Immunohistochemistry on Limited Tissue Samples: Do s and Don ts

Disclosures Immunohistochemistry on Limited Tissue Samples: Do s and Don ts I fretted over this... Andrew M Bellizzi, M.D. Department of Pathology University of Iowa Hospitals and Clinics andrew bellizzi@uiowa.edu Disclosures Last night I was sleepless... in Seattle 1

Disclosures Otherwise, I have nothing to disclose Goals To make an accurate, specific diagnosis with as few immunostains as possible To provide (prognostic and) predictive info To avoid less useful immunostains To perform clinically valid immunohistochemistry To triage tissue for other ancillary studies Outline Competing interests: other ancillary studies Technical aspects of immunocytochemistry Enemies of the state: non specific immunos Next generation immunohistochemistry Favorite markers/panels Carcinoma of unknown origin Lung adenocarcinoma vs. squamous cell carcinoma Mesothelioma Solid pancreatic tumors Sarcoma Lymphoma 2

63 year old woman with liver, lung, and adrenal masses; presumed lung primary based on FNA of liver lesion 4 months prior; EGFR/ALK/ROS1 wild type; progression on platinumbased chemotherapy; biopsy for cancer mutation profiling TTF 1 3

Taher, Thanks for raising this issue in the setting of our increasingly engaging in this era of personalized medicine. This was my case, as staff pathologist. I ordered the TTF 1 because I believed it was necessary to the diagnosis. In fact, in this case, it was negative. I had already ordered it BEFORE your call to the hot seat. So, there was no communication breakdown. I was aware of the prior diagnosis. I read your note while I was evaluating the glass slides. I was aware that the primary driver of this biopsy was to perform NGS. There was actually much more tumor present in the immunostained slide than on the initial H&E, fortunately. I was pessimistic of the prospects for NGS success based on the initial H&E. I was acutely sensitive of the purpose of this biopsy, which is why I limited myself to ONE critical immunostain. Taking one 5 micron section off the tissue block (for IHC) DOES NOT compromise the ability to perform molecular, especially if the sections for molecular are taken simultaneously. The anatomic pathologist is first and foremost responsible that a correct diagnosis is rendered. So, in this case I have to reasonably reassure myself that this is, in fact, metastatic lung adenocarcinoma. In this instance, both purposes should be served. Your note on the consult sheet WAS CRITICAL. If you hadn't made this comment, I would have performed a more extensive immunopanel. As always, communication is key. I would be happy to discuss this issue at greater length or in person. Best, Andrew Tension Between Diagnosis and Predictive Testing is Increasing Use of IHC in pre op lung cytologic specimens Study Period Diagnosis of AdCA IHC Frequency >600% increase in IHC utilization Diagnosis of SCC IHC Frequency 2000 2004 14% (22/156) 11% (5/46) 2005 2010 86% (134/156) 89% (41/46) Ocque R, Tochigi N, Ohori P, Dacic S. Am J Clin Pathol 2011;136:81 7. Ancillary Tests in FNA/Small Biopsy Tumor Type Standard Applications Extended Applications Breast Cancer ER, PR, HER2 Esophagus/Gastric AdCA HER2 Oropharygeal SCC and p16 NGS or HPV ISH Head and Neck SCC of Occult Origin Lung Adenocarcinoma EGFR, ALK ROS1, PD L1 RET, MET, LKB1, PTEN Multigene Colon Cancer KRAS, NRAS, BRAF, MSI PIK3CA, PTEN, EGFR CN Mesothelioma p16 FISH Thyroid Aspirate BRAF, KRAS Pancreatic Cyst Aspirate Cyst fluid analysis KRAS, DNA content Panel Melanoma BRAF KIT Sarcoma FISH or RT/PCR (for specific diagnosis) Hematolymphoid Flow cytometry, IgH/TCR gene rearrangements, FISH (for diagnosis or prognosis) 4

Aisner DL, Sams SB. Diagn Cytopathol 2012;40:511 24. What s Old is New Gailey MP, et al. Cancer Cytopathol 2015. 123;30 9. Knoepp SM, Roh MH. Cancer Cytopathol 2013. 121:120 8. Snow AM, et al. BMC Clin Pathol 2014. 14;30. Hunt JL, et al. Diagn Cytopathol 1998;18:377 80. 5

Lessons Learned Do consider every biopsy of a tumor to be a potential molecular diagnostics specimen Do consider cutting unstained sections up front for molecular testing, if ordering IHC Do communicate with your clinical colleagues about priorities for the specimen Do not give up hope if you ve exhausted a specimen: you may be able to recover DNA from routinely processed material FNA of a Mural Based Gastric Mass Cell block KIT KIT and DOG1 Intensity by FNA Method DOG1 KIT on subsequent resection Cell blocks of 52 GIST, 24 LMS, 10 other No significant difference in number of cells in EUS vs CT cellblocks EUS FNA: FNA collected into CytoLyt (methanol based fixative) CT FNA: FNA collected into RPMI plasma and thrombin added to produce cell block, fixed in 10% formalin, processed for paraffin embedding Hwang DG, et al. Am J Clin Pathol 2011;135:448 53. 6

