Keigo Saito

Biomarkers for the early diagnosis of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the fifth most common cancer and the second leading cause of cancer-related deaths worldwide. Although the prognosis of patients with HCC is generally poor, the 5-year survival rate is > 70% if patients are diagnosed at an early stage. However, early diagnosis of HCC is complicated by the coexistence of inflammation and cirrhosis. Thus, novel biomarkers for the early diagnosis of HCC are required. Currently, the diagnosis of HCC without pathological correlation is achieved by analyzing serum α-fetoprotein levels combined with imaging techniques. Advances in genomics and proteomics platforms and biomarker assay techniques over the last decade have resulted in the identification of numerous novel biomarkers and have improved the diagnosis of HCC. The most promising biomarkers, such as glypican-3, osteopontin, Golgi protein-73 and nucleic acids including microRNAs, are most likely to become clinically validated in the near future. These biomarkers are not only useful for early diagnosis of HCC, but also provide insight into the mechanisms driving oncogenesis. In addition, such molecular insight creates the basis for the development of potentially more effective treatment strategies. In this article, we provide an overview of the biomarkers that are currently used for the early diagnosis of HCC.

Extract of Cordyceps militaris inhibits angiogenesis and suppresses tumor growth of human malignant melanoma cells

Angiogenesis is essential for tumor development and metastasis. Among several angiogenic factors, vascular endothelial growth factor receptor (VEGF) is important for tumor-derived angiogenesis and commonly overexpressed in solid tumors. Thus, many antitumor strategies targeting VEGF have been developed to inhibit cancer angiogenesis, offering insights into the successful treatment of solid cancers. However, there are a number of issues such as harmful effects on normal vascularity in clinical trials. Taking this into consideration, we employed Cordyceps militaris as an antitumor approach due to its biological safety in vivo. The herbal medicinal mushroom Cordyceps militaris has been reported to show potential anticancer properties including anti-angiogenic capacity; however, its concrete properties have yet to be fully demonstrated. In this study, we aimed to elucidate the biological role of Cordyceps militaris extract in tumor cells, especially in regulating angiogenesis and tumor growth of a human malignant melanoma cell line. We demonstrated that Cordyceps militaris extract remarkably suppressed tumor growth via induction of apoptotic cell death in culture that links to the abrogation of VEGF production in melanoma cells. This was followed by mitigation of Akt1 and GSK-3β activation, while p38α phosphorylation levels were increased. Extract treatment in mouse model xenografted with human melanoma cells resulted in a dramatic antitumor effect with down-regulation of VEGF expression. The results suggest that suppression of tumor growth by Cordyceps militaris extract is, at least, mediated by its anti-angiogenicity and apoptosis induction capacities. Cordyceps militaris extract may be a potent antitumor herbal drug for solid tumors.

Coxsackie and adenovirus receptor is a critical regulator for the survival and growth of oral squamous carcinoma cells

Coxsackie and adenovirus receptor (CAR) is essential for adenovirus infection to target cells, and its constitutive expression in various cancerous and normal tissues has been reported. Recently, the biological role of CAR in human cancers of several different origins has been investigated with respect to tumor progression, metastasis and tumorigenesis. However, its biological function in tumor cells remains controversial. Here we report the critical role of CAR in growth regulation of oral squamous cell carcinomas (SCCs) in vitro and in vivo via the specific interaction with Rho-associated protein kinase (ROCK). Loss of endogenous CAR expression by knockdown using specific small interfering RNA (siRNA) against CAR facilitates growth suppression of SCC cells due to cell dissociation, followed by apoptosis. The consequent morphological reaction was reminiscent of anoikis, rather than epithelial–mesenchymal transition, and the dissociation of oral SCC cells was triggered not by lack of contact with extracellular matrix, but by loss of cell-to-cell contact caused by abnormal translocation of E-cadherin from surface membrane to cytoplasm. Immunoprecipitation assays of the CAR-transfected oral SCC cell line, HSC-2, with or without ROCK inhibitor (Y-27632) revealed that CAR directly associates with ROCKI and ROCKII, which results in inhibition of ROCK activity and contributes to maintenance of cell-to-cell adhesion for their growth and survival. Based on these findings, in vivo behavior of CAR-downregulated HSC-2 cells from siRNA knockdown was compared with that of normally CAR-expressing cells in intraperitoneally xenografted mouse models. The mice engrafted with CAR siRNA-pretreated HSC-2 cells showed poor formation of metastatic foci in contrast to those implanted with the control siRNA-pretreated cells. Thus, CAR substantially has an impact on growth and survival of oral SCC cells as a negative regulator of ROCK in vitro and in vivo.

