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Background Immunotherapy for cancer patients has been the subject of attention in recent years. In this study, we investigated whether αβT‐cell therapy causes changes in the host's immune cell profile, and if so, the effect of these changes on prognosis. Methods Peripheral blood mononuclear cells (PBMCs) from 30 gastric cancer patients who had completed one course of αβT‐cell therapy were analyzed. The peripheral blood immune cell profile was established using PBMCs by counting the frequency of CD4+ helper T cells, CD8+ killer T cells, regulatory T cells (Tregs), and myeloid‐derived suppressor cells and measuring the expression of their surface markers. The changes after treatment and their association with response to treatment were investigated. Results Immune cell profiles changed greatly after treatment. The frequency of CD4+ helper T cells decreased, but that of CD8+ killer T cells increased. The frequency of programmed cell death 1 (PD‐1)+ effector Tregs increased significantly, but only in the non‐progressive disease (non‐PD) group, in which it was significantly higher compared with the PD group. Patients in whom the frequency of PD‐1+ effector Tregs increased had a significantly better prognosis than those in whom it decreased. Conclusion Our results suggested that αβT‐cell therapy changes the host's immune cell profile, and an increase in PD‐1+ effector Tregs may help improve prognosis., Our results suggested that αβT‐cell therapy changes the host's immune cell profile. An increase in PD‐1+ effector Tregs may help improve prognosis of advanced gastric cancer.

The interaction between T-cells/fatty acids involved in non-alcoholic fatty liver disease (NAFLD) and liver fibrosis progression is poorly understood. In this study, we conducted a comprehensive analysis of T-cell profiles of NAFLD patients to better understand their relationship with fatty acids and relevance to liver fibrosis. We analyzed the differences in T-cell profiles of peripheral blood mononuclear cells (PBMCs) between 40 NAFLD patients and 5 healthy volunteers (HVs), and their relationship with liver fibrosis stage or progression. Moreover, we analyzed the relationship between T-cell profiles and fatty acid compositions in vivo, and changes in T-cell profiles after treatment with fatty acids in vitro. T-cell profiles of NAFLD patients were different from those of HVs. The CD25+CD45+CD4+ T-cell frequency was increased in NAFLD patients with high liver fibrosis stage and progression, and this indicated immune activation. Despite such a state of immune activation, the PD1+CD4+ T-cell frequency was decreased in the same patients group. The PD1+CD4+ T-cell frequency had a significantly negative correlation with the serum fatty acid composition ratio C16:1n7/C16:0. Moreover, the PD1+CD4+ T-cell frequency was significantly decreased by in vitro treatment with fatty acids. In addition, its rate of frequency change was significantly different between C16:0 and C16:1n7 and decreased by artificially increasing the C16:1n7/C16:0 ratio. The analysis of PBMCs in NAFLD patients showed that T-cell profiles were different from those of HVs. And, it suggested that fatty acids modified T-cell profiles and were involved in liver fibrosis in NAFLD patients.

Dendritic cells (DCs) are antigen-presenting cells with a central role in host immune response. This study analyzed gene expression and DC function in hepatitis B virus (HBV) patients, functions impaired because of HBV, and identified the genes related to these functions. Peripheral blood mononuclear cells from 64 HBV patients and 19 healthy controls were analyzed. Peripheral blood DCs were stained with antibodies against human leukocyte antigen-DR/Lin-1/CD123/CD11c and separated into plasmacytoid DCs (pDCs) and myeloid DCs by fluorescence-activated cell sorting. Using an interferon-gamma enzyme-linked immunospot assay, we analyzed antigen-specific response in HBV-infected patients. Regarding DC function, we analyzed antigen-presenting capacity, cell migration capacity, phagocytic capacity, and cytokine production capacity. DC gene expression was analyzed by microarray to identify genes related to DC function. No difference was found in the number of DCs in peripheral blood between healthy participants and HBV patients. In cell-surface marker analysis, CD80, CD83, CD86, CD40, and C-C motif chemokine receptor 7 expression levels in pDCs were related to the HBV-specific T-cell response. DCs from HBV patients exhibited decreases in antigen-presenting capacity, migration capacity, and cytokine production capacity. In gene expression analysis, immune-related genes with greatly reduced expression levels in chronic hepatitis B patients were identified. Of these genes, interleukin (IL)-6 signal transducer (IL6ST) expression level positively correlated with DC surface marker expression level. Adjustment of IL6ST expression level in DCs and treatment with oncostatin M resulted in recovery of DC function. Conclusion: IL6ST expression was identified as one cause of decline in DC function in HBV patients. Adjustment of IL6 family cytokine signaling may be useful for recovering reduced DC function in HBV infection.

BACKGROUND AND AIMS Accurate diagnosis of non-alcoholic steatohepatitis (NASH) from non-alcoholic fatty liver disease (NAFLD) is clinically important. Therefore, there is a need for easier ways of diagnosing NASH. In this study, we investigated the serum fatty acid composition and evaluated the possibility of using the serum fatty acid composition as a diagnostic marker of NASH. METHODS The subjects were 78 NAFLD patients (non-alcoholic fatty liver [NAFL]: 30, NASH: 48) and 24 healthy individuals. Fatty acids extracted from the liver tissue and serum were identified and quantified by gas chromatography. In addition, we evaluated the relationship between serum and liver tissue fatty acid composition, patient background, and liver histology. The diagnostic performance of NASH was evaluated by calculating the area under the receiver operating characteristic (AUROC). RESULTS The results of the fatty acid analysis showed the C16:1n7/C16:0 ratio to have the strongest correlation between serum and liver tissue (r = 0.865, P

