Nobuhiro Tsuchiya

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.

Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients

ABSTRACT The recurrence rates of Hepatocellular carcinoma (HCC) are high, necessitating novel and effective adjuvant therapies. Therefore, we conducted a phase II study of glypican-3 (GPC3) peptide vaccine as an adjuvant therapy for HCC patients. Forty-one patients with initial HCC who had undergone surgery or radiofrequency ablation (RFA) were analyzed in this phase II, open-label, single-arm trial. Ten vaccinations were performed for 1 y after curative treatment. We also investigated case-control subjects, where selected patients treated surgically during the same period were analyzed. The expression of GPC3 in the available primary tumors was determined by immunohistochemical analysis. Six patients received RFA therapy while 35 received surgery. The recurrence rate tended to be lower in the 35 patients treated with surgery plus vaccination compared to 33 patients who underwent surgery alone (28.6% vs. 54.3% and 39.4% vs. 54.5% at 1 and 2 y, respectively; p = 0.346, 0.983). Twenty-five patients treated with surgery and vaccination had GPC3-positive tumors; the recurrence rate in this group was significantly lower compared to that in 21 GPC3-positive patients who received surgery only (24% vs. 48% and 52.4% vs. 61.9% at 1 and 2 y, respectively; p = 0.047, 0.387). The GPC3 peptide vaccine improved the 1-y recurrence rate in patients with GPC3-positive tumors. This study demonstrated that GPC3 expression by the primary tumor may be used as a biomarker in a putative larger randomized clinical trial to determine the efficacy of the GPC3-derived peptide vaccine.

Potentiality of immunotherapy against hepatocellular carcinoma

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, is the fifth most common cancer worldwide and the second leading cause of cancer-related death. Despite the high incidence, treatment options remain limited for advanced HCC, and as a result prognosis continues to be poor. Current therapeutic options, surgery, chemotherapy and radiotherapy, have only modest efficacy. New treatment modalities to prolong survival and to minimize the risk of adverse response are desperately needed for patients with advanced HCC. Tumor immunotherapy is a promising, novel treatment strategy that may lead to improvements in both treatment-associated toxicity and outcome. The strategies have developed in part through genomic studies that have yielded candidate target molecules and in part through basic biology studies that have defined the pathways and cell types regulating immune response. Here, we summarize the various types of HCC immunotherapy and argue that the new-found field of HCC immunotherapy might provide critical advantages in the effort to improve prognosis of patients with advanced HCC. Already several immunotherapies, such as tumor-associated antigen therapy, immune checkpoint inhibitors and cell transfer immunotherapy, have demonstrated safety and feasibility in HCC patients. Unfortunately, immunotherapy currently has low efficacy in advanced stage HCC patients; overcoming this challenge will place immunotherapy at the forefront of HCC treatment, possibly in the near future.

Perioperative plasma glypican-3 level may enable prediction of the risk of recurrence after surgery in patients with stage I hepatocellular carcinoma.

Glypican-3 (GPC3) is a glycosylphosphatidylinositol-anchored cell surface protein overexpressed in hepatocellular carcinoma(HCC), and its overexpression is associated with poor prognosis. The diagnostic potential of GPC3 as a serum marker has been reported. In the present study, we evaluated the usefulness of plasma GPC3 as a predictor for recurrence after surgical resection in stage I HCC patients by newly developed an enzyme-linked immunosorbent assay (ELISA) system. Current study demonstrated that high levels of preoperative plasma GPC3 patients tended to experience postoperative recurrence. On the other hand, pre- and postoperative plasma GPC3 positivity of non-recurrence patients was very low. Moreover, even after surgery, approximately half of patients who experienced recurrence were positive for plasma GPC3. Postoperative plasma GPC3 positivity was significantly correlated with worse recurrence-free survival. Immuohistochemical analysis also showed positive rate of GPC3-expression in HCC was higher in recurrence patients than in non-recurrence patients. These results suggested that both pre- and postoperative plasma GPC3 levels may be accurate predictors for recurrence after curative resection of early-stage HCC. It should be noted that the current study only examined a small number of cases; thus, a larger sample size is necessary to validate GPC3 as a predictor for HCC recurrence.

