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) 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, 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  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, ckit, keratin, placental alkaline phosphatase, and αFP

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.