Pancreatic ductal adenocarcinoma (PDAC) is the most life-threating disease among all digestive system malignancies. We developed a blood mRNA PDAC screening system using real-time detection PCR to detect the expression of 56 genes, to discriminate PDAC from noncancer subjects. We undertook a clinical study to assess the performance of the developed system. We collected whole blood RNA from 53 PDAC patients, 102 noncancer subjects, 22 patients with chronic pancreatitis, and 23 patients with intraductal papillary mucinous neoplasms in a per protocol analysis. The sensitivity of the system for PDAC diagnosis was 73.6% (95% confidence interval, 59.7%-84.7%). The specificity for noncancer volunteers, chronic pancreatitis, and patients with intraductal papillary mucinous neoplasms was 64.7% (54.6%-73.9%), 63.6% (40.7%-82.8%), and 47.8% (26.8%-69.4%), respectively. Importantly, the sensitivity of this system for both stage I and stage II PDAC was 78.6% (57.1%-100%), suggesting that detection of PDAC by the system is not dependent on the stage of PDAC. These results indicated that the screening system, relying on assessment of changes in mRNA expression in blood cells, is a viable alternative screening strategy for PDAC.

The behaviors and fates of immune cells in cancer patients, such as dysfunction and stem-like states leading to memory formation in T cells, are in intense focus of investigation. Here we show, by post hoc analysis of peripheral blood lymphocytes of hepatocellular carcinoma patients previously undergoing vaccination with tumour-associated antigen-derived peptides in our clinical trials (registration numbers UMIN000003511, UMIN000004540, UMIN000005677, UMIN000003514 and UMIN000005678), that induced peptide-specific T cell responses may persist beyond 10 years following vaccination. Tracking TCR clonotypes at the single cell level reveals in two patients that peptide-specific long-lasting CD8+ T cells acquire an effector memory phenotype that associates with cell cycle-related genes (CCNA2 and CDK1), and are characterized by high expression of IL7R, SELL, and NOSIP along with a later stage promotion of the AP-1 transcription factor network (5 years or more past vaccination). We conclude that effective anti-tumor immunity is governed by potentially proliferative memory T cells, specific to cancer antigens.

Background and Objectives: Although cholangiolocellular carcinoma is considered a combined hepatocellular and cholangiocarcinoma, we feel that this classification is not appropriate. Therefore, we compared the diagnostic imaging findings, surgical prognosis, and pathological features of cholangiolocellular carcinoma with those of other combined hepatocellular and cholangiocarcinoma subtypes, hepatocellular carcinoma, and cholangiocarcinoma. Methods: The study patients included 7 with classical type combined hepatocellular and cholangiocarcinoma; 8 with stem cell feature, intermediate type combined hepatocellular and cholangiocarcinoma; 13 with cholangiolocellular carcinoma; 58 with cholangiocarcinoma; and 359 with hepatocellular carcinoma. All patients underwent hepatectomy or living-related donor liver transplantation from 2001 to 2014. Results: cholangiolocellular carcinoma could be distinguished from hepatocellular carcinom, other combined hepatocellular and cholangiocarcinoma subtypes, and cholangiocarcinoma by the presence of intratumoral Glisson’s pedicle, hepatic vein penetration, and tumor-staining pattern on angiography-assisted CT. Cholangiolocellular carcinoma was associated with a significantly lower SUV-max than that of cholangiocarcinoma on FDG-PET. Hepatocellular carcinoma, classical type, and cholangiolocellular carcinoma had significantly better prognoses than stem cell feature, intermediate type and cholangiocarcinoma. A cholangiocarcinoma component was detected in cholangiolocellular carcinoma that progressed to the hepatic hilum, and the cholangiocarcinoma component was found in perineural invasion and lymph node metastases. Conclusions: From the viewpoint of surgeon, cholangiolocellular carcinoma should be classified as a good-prognosis subtype of biliary tract carcinoma because of its tendency to differentiate into cholangiocarcinoma during its progression, and its distinctive imaging and few recurrence rates different from other combined hepatocellular and cholangiocarcinoma subtypes.

Aim Sequential administration of sorafenib followed by regorafenib or lenvatinib is effective against advanced hepatocellular carcinoma (HCC). In this study, we compared the safety profiles and anti-tumor effects of sequential sorafenib and regorafenib or lenvatinib therapy in patients with HCC. Methods We investigated adverse events, treatment responses and dose intensities in patients with HCC who were consecutively treated with sorafenib followed by regorafenib or lenvatinib at the individual level. Results Each group included 20 patients. The safety profiles of regorafenib and sorafenib were similar. The severity of hypophosphatemia, palmar-plantar erythrodysesthesia syndrome, and decreased neutrophil counts associated with regorafenib or sorafenib was similar in 12 patients. Conversely, the incidences and grades of adverse events differed between sorafenib and lenvatinib treatment. The anti-tumor effects of regorafenib and lenvatinib compared with sorafenib were significantly different for each patient. The response to treatment and progression-free survival were comparable for regorafenib and lenvatinib. The median relative dose intensities during the first 56 days of regorafenib and lenvatinib treatment were 83.6 and 80.0%, respectively. Conclusions Similar adverse events were experienced by patients during consecutive treatment with sorafenib and regorafenib, which was not observed during treatment with sorafenib and lenvatinib. The obtained safety profile of sorafenib provided meaningful insights for selecting sequential therapy for patients with advanced HCC.