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.

Hepatocellular carcinoma cell sensitivity to Vγ9Vδ2 T lymphocyte-mediated killing is increased by zoledronate

The limited efficacy of vaccines in hepatocellular carcinoma (HCC), due to the low frequency of tumor-infiltrating cytotoxic T lymphocytes (CTLs), indicates the importance of innate immune surveillance, which assists acquired immunity by directly recognizing and eliminating HCC. Innate Vγ9Vδ2 T cells have major histocompatibility complex-unrestricted antitumor activity and are activated by phosphoantigens, which are upregulated in cancer cells by the nitrogen-containing bisphosphonate, zoledronate (Zol). A better understanding of HCC susceptibility to Zol and downstream γδ T cell-mediated killing is essential to optimize γδ T cell-mediated immunotherapy. This study systematically examined the interactions between γδ T cells and Zol-treated HCC cell lines (HepG2, HLE, HLF, HuH-1, JHH5, JHH7, and Li-7) in vitro. All HCC cell lines expressed the DNAX accessory molecule-1 ligands, poliovirus receptor, and Nectin-2, and γδ T cell-mediated killing of these cells was significantly enhanced by Zol. Small interfering RNA-mediated knockdown of these ligands did not affect the susceptibility to γδ T cell lysis. This killing activity was partly inhibited by mevastatin, an inhibitor of the mevalonate pathway, and markedly reduced by a monoclonal antibody to γ- and δ-chain T cell receptor, indicating that this is crucial for Zol-induced HCC killing. In addition, Zol-treated HCC cell lines triggered γδ T cell proliferation and induced production of Th1 and Th2, but not Th17, cytokines. The Zol concentration that enhanced HCC cell susceptibility to γδ T cell killing was lower than that required to directly inhibit HCC proliferation. Thus, γδ T cells may be important effector cells in the presence of Zol, especially where there are insufficient number of cancer antigen-specific CTLs to eliminate HCC. Our in vitro data support the proposal that Zol-treatment, combined with adaptive γδ T cell immunotherapy, may provide a feasible and effective approach for treatment of HCC.

Phase I study of glypican-3-derived peptide vaccine therapy for patients with refractory pediatric solid tumors

ABSTRACT The carcinoembryonic antigen glypican-3 (GPC3) is a good target of anticancer immunotherapy against pediatric solid tumors expressing GPC3. In this non-randomized, open-label, phase I clinical trial, we analyzed the safety and efficacy of GPC3-peptide vaccination in patients with pediatric solid tumors. Eighteen patients with pediatric solid tumors expressing GPC3 underwent GPC3-peptide vaccination (intradermal injections every 2 weeks), with the primary endpoint being the safety of GPC3-peptide vaccination and the secondary endpoints being immune response, as measured by interferon (IFN)-γ enzyme-linked immunospot assay and Dextramer staining, and the clinical outcomes of tumor response, progression free survival (PFS), and overall survival (OS). Our findings indicated that GPC3 vaccination was well tolerated. We observed disease-control rates [complete response (CR)+partial response+stable disease] of 66.7% after 2 months, and although patients in the progression group unable to induce GPC3-peptide-specific cytotoxic T lymphocytes (CTLs) received poor prognoses, patients in the partial-remission and remission groups or those with hepatoblastoma received good prognoses. The GPC3-peptide vaccine induced a GPC3-specific CTL response in seven patients, with PFS and OS significantly longer in patients with high GPC3-specific CTL frequencies than in those with low frequencies. Furthermore, we established GPC3-peptide-specific CTL clones from a resected-recurrent tumor from one patient, with these cells exhibiting GPC3-peptide-specific cytokine secretion. The results of this trial demonstrated that the GPC3-peptide-specific CTLs induced by the GPC3-peptide vaccine infiltrated tumor tissue, and use of the GPC3-peptide vaccine might prevent the recurrence of pediatric solid tumors, especially hepatoblastomas, after a second CR.