The staining of cytologic preparations with a battery of antibodies to the various cell components or products is very costly and rarely rewarding... The results of ICC vary according to the batch of antibodies and the technical skills of the laboratories, the interpretation of the results is not always easy, and the problem with borderline positive stains is often perplexing. We This Might Be So... Surgical Pathologists order IHC on FFPE tissue Cytopathologists may request IHC on: Direct Smears Air dried Unfixed Post fixed Alcohol fixed Unstained Stained Destained Cytospins Monolayer Preparations Cell Blocks Koss LG. The future of cytology. The Wachtel lecture for 1988. Acta Cytol 1990;34:1 9. Cell Block Mandelbaum FS. Diagnosis of malignant tumors by paraffin sections of centrifuged exudates. J Lab Clin Med. 1917; 2:580. Cell Blocks: Advantages and Disadvantages Advantages Processed similarly to surgical pathology material Facilitates multiple sections Easy to store Disadvantages Not amenable to on site adequacy assessment May be pauci/acellular 10.6% of 246 lung/thoracic FNA s (Rafael OC, et al 2014) 57% of 76 consecutive EBUS FNA s (Knoepp, Roh 2013) 7

Performance in Other Cytology Specimen Types Potential Sources of Pre Analytic Variation Delay in fixation Type, concentration, ph of fixative Fixation time Tissue Processing Paraffin impregnation (paraffin melting point) Block/slide storage Engel KB, Moore HM. Arch Pathol Lab Med 2011;135:537 43. Potential Sources of Analytic Variation Antigen retrieval Yes or no Enzyme or heat induced For heat induced: buffer, ph, heat source Primary antibody dilution Duration of primary antibody incubation Detection chemistry (ABC, polymer based) Optimization and Validation Optimization: determination of provisional assay conditions, which is most often involves staining a single case or small number of cases at varying assay conditions Validation: testing and appropriate tissue set to determine analytic sensitivity and specificity, to reasonably assure that the test performs as expected Goldsmith J. CAP Today Q&A. July 13, 2013. Goldsmith J. CAP Today Q&A. July 13, 2013. Fitzgibbons PL, et al. Arch Pathol Lab Med 2014;138:1432 43. 8

Recommendation Tissue should be fixed in 10% neutral ph, phosphate buffered formalin for a minimum of 8 hours. Non formalin based fixatives and or alternative fixation methodologies are strongly discouraged in regard to IHC, in large part because performance data are limited and extrapolation from formalin fixed data is unreliable. Goldstein NS, et al. Appl Immunohistochem Mol Morphol 2007;15:124 33. Recommendation Antigen retrieval (AR) is presumed to restore the antigenicity after the formalin fixation. The parameters of an AR protocol must be balanced to match the unique length and type of tissue fixation of the individual laboratory and the characteristics of the individual antibody. Antigen Retrieval Air dried smears theoretically should require (less or) no AR Alcohol fixed smears (coagulation, rather than cross linking, fixation) theoretically should require (less or) no AR Over AR produces high background staining and strong edge staining Under AR produces false negative IHC 9

Recommendations Laboratories must validate all IHC tests before placing them into clinical service For initial analytic validation of nonpredictive factor assays, laboratories should test a minimum of 10 positive and 10 negative tissues Fitzgibbons PL, et al. Arch Pathol Lab Med 2014;138:1432 43. Recommendations If IHC is regularly done on cytologic specimens that are not processed in the same manner as the tissues used for assay validation (eg, alcohol fixed cell blocks, air dried smears, formalin postfixed specimens), laboratories should test a sufficient number of such cases to ensure that assays consistently achieve expected results... The strength of evidence was inadequate to address the criteria and number of samples needed for validation with cytology specimens. If an assay has not been fully validated on cytologic specimens, laboratories may include a disclaimer in their report that results should be interpreted with caution. Controls Unless the laboratory has a large bank of similarly prepared cytology material for positive and negative IC controls (nonformalin fixed cytology specimen by the exact same preparation method as the current sample being tested), then criteria for having proper controls is not met and any interpretation of IC results should be suspect. Fowler LJ, Lachar WA. Arch Pathol Lab Med 2008;132:372 83. 10

