From the *Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL; and Laboratory of

1. Glypican 3: A Novel Marker in TesticularGerm Cell TumorsDebra L. Zynger,MD,* Nikolay D. Dimov,MD,* Chunyan Luan,MS,* Bin Tean Teh,MD, PhD,wand Ximing J. Yang, MD, PhD* From the *Department of Pathology, Feinberg School of Medicine, Northwestern University, Chicago, IL; and Laboratory of Cancer Genetics, Van Andel Research Institute American Journal of Surgical Pathology 2006;30:1570–1575 指導老師: 方嘉郎醫師 Intern林培豐

2. Introduction-1 • Glypicans(GPC) • A family of heparin sulfate proteoglycans. 6 members in mouse and human. • Extracellular proteins bound to the cell surface by a glycophosphatidylinositol anchor • glypican 3(GPC3) • Regulate growth through interactions with morphogenic or growth factorsWnt5a, fibroblast growth factor 2, bone morphogenic protein 7, and tissue factor pathway inhibitor

3. Introduction-2 • Normally expressed in trophoblasts and fetal tissues • Limited expression in adult tissues: ovary, breast, lung, and mesothelium • Mammary carcinoma, ovarian carcinoma, lung adenocarcinoma, and malignant mesothelioma GPC3 mRNA level ↓ • Mutations Simpson-Golabi-Behmel (SGB) syndrome, a rare X-linked overgrowth syndrome characterized by numerous craniofacial, skeletal, and genitourinary abnormalities.

4. Introduction-3 Maybe an oncofetal protein • GPC3 mRNA level↑hepatocellular carcinoma, hepatoblastoma, Wilms tumor, neuroblastoma, rhabdomyosarcoma, Wilms tumor, hepatocellular carcinoma, and hepatoblastoma.

5. Testicular germ cell tumors • Common cell of origin, but differ with degree of differentiation.

6. MATERIALS-1 • 71 primary testicular germ cell tumors between 1996 and 2005 were obtained from Northwestern Memorial Hospital • 46 mixed tumors; 25 pure seminomas

7. MATERIALS-2 • Mixed tumors: 17 to 60 years (mean=30.9 y) • The mixed germ cell tumors • 37stage I • 5stage II • 4stage III • Pure seminomas：24 to 55 years (mean=35.6 y). • 24 stage I • 1 stage III • Benign testicular tissues from 58 cases  negative control • Immature and mature placental tissues positive control

8. METHONDS • Sections (5 mm) from 1 to 3 representative blocks deparaffinized, rehydrated in graded alcohols, and epitope retrieval in 0.1M citrate buffer at pH 6.0 in a microwave for 20 minutes • Primary monoclonal antibody of 1:200 for 1 hour at room temperature • Rabbit antimouse secondary antibody • semiquantitatively evaluated as • negative (0, <5% of cells stained) • focally positive (1+, 5% to 10% of cells stained) • positive (2+, 11% to 50% of cells stained) • diffusely positive (3+, >50% of cells stained

9. RESULTS-1 • Positive control : strong GPC3 (+) in syncytiotrophoblasts; weaker staining in cytotrophoblasts. • Negative control germ cells, Sertoli cells, Leydig cells, blood vessels, fibroblasts, and hematopoietic cells, intratubular germ cell neoplasia  GPC(-)

10. RESULTS-2

11. Yolk sac tumor hematoxylin and eosin (H&E) and diffuse, strong GPC3 immunoreactivity.

12. C: GPC3 immunostaining, Yolk sac tumor and Embryonal carcinoma D, GPC3 immunostaining, yolk sac tumor and teratoma with mature elements

13. E and F, Choriocarcinoma H&E and GPC3 immunostaining showing strong immunoreactivity in syncytiotrophoblasts and weaker staining in cytotrophoblasts.

14. A and B, Teratoma with immature neuroectodermal elements H&E and GPC3 immunostaining showing strong immunoreactivity.

