Glypican 3: A Novel Marker in Testicular Germ Cell Tumors Debra L Glypican 3: A Novel Marker in Testicular Germ Cell Tumors Debra L. Zynger,MD,* Nikolay D. Dimov,MD,* Chunyan Luan,MS,* Bin Tean Teh,MD, PhD,w and 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

Introduction-1 Glypicans(GPC) glypican 3(GPC3) 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

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.

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

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

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

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

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

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(-)

RESULTS-2

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

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

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

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

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

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

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

Seminoma

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 ↓

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

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.

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

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.

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

Discussion-6 α-FP(+) in 74~100% of yolk sac tumors and 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.

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

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

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.