Thomas M. Ulbright, MD Daniel M. Berney, FRCP Satish K. Tickoo, MD John R. Srigley, MD

1. 2013 International Society of Urologic Pathology Conference on Best Practices Recommendations in the Application of Immunohistochemistry in Diagnostic Urologic Pathology: The Role of Immunohistochemistry in Testicular Neoplasms • Thomas M. Ulbright, MD • Daniel M. Berney, FRCP • Satish K. Tickoo, MD • John R. Srigley, MD

2. Principles of IHC in Neoplasms of the Testis • There are recurring differential diagnostic situations in testicular neoplasia • IHC is not a diagnostic tool but a differential diagnostic tool • Clinical, serological and light microscopic features are critical to the generation of reasonable and limited numbers of differential diagnoses • Exceptions to expected IHC reactivities are invariable and emphasize the need to correlate IHC with morphologic and clinical features • If a pathologist cannot narrow the differential diagnostic considerations to 2 or 3 entities without IHC, “expert” consultation should be sought

3. IHC is very helpful to resolve between differential diagnostic considerations and should be applied in a conservative fashion, ideally utilizing 2 or 3 immunostains with different patterns of reactivity for the differential diagnoses under consideration Diagnostic algorithms for specific differential diagnostic considerations should therefore be utilized There are currently no clinically utilized prognostic IHC markers for testicular neoplasms. Principles of IHC in Neoplasms of the Testis (con’t)

4. Useful Antibodies for Testicular Neoplasms: OCT4 (OCT3/4, POU5F1) • Nuclear protein critical for pluripotency of embryonic stem cells • ~100% sensitive for IGCNU, seminoma, embryonal carcinoma (EC) • Negative in other testis tumors • Other + tumors: rarely, lung & kidney ca, large cell lymphoma • Caveat: Post-chemo ECs may be negative • Overview: Very valuable for seminoma vs mimics (yolk sac tumor [YST], Sertoli cell tumor [SCT]) and to support Dx of seminoma and EC in Bxs (mets).

5. Useful Antibodies for Testicular Neoplasms: CD117 (cKIT) • Receptor tyrosine kinase in stem cells • Membranous expression occurs in 95-100% of seminomas & IGCNU • Variably + in YSTs and spermatocytic seminomas (SS) • ~ negative in ECs & choriocarcinomas (CC) • Other + tumors: Numerous • Caveat: Spermatogonia may be +; therefore not helpful for IGCNU • Overview: Main utility is assisting with the Dx of seminoma vs EC

6. Useful Antibodies for Testicular Neoplasms: Podoplanin (D2-40, M2A) • Transmembrane mucoprotein expressed on fetal germ cells, lymphatic endothelium & mesothelium • ~100% of IGCNU & seminomas show membranous positivity • YSTs, CCs, SSs, non-neoplastic GCs ~negative • Other + tumors: Numerous (gliomas, meningiomas, mesothelial, lymphatic, adenocas etc.) • Caveat: Up to 30% of ECs show some positivity limited to apical surfaces • Overview: Useful for seminoma vs solid YST (unlike CD117) and EC (beware of apical staining). Better for IGCNU than CD117

7. Useful Antibodies for Testicular Neoplasms: SOX17 (SRY-box 17) • Member of the SOX family of nuclear transcription factors involved in embryonic development • Positive in seminomas (95%), IGCNU (~100%), YSTs (50%), some teratomas • Negative in ECs, CCs & SSs (with limited experience in CC & SS). • Other + tumors: ? • Caveat: Non-neoplastic germ cells are + • Overview: Mostly useful for seminoma vs EC. Not helpful for IGCNU. May become best marker for seminoma vs EC but more experience needed

8. Useful Antibodies for Testicular Neoplasms: CD30 (Ki-1,Ber-H2) • Protein in the TNF receptor family with membranous and Golgi-zone staining • 93-100% of ECs are positive • Negative or, at most, stains rare cells in other GCTs • Other + tumors: lymphomas, soft tissue tumors, melanomas & infrequent carcinomas • Caveats: Intensity in EC can be variable requiring close examination. Loss occurs in some post-Rx ECs. • Overview: Very useful for EC vs seminoma or solid YST.