Immunological efficacy of glypican-3 peptide vaccine in patients with advanced hepatocellular carcinoma

ABSTRACT We have previously conducted a phase I trial to test the efficacy of a glypican-3 (GPC3) peptide vaccine in patients with advanced hepatocellular carcinoma (HCC); however, its immunological mechanism of action remains unclear. Here, we report a pilot study conducted to evaluate the immunological mechanisms of action of this GPC3 peptide vaccine (UMIN-CTR number 000005093). Eleven patients with advanced HCC were vaccinated with the GPC3 peptide in this trial. The primary end point was GPC3 peptide-specific immune response induced by the GPC3 peptide vaccination. The secondary endpoints were clinical and biologic outcomes. We demonstrated that the present vaccine induced GPC3 peptide-specific cytotoxic T lymphocytes (CTLs), which were found to infiltrate into the tumor. Moreover, we established GPC3 peptide-specific CTL clones from a biopsy specimen: these cells exhibited GPC3 peptide-specific cytokine secretion and cell cytotoxicity. The plasma GPC3 level tended to decrease temporarily at least once during the follow-up period. The GPC3-specific CTL frequency after vaccination was correlated with overall survival. The degree of skin reactions at the injection site correlated with the GPC3 peptide-specific CTLs. Furthermore, we sequenced the T cell receptors (TCRs) of tumor-infiltrating lymphocyte (TIL) clones, and confirmed the existence of this TCR repertoire in both tumor tissue and PBMCs. In response to these data, we are developing TCR-engineered T cell therapy using TCR sequences obtained from GPC3 peptide-specific CTL clones for improved efficacy in patients with advanced HCC.

Critical analysis of the potential of targeting GPC3 in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is a leading cause of cancer-related deaths worldwide. The treatment options for patients with advanced HCC are limited, and novel treatment strategies are required urgently. Glypican-3 (GPC3), a member of the glypican family of heparan sulfate proteoglycans, is overexpressed in 72%−81% of HCC cases, and is correlated with a poor prognosis. GPC3 regulates both stimulatory and inhibitory signals, and plays a key role in regulating cancer cell growth. GPC3 is released into the serum, and so might be a useful diagnostic marker for HCC. GPC3 is also used as an immunotherapeutic target in HCC. A Phase I study of a humanized anti-GPC3 monoclonal antibody, GC33, revealed a good safety profile and potential antitumor activity, and a Phase II trial is currently ongoing. In addition, the authors’ investigator-initiated Phase I study of a GPC3-derived peptide vaccine showed good safety and tolerability, and demonstrated that the GPC3 peptide-specific cytotoxic T-lymphocyte frequency in peripheral blood correlated with overall survival in HCC patients. A sponsor-initiated Phase I clinical trial of a three-peptide cocktail vaccine, which includes a GPC3-derived peptide, is also underway. GPC3 is currently recognized as a promising therapeutic target and diagnostic marker for HCC. This review introduces the recent progress in GPC3 research, from biology to clinical impact.

Abstract 573: Glypican-3 is a predictive marker for recurrence of hepatocellular carcinoma after surgical resection, especially in early stage