Cancer immunotherapy‐targeted glypican‐3 or neoantigens

Immune checkpoint inhibitors have ushered in a new era in cancer therapy, although other therapies or combinations thereof are still needed for many patients for whom these drugs are ineffective. In this light, we have identified glypican‐3 an HLA‐24, HLA‐A2 restriction peptide with extreme cancer specificity. In this paper, we summarize results from a number of related clinical trials showing that glypican‐3 peptide vaccines induce specific CTLs in most patients (UMIN Clinical Trials Registry: UMIN000001395, UMIN000005093, UMIN000002614, UMN000003696, and UMIN000006357). We also describe the current state of personalized cancer immunotherapy based on neoantigens, and assess, based on our own research and experience, the potential of such therapy to elicit cancer regression. Finally, we discuss the future direction of cancer immunotherapy.

Abstract CT115: Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients

Background and purpose: In a phase I trial for advanced hepatocellular carcinoma (HCC), glypican-3(GPC3)-derived peptide vaccination was well-tolerated, and immune responses and antitumor efficacy were noted. The recurrence rates of HCC are still high and immunotherapy, as an adjuvant therapy, is expected. Therefore, we have conducted a phase II study of GPC3 peptide vaccine as an adjuvant therapy for HCC patients. Study design: Forty one patients with initial HCC, who had undergone surgery or radiofrequency ablation (RFA), were analyzed in this phase II, open-label, single-arm trial.Ten vaccinations were performed for one year after curative treatment. The primary endpoints were 1- and 2-year recurrence rates. The secondary endpoints were safety and immune responses. We have evaluated GPC3-specific CTL response in PBMCs by IFN-γ enzyme-linked immunospot (ELISPOT) assay. We have evaluated the expression of GPC3 in the 33 primary tumors and 11 recurrence tumors, that could be obtained, by immunohistochemical analysis, and the phenotype of CTLs in recurrence tumor and vaccine site by flow cytometry. Results: GPC3 peptide vaccine showed no severe adverse events. 1-year and 2-year recurrence rates of the 41 patients treated with the vaccination were 24.4% and 53.7%. In this study, 35 patients had received surgery and 6 patients RFA therapy. We analyzed the case-control study to evaluate the reduction in the risk of post-operative recurrence by vaccination. Thirty three patients with initial HCC who underwent curative resection in the same period were selected as control group. The recurrence rate tended to be lower in 35 patients treated with surgery and the vaccination than in 33 patients with surgery alone (28.6% and 54.3% vs 39.4% and 54.5% at 1 year and 2 year, p = 0.346, 0.983). In the patients with GPC3 positive tumor, the recurrence rate was significantly lower in 25 patients treated with surgery and the vaccination than in 21 patients with surgery alone (24% and 48% vs 52.4% and 61.9% at 1 year and 2 year, p = 0.047, 0.387), and there was no significant difference in clinical background. There were not the correlation between the relapse free survival and the antigen-specific immune response as measured by IFN-γ ELISPOT assay. Two of 11 case appeared to lack GPC3 expression in the recurrence tumor, although GPC3 was expressed in the primary HCC tissue before GPC3 peptide vaccine. In the other case, GPC3 peptide specific CTLs had infiltrated into recurrence tumor, and the expression of PD-1 among CD8 positive T cells, was higher in recurrence tumor and vaccine site than in PBMCs. Conclusions: GPC3 peptide vaccine for patient with GPC3 positive tumor could improve 1-year recurrence rates. This study showed GPC3 expression of the primary tumor could be the biomarker for a larger randomized clinical trial to determine the efficacy of the GPC3-derived peptide vaccine. Citation Format: Yu Sawada, Toshiaki Yoshikawa, Kazuya Ofuji, Mayuko Yoshimura, Nobuhiro Tsuchiya, Mari Takahashi, Daisuke Nobuoka, Shoichi Mizuno, Itaru Endo, Tetsuya Nakatsura. Phase II study of the GPC3-derived peptide vaccine as an adjuvant therapy for hepatocellular carcinoma patients. [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 CT115. doi:10.1158/1538-7445.AM2015-CT115