Background aims Immunotherapy is effective for many types of cancer, but its benefits in advanced pancreatic cancer, which has a poor prognosis, are not well established. In this study, the authors examined the effects of adoptive T-cell immunotherapy (ATI) on immune cell profiles and prognosis in patients with unresectable advanced pancreatic cancer. Methods Seventy-seven patients with unresectable advanced pancreatic cancer were treated with six cycles of αβ T cells alone or in combination with chemotherapy or chemoradiation. Immune cell profiles in peripheral blood samples obtained before and after treatment were comprehensively evaluated by flow cytometry. Furthermore, associations between changes in immune cell frequencies and prognosis were determined. Results ATI prolonged survival to 18.7 months compared with previous estimates of 6.2–11.1 months for patients treated with chemotherapy alone. ATI decreased CD3+CD4+CD8− T cell frequency in peripheral blood and increased CD3+CD4−CD8+ T cell frequency. An increase in CD3+ T cells and CD3+TCRγδ− T cells in peripheral blood after treatment was associated with a good prognosis. Conclusions ATI altered the immune profile in peripheral blood, including CD3+CD4−CD8+ T cells, and improved prognosis in pancreatic cancer.

A 79-year-old man with metastatic melanoma of the right maxillary sinus and multiple liver metastases received a single dose of nivolumab. Eight days later, he experienced impaired consciousness, accompanied by abnormal laboratory and electrocardiographic findings. He was therefore diagnosed with tumor lysis syndrome (TLS). Laboratory and electrocardiographic findings improved immediately after continuous hemodiafiltration; however, he died 22 days after receiving nivolumab. Autopsy revealed massive tumor necrosis in the liver. There are few case reports of TLS associated with immune checkpoint inhibitors, indicating that we should be prepared to manage especially in a patient with liver involvement of high tumor burden.

α-Fetoprotein (AFP) is considered a good target for immunotherapy strategies against hepatocellular carcinoma (HCC); however, no immunodominant AFP-derived MHC class II-restricted helper T-lymphocyte (HTL) epitope has been reported. Therefore, we identified novel AFP-derived HTL epitopes possessing high immunogenicity. HTL epitopes were predicted using the online service, and peptides were subsequently synthesized. Four newly synthesized peptides showed positive reactivity in >20% patients on ELISPOT using peripheral blood mononuclear cells (PBMCs). Among these, the highest rate was shown by AFP1 (MKWVESIFLIFLLNFTESRT), which also showed the highest positive rate in cell proliferation assays. Binding assays demonstrated that AFP1 had strong binding properties toward MHC molecules. Further, blocking assays performed using an anti-HLA-DR antibody showed that immune response decreased, confirming the binding of AFP1 to HLA-DR molecules. Furthermore, the survival rates of patients with stages II–IV HCC indicated that T cell response against AFP1 led to significantly greater survival that of patients without T cell response. When evaluating immune response against AFP1 before and after HCC treatment, an increase in the frequency of peptide-specific T cells was observed after treatment in patients with HLA-DRB1*1502, *0405, and *0901 alleles. In conclusion, the identified epitopes may be useful for immunotherapy strategies against HCC.

Host antitumor immune responses may be different between hepatocellular carcinoma (HCC) caused by metabolic disorders and HCC associated with hepatitis virus infection. In this study, we examined the immune response of tumor-associated antigen (TAA)-specific T cells and immune cell profile in patients with HCC separated by cause. Thirty-two patients with hepatitis B virus (HBV)-related HCC, 42 patients with hepatitis C virus-related HCC, and 18 patients with nonalcoholic steatohepatitis (NASH)-related HCC were analyzed. The frequencies of TAA-specific T cells, the expression levels of surface markers on each immune cell, and the expression of each TAA in HCC tissue were measured. The immune response to TAA and immune cell profile were markedly different among the three groups. The immune response to TAA in the NASH-related HCC group was weaker than the responses in the other two groups. In patients with NASH-related HCC, the frequencies of effector regulatory T cells (eTregs) and cluster of differentiation 8-positive (CD8+ ) T cells strongly expressing cytotoxic T-lymphocyte antigen (CTLA)-4 were high. The frequency of CD8+ T cells strongly expressing programmed cell death 1 was the highest in patients with HBV-related HCC. Among these immune cell profiles, the frequencies of C-X-C motif chemokine receptor 3+ eTregs and CTLA-4+ CD8+ T cells were inversely correlated with the strength of the TAA-specific T-cell immune response, and the restoration of TAA-specific T-cell responses by anti-CTLA-4 antibody was observed. Conclusion: The immune response to TAA were markedly different among the three groups, and a correlation with the immune cell profile was observed, suggesting that development of immunotherapy based on the etiology of HCC may lead to more effective treatment outcomes.

We report three cases of genotype 1b hepatitis C virus (HCV) reinfection after liver transplantation. When antiviral treatment was considered, all three patients had renal dysfunction and had been treated with immunosuppressive agents for a long time; one with tacrolimus (TAC) and the others with cyclosporine A (CyA). Therefore, the possible antiviral regimens among direct-acting antivirals (DAA) were limited and so we treated all three patients with ombitasvir/paritaprevir/ritonavir (OBV/PTV/r). Because ritonavir is known to markedly increase the blood concentration of TAC and CyA through drug-drug interactions, close monitoring of blood concentrations of TAC or CyA and dose adjustments of immunosuppressive agents were needed. Sustained virus response was achieved in all the patients treated, and there were no adverse effects or transplant rejection. OBV/PTV/r might be a useful DAA regimen for patients with genotype 1 HCV reinfection in the setting of renal dysfunction.

