TATA Memorial Cancer Center Mumbai, India Dec 21, 2011

Acknowledgement Shidham & Atkinson Cytopathologic Diagnosis of Serous Fluids Elsevier (W. B. Saunders Company) Some of the sketches and tables used are from the following reference.

2 Outline Immunocytochemistry of effusion fluids: SCIP (Subtractive Coordinate Immunoreactivity Pattern) approach Evaluation of unknown primary sites of origin- Where do they come from?

Immunocytochemistry of effusion fluids The most important issue to be considered when applying immunocytochemistry to effusion fluids is- Potential variation in immunoreactivity patterns Secondary to associated variables- from the time of collection of the specimen to its final immunostaining 3

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 correlates with metastatic cancer with objectivity. UNIQUENESS OF EFFUSION IMMUNOCYTOCHEMISTRY ► Know the sequence of these serial sections (to evaluate their co-ordinate immunoreactivity pattern). It is crucial to : ► Immunocytochemistry usually does not have significant role in evaluation of peritoneal washings .

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)

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 (continued)

It is prudent to be conservative and recommend to repeat. Malignant effusions usually re-accumulate quickly . Acquiring a new sample is generally not a challenge. 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 adequately cellular material in cell-block sections for elective immunocytochemical evaluation ”. Immunocytochemistry of effusion fluids (continued)

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)

PGM1 (CD68) WT-1 HBME-1 EMA LCA (CD45) Vimentin Cytokeratin* D2-40 Calretinin Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker Immunocytochemistry of effusion fluids (continued)

PGM1 (CD68) WT-1 HBME-1 EMA LCA (CD45) Vimentin Cytokeratin* D2-40 Calretinin Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker Immunocytochemistry of effusion fluids (continued) X X

PGM1 (CD68) WT-1 HBME-1 EMA LCA (CD45) Vimentin Cytokeratin* D2-40 Calretinin Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker Immunocytochemistry of effusion fluids (continued) X X X X

PGM1 (CD68) WT-1 HBME-1 EMA LCA (CD45) Vimentin Cytokeratin* D2-40 Calretinin Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker Immunocytochemistry of effusion fluids (continued) X X X X X X AdCa X AdCa X X X

PGM1 (CD68) WT-1 HBME-1 EMA LCA (CD45) Vimentin Cytokeratin* D2-40 Calretinin Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker Immunocytochemistry of effusion fluids (continued) X X X X X X AdCa X AdCa X X X X

PGM1 (CD68) WT-1 HBME-1 EMA LCA (CD45) Vimentin Cytokeratin* D2-40 Calretinin Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker Immunocytochemistry of effusion fluids (continued) X X X X X X AdCa X AdCa X X X X X meso X meso

Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker MOC-31 BerEP4 CA19.9 Ttf-1 Cadherins CD44S Mesothelin CD15 (Lue-M1) CK 5/6 B72.3 mCEA Immunocytochemistry of effusion fluids (continued)

Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker MOC-31 BerEP4 CA19.9 Ttf-1 Cadherins CD44S Mesothelin CD15 (Lue-M1) CK 5/6 B72.3 mCEA Immunocytochemistry of effusion fluids (continued) X

Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker MOC-31 BerEP4 CA19.9 Ttf-1 Cadherins CD44S Mesothelin CD15 (Lue-M1) CK 5/6 B72.3 mCEA Immunocytochemistry of effusion fluids (continued) X X X X X X

Microvillous Membranous Cytoplasmic Nuclear & cytoplasmic Nuclear None Immunostaining pattern 9 Immunomarker MOC-31 BerEP4 CA19.9 Ttf-1 Cadherins CD44S Mesothelin CD15 (Lue-M1) CK 5/6 B72.3 mCEA Immunocytochemistry of effusion fluids (continued) X X X X X X X X X X X

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. Immunocytochemistry of effusion fluids (continued) 1 2 a b Calretinin Calretinin

D2-40 (Podoplanin) immunoreactivity pattern (Omental Bx). Malignant mesothelial cells show microvillous immunoreactivity pattern. (Malignant mesothelioma)

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. Immunocytochemistry of effusion fluids (continued)

Cytokeratin 7 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Neoplastic mesothelial cells with cytoplasmic immunostaining. Note the bushy microvilli (arrowhead). Immunocytochemistry of effusion fluids (continued) Cytokeratin 7

EMA Immunoreactivity pattern with EMA (epithelioid mesothelioma, pleural fluid). 11 Immunocytochemistry of effusion fluids (continued) [Immunostained cell-block section (100XZoomed)].

