Risa Omori

Cyclooxygenase‐2 gene promoter polymorphisms affect susceptibility to hepatitis C virus infection and disease progression

Aim:  Because polymorphisms of cyclooxygenase‐2 (COX‐2) and osteopontin (OPN) promoter regions and a promoter/enhancer region of forkhead box protein 3 (FOXP3) gene are known to affect immune responses, we examined whether these polymorphisms can influence susceptibility to hepatitis C virus (HCV) infection and progression of liver disease.

Immune Response of Cytotoxic T Lymphocytes and Possibility of Vaccine Development for Hepatitis C Virus Infection

Immune responses of cytotoxic T lymphocytes (CTLs) are implicated in viral eradication and the pathogenesis of hepatitis C. Weak CTL response against hepatitis C virus (HCV) may lead to a persistent infection. HCV infection impairs the function of HCV-specific CTLs; HCV proteins are thought to actively suppress host immune responses, including CTLs. Induction of a strong HCV-specific CTL response in HCV-infected patients can facilitate complete HCV clearance. Thus, the development of a vaccine that can induce potent CTL response against HCV is strongly expected. We investigated HCV-specific CTL responses by enzyme-linked immuno-spot assay and/or synthetic peptides and identified over 40 novel CTL epitopes in the HCV protein. Our findings may contribute to the development of the HCV vaccine. In this paper, we describe the CTL responses in HCV infection and the attempts at vaccine development based on recent scientific articles.

Interleukin-4 and CpG oligonucleotide therapy suppresses the outgrowth of tumors by activating tumor-specific Th1-type immune responses.

Because IL-4 and CpG oligodeoxynucleotides (CpG-ODNs) are immune stimulants, we evaluated the antitumor effects of IL-4 gene therapy and CpG-ODN treatment in a poorly immunogenic murine cancer model. We used a murine colorectal cancer MC38 cell line overexpressing the IL-4 gene (MC38-IL4). Incubation with MC38-IL4 and CpG-ODN enhanced bone marrow-derived dendritic cell (DC) maturation in vitro. In addition, interferon (IFN)-γ production was significantly increased in naïve splenocytes after they were coincubated with MC38-IL4 and CpG-ODN. When mice bearing MC38 wild-type tumors were inoculated subcutaneously with MC38-IL4 cells and CpG-ODN, the outgrowth of established parental tumors was significantly suppressed compared to those in the MC38-IL4-treated group (IL-4 vs. IL-4 + CpG-ODN, p=0.015). A marked infiltration of CD8+ cells in the established parental tumors of mice treated with MC38-IL4 and CpG-ODN was confirmed by immunohistochemical analyses (MC38-IL4, 2.8 ± 1.9 cells/field vs. MC38-IL4 + CpG-ODN, 20.7 ± 15.3 cells/field, p=0.027). Significant tumor-specific cytolysis was detected when splenocytes of MC38-IL4 + CpG-ODN-treated mice were stimulated by γ-irradiated MC38-IL4 cells and CpG-ODN twice weekly in vitro and used as effector cells in a chromium-release assay (32.2 ± 3.5% for MC38 cells vs. 3.2 ± 1.1% for YAC-1 cells; at an effector to target ratio of 40). These results suggest that IL-4 and CpG-ODN treatment promotes potent Th1-type antitumor immune responses. Therefore, the combination of IL-4 gene therapy and CpG-ODN treatment for cancer should be evaluated in clinical trials.

Efficacy of direct-acting antiviral treatment for chronic hepatitis C: A single hospital experience

AIM To evaluate the efficacy of direct-acting antivirals (DAAs) in Kanto Rosai Hospital. METHODS All patients with hepatitis C virus (HCV) who underwent DAA prescription were enrolled in this study. The present study was a single center retrospective analysis using patients infected with HCV genotype 1 or 2. Resistance analysis was performed by using direct sequencing and cycleave PCR in genotype 1 patients treated with interferon (IFN)-free DAA. The primary endpoint was sustained virologic response at 12 wk after therapy (SVR12). RESULTS A total of 117 patients participated in the study, including 135 with genotype 1 and 42 with genotype 2. Of the 135 patients with genotype 1, 16 received protease inhibitor + IFN + ribavirin and all achieved SVR. Of the 119 patients who received IFN-free DAA (in different combinations), 102 achieved SVR and 9 failed (7/9 were on daclatasvir/asunaprevir and 2/9 on ledipasvir/sofosbuvir). Efficacy analysis was done only for 43 patients who received daclatasvir/asunaprevir. From this analysis, Y93 resistance-associated substitutions were significantly correlated with SVR. CONCLUSION The SVR rate was 98% for genotype 1 and 100% for genotype 2. However, caution is needed for HCV NS5A resistance-associated substitutions that are selected by HCV NS5A inhibitors because cerebrovascular adverse events are induced by some DAA drugs.