Background and aims Human telomerase reverse transcriptase is a catalytic enzyme involved in telomere elongation. It is expressed in many tumours, including hepatocellular carcinoma. The purpose of the present study was to identify major histocompatibility complex class II-restricted helper T cell epitopes derived from human telomerase reverse transcriptase in patients with hepatocellular carcinoma. Methods TEPITOPE software was used to predict helper T cell epitopes based on the entire amino acid sequence of human telomerase reverse transcriptase, and peptides were synthesized based on the predicted sequence. Interferon (IFN)-γ enzyme linked immunospot assay was performed to examine the T cell response to each of the synthesized peptides in peripheral blood mononuclear cells. Furthermore, the peptides were labelled with fluorescein isothiocyanate to test their binding affinity for major histocompatibility complex class II molecules. Lastly, the association between patient characteristics and the level of immune response to these epitopes was examined. Results Positive T cell response (>10% enzyme linked immunospot positivity) was detected against 4 of 10 peptides. Among all peptides, positive T cell response to the hTERT68 peptide was detected most frequently. While hTERT68 was HLA-DRB1*0405-restricted, it also bound to other MCH class II molecules. Positive helper T cell response was detected most frequently in hepatocellular carcinoma patients with a low serum alpha-foetoprotein level. Several treatments for hepatocellular carcinoma enhanced the immune response against the peptides. Conclusion Our findings indicate that helper T cell epitopes identified in the present study may be useful to investigate immune responses and for immunotherapy in hepatocellular carcinoma patients.

BackgroundPortal vein thrombosis (PVT) is a common complication of cirrhosis. However, in patients with PVT and cirrhosis, there is no clear evidence supporting effective treatment modalities. In this study, we examined the effectiveness and safety of anticoagulation therapy using danaparoid sodium for PVT in patients with cirrhosis.MethodsThis retrospective study assessed 52 cirrhotic patients with PVT treated with danaparoid sodium for 2 weeks between November 2008 and September 2018. The primary outcome measure was the post-treatment status of PVT assessed by reduction in thrombus volume and safety of the therapeutic intervention. PVT status was evaluated with contrast-enhanced computed tomography (CECT). All patients received 1250 units of danaparoid sodium twice daily by intravenous injection for 14 days. Patients on antithrombin III (AT-III) combination therapy were additionally administered 1500 units of AT-III on days 1–5 and days 8–12. Effectiveness was evaluated by CECT from between days 13 and 18. The secondary outcome measure was the prognosis of PVT.ResultsAll patients showed reduction in PVT volume without complications. Return of plasma AT-III level to > 70% during the treatment period contributes to ≥75% reduction of PVT volume. The prognosis in PVT patients depends on hepatic reserve capacity. When limited to Child-Pugh B and C liver cirrhosis patients, a ≥ 75% reduction of PVT volume improved the prognosis.ConclusionsDanaparoid sodium-based anticoagulation therapy was effective and safe for PVT in patients with cirrhosis. Return of plasma AT-III level to the normal range during the treatment period contributes to reduction of PVT volume. A reduction of ≥75% in PVT volume may improve the prognosis of Child-Pugh B and C decompensated cirrhosis patients with PVT.

BACKGROUND Hepatocellular carcinoma (HCC) is a worldwide health concern because of a continued increase in cases globally; furthermore, the prognosis for patients with HCC remains poor. Transarterial chemoembolization (TACE) has been established as the standard of care for the intermediate stage of HCC; however, no therapeutic agents are available to reduce the high rate of recurrence. OBJECTIVE This study aims to evaluate the safety of alpha-fetoprotein (AFP)-derived peptides for patients with HCC post-TACE. METHODS This will be an open-label, single-arm, multicenter study to evaluate the safety of AFP-derived peptides (AFP 357 and AFP 403), which contain histocompatibility antigen-A24-restricted cytotoxic T lymphocyte epitopes from tumor antigens expressed in HCC and is recognized at a high rate by lymphocytes in patients with HCC. Protocol treatment will consist of six courses of the subcutaneous administration of 3 mg each of AFP 357 and AFP 403. A total of 14 patients will be included in this study, the first 6 as a main analysis target group and an additional 8 as an extended cohort from three institutions in Japan. The primary endpoint will be the occurrence of serious adverse events (safety profile). The secondary endpoints will include time to progression, overall survival, completion rate, and adverse events (efficacy profile). RESULTS We have recruited 14 patients with HCC as of December 2019. The final follow-up will be completed by March 2020. CONCLUSIONS In this study, we will evaluate the safety profile of AFP-derived peptides for patients with HCC post-TACE. We believe that this study will provide useful information and will help to design a subsequent phase II trial based on the results. CLINICALTRIAL Japan Registry of Clinical Trials jRCTs041180155; https://jrct.niph.go.jp/latest-detail/jRCTs041180155 INTERNATIONAL REGISTERED REPORT DERR1-10.2196/17082

Dendritic cell (DC)–based immunotherapies are believed to help eradicate residual tumor cells, including hepatocellular carcinoma (HCC). Here, we assessed the safety and clinical response to OK432-stimulated monocyte-derived DCs (MoDCs) in treating HCC after radiofrequency ablation (RFA). MoDCs were derived from 30 HCC patients in the presence of interleukin-4 and granulocyte-macrophage colony stimulating factor for 5 days and then cultured for 2 more days in the medium (basic protocol) or stimulated with OK432. On day 7, DCs were harvested and percutaneously injected into HCC tumors after RFA. We observed no grade 3 or 4 National Cancer Institute Common Toxicity Criteria adverse events. Kaplan-Meier analysis indicated that patients treated with RFA + OK432-stimulated DCs transfer had longer recurrence-free survival than those treated with RFA + basic-protocol DCs (median: 24.8 vs 13.0 months; P = .003). RFA with DC infusion can enhance various tumor-associated antigen (TAA)–specific T-cell responses. Additionally, the 5-year RFS rate for patients with significantly increased TAA-specific T-cell responses was much higher than for other patients (50.0% vs. 7.7%; P = .030). Our study provides useful information for development of HCC immunotherapies (trial registration: UMIN000001701).

