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- Materials and Methods
The relationship between overexpression of glypican (GPC)-3 that is specific for hepatocellular carcinoma (HCC) and the prognosis has not yet been clarified. We attempted to determine the expression profile of GPC3 in association with the clinicopathological factors by immunohistochemical analysis in HCC patients and investigated the potential prognostic value of GPC3 by comparing the survival rate between the GPC3-positive and GPC3-negative HCC patients. Primary HCC tissue samples (n = 107) obtained from patients who had undergone hepatectomy between 2000 and 2001 were analyzed. GPC3 expression was less frequently observed in well-differentiated HCC than in moderately and poorly differentiated HCC, the difference in the frequency being statistically significant. GPC3-positive HCC patients had a significantly lower 5-year survival rate than the GPC3-negative HCC patients (54.5 vs 87.7%, P = 0.031). Among 80 of the 107 (74.6%) patients with initial treatment who underwent hepatectomy, none of GPC3-negative HCC patients (n = 16, 20.0%) died during the follow-up period. No deaths were noted in the GPC3-negative HCC patients among the 71 (88.7%) patients with moderately and poorly differentiated HCC. Multivariate analysis identified GPC3 expression (P = 0.034) as an independent prognostic factor for the overall survival. We showed that GPC3 expression is correlated with a poor prognosis in HCC patients. (Cancer Sci 2009)
Hepatocellular carcinoma (HCC) is one of the most common malignancies and is ranked as the third most common cause of cancer-related death worldwide. HCC is generally associated with a poor prognosis, the 5-year survival rate after surgery has been reported to be as low as 25–39%, and systemic therapy with cytotoxic agents provides only marginal benefit.(1) Even in those patients in whom the tumor has been successfully removed, the 2-year recurrence rate can be as high as 50%.(2,3) Several clinicopathological factors including poor levels of differentiation of the cancer cells, large size of the tumor, portal venous invasion, and intrahepatic metastasis have been shown to contribute to the poor prognosis in patients of HCC. Despite the critical need for better methods for the diagnosis and treatment of HCC, the mechanisms underlying the development of HCC remain unclear.
Glypican (GPC)-3 was discovered as a potential serological and histochemical marker that is specific for HCC. GPC3 is a member of the glypican family and belongs to a group of heparan sulfate proteoglycans bound to the outer surface of the cell membrane through a glycosylphosphatidylinositol anchor.(4) In mammals, this family comprises six members, GPC1 to GPC6. GPC are released from the cell surface by a lipase called Notum to regulate the signaling of Wnts, Hedgehogs, fibroblast growth factors, and bone morphogenetic proteins.(5–9) Depending on the context, their functions exerted may either be stimulatory or inhibitory through these pathways. GPC3 has been detected in the placenta and fetal liver, but not in other adult organs. During hepatic carcinogenesis, GPC3 appears in the HCC tissue and is released into the serum.(10–12) In addition, its expression has also been reported in melanoma.(13–15)
A dramatic elevation of GPC3 expression has been reported in a large proportion of HCC, as determined by cDNA microarray analysis, whereas its expression has been shown to be less frequent in preneoplastic or entirely absent in non-neoplastic liver tissue.(16–18) This has led to the notion that GPC3 may have diagnostic usefulness as a marker of differentiation or a specific tumor marker in the case of HCC. However, until now, the relationship between GPC3 overexpression and the prognosis of HCC has not been clarified.
In the present study, we attempted to determine the tumor expression profile of GPC3 in association with clinicopathological factors in HCC patients by immunohistochemical analysis. We also investigated the potential prognostic value of GPC3 by analyzing the survival rate of GPC3-positive and GPC3-negative HCC patients. By elucidating the association between the GPC3 expression level in HCC tumors and the survival rate of the patients, we concluded that the GPC3 expression level is correlated with a poor prognosis in HCC patients.