9. Useful Antibodies for Testicular Neoplasms: AE1/AE3 cytokeratin • Antikeratin cocktail specific for CKs 1-8, 10, 14, 15, 16 & 19. • Generally positive (cytoplasmic) in all non-seminomatous tumors • Usual seminomas are mostly non-reactive, with some positivity in 20-36%, mostly paranuclear & dot-like. • Other + tumors: Numerous (carcinomas & others) • Caveat: Marks trophoblast cells in seminomas, & these cases are still seminomas. • Overview: Widely available Ab mostly helpful to distinguish seminoma vs. EC

10. Useful Antibodies for Testicular Neoplasms: SOX2 (SRY-box 2) • Member of the SOX family of nuclear transcription factors involved in embryonic development; needed forpluripotency of undifferentiated embryonic stem cells • Positive in 96% of ECs & <1% of seminomas • Negative in YSTs, CCs & IGCNU • Other nuclear + tumors: immature elements in teratoma, melanoma & rhabdoid tumors • Caveat: Non-neoplastic Sertoli cells are + • Overview: Mostly useful for seminoma vs EC. May become a preferred marker for seminoma vs EC but the panelists considered it currently technically difficult

11. Useful Antibodies for Testicular Neoplasms: Glypican 3 (GPC3) • Membrane anchored heparan sulfate proteoglycan • Positive in YSTs (100%), CCs (80%), teratomas (“immature”) (17%) & rare ECs (5%) • Negative in IGCNU & seminoma • Other + tumors: : hepatocellular and gastric cancers • Caveats: Syncytiotrophoblast cells are often positive (71%) • Overview: More sensitive but less specific for YST among testis GCTs than AFP

12. Useful Antibodies for Testicular Neoplasms: Alpha-fetoprotein (AFP) • A major plasma protein produced by the yolk sac & liver during fetal life • YSTs are variably and often focally positive (overall, ~80%) • AFP is negative in the other GCTs, except for glands and luminal secretions of some teratomas • Other + tumors: hepatocellular neoplasms, hepatoid carcinomas & occasional other non-GCTs • Caveat: Negative AFP does not exclude YST • Overview: Wide availability & relative YST specificity make it helpful for Dx of YST but has limited sensitivity

13. Useful Antibodies for Testicular Neoplasms: Human Chorionic Gonadotropin (hCG) • A dimeric glycoprotein produced by placental trophoblast cells, mostly syncytiotrophoblasts; α subunit is shared by LH, TSH & FSH but the β is unique • Primary CC is positive (100%) for βhCG as are all non-CCs with syncytiotrophoblast cells • Other + tumors: any non-germ cell tumor with trophoblastic differentiation • Caveat: CCs after Rx may lose syncytiotrophoblasts and show scant to absent reactivity for βhCG • Overview: Useful for supporting Dx of CC but usually not necessary

14. Useful Antibodies for Testicular Neoplasms: Placental Alkaline Phosphatase (PLAP) • An allosteric enzyme in placental trophoblast • + in IGCNU (83-100%) & <1% of non-neoplastic germ cells • + in 90-100% of usual seminomas with a membranous pattern. Most ECs & ~ 50% of YSTs & CCs are + • SS & SCT are negative • Other + tumors: many adenocas (ovary, colon, endometrium, lung) • Caveat: Not a specific GCT marker • Overview: Mostly helpful for IGCNU; useful for usual seminoma vs SS or SCT

15. Immunohistochemistry Algorithm #1 for Testicular Neoplasia: Germ Cell Tumor Subtyping Preferred markers in bold; alternatives in parentheses

16. Proposed ISUP Recommendations:Germ Cell Tumor Subtyping • A reasonable and efficient initial panel is: OCT4, CD117, CD30 & GPC3 • This panel may be reduced depending on the light microscopic differential, for instance omitting OCT4 & GPC3 if the question by morphology is limited to seminoma versus embryonal carcinoma

17. Solid YST with clear cells, potentially mimicking seminoma

18. Solid YST – OCT4 Seminoma – OCT4

19. Solid YST – glypican 3

20. Seminoma & EC

21. Seminoma & EC – CD30

22. Seminoma & EC – CD117

23. Useful Antibodies for Testicular Neoplasms: SALL4 • Zinc finger nuclear transcription factor with role in embryonic development • ~100% sensitive for IGCNU, seminoma, EC & YST; 69% of CCs & 52% of teratomas • Negative in other testis tumors • Other + tumors: ALCL, rhabdoid tumor, Wilms tumor, precursor B-cell ALL, AML & ~ 5% of GI tract adenoca • Caveat: Non-neoplastic germ cells are + • Overview: Sensitive general GCT marker valuable for GCT vs non-GCT of testis and in DX of metastatic GCTs; sensitive YST marker, unlike OCT4