金沢大学先進予防医学研究センター / 金沢大学医薬保健研究域医学系, Multidrug resistance-associated protein 3 (MRP3) is a carrier-type transport protein belonging to the ABC transporters. In this study, we investigated the safety and immunogenicity of a MRP3-derived peptide (MRP3765) as a vaccine and characterized the MRP3-specific T cell responses induced. Twelve hepatocellular carcinoma (HCC) patients treated with hepatic arterial infusion chemotherapy (HAIC) were enrolled. The MRP3-derived peptide was emulsified in incomplete Freund's adjuvant and administered via subcutaneous immunization three times weekly. No serious adverse drug reactions to the peptide vaccine were observed, and the vaccination was well tolerated. The vaccination induced MRP3-specific immunity in 72.7% of the patients. In a phenotypic analysis, the largest post-vaccinated increase in MRP3-specific T cells was due to an increase in cells with the effector memory phenotype. Among the 12 patients, one patient showed a partial response, nine showed a stable disease, and two showed a progressive disease. The median overall survival time was 14.0 months. In conclusion, the safety, effects of immune boosting, and possible prolongation of overall survival by the MRP3-derived peptide demonstrate the potential of the peptide to provide clinical benefit in HCC patients. © 2015.

Aim Alcoholic hepatocellular carcinoma (ALD-HCC) accounts for the majority of non-B non-C HCC (NBNC-HCC) cases. Although alcohol is a potent carcinogen, there have been few reports on the influence of modest alcohol consumption in NBNC-HCC. This study aimed to investigate the clinical characteristics and prognosis of NBNC-HCC patients with modest alcohol consumption. Methods From 2007 to 2010, 2283 HCC patients were evaluated at 10 hospitals. We collected detailed etiology data of 588 NBNC-HCC patients and compared the clinical characteristics and prognosis between ALD-HCC and modest alcohol-HCC patients. Results There were 69 HCC patients with modest alcohol consumption, accounting for 3% of all HCC patients evaluated. This patient group had significantly more women and higher prevalence of Child–Pugh class A, hypertension and advanced disease stage, and were diagnosed with HCC at an older age than the ALD-HCC group (266 patients). Additionally, among the modest alcohol-HCC patients, diabetes was significantly more common in the anti-hepatitis B core (HBc) negative subgroup than in the anti-HBc positive subgroup. However, no significant difference in survival was observed between the two patient groups regardless of significant differences in tumor staging. Alcohol consumption and metabolic factors were not significant independent predictors of survival. Conclusion The clinical characteristics of modest alcohol-HCC included advanced staging, favorable liver reserve capacity and older age at diagnosis. HCC development in patients with modest alcohol consumption may relate to metabolic factors. Although approximately 30% of the evaluated HCC cases were in advanced stages, the prognosis of NBNC-HCC patients with modest alcohol consumption was relatively favorable.

Human telomerase reverse transcriptase (hTERT) is a catalytic enzyme required for telomere elongation. In this study, we investigated the safety and immunogenicity of an hTERT-derived peptide (hTERT461) as a vaccine and characterized the hTERT-specific T cell responses induced. Fourteen hepatocellular carcinoma (HCC) patients were enrolled in the study. The hTERT-derived peptide was emulsified in incomplete Freund's adjuvant and administered via subcutaneous immunization three times biweekly. The maximum toxicity observed was grade 2 according to the common terminology criteria and mainly consisted of skin reactions at the site of vaccination. The vaccination induced hTERT-specific immunity in 71.4% of patients and 57.1% of patients administered with hTERT461 peptide-specific T cells could prevent HCC recurrence after vaccination. In phenotypic analysis, the post-vaccinated increase in hTERT-specific T cells was due to an increase in cells with the effector memory phenotype, with the potential to produce multiple cytokines. Seven hTERT-specific T cell receptors were obtained from the vaccinated patients, showing their cytotoxic activities to hTERT-derived peptide-bearing cells. In conclusion, the safety and effects of immune boosting by hTERT461 peptide have shown the potential of the peptide to provide clinical benefits in HCC patients.

Background & aims Immunotherapy is a promising treatment option for cholangiocarcinoma. We compared cytotoxic T lymphocyte (CTL) responses against several tumour-associated antigen (TAA)-derived epitopes in cholangiocarcinoma patients to identify candidate epitopes for immunotherapy. Methods Twenty-six TAAs were selected, and the expression of TAAs in 6 cholangiocarcinoma cell lines and 9 specimens were measured using real-time polymerase chain reaction (PCR). CTL responses against 38 TAA-derived epitopes were measured using samples from 26 cholangiocarcinoma patients by interferon-γ enzyme linked immunospot (ELISPOT)-assay. Results Most TAAs were expressed in cholangiocarcinoma cell lines and specimens in PCR. Epitopes that stimulated a specific immune response were defined as those that elicited a CTL response in more than 3 patients and little response in healthy volunteers, as measured by ELISPOT-assay. Based on these criteria, there were 18 epitopes that stimulated specific immune responses: squamous cell carcinoma antigen recognized by T cells (SART)1690 , P53161 , multidrug resistance-associated protein (MRP)3503 , Survivin2B80 , melanoma-associated antigen (MAGE)-A4143 , receptor tyrosine kinase ErbB-2/neu (Her2/neu)63 , Wilms tumour (WT1)235 , WT1417 , β-catenin29 , carcinoembryonic antigen (CEA)268 , CEA652 , epithelial cell adhesion molecule (EpCAM)173 , enhancer of zeste homolog (EZH)2291 , mucin 5AC (MUC5AC)716 , glypican-3 (GPC3)298 and kinesin family member 20A (KIF20A)66 . Furthermore, the absolute number of lymphocytes in peripheral blood was significantly correlated with the TAA-specific response. Lastly, the overall survival was significantly prolonged in patients with 2 or more TAA-specific CTL responses compared with none to one. Conclusions These results demonstrated several TAAs may be promising for immunotherapy for cholangiocarcinoma, and patients with high lymphocyte counts may benefit more from immunotherapy.

