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.

Purpose: The carcinoembryonic antigen glypican-3 (GPC3) is an ideal target of anticancer immunotherapy against hepatocellular carcinoma (HCC). In this nonrandomized, open-label, phase I clinical trial, we analyzed the safety and efficacy of GPC3 peptide vaccination in patients with advanced HCC. Experimental Design: Thirty-three patients with advanced HCC underwent GPC3 peptide vaccination (intradermal injections on days 1, 15, and 29 with dose escalation). The primary endpoint was the safety of GPC3 peptide vaccination. The secondary endpoints were immune response, as measured by IFN-γ ELISPOT assay, and the clinical outcomes tumor response, time to tumor progression, and overall survival (OS). Results: GPC3 vaccination was well-tolerated. One patient showed a partial response, and 19 patients showed stable disease 2 months after initiation of treatment. Four of the 19 patients with stable disease had tumor necrosis or regression that did not meet the criteria for a partial response. Levels of the tumor markers α-fetoprotein and/or des-γ-carboxy prothrombin temporarily decreased in nine patients. The GPC3 peptide vaccine induced a GPC3-specific CTL response in 30 patients. Furthermore, GPC3-specific CTL frequency after vaccination correlated with OS. OS was significantly longer in patients with high GPC3-specific CTL frequencies (N = 15) than in those with low frequencies (N = 18; P = 0.033). Conclusions: GPC3-derived peptide vaccination was well-tolerated, and measurable immune responses and antitumor efficacy were noted. This is the first study to show that peptide-specific CTL frequency can be a predictive marker of OS in patients with HCC receiving peptide vaccination. Clin Cancer Res; 18(13); 3686–96. ©2012 AACR.

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)

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.

The use of allogeneic, pluripotent stem‐cell‐derived immune cells for cancer immunotherapy has been the subject of recent clinical trials. In Japan, investigator‐initiated clinical trials will soon begin for ovarian cancer treatment using human leukocyte antigen (HLA)‐homozygous‐induced pluripotent stem cell (iPSC)‐derived anti–glypican‐3 (GPC3) chimeric antigen receptor (CAR)‐expressing natural killer/innate lymphoid cells (NK/ILC). Using pluripotent stem cells as the source for allogeneic immune cells facilitates stringent quality control of the final product, in terms of efficacy, safety and producibility. In this paper, we describe our methods for the stable, feeder‐free production of CAR‐expressing NK/ILC cells from CAR‐transduced iPSC with clinically relevant scale and materials. The average number of cells that could be differentiated from 1.8‐3.6 × 10⁶ iPSC within 7 weeks was 1.8‐4.0 × 10⁹. These cells showed stable CD45/CD7/CAR expression, effector functions of cytotoxicity and interferon gamma (IFN‐γ) production against GPC3‐expressing tumor cells. When the CAR‐NK/ILC cells were injected into a GPC3‐positive, ovarian‐tumor‐bearing, immunodeficient mouse model, we observed a significant therapeutic effect that prolonged the survival of the animals. When the cells were injected into immunodeficient mice during non–clinical safety tests, no acute systemic toxicity or tumorigenicity of the final product or residual iPSC was observed. In addition, our test results for the CAR‐NK/ILC cells generated with clinical manufacturing standards are encouraging, and these methods should accelerate the development of allogeneic pluripotent stem cell‐based immune cell cancer therapies.

Novel treatment modalities are required urgently in patients with hepatocellular carcinoma (HCC). A vaccine that induces cytotoxic T lymphocytes (CTLs) is an ideal strategy for cancer, and glypican-3 (GPC3) is a potential option for HCC. Blocking the programmed death-1 (PD-1)/PD-L1 pathway is a rational strategy to overcome tumor escape and tolerance toward CTLs. In the present study, we investigated whether anti-PD-1 blocking antibodies (αPD-1 Ab) enhanced the number of vaccine-induced peptide-specific CTLs in peripheral blood mononuclear cells (PBMCs) following the administration of GPC3 peptide vaccine to both patients and in a mouse model. The inhibitory receptor PD-1 was highly expressed in ex vivo GPC3-specific CTLs isolated from the PBMCs of vaccinated HCC patients. In vitro, interferon-γ induced PD-L1 expression in liver cancer cell lines. In addition, PD-1 blockade increased the number of GPC3-specific CTLs, which degranulate against liver cancer cell lines. In vivo experiments using tumor-bearing mouse models showed that the combination therapy of peptide vaccine and αPD-1 Ab suppressed tumor growth synergistically. PD-1 blockade increased the number of peptide-specific tumor-infiltrating T cells (TILs) and decreased the expression of inhibitory receptors on TILs. This study demonstrated that PD-1/PD-L1 blockade augmented the antitumor effects of a peptide vaccine by increasing the immune response of vaccine-induced CTLs, and provided a foundation for the clinical development of a combination therapy using a GPC3 peptide vaccine and αPD-1 Ab.