Controls In our experience, unstained direct smears in high quantities can be prepared using centrifuged cellular material for effusion specimens and these can be used for positive control ICC reactions. Multiplexing Double staining, triple staining, etc. Performing a second IHC on a previously IHCstained slide (if negative) Knoepp SM, Roh MH. Cancer Cytopathol 2013. 121:120 8. Dabbs DJ, Wang X. Diagn Cytopathol 1998;18:166 9. Lessons Learned Do perform ICC on cell blocks, if possible Do examine cell block technique if ICC performing suboptimally Do consider including cytology specimens in clinical IHC validations Do include similarly processed positive controls on ICC runs Do not perform ICC on other cytology specimen types unless the procedure has been specifically optimized for them (esp. antibody dilution, antigen retrieval) 11

Myths CA125 is specific for Müllerian origin CA19 9 is specific for pancreatic origin CK19 is specific for pancreatobiliary origin MOC 31 is an adenocarcinoma marker RCC is specific for renal origin Vimentin is specific for sarcoma CA125 Expression Tumor Site (adenocarcinoma unless otherwise noted) Ovary 98 Endometrium 90 Cervix 53 Pancreas 82 Gallbladder 63 Lung 56 Cholangiocarcinoma 51 Head and Neck (SCC) 40 Thyroid 39 Breast 27 Lung (SCC) 23 Melanoma, lymphoma, sarcoma, 0 neuroendocrine, hepatocellular % Positive (at 10% cells staining with moderate intensity) Gremel G, et al. Histopathology 2014;64:293 305. CA19 9 Expression Tumor Site (n) % Positive (adenocarcinoma unless otherwise noted) Pancreas (26) 85 Breast, ductal (119) 48 Breast, lobular (10) 10 Lung (35) 69 Stomach (39) 56 Colon (25) 76 Ovary (29) 41 Kidney (45) 27 Mesothelioma <<1 Kaufmann O, et al. Histopathology 1996;29:233 40. 12

CK19 Expression Tumor Site (adenocarcinoma unless otherwise noted) Cholangiocarcinoma 100 Pancreas 100 Gallbladder 100 Lung 100 Ovary 98 Breast 98 Colon 98 Stomach 98 Urothelial 95 Cervix 93 Lung (SCC) 91 Melanoma, lymphoma, hepatocellular 0 % Positive (at 10% cells staining with moderate intensity) Gremel G, et al. Histopathology 2014;64:293 305. MOC 31 MOC 31 Expression Dx Algorithm: Poorly Differentiated Carcinoma in the Liver Tumor Type (n) % Positive Squamous cell carcinoma (110) 41 Mesothelioma (10) 0 Hepatocellular carcinoma (10) 20 Neuroendocrine carcinoma (5) 60 Lung adenocarcinoma (6) 100 Colon adenocarcinoma (6) 100 Urothelial carcinoma (11) 55 Neuroendocrine tumor, midgut (5) 100 Neuroendocrine tumor, pancreas (5) 80 Savage EC, Gailey MP, Bellizzi AM. Abstract at 2014 USCAP Annual Meeting. HCC vs. ICC vs. Metastasis Perform Hep Par 1, MOC 31 (alternatively Arginase 1, Claudin 4) Hep Par 1 (+)/ MOC 31 ( ) HCC Hep Par 1 ( )/ MOC 31 (+) ICC or Metastasis IHC menu for this application: HCC Markers: Hep Par 1 GPC3 pcea/cd10 (canalicular) Arginase 1 Non HCC Markers: MOC 31 Claudin 4 13

Hepatocellular carcinoma 73 year old man with liver lesion, subtotally necrotic Hep Par 1 MOC 31 Large right retroperitoneal tumor 14

13 cm tumor spanning the kidney and adrenal, expresses CD10, melan A, and inhibin PAX8 vs PAX2 Expression Tumor Type PAX8 (% Positive) PAX2 (% Positive) Clear cell 97 95 Papillary 100 76 Chromophobe 88 56 Collecting duct 71 43 Serous 98 55 Endometrioid 94 25 Clear cell 100 19 Transitional 67 11 Thyroid 91 0 PAX8 Ozcan A, et al. Am J Surg Pathol 2011;35:1837 47. Ozcan A, et al. Arch Pathol Lab Med 2012;136:1541 51. Laury AR, et al. Am J Surg Pathol 2011;35:816 26. 15

Melanoma Vimentin Burkitt lymphoma Vimentin Endometrioid Endometrial vs. Endocervical AdCA Endometrioid Endometrial (n=30: ER, vim, CEA) (n=29: p16) Endocervical (n=26: ER, vim, CEA) (n=23: p16) ER 93% 38% Vimentin 97% 8% CEA 70% 96% (usually in squamous foci) p16 97% (any staining) 27% ( 50% cells stain) 10% (100% cells stain) 96% (any staining) 96% (100% cells stain) McCluggage WG, et al. Int J Gynecol Pathol. 2002 Jan;21(1):11 5. McCluggage WG, Jenkins D. Int J Gynecol Pathol. 2003 Jul;22(3):231 5. Results of immunopanel favor endometrial origin Lessons Learned Uterine cervical mass ER, PR Do not extrapolate the results of differentialspecific markers beyond the differential (e.g., CK19, MOC 31) Do not order CA125, CA19 9, RCC (No No Never) Vimentin??? If you must... sparingly... But please don t tell me that you did... Vimentin p16 16