The Effect of New Therapeutic and Diagnostic Agents on the Prognosis of Hepatocellular Carcinoma in Japan – An Analysis of Data from the Kanagawa Cancer Registry

Objective: Notable advances in diagnostic imaging modalities and therapeutic agents have contributed to improvement in the prognosis of hepatocellular carcinoma (HCC) over the past decade. However, knowledge concerning their epidemiological contribution remains limited. The present study investigated the effect of emerging diagnostic and therapeutic agents on HCC prognosis, using the largest regional cancer registry in Japan. Methods: Using data from the Kanagawa Cancer Registry, the five-year survival rate of patients with liver cancer was estimated according to the International Statistical Classification of Diseases and Related Health Problems (10th Edition). Result: A total of 40,276 cases of HCC (from 1976 to 2013) were identified. The prognosis markedly improved after the introduction of new devices into the diagnosis and treatment of HCC (p<0.01). The trend of survival rate varied significantly between institutions with many registered patients (high-volume centers) (p<0.01). Conclusion: The five-year survival rate of patients with HCC in Kanagawa has markedly improved in recent years. This improvement in survival may be attributed to the advances in surveillance and intervention for the treatment of HCC.

Increased expression of immuno-inhibitory molecules on peripheral blood lymphocytes may suppress disease progression in autoimmune hepatitis.

Accumulation of activated lymphocytes in the liver has been reported in patients with autoimmune liver diseases; the activity of these cells may be related to the disease immunopathogenesis. Although immuno-inhibitory molecules on lymphocytes are thought to play an important role in immunomodulation, there are only a few reports on the status of these molecules in autoimmune liver diseases. We focused on representative immunoinhibitory molecules, such as tumor necrosis factorrelated apoptosis-inducing ligand receptor 2/death receptor 5 (DR-5)/CD262, Fas/APO-1/CD95, programmed cell death-1 (PD-1)/CD279 and cytotoxic T-lymphocyte antigen-4 (CTLA-4)/CD152 on the surface of Tlymphocytes in patients with autoimmune liver disease, and investigated the relationship between inhibitory molecules on lymphocytes and the clinical features of patients with autoimmune liver diseases A limited number of patients with autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC) and chronic hepatitis C (CHC) (n=10/group) and 10 healthy volunteers (control) were enrolled in this study. All patients and healthy volunteers gave written informed consent according to a protocol approved by the ethics committee of Showa University. Expression of inhibitory molecules DR5, Fas, PD-1 and CTLA-4 on the surface of peripheral CD4 and CD8 cells was assessed by staining withmonoclonal antibodies and by analyzing with flow cytometry. Differences between groups were assessed by ANOVA with Tukey’s honestly significant difference test and were considered statistically significant at P<0.05. Death receptor 5 expression was significantly upregulated on CD4 cells, but not on CD8 cells of patients with AIH compared with the controls (Fig. 1a). We found no differences in the expression of Fas molecules between patients and controls (Fig. 1b). PD-1 expression was more common on CD4 and CD8 cells of patients with AIH than on the control cells (Fig. 1c). The CD4 and CD8 cells of patients with AIH expressed CTLA-4 molecules more frequently than those of the controls, as shown in Figure 1(d). By comparison of the groups, significant difference could not be found between the control group and the disease groups other than AIH. In three AIH patients, the frequency of CTLA-4 positive cells was extremely high (Fig. 1d). The characteristics of these patients are presented in Table 1. They presented high frequencies of DR-5 and PD-1 expression as well. We noticed no signs of hepatitis activation such as elevated serum transaminase levels during the observation period. The other seven AIH patients had been treated with more than 5mg/day of prednisolone to control AIH. High expression of inhibitory molecules on CD4 and CD8 cells may suppress the disease activity of AIH. Upregulation of these immuno-inhibitory molecules may be a strategy for controlling AIH. We have reported that AIH patients have fewer intrahepatic regulatory T cells than do PBC patients. Immuno-inhibitory molecules also play important roles in immune regulation to prevent excessive inflammation. It has been reported that intrahepatic expression of PD-1 may play a crucial role in immunopathogenesis of AIH. Comprehensive study examining expression of inhibitory molecules on the surface of peripheral lymphocytes has not been reported, and it remains unclear whether overexpression of each molecule affects disease progression. Although each characteristic detail of inhibitory molecules has not been defined, several reports have compared CTLA-4 and PD-1. Each molecule inhibits T-cell activation by a different mechanism. PD-1 may be antigen-specific. Elucidation of the role of these molecules in immunerelated diseases would contribute to our understanding of the immunopathogenesis and the development of the treatment strategy. Further investigations including functional assay of inhibitory moleculeoverexpressing cells are needed.