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- Materials and Methods
In this study, we characterized the association between the expression level of GPC3 and the malignancy grade, and the prognostic value of GPC3 in HCC. Higher levels of GPC3 expression were observed in moderately or poorly differentiated tumor cells, which was in agreement with previous reports.(19) Our contingency table analysis showed that the GPC3 expression level was correlated with the tumor differentiation level. In addition, Kaplan–Meier survival analysis revealed that GPC3 expression was significantly linked to a poor prognosis after surgical resection in HCC patients. Moreover, univariate analysis indicated that GPC3 expression is associated with an increased risk of death from HCC, and this risk factor could still be extracted in a multivariate setting. On the other hand, multivariate analysis did not identify the tumor differentiation level as an independent predictive factor of the prognosis. Among the 80 HCC patients who underwent initial surgical treatment, the GPC3-negative patients showed better prognosis than the GPC3-positive patients. Patients with well-differentiated HCC also showed a better prognosis than those with moderately and poorly differentiated HCC. Furthermore, we confirmed that among the previously treated subjects, the GPC3-negative group had a better prognosis than the GPC3-positive group with moderately and poorly differentiated HCC tumors.
In this study, the patients who were HCV positive, had higher ICG-R15 values, or portal vein involvement showed longer survival times, especially the patients who were HCV-positive or had higher ICG-R15 values, showed statistical significance in the univariate analysis. However, there was no statistical significance in these variables in the multivariate analysis. The reasons for these contradictive results in the univariate analysis are unclear.
In contrast, subgroup analysis did not reveal any significant difference in the disease-free survival rate between the GPC3-positive and GPC3-negative HCC patients (data not shown). The rate of recurrence in patients after surgery was 63.8% within the first 2 years after surgery among the previously treated patients in this study. Tumor recurrence in the GPC3-positive HCC patients occurred earlier than that in the GPC3-negative HCC patients until 9.7 months after the surgery among the patients who had received previous treatment. Two mechanisms of postoperative recurrence of HCC have been suggested: one is intrahepatic metastasis in the residual liver in a metachronous manner, and the other is multicentric hepatocarcinogenesis based on chronic hepatitis.(20–23) Some authors have suggested that early recurrence arises most often from intrahepatic metastases, whereas late recurrence is more likely to be multicentric in origin. Poon et al. and Portolani et al. reported that tumor factors like neoplastic vascular infiltration, but not host factors, were linked to early recurrence, whereas the risk of late recurrence was dependent on the underlying liver status.(21,22) These results indicate that GPC3 expression may indicate a high risk of intrahepatic recurrence.
Most of the GPC3 expression patterns in HCC cells showed the cytoplasmic pattern. There was no case that showed only the membrane pattern. Almost half of the HCC cases showed the mixed pattern (cytoplasm and membrane) and the other half showed only the cytoplasmic pattern.
There was no statistical significance between the mixed pattern (cytoplasm and membrane) and cytoplasmic pattern (P = 0.297) in Kaplan–Meier survival analysis. The functional difference between cytoplasmic GPC3 and membrane GPC3 is unknown, so further investigations are needed to clarify whether the different localization of staining has a different significance.
In addition to the investigation of its role as a prognostic indicator, a phase I clinical trial of a GPC3-derived peptide vaccine for advanced HCC is now underway; GPC3 is an ideal target for this therapy because it is more effective in patients with increased expression of GPC3, which is frequently observed in the later stages of HCC, as shown in the present study. The poor prognosis of patients with GPC3-positive HCC also prompted us to develop a strategy of anticancer immunotherapy,(24,25) that is, we may expect the effect of hepatocarcinogenesis prevention after surgery in patients with GPC3-positive HCC.
In summary, our study evaluated the prognostic significance of GPC3 expression at the protein level in clinical tissue specimens of HCC. The overall survival rate was significantly poorer in patients with elevated GPC3 expression in the tumor than in those with lower levels of GPC3 expression. Further functional characterization of GPC3 may be expected to lead to a better understanding of the molecular mechanisms underlying the development and progression of HCC.