Next Generation Immunohistochemistry Gene Expression Profiling Comparing Urothelial, Kidney, and Prostate Cancer Mine developmental biology and molecular genetic literature to find: Lineage restricted transcription factors Markers identified by gene expression profiling Protein correlates of molecular genetic events Bottom line: our markers keep getting better Higgins JPT, et al. Am J Surg Pathol. 2007 May;31(5):673 80. Historically, diagnostic armamentarium geared toward cytoplasmic or membranous differentiation markers; reduced sensitivity in poorly differentiated tumors Primacy of lineage restricted transcription factors Breast cancer Mammaglobin GCDFP 15 GATA 3: highly expressed, regardless of differentiation 17

Regional Differential Diagnosis Brain (carcinoma, melanoma, lymphoma, glioma) Lymph node (lymphoma vs. other) Supraclavicular (anything), Periaortic (germ cell tumor) Mediastinum (lung, lung, lung, thymic [KIT,CD5], germ cell) Visceral organ (1 vs metastasis) Lung (AdCA vs SCC vs metastasis) Liver (HCC vs pancreatobiliary vs other) Pleural effusion (AdCA vs mesothelioma) Peritoneum/Ovary (1 surface epithelial vs metastatic GI) Retroperitoneum (well and dedifferentiated liposarcoma) Somatic soft tissue (sarcoma vs. metastasis) Bone (blastic prostate, breast; lytic RCC, thyroid; mixed lung) Diagnosis of Broad Tumor Class Carcinoma (broad spectrum keratins, EMA [aka MUC1]) Melanoma (S 100, SOX10, melan A, HMB 45, MiTF, BRAF) Lymphoma (CD45; CD43, CD79a, MUM1, ALK1, CD30 if LCA ) Sarcoma (CD34, MDM2/CDK4 DDLPS, add l based on morph.) Mesothelioma (WT 1, calretinin, CK5/6, D2 40) Germ cell tumor (SALL4, PLAP) Non epithelial neuroendocrine neoplasm (CG, SYN) Adenocarcinoma (gland forming/ mucin producing) see next algorithm SCC CK7 var. GATA3? Diagnosis of Carcinoma Type UC CK7/CK20+ GATA3+ Squamotransitional (p63, p40, CK5/6+) Carcinoma (cohesive, keratin and/or EMA+) HCC Hep Par 1 + GPC3+ Large polygonal cell (CK7/CK20 ) RCC PAX8+ AdCC Mel A+ Inhibin+ SYN+ SF1+ Neuroendocrine (CG and/or SYN+) NET Ki 67 20% NEC Ki 67 >20% Which Screening Keratin Should I Use? Clone 1 2 3 4 5 6 7 8 9 1 0 AE1/AE3 X X X X X X X X X X X X OSCAR X X X X MAK 6 X X X X X X MNF116 X X X X CAM5.2 X X KL1 X X X X X X X X X X X 34βE12 X X X X In general, any of these are acceptable It s not the number of keratins, per se, but rather the affinity (e.g., CAM5.2 vs AE1/AE3 in HCC/RCC) Stratified epithelia: K1 6, 9 17 Simple epithelia: 7, 8, 18, 19, 20 1 1 1 2 1 3 Ordóñez NG. Hum Pathol. 2013 Jul;44(7):1195 215. 1 4 1 5 1 6 1 7 1 8 1 9 18

Coordinate Expression of CK7/CK20 Site (tumor) CK7 CK20 Prostate, HCC, AdCC, RCC - - Lung, Breast, Müllerian, Pancreatobiliary (PB), Upper GI (UGI) + - Bladder, UGI, PB, Mucinous Ovarian, Colon (esp. Rectum), Occ. Lung + + Colon, Merkel cell, Occ. UGI - + Cancer Epidemiology and Morphology Based Site of Origin Generator Most Common Primary Sites in Men (Rank Order) Prostate (25%) (AdCA) Lung (15%) (AdCA, SCC, NEC) Colorectum (10%) (AdCA) Morphology Characteristic nuclear features regardless of Gleason grade: monomorphous and prominent nucleoli Most Common Primary Sites in Women (Rank Order) Breast (26%) (AdCA) Variable (AdCA) Lung (14%) (AdCA, SCC, NEC) Characteristic cytoarchitectural features: tall, dark, and dirty Colorectum (10%) (AdCA) Morphology Variable Variable (AdCA) Characteristic cytoarchitectural features: tall, dark, and dirty IHC to Assign AdCA Site of Origin: Ranked from Most to Least Ordered (2011) CDX2 (enteric differentiation) TTF 1 (lung, thyroid) ER and PR (breast, Müllerian) PAX8 (kidney, Müllerian, thyroid) Transcription factors in bold p53and WT 1 (serous carcinoma, latter also exp. by mesothelioma) Napsin A (lung, papillary RCC) PSA and PSAP (prostate) GCDFP 15 and mammaglobin (breast) Thyroglobulin (thyroid) GATA 3 (breast; also urothelial) NKX3.1 (prostate) Lung Adenocarcinoma vs Squamous Cell Carcinoma Marker AdCA (% positive) CK7 100 68 TTF 1 86 21 p63 31 100 CK5/6 21 100 SCC (% positive) p63, CK5/6 have a specificity problem TTF 1 has a sensitivity problem Ocque R, Tochigi N, Ohori P, Dacic S. Am J Clin Pathol 2011;136:81 7. 19

