Novel Glycobiomarker for Ovarian Cancer That Detects Clear Cell Carcinoma

Epithelial ovarian cancer (EOC) is often asymptomatic and thus diagnosed at advanced stages with a poor prognosis. False-negative results for the conventional marker CA125 frequently occur in cases of clear cell carcinoma (CCC), a type of EOC; therefore, it is necessary to develop biomarkers with greater sensitivity. We previously reported a strategy to discover glycobiomarker candidates by combined lectin microarray and IGOT–LC/MS analysis. We have now optimized this strategy for discovering EOC biomarkers. Glycopeptides possessing cancerous glycans were enriched from the ascites fluids and culture supernatants of cancer cell lines with a fucose-binding lectin, AAL. IGOT–LC/MS analysis of CCC samples yielded 144 candidate glycoproteins. We selected WFA by lectin microarray as the optimal lectin to distinguish EOC from gastric and colon cancer. The candidates were narrowed by Western analysis of the WFA-bound fraction of ascites fluids. One of the final candidates, WFA-reactive ceruloplasmin, produced higher signals in the ascites fluids of EOC patients, including CCC, in comparison with the benign samples, while CA125 levels were comparable in the sandwich ELISA. Thus, our glycoproteomic strategy featuring efficient enrichment of glycans with disease-related alterations is applicable to various diseases

Glycoproteomic Discovery of Serological Biomarker Candidates for HCV/HBV Infection-Associated Liver Fibrosis and Hepatocellular Carcinoma

We previously proposed a high-throughput strategy to discover serological biomarker candidates of cancer. This strategy focuses on a series of candidate glycoproteins that are specifically expressed in the original tissues (cells) of the target cancer and that carry glycan structures associated with carcinogenesis [Narimatsu, H., et al. <i>FEBS J.</i> <b>2010</b>, <i>277</i>(1), 95–105]. Here, we examined the effectiveness of our strategy in identifying biomarkers to assess progression of liver fibrosis and for the early detection of hepatocellular carcinoma (HCC). On the basis of the results of lectin array analyses in culture media of hepatoma cell lines, we captured glycopeptides carrying AAL-ligands (fucosylated glycans) or DSA-ligands (branched glycans) from digests of culture media proteins and sera from HCC patients with a background of liver cirrhosis (LC). Glycoproteins were identified by the IGOT-LC–MS method. In all, 21 candidates were selected from 744 AAL-bound glycoproteins for further verification according to (i) their abundance in serum, (ii) their specific expression in liver, and (iii) the availability of antibodies to the glycoproteins. All selected candidates showed enhancement of AAL-reactivity in sera of HCC patients compared with that of healthy volunteers (HV). These results indicate that our glycoproteomic strategy is effective for identifying multiple glyco-biomarker candidates in a high-throughput manner