24. Useful Antibodies for Testicular Neoplasms: Inhibin • A dimeric glycoprotein in the transforming growth factor-ß superfamily. Inhibits production or secretion of pituitary gonadotropins. Ab vs the α subunit is used • Positive in sex cord-stromal tumors (SCST); ~100% of Leydig cell tumors (LCT) & 30-91% SCTs • Positive in syncytiotrophoblast cells but GCTs are otherwise negative • Other + tumors: any with trophoblast cells, adrenal cortical, hemangioblastoma, some CCRCCs, rare soft tissue • Caveat: Negative α-inhibin does not exclude SCST • Overview: Helpful positive stain for SCST vs GCT

25. Useful Antibodies for Testicular Neoplasms: Calretinin • A 29 kDa calcium-binding protein of the E-F hand protein family. • Positive in SCSTs, including ~ 100% of LCTs but only a minority of SCTs • GCTs are negative • Other + tumors: mesothelioma, adenomatoid tumors and many others • Caveat: Negative calretinin does not exclude SCST • Overview: Helpful positive stain for SCST vs GCT

26. Immunohistochemistry Algorithm #2a for Testicular Neoplasia: Germ Cell Tumor vs Sex Cord-Stromal Tumor

27. Immunohistochemistry Algorithm #2b* for Testicular Neoplasia: Germ Cell Tumor vs Sex Cord-Stromal Tumor * Alternative algorithm if SALL4 not available

28. Proposed ISUP Recommendations:Germ Cell Tumor versus Sex Cord-Stromal Tumor • A reasonable and efficient initial panel is: SALL4, Inhibin & Calretinin • An alternative panel is OCT4, GPC3, Inhibin & Calretinin

29. Immunohistochemistry Algorithm #3a for Testicular Neoplasia: Germ Cell Tumor vs Large Cell Lymphoma * Some lymphoblastic & ALCLs & myeloid leukemias are SALL4 +.

30. Immunohistochemistry Algorithm #3b* for Testicular Neoplasia: Germ Cell Tumor vs Large Cell Lymphoma * Alternative algorithm if SALL4 not available; †rare large cell lymphomas are OCT4 +

31. Proposed ISUP Recommendations:Germ Cell Tumor versus Large Cell Lymphoma • A reasonable and efficient initial panel is: SALL4, CD45, CD20 & CD3 • An alternative initial panel is OCT4, GPC3, AE1/AE3, CD45, CD20 & CD3

32. Useful Antibodies for Testicular Neoplasms: Epithelial Membrane Antigen (EMA) • Glycoprotein in human milk fat globule membranes • Positive in most carcinomas and some sarcomas & lymphomas (ALCL) • Rarely positive in seminomas (2%), YSTs (2%) & ECs (2-12%) • Caveat: Negative GCT marker; not entirely specific in DX of GCT vs non-GCT • Overview: Useful in the differential of GCT versus somatic carcinoma.

33. Useful Antibodies for Testicular Neoplasms: Cytokeratin 7 (CK7) • Type II keratin of simple nonkeratinizing epithelium • Positive in many carcinomas • Negative in YST • May be positive in non-YST GCTs • Caveat: A negative YST marker & some carcinomas are negative (prostate, colon, etc.) • Overview: Mostly useful in the differential of YST vs somatic carcinoma

34. Immunohistochemistry Algorithm #4a for Testicular Neoplasia: Germ Cell Tumor vs Metastatic High Grade Carcinoma *Embryonal carcinoma and Seminoma only

35. Immunohistochemistry Algorithm #4b* for Testicular Neoplasia: Germ Cell Tumor vs Metastatic High Grade Carcinoma * Alternative algorithm if SALL4 not available † Hepatocellular carcinomas, hepatoid carcinomas of other organs and squamous cell carcinomas may be glypican 3 positive.

36. Proposed ISUP Recommendations:Germ Cell Tumor versus Metastatic High Grade Carcinoma • A reasonable and efficient initial panel is: SALL4, OCT4 & EMA • An alternative panel is OCT4, GPC3, EMA & CK7

37. Immunohistochemistry Algorithm #5 for Testicular Neoplasia: Seminoma with Syncytiotrophoblasts vs Choriocarcinoma Preferred markers in bold; alternatives in parentheses

38. Seminoma+SynT

39. Seminoma+SynT – OCT4

40. Immunohistochemistry Algorithm #6 for Testicular Neoplasia: Intratubular Germ Cell Neoplasia Unclassified (IGCNU) vs Non-neoplastic Atypical Germ Cells Preferred marker in bold