Abstract 4386: Effects of interferon-α-transduced tumor cell vaccines and blockade of programmed cell death 1 on the growth of established tumors

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Progress in treatments for cancer has improved the prognosis of patients with colorectal cancer. However, there is a strong need for the development of a new intervention therapy that suppresses the occurrence or recurrence effectively with fewer side effects. Immunotherapy may be such a treatment and gene therapy using tumor cells that are genetically modified to produce cytokines has been studied in several therapeutic models. We have previously reported that the interferon (IFN)-α gene-transduced tumor-based vaccination therapy suppresses the outgrowth of established tumors. Although the suppressive effects on established tumors were observed, we did not see reductions in the size of all of the parental tumors. Therefore, further improvements in the treatment are needed before clinical application, we focused on programmed cell death 1 (PD-1), which has been identified as a marker of exhausted T cells. In this study, we evaluated the efficacy of the combination of IFN-γ-transduced tumor cell vaccines and PD-1 blockade, and investigated the mechanisms of the antitumor effects of the combined therapy. A poorly immunogenic murine colorectal cancer cell line, MC38, was transduced to overexpress IFN-γ (MC38-IFNα). In a therapeutic model, parental tumor-bearing mice were inoculated with MC38-IFNα cells and an anti-PD-1 antagonistic antibody. Significant suppression of outgrowth of the established tumors was observed in the IFN-γ and anti-PD-1 combination treatment group (IFN+ anti-PD-1, 174.17 ± 35.54 mm2 vs control, 328.67 ± 26.36 mm2 on day 28, P = 0.0114 vs controls). Immunohistochemical analyses showed marked infiltration of CD4+ cells as well as CD8+ cells in the established tumors of mice treated with both IFN-γ and anti-PD-1. To investigate induction of tumor-specific immune responses, we stimulated splenocytes of IFN-γ or/and anti-PD-1 treated mice twice weekly by DCs in vitro. Significant tumor-specific cytolysis was detected when splenocytes of mice treated with both IFN-γ and anti-PD-1 were used as effector cells (58.1% ± 6.7% for MC38 and 14.1% ± 1.7% for YAC-1, effector:target = 20, P < 0.001). Our findings suggest that blockade of the PD-1 PD-ligand enhanced the Th1-type antitumor immune responses induced by IFN-γ. The combination of IFN-γ gene-transduced tumor cell vaccines and PD-1 blockade may be a possible candidate for a cancer vaccine for clinical trials. 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 4386. doi:1538-7445.AM2012-4386

Abstract 524: Cytokine therapy by allogenic IFN-alpha-expressing murine colorectal cancer cells suppresses outgrowth of established tumors in a murine hepatocellular carcinoma model

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Although several effective therapies for hepatocellular carcinoma (HCC) have been developed, many patients with both advanced cirrhosis and HCC are not able to receive those treatments because of their poor liver function. Thus, it is needed to establish a new effective therapy that has lesser side effects for those patients. We previously reported that immunotherapy by interferon-alpha (IFNa) and dendritic cells effectively suppressed outgrowth of established tumors and showed preventive effects in the murine colorectal cancer, MC38 cell model. We also revealed that tumor-specific cytotoxic T cell responses were important for these anti-tumor effects. In this study, we investigated the antitumor effect of allogenic cytokine (IFNa and IL-4)- expressing MC38 cells in the murine hepatocellular carcinoma, BNL model. There were no differences in cell growth between BNL cells and BNL cells co-incubated with cytokine-expressing MC38 cells in vitro. Balb/c mice were injected with 5x105 of BNL cells on their flank. Once BNL tumors were established, these mice received therapeutic injection of cytokine-expressing MC38 cells (2.5x105/mouse or 5x105/mouse) and tumor size was measured twice weekly. While tumor growth of the mice injected with MC38-IL4 was not suppressed (control; 421.0±146.9mm2, MC38-IL4 (5x105); 351.3±126.1mm2, p=0.33), injection of MC38-IFNa cells significantly suppressed the tumor growth by dose-dependent manner (MC38-IFNa: 5x105 cells; 183.5±46.2mm2, p=0.02). When we injected mice with both IFNa cells and IL-4 cells, any additive anti-tumor effects were not observed (mice injected both modified MC38: 5x105 cells each; 182±44.6 mm2, p=0.01). A BNL-specific cytolysis was detected when splenocytes of mice injected with MC38-IFNa were used as effector cells in a chromium-release assay. Furthermore, immunohistocemistrical analysis revealed that CD4+ T cells, CD8+ T cells and especially Gr-1+ cells infiltrated established BNL tumors of mice injected with MC38-IFNa. Our results suggest that the immunotherapy with allogenic IFNa-expressing cells has potent antitumor effects, and that it would be applicable for treatment to advanced HCC patients. 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 524. doi:1538-7445.AM2012-524