EMA Immunoreactivity pattern with EMA (epithelioid mesothelioma, pleural fluid). Mesothelioma cells with membranous (arrow) 11 Immunocytochemistry of effusion fluids (continued) [Immunostained cell-block section (100XZoomed)].

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

HBME-1 immunoreactivity pattern (epithelioid mesothelioma, pleural fluid). Mesothelioma cells with membranous (arrow in a) and cytoplasmic immunostaining. Note the microvilli (arrowhead in b). Immunocytochemistry of effusion fluids (continued) a b HBME-1 HBME-1

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. Immunocytochemistry of effusion fluids (continued) Pan-cytokeratin

RM vimentin Vimentin immunoreactivity pattern (peritoneal wash). Reactive mesothelial cells (arrow RM) show cytoplasmic immunoreactivity pattern (arrowhead in inset). Immunocytochemistry of effusion fluids (continued)

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). Immunocytochemistry of effusion fluids (continued) RM LCA

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. Immunocytochemistry of effusion fluids (continued) H NC H H H CD68

B72.3 immunoreactivity pattern (Metastatic mammary adenocarcinoma, pleural fluid). Metastatic adenocarcinoma cells (red arrow NC) show a cytoplasmic immunoreactivity pattern. Immunocytochemistry of effusion fluids (continued) NC B72.3

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). Immunocytochemistry of effusion fluids (continued) a NC NC b BerEP4 BerEP4 NC

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. Immunocytochemistry of effusion fluids (continued) BerEP4 c a b d BerEP4 B72.3 B72.3 NC NC NC NC NC NC

MOC-31 immunoreactivity pattern (Metastatic mammary carcinoma, pleural fluid). The adenocarcinoma cells show predominantly membranous (m) with cytoplasmic (c) immunoreactivity. Immunocytochemistry of effusion fluids (continued) MOC-31 c m

mCEA c m Monoclonal CEA (mCEA) immunoreactivity pattern (Metastatic ovarian carcinoma, peritoneal fluid). Metastatic adenocarcinoma cells show cytoplasmic (c) and membranous (m) immunostaining. Immunocytochemistry of effusion fluids (continued)

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). Immunocytochemistry of effusion fluids (continued) CDX2 NC

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). Immunocytochemistry of effusion fluids (continued) TTF-1 NC NC

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). Immunocytochemistry of effusion fluids (continued) EMA a b c d e HBME-1

Adenocarcinoma, peritoneal fluid. Cytoplasmic immunostaining pattern (arrows) with focal blotchy immunostaining for EMA (a) and HBME-1 (b) along the cell membrane . Immunocytochemistry of effusion fluids (continued) a b EMA HBME-1

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 Immunocytochemistry of effusion fluids (continued)

Evaluation of ‘ Subtractive Coordinate Immunoreactivity Pattern’ (SCIP) Immunocytochemistry of effusion fluids (continued)

SCIP approach 31 Immunocytochemistry of effusion fluids (continued) Immunocytochemistry of effusion fluids (continued) 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

Immunocytochemistry of effusion fluids (continued)

SCIP approach Immunocytochemistry of effusion fluids (continued) a b

SCIP approach Metastatic colonic adenocarcinoma, (peritoneal fluid). 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

SCIP approach (continued) Metastatic ovarian carcinoma, (peritoneal fluid). 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

SCIP approach Metastatic mammary adenocarcinoma, (pleural effusion). 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

Metastatic mammary adenocarcinoma, (pleural effusion). SCIP approach (continued) 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

Metastatic small cell carcinoma, (pleural fluid). SCIP approach (continued) 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

Large B-cell lymphoma, (peritoneal fluid). SCIP approach (continued) 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

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). Immunocytochemistry of effusion fluids (continued)

SCIP with dual staining method a b c Calretinin (Brown)-BerEP4 (Red) Vimentin (Brown)-Cytokeratin 7 (Red) Metastatic mammary adenocarcinoma, pleural fluid. Immunocytochemistry of effusion fluids (continued)

SCIP with dual staining method a b c Calretinin (Brown)-BerEP4 (Red) Vimentin (Brown)-Cytokeratin 7 (Rred) Metastatic gastric adenocarcinoma, peritoneal fluid. Immunocytochemistry of effusion fluids (continued)