Background & aims Immunotherapy using dendritic cells (DCs) is a promising cancer therapy. The success of this therapy depends on the function of induced DCs. However, there has been no consensus on optimal conditions for DC preparation in vitro for immunotherapy of hepatocellular carcinoma (HCC) patients. To address relevant issues, we evaluated the procedures to induce DCs that efficiently function in hepatitis C virus (HCV)-related HCC. Methods We studied immunological data from 14 HCC patients. The DC preparation and the surface markers were assessed by flow cytometric analysis. Four different additional activation stimuli (Method I, medium alone; Method II, with OK-432; Method III, with IL-1β + IL-6 + TNF-α; Method IV, with IL-1β + IL-6 + TNF-α + PGE2) were tested and the functions of DCs were confirmed by examination of the ability of phagocytosis, cytokine production and allogeneic mixed lymphocyte reaction (MLR). Results The numbers of DCs induced and their cytokine production ability were not different between healthy controls and HCC patients. T-cell stimulatory activity of DCs in MLR was significantly lower in HCC patients than in healthy controls. The maturation of DCs with OK-432 boosted production of cytokines and chemokines, such as IL-2, IL-12p70, IFN-γ, TNF-α, IL-13 and MIP1α, and restored T-cell stimulatory activity of DCs in MLR. Conclusions The clinically approved compound OK-432 is a candidate for highly immunocompetent DC preparation and may be considered as a key drug for immunotherapy of HCV-related HCC patients. © 2014 Published by Elsevier B.V.

Aim Sorafenib is the standard treatment for advanced hepatocellular carcinoma (HCC). However, although there is no proven therapeutic procedure following the termination of sorafenib, hepatic arterial infusion chemotherapy (HAIC) may be a treatment option in advanced HCC. The aim of this study was to evaluate feasibility and efficacy of HAIC for patients with advanced HCC as subsequent therapy. Methods We retrospectively evaluated 27 consecutive patients with advanced HCC who were treated with HAIC following sorafenib between June 2009 and December 2012 at our hospital. Cisplatin (20 mg/m2 per day) was administered via the hepatic artery for 10 min, prior to the continuous administration of 5-fluorouracil (330 mg/m2 per day) over 24 h from days 1–5 and 8–12 and the s.c. administration of pegylated interferon α-2b (1 μg/kg) on days 1, 8, 15, and 22. A treatment cycle consisted of 28 days of drug administration followed by 14 days of rest. Results The toxicity profile showed that hematological toxicities were common, and grade 3/4 neutropenia and thrombocytopenia were observed (51.9% and 48.1%, respectively). Five patients (18.5%) experienced device-related complications. No unexpected adverse reactions and no treatment-related deaths were observed. Partial response was obtained in eight patients (29.6%), and stable disease was noted in nine patients (33.3%). Median progression-free survival and median survival time from initiation of HAIC were 4.0 and 7.6 months, respectively. Conclusions Because HAIC was well tolerated and exhibited moderate antitumor activity, it is a potentially useful treatment procedure in patients with advanced HCC even after failure of sorafenib.

金沢大学先進予防医学研究センター / 金沢大学医薬保健研究域医学系, Radiofrequency ablation therapy (RFA) is a radical treatment for liver cancers and induces tumor antigen-specific immune responses. In the present study, we examined the antitumor effects of focal OK-432-stimulated dendritic cell (DC) transfer combined with RFA and analyzed the functional mechanisms involved using a murine model. C57BL/6 mice were injected subcutaneously with colon cancer cells (MC38) in their bilateral flanks. After the establishment of tumors, the subcutaneous tumor on one flank was treated using RFA, and then OK-432-stimulated DCs were injected locally. The antitumor effect of the treatment was evaluated by measuring the size of the tumor on the opposite flank, and the immunological responses were assessed using tumor-infiltrating lymphocytes, splenocytes and draining lymph nodes. Tumor growth was strongly inhibited in mice that exhibited efficient DC migration after RFA and OK-432-stimulated DC transfer, as compared to mice treated with RFA alone or treatment involving immature DC transfer. We also demonstrated that the antitumor effect of this treatment depended on both CD8-positive and CD4-positive cells. On the basis of our findings, we believe that combination therapy for metastatic liver cancer consisting of OK-432-stimulated DCs in combination with RFA can proceed to clinical trials, and it is anticipated to be markedly superior to RFA single therapy. © 2013 Springer-Verlag Berlin Heidelberg.

金沢大学医薬保健研究域医学系, Despite curative locoregional treatments for hepatocellular carcinoma (HCC), tumour recurrence rates remain high. The current study was designed to assess the safety and bioactivity of infusion of dendritic cells (DCs) stimulated with OK432, a streptococcus-derived anti-cancer immunotherapeutic agent, into tumour tissues following transcatheter hepatic arterial embolization (TAE) treatment in patients with HCC. DCs were derived from peripheral blood monocytes of patients with hepatitis C virus-related cirrhosis and HCC in the presence of interleukin (IL)-4 and granulocyte-macrophage colony-stimulating factor and stimulated with 0.1 KE/ml OK432 for 2 days. Thirteen patients were administered with 5 × 106 of DCs through arterial catheter during the procedures of TAE treatment on day 7. The immunomodulatory effects and clinical responses were evaluated in comparison with a group of 22 historical controls treated with TAE but without DC transfer. OK432 stimulation of immature DCs promoted their maturation towards cells with activated phenotypes, high expression of a homing receptor, fairly well-preserved phagocytic capacity, greatly enhanced cytokine production and effective tumoricidal activity. Administration of OK432-stimulated DCs to patients was found to be feasible and safe. Kaplan-Meier analysis revealed prolonged recurrence-free survival of patients treated in this manner compared with the historical controls (P = 0.046, log-rank test). The bioactivity of the transferred DCs was reflected in higher serum concentrations of the cytokines IL-9, IL-15 and tumour necrosis factor-α and the chemokines CCL4 and CCL11. Collectively, this study suggests that a DC-based, active immunotherapeutic strategy in combination with locoregional treatments exerts beneficial anti-tumour effects against liver cancer. © 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.