p40 (ΔNp63) Napsin A Marker AdCA (% positive) SCC (% positive) p63 31 100 54 p40 3* 100 0 * Each 1 5% cells staining TTF 1+/p40 AdCA TTF 1 /p40 AdCA Bishop TTF 1 /p40+ JA, et al. Mod Pathol 2012;25:405 15. SCC Large Cell Lymphoma (% positive) Marker Lung AdCA (% positive) Napsin A 87 (n=303) SCC (% positive) 3 (n=200) TTF 1 64 (n=94) 2 (n=94) Lower rate of TTF 1+ AdCA than literature (75 85%) Napsin A frequently expressed by RCC (esp. papillary) and clear cell carcinoma Turner BM, et al. Arch Pathol Lab Med 2012;136:163 71. Soft Tissue: IHC to Assign Lineage Line of Differentiation Characteristic Historically Useful Immunostains Adipocytic Fatty S 100 Fibroblastic/myofibroblastic Spindled; light pink SMA, CD34, ALK So called fibrohistiocytic Shrinking group (e.g., TSGCT) None especially Smooth muscle Spindled; brightly eosinophilic SMA, desmin Pericytic Spindled; circumferential SMA perivascular growth pattern Skeletal muscle Rhabdomyo(sarco)ma Desmin, myogenin Vascular Vascular channels CD34, CD31, Factor VIII Chondro osseous Cartilage, osteoid S 100 Interstitial cell of Cajal GIST KIT, CD34 Nerve sheath Spindle cell; wavy nuclei S 100, GFAP Uncertain Distinctive tumor types (e.g., synovial sarcoma, epithelioid sarcoma, PEComa) Keratins, EMA, S 100, (TFE3, HMB 45, WT 1) Undifferentiated/unclassified Formerly MFH Diagnosis of exclusion Name Beta catenin Soft Tissue Markers Validated at U of Iowa in Last 3 Years MDM2 and CDK4 (MDM2 FISH) TLE1 MUC4 ERG STAT6 HHV8 Diagnostic Application Desmoid fibromatosis (70%), solid pseudopapillary neoplasm, pancreatoblastoma; hepatoblastoma (50 90%), fetal type lung AdCA, colonic adenoma/adca Well and dedifferentiated liposarcoma Synovial sarcoma; Pitfalls: weaker staining in other sarcomas, frequently positive in carcinomas Low grade fibromyxoid sarcoma, sclerosing epithelioid fibrosarcoma (75%); also broadly expressed by epithelia Vascular tumors,prostate cancer (50%); occ. myeloid leukemias and Ewing sarcoma (10%) Solitary fibrous tumor (NAB2 STAT6 translocation) Kaposi sarcoma, primary effusion lymphoma, multicentric Castleman disease, plasmablastic lymphoma arising in plasmablastic lymphoma 20

Name Soft Tissue Markers Validated at U of Iowa in Last 3 Years Diagnostic Application SOX10 INI1 Melanoma, MPNST (50%), clear cell sarcoma, myoepithelial differentiation; superior in sensitivity and specificity to S 100 Epithelioid sarcoma (90%), malignant rhabdoid tumors of soft tissue, kidney, CNS (>95%), medullary carcinoma of the kidney, other INI1 deficient tumors (some epithelioid MPNST, myoepithelial CA of soft tissue, extraskeletal myxoid chondrosarc.) Burkitt lymphoma DLBCL c Myc c Myc Advantages of ICC over Flow Cytometry Hodgkin lymphoma Burkitt lymphoma vs DLBCL, GC Type BL: c Myc+, Bcl 2 DLBCL: c Myc, Bcl 2+ or CD20 Tumor Type CD79a PAX5 Tumors composed of small cells Extranodal marginal zone lymphoma of mucosa associated lymphoid tissues (MALT lymphoma) CD10 Bcl 6 CD5 + CD43 Cyclin D1 Bcl 2 Occ. (30%) Var. Mantle cell lymphoma + + + + + Other Useful Markers/Notes Kappa/lambda light chain restriction (occ.); keratins to highlight lymphoepithelial lesions Ki 67 proliferation index >40 60% associated with poor prognosis; rarely CD5 or cyclin D1 Follicular lymphoma + + + Higher grade tumors more likely to show aberrant IHC (e.g., CD10 or CD43+) Chronic lymphocytic leukemia/small lymphocytic lymphoma + + + + CD23+ 21