Background and Objective: Even in early stages, recurrence risk of hepatocellular carcinoma (HCC) is high. Glypican-3 (GPC3) is specifically overexpressed in HCC, and its overexpression is correlated with poor prognosis in patients with HCC. Recent studies have shown the utility of GPC3 as a serum and immunohistochemical marker for the diagnosis of HCC. However, the diagnostic value of GPC3 as a predictive marker for recurrence of early-stage HCC is not well understood. Thus, in the present study, we evaluated the usefulness of GPC3 as a predictive marker for recurrence after surgical resection, especially in early-stage HCC. Methods: Plasma samples were collected from 27 patients with stageI HCC who underwent for surgical resection during May 2008 to October 2010. GPC3-expression of surgical specimens was analyzed by immunohistochemical staining. The novel anti-GPC3 mouse monoclonal antibodies were generated and the sandwich enzyme-linked immunosorbent assay (ELISA) system for detection of plasma GPC3 levels was established. Plasma GPC3 concentrations were measured by the developed novel sandwich ELISA system. Plasma samples of age-matched 39 non-liver disease patients were used as control. Results: During follow-up period (median, 738 days), recurrence was observed in 14 cases after surgical resection (51.8%). GPC3 positive rate by the immunohistochemical staining of resected specimen was 53% (13/23). There was a tendency towards a shorter recurrence-free survival (RFS) in GPC3 positive cases than in GPC3 negative cases (p = 0.233). The mean ± standard deviation (SD) of plasma GPC3 concentrations in control subjects was 110.12 ± 37.70 ng/ml, and cut-off value was determined to be 132 ng/ml (mean + 1.5 SD). In stageI HCC, the sensitivity and specificity were 40.7% and 92%, respectively. In the recurrence group, pre-operation plasma GPC3 levels were significantly higher than in the non-recurrence group(median, 191.7 ng/ml vs 29.7 ng/ml, p = 0.029). There was a tendency of shorter RFS in the pre-operation plasma GPC3 positive patients group compared with the negative group (median RFS, 16 months vs not reached), although this difference was not significant (p = 0.127). Among 11 cases remained recurrence-free for over 4 years of follow-up, pre-operation plasma GPC3 positive was only one case. The recurrence rate of patients with post-operation plasma GPC3 positive was significantly higher than GPC3 negative patients (p = 0.033). Conclusions: We have developed a novel ELISA system for detecting plasma GPC3 levels and determined utility of GPC3 as diagnostic marker of HCC. This study suggests GPC3 expression and plasma GPC3 levels might help in identification of patients at high risk of recurrence of HCC after surgical resection, especially in early stage. Citation Format: Kazuya Ofuji, Keigo Saito, Yasunari Nakamoto, Tetsuya Nakatsura. Glypican-3 is a predictive marker for recurrence of hepatocellular carcinoma after surgical resection, especially in early stage. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 573. doi:10.1158/1538-7445.AM2015-573

Abstract 5371: Immune correlates of survival in advanced hepatocellular carcinoma patients treated with glypican-3-derived peptide vaccine: Results from phase I trial

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Introduction: Glypican-3 (GPC3), a carcinoembryonic antigen, is an ideal target of anticancer immunotherapy against hepatocellular carcinoma (HCC), because it is overexpressed specifically in HCC and correlates to a poor prognosis. In a preclinical study using a mouse model, we identified HLA-A24:02-restricted GPC3298-306 (EYILSLEEL) and HLA-A02:01-restricted GPC3144-152 (FVGEFFTDV) peptides, both of which can induce GPC3-reactive cytotoxic T-lymphocytes (CTLs) without inducing autoimmunity. On the basis of these results, we conducted a clinical trial of GPC3-derived peptide vaccine for advanced HCC patients to evaluate the vaccines safety, tolerability, and immunological and clinical responses. Methods: Thirty-three advanced HCC patients were enrolled in this non-randomized, open-label, dose escalation phase I trial. GPC3-derived peptide was administered in a liquid form emulsified with incomplete Freunds adjuvant, and intradermally injected on days 1, 15, and 29. PBMCs, obtained from all patients before and after vaccination, were examined by ex vivo IFN-γ enzyme-linked immunospot (ELISPOT) assay. The primary endpoint was the safety of GPC3 peptide vaccination. The secondary endpoints were immune response, as measured by ex vivo IFN-γ ELISPOT assay, and clinical outcomes, as measured by tumor response, time to tumor progression, and overall survival (OS). Results: GPC3 vaccination was well-tolerated. One patient showed a partial response, and 18 patients showed stable disease 2 months after initiation of treatment. Four patients, of which 18 had stable disease, had tumor regression or necrosis not meeting the criteria for partial response. The median time to tumor progression was 3.4 months (95% CI, 2.1 to 4.6). The median OS was 9.0 months (95% CI, 8.0 to 10.0). Tumor biopsy was performed with adequate informed consent in 7 patients for the evaluation of the therapeutic effect after vaccination. In 5 out of 7 cases, infiltration of CD8-positive T cells into the tumor was increased after vaccination. GPC3 peptide vaccine could induce the GPC3-specific CTL response in 30 of 33 patients by ex vivo IFN-γ ELISPOT assay, and the GPC3-specific CTL frequency after vaccination correlated with overall survival. The analysis of all 33 patients showed that the median OS was 12.2 months (95% CI, 6.5 to 18.0) in patients with high frequency of GPC3-specfic CTL, compared with 8.5 months (95% CI, 3.7 to 13.1) in those with low frequency of GPC3-specfic CTL (p = 0.033). Conclusions: GPC3-derived peptide vaccination was well-tolerated, and measurable immune responses and antitumor efficacy were noted. Furthermore, this study shows that peptide-specific CTL frequency can be a predictive marker for overall survival in HCC patients receiving peptide vaccination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5371. doi:1538-7445.AM2012-5371