Background & aims Many individuals are infected with hepatitis B virus (HBV) worldwide, and this virus is commonly controlled by treatments with interferon (IFN)-alpha and nucleoside analogues (NA). However, the complete elimination of HBV by these treatments is difficult and, thus, the development of new treatments is needed. Host immune responses are closely involved in the elimination of HBV, suggesting the usefulness of immunotherapy. In the present study, we attempted to identify novel cytotoxic T-lymphocyte (CTL) epitopes that are useful for immunotherapy against HBV. Methods CTL epitopes were predicted using computer software. Immune responses to each peptide were evaluated by IFN-γ ELISPOT and cytotoxic assays. The relationships between the immune responses to these newly identified CTL epitopes and the clinical backgrounds of patients and administration of NA were analyzed. Peptides were administered to mice as vaccines and peptide-specific T-cell induction was measured in vivo. Results Positive reactions to 10 synthesized peptides were detected in 3 or more patients using the IFN-γ ELISPOT assay, and concentration-dependent cytotoxicity against 2 of these peptides was observed in the cytotoxic assay. Some peptides that correlated with serum ALT, HBsAg, and HBV core-related antigen (HBcrAg) levels were identified. Immune reactions against some peptides were enhanced by the administration of NA. Regarding their effects as a vaccine, peptide-specific T-cells were induced by four peptides in vivo. Conclusions Novel HBV epitopes that correlated with HBsAg and HBcrAg levels were identified. These newly identified epitopes may be useful in the analysis of immune responses to HBV and development of immunotherapy against HBV.

Summary The curative treatments for hepatocellular carcinoma (HCC), including surgical resection and radiofrequency ablation (RFA), do not prevent tumour recurrence effectively. Dendritic cell (DC)-based immunotherapies are believed to contribute to the eradication of the residual and recurrent tumour cells. The current study was designed to assess the safety and bioactivity of DC infusion into tumour tissues following transcatheter hepatic arterial embolization (TAE) for patients with cirrhosis and HCC. Peripheral blood mononuclear cells (PBMCs) were differentiated into phenotypically confirmed DCs. Ten patients were administered autologous DCs through an arterial catheter during TAE treatment. Shortly thereafter, some HCC nodules were treated additionally to achieve the curative local therapeutic effects. There was no clinical or serological evidence of adverse events, including hepatic failure or autoimmune responses in any patients, in addition to those due to TAE. Following the infusion of 111Indium-labelled DCs, DCs were detectable inside and around the HCC nodules for up to 17 days, and were associated with lymphocyte and monocyte infiltration. Interestingly, T lymphocyte responses were induced against peptides derived from the tumour antigens, Her-2/neu, MRP3, hTERT and AFP, 4 weeks after the infusion in some patients. The cumulative survival rates were not significantly changed by this strategy. These results demonstrate that transcatheter arterial DC infusion into tumour tissues following TAE treatment is feasible and safe for patients with cirrhosis and HCC. Furthermore, the antigen-non-specific, immature DC infusion may induce immune responses to unprimed tumour antigens, providing a plausible strategy to enhance tumour immunity.

金沢大学先進予防医学研究センター / 金沢大学医薬保健研究域医学系, Hepatic arterial infusion chemotherapy (HAIC) has been employed as an alternative therapy to sorafenib for the patients with advanced hepatocellular carcinoma (HCC). In this study, we performed a comparative analysis of various immune cell responses including tumor-associated antigen (TAA)-specific T cells, regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs) in advanced HCC patients treated with HAIC. Thirty-six HCC patients were examined in the study. Interferon gamma enzyme-linked immunospot assays were performed to examine the frequency of TAA-specific T cells. The frequencies of Tregs and MDSCs were examined by multicolor fluorescence-activated cell sorting analysis. The treatment with HAIC using interferon (IFN)/5-fluorouracil (FU) or IFN/FU + cisplatin modulated the frequencies of various immune cells. In 22.2 % of patients, the frequency of TAA-specific T cells increased after HAIC. Although the frequency of Tregs decreased after HAIC, it was not associated with the prognosis of patients. An analysis of prognostic factors for overall survival identified diameter of the tumor (

A survey of 265 Japanese patients with Meniere's disease revealed bilateral involvement in approximately 29% of all these patients. Somatic and psychiatric aspects of bilateral Meniere's disease were as follows. (1) The duration of the disease in cases of bilateral involvement was significantly longer than cases of unilateral involvement. (2) An abnormal general condition, a more extensive degree of hearing loss and neurotic type were frequently diagnosed in cases of bilateral involvement. Based on these results, it was concluded that psychiatric management and prevention of aggravation of deafness should be part of the management of treatment in bilateral Meniere's disease.

