Glypican-3 as a Serum Marker for Hepatocellular Carcinoma

To the Editor: Hippo et al. (1) have recently reported in an article published by Cancer Research that glypican-3 (GPC3) is a serologic marker of hepatocellular carcinoma (HCC). The results published by Hippo et al. basically confirm the findings reported previously by our laboratory (2) and by Nakatsura et al. (3). The fact that three independent laboratories using different anti-GPC3 antibodies have reached similar results strongly suggests that GPC3 is a useful marker for HCC. Furthermore, the three laboratories have also found that, due to the lack of correlation between serologic concentrations of GPC3 and a-fetoprotein in HCC patients, the simultaneous use of both markers significantly increases the sensitivity of the test. Despite our general agreement with the results of Hippo et al., we disagree with the conclusions reached by these authors with regard to the biochemical analysis of the results. Basically, their claim that only the NH2-terminal portion of GPC3 is present in the sera of HCC patients is not correct. It is well established that GPC3 is composed of two subunits that are linked by one or more disulfide bonds (4, 5). The two subunits are produced by cleavage of GPC3 at residue R358 by a convertase (5), which generates a NH2-terminal fragment of f40 kDa and a COOH-terminal fragment of f30 kDa. It is important to note that in most cases the COOH-terminal fragment of GPC3 will carry the heparan sulfate chains and will produce a high molecular weight smear instead of a band in a Western blot. If GPC3 is run through a reducing gel, the two GPC3 subunits will be separated, and an antibody directed against the NH2 terminus will only detect the 40-kDa band, which does not have the heparan sulfate chains, as was shown by Hippo et al. Currently, we do not have an explanation as to why Hippo et al. did not detect GPC3 in their Western blot analysis of serum from HCC patients and in the supernatant of HCC cell lines when they used their own anti-GPC3 monoclonal antibody against the COOHterminal antibody. We have already shown that our COOH-terminal antibody detects the smear corresponding to glycanated GPC3 both in the conditioned medium from HCC cells (2) and in the serum of HCC patients (Fig. 1). In summary, we believe that the GPC3 ELISA tests done by Hippo et al. and by our laboratory in the serum of HCC patients are detecting the same biochemical entity, although we have used an antibody that recognizes the COOH-terminal fragment and Hippo et al. used an antibody against the NH2-terminal portion of GPC3. This conclusion is clearly supported by the fact that both laboratories found a similar proportion of GPC3-positive sera (51% in the case of Hippo et al. and 53% in our laboratory).