Tumor Type CD20 CD79a PAX5 CD10 Bcl 6 Tumors composed of intermediate/large cells Diffuse large B cell lymphoma + CD10 Var. (30 60%) Bcl 6+ (60 90%) CD5 CD43 Cyclin D1 Bcl 2 Other Useful Markers/Notes Rare (10%) Occ. (25%) Var. MUM1 (35 65%) Burkitt lymphoma + + + Plasmablastic lymphoma Granulocytic Sarcoma CD20/PAX5 CD79a+ (50 85%) + + + Ki 67 proliferation index approaches 100%; TdT ; c Myc+; Bcl 2 occ. weak + EBV EBER (60 75%); CD45 ; MUM1/CD138/CD38+; EMA/CD30 var.; association with HIV + CD68/KIT/CD99/ CD34/TdT Var. Hematolymphoid Markers Validated at U of Iowa in Last 2 Years Name Tryptase CD163 c MYC SOX11 Diagnostic Application Mast cells Monocyte macrophage lineage marker (more specific than CD68) Burkitt lymphoma and c MYC activated DLBCL Mantle cell lymphoma (w/ emphasis on identifying CycD1 cases) B lymphoblastic lymphoma CD20 Var. (25 50%) CD79a/PAX5+ CD10+ (60%) Bcl 6 + (67%) + TdT (95%), CD34/CD99+; CD13/CD33 Occ. T lymphoblastic lymphoma CD20/ PAX5 CD79a Rare (5 10%) CD10 Var. Bcl 6 Var. + (90%) + TdT (95%), CD3/CD99+; CD4/CD8 double+ (70%); CD1a (67%); CD34 Var.; CD13/CD33 Occ. Lessons Learned Do perform a limited panel of IHC to assign tumor type/site of origin based on clinical and morphologic differential ( big 3 + appropriate next gen markers) Do not equate p63+ with SCC; do consider p40 Do consider using next gen markers to make specific sarcoma and lymphoma diagnoses Thank you!!! 22

U of Iowa Development Queue Name Diagnostic Application Androgen receptor Sebaceous CA, salivary duct CA, prostate CA; Pitfall: low specificity Arginase Hepatocellular CA; Pitfall: occ. stains AdCA Claudin 4 Broad spectrum epithelial marker not expressed by hepatocytes/meso s Clusterin Follicular dendritic cell sarcoma, ALCL, tenosynovial GCT, non ileal NET D2 40 Mesothelioma (inc. sarc.), lymphatic endothelium, seminoma, others Glutamine synthetase Focal nodular hyperplasia, BCAT HA, HCC vs. B9 liver: Pitfall: other CAs + Mammaglobin Breast CA;surrogate for ETV6 FISH in mammary analogue secretory CA NKI C3 (CD63) Cellular neurothekeoma; + in many other tumors inc. melanoma NUT NUT midline carcinoma (undifferentiated CA with abrupt keratinization) p40 (ΔNp63) Squamous cell carcinoma (superior spec. to p63); basal cells and myoep s SATB2 Colon AdCA; osteoblastic differentiation; rectal NET SDHB SDHB deficient GIST, paraganglioma/pheochromocytoma, other SMAD4 Pancreatic ductal AdCA (50% demonstrate loss); midgut NET (40%) SF1 Adrenal cortical neoplasms, sex cord stromal tumors Diagnosis of Broad Tumor Class Morphologic Boxes Cohesive, poorly cohesive, dyscohesive Spindle cell (sarcoma, sarcomatoid carcinoma) Pleomorphic (anything) Round cell (lymphoma, sarcoma) Epithelioid (carcinoma, melanoma) Pleomorphic and High Grade are NOT Synonymous Monomorphous or pleomorphic 23

Clear cell sarcoma Melanoma Monomorphous Pleomorphic CDX2 homogenous: LGI CDX2 heterogenous: UGI, mucinous ovarian, PB (20%) NET Site of Origin Algorithm CK20 diffuse, strong: LGI CK20 weak, patchy: UGI, mucinous ovarian, PB ( 40%) Maxwell JE, Sherman SK, Stashek KM, O Dorisio TM, Bellizzi AM, Howe JR. Surgery. 2014 Dec;156(6):1359 66. 24