This study evaluated the efficacy of endolymphatic sac-expanding surgery in comparison with medical treatment for intractable Meniere's disease. The outcomes of 47 patients with intractable Meniere's disease who underwent endolymphatic sac-expanding surgery were retrospectively compared with those of 22 patients who were offered surgery but preferred to continue medical treatment instead. Age, gender, disease duration, frequency of attacks and stage of hearing before treatment were almost equally distributed in both the surgically and medically treated groups. The outcomes were analyzed according to the 1995 AAO-HNS reporting guidelines. Definitive spells were completely controlled in approximately 90% of cases every year and hearing was improved in approximately 30% of cases for 0.5 to 6 years after surgery. Statistical analysis demonstrated that endolymphatic sac-expanding surgery is superior to medical treatment, at least from the perspective of immediate improvement in the quality of life. However, the proportion of cases showing hearing deterioration increased from 4% to 26% over 6 years after surgery. We would like to emphasize that further effort to improve and avoid deterioration in hearing remains urgent.

Background & aims The modest consumption of alcohol has been reported to decrease the incidence of fatty liver or prevalence of steatohepatitis. In this study, we investigated the effect of light alcohol consumption on liver function and gene expression in patients with non-alcoholic fatty liver disease (NAFLD). Methods The study group was formed of 178 patients diagnosed with non-alcoholic fatty liver disease, subclassified into two groups for analysis based on the daily alcohol consumption: non-alcohol group and light alcohol consumer group (≤20 g of ethanol/day). Clinical characteristics, liver histological features, gene expression, comprehensively analyzed using microarrays (BRB-Array tools), and molecular network were evaluated and compared between the two groups. Results No significant differences in steatosis or inflammation score were noted among the groups. However, the ballooning and fibrosis scores were significantly lower in the light alcohol consumer group than in the non-alcohol group. Gene expression analysis revealed a marked inhibition of the pathways involved in the immune response in the light alcohol group compared to that in the non-alcohol group. Conclusions Light alcohol consumption might suppress activity of non-alcoholic steatohepatitis by reducing gene expression levels involved in the immune response. This inhibition in gene expression was associated with a lowering of liver fibrosis and hepatocellular injury.

Background & Aims Levels of α-fetoprotein (AFP) are measured for surveillance and diagnosis of hepatocellular carcinoma (HCC). We performed a phase 1 trial to evaluate the safety and efficacy of AFP-derived peptides as an anti-tumor vaccine for patients with advanced HCC, and characterized induction of AFP-specific T-cell receptors (TCRs). Methods We performed a prospective study of 15 patients with HCC seen at Kanazawa University Hospital in Japan from March 2010 through March 2012. Each patient was given a subcutaneous injection of 3 mg AFP-derived peptides (AFP 357 and AFP 403 ) in an emulsion with incomplete Freund's adjuvant every other week for at least 6 weeks. Patients were evaluated every 8 weeks by radiologic imaging; adverse events and toxicities were categorized and graded using the common terminology criteria for adverse events. Criteria for discontinuation included unacceptable toxicities and disease progression defined as progressive disease using the Response Evaluation Criteria In Solid Tumors criteria. Patients' immune responses were monitored using an interferon-gamma enzyme-linked immunospot assay. Peptide-specific TCRs were assessed using a rapid TCR cloning and evaluation system. The observation period was 730 days. A complete response was defined as the disappearance of all tumors; stable disease was defined as tumors whose total diameter remained between >70% and Results We did not observe any serious adverse reactions to the peptides, which were well tolerated. Of the 15 patients who received at least 3 injections, 5 (33%) had an immune response to the peptides. One of the 15 patients had a complete response and disease stabilized in 8 patients. In 4 of the 15 patients, we detected AFP 357 -specific CD8 T cells; we cloned 14 different TCRs with different avidities for the peptide. A TCR with the highest avidity was observed in the patient who achieved a complete response for more than 2 years. Conclusions In a phase 1 trial, administration of AFP-derived peptides to 15 patients with HCC did not cause adverse events and produced T cells with receptors that reacted to the peptides; 1 patient had a complete response and tumor growth slowed in 8 patients. T cells from the patient with a complete response expressed a highly functional TCR induced by the peptide vaccines. UMIN-CTR no: UMIN000003514.

Veno-occlusive disease/sinusoidal obstruction syndrome (VOD/SOS) occurring after liver transplantation is a relatively rare complication but it often takes a life-threatening course. However, the detailed etiology and mechanism of VOD/SOS after liver transplantation (LT) remains unclear. We report two cases with rapidly progressive VOD/SOS after ABO-identical LT resistant to various therapies. In case 1, in which the patient underwent deceased-donor LT, the first episode of acute allograft rejection was triggered VOD/SOS, and the presence of donor non-specific anti-HLA antibodies was confirmed. The recipient died with graft failure on day 46 after transplantation. Case 2, in which the patient underwent living-donor LT from the mother, had neither rejection nor mechanical venous obstruction, but condition of the patient rapidly worsened and he died on day 13 after transplantation. This recipient's direct cross-match test for the donor's B lymphocyte was strongly positive, but that for T lymphocyte was negative. In both cases, neither stenosis of hepatic vein outflow tract nor C4d deposition in post-transplantation liver biopsy specimens and autopsy specimen was found. On the other hand, in both cases, the patient was transfusion unresponsive thrombocytopenia and hyperbilirubinemia persisted postoperatively, and glycoprotein Ⅰ bα was strongly stained in the neighboring centrilobular area (zone 3), especially in the space of Disse, and platelet phagocytosis was observed in Kupffer cells and hepatocytes around zone 3 such as clinical xenotransplantation of the liver in post-transplantation liver biopsy specimens. From the viewpoint of graft injury, VOD/SOS was considered that sustained sinusoidal endothelial cells injury resulted in bleeding in the space of Disse and led to around centrilobular hemorrhagic necrosis, and the fundamental cause was damage around centrilobular area including sinusoid by acute cellular rejection, antibody-mediated rejection or ischemic reperfusion injury. The extrasinusoidal platelet activation, aggregation, and phagocytosis of platelets were some of the main reasons for VOD/SOS and transfusion-resistant thrombocytopenia.