15. C and D, Teratoma with mature squamous epithelium H&E and negative GPC3.

16. E and F, Embryonal carcinoma H&E and GPC3 immunostaining without reactivity or background.

17. G and H, Embryonal carcinoma H&E and GPC3 immunostaining demonstrating weak, focal positivity • Peripheral teratoma: mature glandular epithelium, GPC3(-)

18. Seminoma

19. DISCUSSION-1 • Previously reported by Sugimura et al Testicular germ cell tumors revealed differential expression of a large number of genes. • GPC3: 3rd most overexpressed gene in yolk sac tumors mRNA level 4.2-fold ↑ comparing other non-seminoma  embryonal carcinoma 10-fold ↓

20. DISCUSSION-2 • In current studies • GPC3 expression (+): yolk sac tumor,choriocarcinoma • GPC3 expression(-): non-neoplastic testicular parenchyma, intratubular germ cell neoplasia, seminomas, teratomas with mature elements, and the majority of embryonal carcinomas and teratomas with immature elements • In this study diagnostic value in identifying yolk sac tumor from other germ cell tumor subtypes

21. DISCUSSION-3 • GPC3 mutations  SGB syndrome in prenatal and postnatal overgrowth correlated with GPC3-deficient mice models • GPC3 widely expressed in fetal tissues, esp. in liver, lung, kidney and placental tissues • In adult, GPC3 expression is restricted in the epithelium of breast, ovary, lung, and mesothelium • In mammary carcinoma, ovarian carcinoma, lung adenocarcinoma, and malignant mesothelioma, GPC3 expression was reduced or silenced • GPC3: negative regular of growth and tissue-specific tumor supressor activity • Loss of function or reduced expression contribute to the malignant phenotype of certain tumors.

22. DISCUSSION-4 • 10% cases of SGB syndromes acquire a malignancy, including neuroblastoma, gonadoblastoma, Wilms tumor, hepatoblastoma, or hepatocellular carcinoma. • Sporadic forms of these tumor have increased GPC3 expression • GPC3 conversely acted as oncofetal protein GPC3 is a growth inhibitor or oncofetal protein depending upon the tissue

23. Discussion-5 • Interestingly, the expression of GPC3 in other embryonal tumors varies • Rhabdomyosarcoma: GPC 3 protein(+) • But Ewing sarcoma or medulloblastoma: GPC3 protein(-) • UnclearGPC3 expression ↑ plays a critical role in tumor development • Perhaps GPC3 is expressed in tumors with a certain level of fetal differentiation, but is negative in poorly differentiated embryonal malignancies.

24. GPC3 may play a role in lineage or stage specific germ cell tumor differentiation.

26. Discussion-6 • Yolk sac tumor：most frequently overlooked and close association with embryonal carcinoma clinical significance in metastasis, recurrence • GPC3 immunostaining makes it easy to distinguish, whereas more than 90% of embryonal carcinomas were negative. • α-FP(+) in 74~100% of yolk sac tumors and 0% ~33% of embryonal carcinomas. • Further experiments are needed to compare the sensitivity and specificity of GPC3 and α-FP in yolk sac tumor.

27. Discussion-7 • Certain patterns of yolk sac tumor may seem similar to seminoma. • This study revealed that all seminomatous components and intratubular germ cell neoplasia were GPC(-) • Future studiessensitivity and specificity of GPC3 in distinguishing yolk sac tumor from seminoma in comparison with other markers such as OCT4, c-kit, keratin, placental alkaline phosphatase, and α-FP

28. Discussion-8 • Choriocarcinoma：GPC3 exhibited a similar pattern as β-HCG • more intense staining in syncytiotrophoblasts than in cytotrophoblasts. • Serum test for GPC3 in non-seminoma germ cell tumors • Recent studies, GPC3 more sensitive and specific serum marker for hepatocellular carcinoma than α-FP • Potential therapeutic target

29. CONCLUSION • GPC3 is a novel marker in all yolk sac tumors and choriocarcinomas, but not in seminomas, teratomas with mature elements, and the majority of embryonal carcinomas and teratomas with immature elements. • This study not only suggests a possible role of GPC3 in germ cell tumor lineage-specific differentiation, but also provides evidence for a new promising diagnostic marker of testicular germ cell tumors.