Midgut NETs have a characteristic histologic appearance and nearly always express CDX2 Pancreatic NETs are of diverse histologic appearance and nearly always express ISL1 Merkel cell carcinoma CK20+/TTF 1 ( 90% Merkel cell carcinoma Small cell lung carcinoma TTF 1+/CK20 ( 90% SCLC, 45% visceral NEC) Transcription Factor Infidelity in NEC Frequently Expressed Transcription Factors (%) Merkel Cell Small Cell Extrapulmonary Carcinoma Lung Carcinoma Visceral NEC (n=40) (n=24) (n=19) FLI1 90 88 78 GATA3 24 8 40 Islet 1 97 88 58 Myc 29 25 61 PAX6 78 29 26 PLAG1 58 79 72 SATB2 63 42 89 SOX2 100 96 89 TTF1 5 96 44 NECs expressed a median of 8 TFs (range 0 18) out of 38 examined Czeczok TW, Gailey MP, Hornick JL, Bellizzi AM. Mod Pathol. 2014 Feb;27(S2):152A. 25

76 year old man with past tobacco use and asbestos exposure p/w pleural effusion; at thoracotomy diaphragm encased by nodular fibrous tissue; multiple plaque like areas throughout pleural cavity Pan keratin Calretinin, WT 1, CK5/6, MOC 31, Ber EP4, STAT6 Mesothelioma Marker Expression in Sarcomatoid Mesothelioma AE1/AE3 75% (18/24) CAM5.2 96% (23/24) MNF 116 100% (21/21) Calretinin 25% (6/24) WT 1 33% (8/24) D2 40 100% (24/24) Chirieac LR, et al. Am J Cancer Res. 2011;1(1):14 24. All of these were prospectively dx d as pancreatic neuroendocrine tumor... only one of them is 26

Cellular Epithelioid Neoplasms of the Pancreas Solid neoplasms composed predominantly of neoplastic elements with little stroma Pancreatic neuroendocrine tumor (PNET) Solid pseudopapillary neoplasm (SPN) Acinar cell carcinoma (ACC) Pancreatoblastoma (PB) On cytology: dyshesive, monomorphic SPN: 3 of 6 misdiagnosed as PNET (Bardales et al 04) ACC: 2 of 4 misdiagnosed as PNET (Stelow et al 06) ACC: 14/29 misdiagnosed, 5 as PNET, 5 as ductal AdCA (Sigel et al 13) Immunophenotype of Cellular Epithelioid Neoplasms of the Pancreas Broad Spectrum Keratins PNET SPN ACC PB + 70% + + Synaptophysin 95% 20 70% Rare cells 80% Chromogranin 90% Rare cells 80% Trypsin Rare cells 95% 95% β catenin <5% >95% 10% >90% BCAT Trypsin BCAT Trypsin Core biopsy from a 40 cm retroperitoneal tumor demonstrates undifferentiated neoplasm composed of sheets of epithelioid cells. After performing 17 immunostains a diagnosis of malignant neoplasm indeterminate for sarcoma, carcinoma, or lymphoma was rendered. 27

Most consistent with dedifferentiated liposarcoma Resection of similar case Well differentiated component MDM2, CDK4 Abrupt transition Dedifferentiated component Keratin and/or EMA positivity do not assure a diagnosis of carcinoma Potential Pitfall #1 28

Leiomyosarcoma Smooth muscle actin, desmin+ Keratin AE1/AE3+: keratin, EMA expression in 30 40% LMS 33 year old woman with 1 year h/o R hip pain; large SQ mass EMA+, AE1/AE3+, GATA 3 focal + p63, CK5/6, ER, PR, TTF 1, WT 1, CD31, S 100, HMB45 all Conclusion: Favor metastatic carcinoma,? breast or urothelial 29

INI1: absent expression epithelioid sarcoma, proximal type 63 year old man with increasing hip pain x 1 month; proximal femur lesion with soft tissue extension Undifferentiated Malignant Neoplasm with Osteoclast like Giant Cells Undifferentiated/anaplastic carcinoma Keratin AE1/AE3 CDX2, PAX8, TTF 1 Osteosarcoma SATB2 Leiomyosarcoma Desmin, SMA, caldesmon Keratin AE1/AE3+ (desmin, SMA ) Conclusion: Favor undifferentiated carcinoma 30

