Glypican-3, overexpressed specifically in human hepatocellular carcinoma, is a novel tumor marker

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Abstract

With the global pandemic of hepatitis B and C infections, the incidence of Hepatocellular carcinoma (HCC) is rapidly increasing world wide. We identified glypican-3 (GPC3), a novel oncofetal gene over-expressed specifically in human HCC, as based on data of cDNA microarrays. As GPC3 is a GPI-anchored membrane protein and could be secreted, we attempted to detect secreted GPC3 protein in sera from HCC patients using Western blotting and ELISA. GPC3 protein was positive in sera of 40.0% (16/40) of HCC patients, and negative in sera from subjects with liver cirrhosis (LC) (0/13), chronic hepatitis (CH) (0/34), and healthy donors (0/60). All subjects were Japanese. Although 12 of 40 HCC patients were negative for both α-fetoprotein (AFP) and PIVKA-II well known tumor markers of HCC, four of these were GPC3-positive in the sera. We also observed vanishing GPC3 protein in the sera of three patients after the surgical treatment for HCC. On the other hand, immunohistochemical analysis revealed that HCC expressed GPC3 protein in all 14 HCC patients tested. In conclusion, GPC3, as defined in this study was shown to be a useful tumor marker for cancer-diagnosis for large numbers of patients with HCC.

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Materials and methods

cDNA microarrays. Profiling of gene expression by cDNA microarrays was done, as reported [4]. Primary HCCs and corresponding non-cancerous liver tissues were obtained with informed consent from 20 Japanese patients who underwent hepatectomy in the Department of Gastroenterological Surgery, Kyoto University Graduate School of Medicine. These specimens were used only for cDNA microarray analysis, as reported [4]. Poly(A)+ RNAs isolated from human bone marrow, brain, heart, kidney, liver, lung,

Identification of the GPC3 gene over-expressed specifically in HCC

We obtained data comparing expression profiles between 20 HCCs (10 cases were hepatitis B virus (HBV)-positive and 10 were hepatitis C virus (HCV)-positive) and their corresponding non-cancerous liver tissues [4] and in various normal human tissues [6], using cDNA microarrays. We then searched for genes over-expressed specifically in HCC using these data and we identified GPC3. In 16 cases of the 20 HCCs, the expression of GPC3 mRNA in the cancer tissue was 5 or more times higher than that in

Discussion

In 1996, Pilia et al. reported that GPC3, which encodes one member of the glypican family, is mutated in patients with Simpson–Golabi–Behmel syndrome (SGBS) [9]. SGBS is an X-linked disorder characterized by pre- and postnatal overgrowth, and a broad spectrum of clinical manifestations which vary from a very mild phenotype in carrier females to infantile lethal forms in some males [10]. The list of clinical manifestations of SGBS includes a distinct facial appearance, cleft palate, syndactyly,

Acknowledgements

This work was supported in part by Grants-in-Aid 60715 and 12213111 from the Ministry of Education, Science, Technology, Sports and Culture, Japan, and by a grant from the Sagawa Science and Technology Promotion Foundation, Japan. We thank T. Kubo (Department of Molecular Pathology, Kumamoto University) for technical assistance of immunohistochemical analyses and M. Ohara (Fukuoka) for helpful comments.

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    Abbreviations: HCC, hepatocellular carcinoma; GPC3, glypican-3; HD, healthy donor; LC, liver cirrhosis; CH, chronic hepatitis; AIH, autoimmune hepatitis; PBC, primary biliary cirrhosis; AFP, α-fetoprotein; PIVKA-II, protein induced by vitamin K absence or antagonist-II.

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