Effusion cytology 1 Negative for malignant cells 3 Cytopathology & immunocytochemistry of effusion fluids Unequivocal for malignant cells 4 Equivocal for malignant cells 2

Cell-block Not available Or insufficient 9 Equivocal for malignant cells 2 5 Immunocytochemistry of effusion fluids (continued) Available 8

Cell-block Equivocal for malignant cells 2 5 Immunocytochemistry of effusion fluids (continued) 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

Cell-block Equivocal for malignant cells 2 5 Immunocytochemistry of effusion fluids (continued) 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

Cell-block Equivocal for malignant cells 2 5 Immunocytochemistry of effusion fluids (continued) 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

Cell-block Equivocal for malignant cells 2 5 Immunocytochemistry of effusion fluids (continued) 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

Immunocytochemistry of effusion fluids (continued) Negative for malignant cells 3

Immunocytochemistry of effusion fluids (continued) Negative for malignant cells 3 Negative for malignant cells Report 6

Evaluation of unknown primary sites of origin- Where do they come from? (continued) Unequivocal for malignant cells 4

Clinical correlation c 7 Not possible 11 Possible 10 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Unequivocal for malignant cells 4

Clinical correlation c 7 Not possible 11 Possible 10 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Cell-block 20 Unequivocal for malignant cells 4

Clinical correlation c 7 Not possible 11 Possible 10 Comparative review of primary lesion 14 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Cell-block 20 Unequivocal for malignant cells 4

Clinical correlation c 7 Not possible 11 Possible 10 Cytomorphology consistent with primary site 15 Comparative review of primary lesion 14 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Cell-block 20 Unequivocal for malignant cells 4 Positive for malignant cells, consistent with metastatic cancer from previous neoplasm . Report 19

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 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Cell-block 20 Unequivocal for malignant cells 4 Positive for malignant cells, consistent with metastatic cancer from previous neoplasm . Report 19

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 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Cell-block 20 Unequivocal for malignant cells 4 Positive for malignant cells, consistent with metastatic cancer from previous neoplasm . Report 19

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 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

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 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 Immunocytochemical characterization (See next slide) 23 Unequivocal for malignant cells 4 Positive for malignant cells, consistent with metastatic cancer from previous neoplasm . Report 19

Evaluation of unknown primary sites of origin- Where do they come from? (continued) Immunocytochemical characterization 23

Immunoprofile of the second population is consistent with primary neoplasm. 24 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Immunocytochemical characterization 23

Immunoprofile of the second population is consistent with primary neoplasm. 24 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Immunocytochemical characterization 23 Positive for malignant cells, consistent with XYZ primary. Report 27

Immunoprofile of the second population is not distinct for primary neoplasm. 25 Immunoprofile of the second population is consistent with primary neoplasm. 24 Evaluation of unknown primary sites of origin- Where do they come from? (continued) Immunocytochemical characterization 23 Positive for malignant cells, consistent with XYZ primary. Report 27

Immunoprofile of the second population is not distinct for primary neoplasm. 25 Immunoprofile of the second population is consistent with primary neoplasm. 24 Evaluation of unknown primary sites of origin- Where do they come from? (continued) 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

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population CK-,vim+ CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population CK-,vim+ Carcinoma 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 CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population CK-,vim+ LCA+ LCA – Carcinoma 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 CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population CK-,vim+ Lymphoma LCA+ LCA – Carcinoma 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 CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population CK-,vim+ Lymphoma Melanoma/ 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 CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population CK-,vim+ Lymphoma Melanoma/ 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 + CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population 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 + – CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

Basic panel for evaluation by SCIP (vimentin, PanCK, LCA, Calretinin, WT-1) Without ‘ second-foreign ’ population With ‘ second-foreign ’ population 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 CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

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 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 CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

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 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 CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

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 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 Calret, D2-40, EMA/HBME-1: Microvillous pattern B72.3 – , BerEP4– CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

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 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 Calret, D2-40, EMA/HBME-1: Microvillous pattern B72.3 – , BerEP4– CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

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 Calret, D2-40, EMA/HBME-1: Microvillous pattern B72.3 – , BerEP4– CK+,vim –/+ Immunocytochemistry of effusion fluids Summary

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Shidham- Immunohistochemistry in Serous Fluid Cytopathology

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