Musculoskeletal radiologist sug. presence of chondroid matrix (MRI) Subsequent femoral head resection for pathologic fracture Abrupt transition from WD chondrosarcoma to undifferentiated neoplasm dedifferentiated chondrosarcoma Keratin Positive Soft Tissue Tumors EMA Positive Soft Tissue Tumors Chondroid lipoma Pleomorphic liposarcoma Desmoplastic fibroblastoma Solitary fibrous tumor Inflammatory myofibroblastic tumor Myxoinflammatory fibroblastic sarcoma Leiomyosarcoma Rhabdomyosarcoma Schwannoma (cross reactivity with GFAP) Epithelioid hemangioma Pseudomyogenic hemangioendothelioma Epithelioid hemangioendothelioma Angiosarcoma Gastrointestinal stromal tumor Sclerosing perineurioma Dermal nerve sheath myxoma Epithelioid MPNST Ectopic hamartomatous thymoma Ossifying fibromyxoid tumor Myoepithelial tumors of soft tissue Synovial sarcoma Epithelioid sarcoma Desmoplastic small round cell tumor Extrarenal rhabdoid tumor Undifferentiated/unclassified sarcoma Chondroblastoma Dedifferentiated chondrosarcoma Conventional osteosarcoma Ewing sarcoma Chordoma Adamantinoma Osteofibrous dysplasia Pleomorphic liposarcoma Calcifying aponeurotic fibroma Lipofibromatosis Dermatofibrosarcoma protuberans Solitary fibrous tumor Low grade fibromyxoid sarcoma Sclerosing epithelioid fibrosarcoma Leiomyosarcoma Pleomorphic rhabdomyosarcoma Epithelioid hemangioma Epithelioid hemangioendothelioma Angiosarcoma Neurofibroma Perineurioma Dermal nerve sheath myxoma Solitary circumscribed neuroma Meningioma Hybrid nerve sheath tumor Acral fibromyxoma Angiomatoid fibrous histiocytoma Myoepithelial tumors of soft tissue Synovial sarcoma Epithelioid sarcoma Desmoplastic small round cell tumor Extrarenal rhabdoid tumor Undifferentiated/unclassified sarcoma Conventional osteosarcoma Chordoma Epithelioid hemangioma Adamantinoma 31

Dr. Bellizzi, what kind of S 100 positive carcinoma is this? 78 year old man p/w word finding difficulty: L temporal lobe mass Pan keratin S 100 S 100 Expression in Adenocarcinoma Primary Tumors Metastatic Tumors Salivary gland 80% (n=15) 75% (n=4) Lung 7% (n=27) 12% (n=25) Breast 60% (n=20) 62% (n=8) Esophagus 0% (n=8) 0% (n=2) Stomach 20% (n=10) 25% (n=8) Gallbladder 0% (n=1) 0% (n=1) Colorectum 25% (n=28) 23% (n=13) Pancreas 0% (n=8) 0% (n=5) Kidney 65% (n=23) 66% (n=3) Endometrium 78% (n=36) 64% (n=14) Ovary 84% (n=24) 87% (n=22) Prostate 0% (n=27) 0% (n=8) Unknown origin 22% (n=9) Total 43% (n=228) 39% (n=122) Herrera GA, et al. Am J Clin Pathol. 1988 Feb;89(2):168 76. Melan A (A103) MiTF HMB 45 Conclusion: metastatic melanoma BRAF mutation testing: wild type 32

Keratin Positivity in Melanoma Primary cutaneous melanoma (n=62) Vimentin 100% 100% S 100 95% 95% NSE 87% 77% HMB45 97% 64% NKI C3 97% 95% Cytokeratin (KL1, CAM5.2, 35βH11) 0% 23% Achilles E, Schröder S. Pathologe. 1994 Aug;15(4):235 41. Recurrent or metastatic melanoma (n=22) Keratin Positivity in Lymphoma Anaplastic large cell lymphoma (5/18; 28%) 1 13/866 (1.5%) hematolymphoid tumors in TMA 2 5/18 (28%) mantle cell lymphomas Otherwise, case reports and small series 1. Gustmann C, et al. Am J Pathol. 1991 Jun;138(6):1413 22. 2. Adams H, et al. Pathol Res Pract. 2008;204(8):569 73. EMA positivity in Hematolymphoid Neoplasms Anaplastic large cell lymphoma (50 95%) Plasma cell neoplasms (most) Diffuse large B cell lymphoma TC/HR, ALK+, plasmablastic, primary effusion lymphoma T cell lymphoma (20%) Nodular lymphocyte predominant Hodgkin (L&H) Follicular dendritic cell sarcoma (var.) Beware of LCA negative hematolymphoid neoplasms Potential Pitfall #2 33

LCA Negative Hematolymphoid Neoplasms Acute megakaryoblastic leukemia B cell acute lymphoblastic leukemia/lymphoma ALK positive large B cell lymphoma EMA+, CK /+; CD20, CD79a Plasmablastic lymphoma (EBER usually +) EMA+/ ; CD20, PAX5 Anaplastic large cell lymphoma, ALK positive (some) Classical Hodgkin lymphoma Follicular dendritic cell sarcoma 34