Effects of adoptive T-cell immunotherapy on immune cell profiles and prognosis of patients with unresectable or recurrent cholangiocarcinoma.

The identification of immune cell profiles (ICP) involved in anti-tumor immunity is crucial for immunotherapy. Therefore, we herein investigated cholangiocarcinoma patients (CCA) who received adoptive T-cell immunotherapy (ATI). Eighteen unresectable or recurrent CCA received ATI of αß T cells alone or combined with chemotherapy. ICP were evaluated by flow cytometry. There were 14 patients with intrahepatic cholangiocarcinoma (iCCA) and four with distal cholangiocarcinoma (dCCA). After one course of treatment, nine iCCA and four dCCA had progressive disease (PD), while five iCCA had stable disease (SD). Median overall survival (OS) was prolonged to 21.9 months. No significant differences were observed in OS between the PD and SD groups of iCCA. The frequency of helper T cells (HT) in iCCA decreased from 70.3% to 65.5% (P = .008), while that of killer T cells (KT) increased from 27.0% to 30.6% (P = .005). dCCA showed no significant changes of immune cells. OS was prolonged in iCCA with increased frequencies of CD3+ T cells (CD3) (P = .039) and αß T cells (αß) (P = .039). dCCA showed no immune cells associated with OS. The frequencies of CD3+ T cells and αß T cells in the PD group for iCCA decreased from 63.5% to 53% (P = .038) and from 61.6% to 52.2% (P = .028), respectively. In the SD group, the frequency of HT decreased from 65.8% to 56.9% (P = .043), whereas that of KT increased from 30.1% to 38.3% (P = .043). In conclusions, ATI affected ICP and prolonged OS. Immune cells involved in treatment effects differed according to the site of cholangiocarcinoma.

Safety evaluation of immune-cell therapy for malignant tumor in the Cancer Immune-cell Therapy Evaluation Group (CITEG).

BACKGROUND AIMS: With the aim of strengthening the scientific evidence of immune-cell therapy for cancer and further examining its safety, in October 2015, our hospital jointly established the Cancer Immune-Cell Therapy Evaluation Group (CITEG) with 39 medical facilities nationwide. METHODS: Medical information, such as patients' background characteristics, clinical efficacy and therapeutic cell types obtained from each facility, has been accumulated, analyzed and evaluated by CITEG. In this prospective study, we analyzed the adverse events associated with immune-cell therapy until the end of September 2022, and we presented our interim safety evaluation. RESULTS: A total of 3839 patients with malignant tumor were treated with immune-cell therapy, with a median age of 64 years (range, 13-97 years) and a male-to-female ratio of 1:1.08 (1846:1993). Most patients' performance status was 0 or 1 (86.8%) at the first visit, and 3234 cases (84.2%) were advanced or recurrent cases, which accounted for the majority. The total number of administrations reported in CITEG was 31890, of which 960 (3.0%) showed adverse events. The numbers of adverse events caused by treatment were 363 (1.8%) of 19661 administrations of αßT cell therapy, 9 of 845 administrations of γδT-cell therapy (1.1%) and 10 of 626 administrations of natural killer cell therapy (1.6%). The number of adverse events caused by dendritic cell (DC) vaccine therapy was 578 of 10748 administrations (5.4%), which was significantly larger than those for other treatments. Multivariate analysis revealed that αßT cell therapy had a significantly greater risk of adverse events at performance status 1 or higher, and patients younger than 64 years, women or adjuvant immune-cell therapy had a greater risk of adverse events in DC vaccine therapy. Injection-site reactions were the most frequently reported adverse events, with 449 events, the majority of which were associated with DC vaccine therapy. Among all other adverse events, fever (228 events), fatigue (141 events) and itching (131 events) were frequently reported. In contrast, three patients had adverse events (fever, abdominal pain and interstitial pneumonia) that required hospitalization, although they were weakly related to this therapy; rather, it was considered to be the effect of treatment for the primary disease. CONCLUSIONS: Immune-cell therapy for cancer was considered to be a safe treatment without serious adverse events.

Peptide vaccine-treated, long-term surviving cancer patients harbor self-renewing tumor-specific CD8+ T cells.

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.

Effect of adoptive T-cell immunotherapy on immunological parameters and prognosis in patients with advanced pancreatic cancer.

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 αß 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.

Safety and efficacy of sorafenib followed by regorafenib or lenvatinib in patients with hepatocellular carcinoma.

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.

Clinical features and diagnostic imaging of cholangiolocellular carcinoma compared with other primary liver cancers: a surgical perspective.

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.

Tumor lysis syndrome in a patient with metastatic melanoma treated with nivolumab.

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.

Effects of adaptive immune cell therapy on the immune cell profile in patients with advanced gastric cancer.

BACKGROUND: Immunotherapy for cancer patients has been the subject of attention in recent years. In this study, we investigated whether αßT-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 αßT-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 αßT-cell therapy changes the host's immune cell profile, and an increase in PD-1+ effector Tregs may help improve prognosis.

Safety and Long-Term Outcome of Intratumoral Injection of OK432-Stimulated Dendritic Cells for Hepatocellular Carcinomas After Radiofrequency Ablation.

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).

A novel α-fetoprotein-derived helper T-lymphocyte epitope with strong immunogenicity in patients with hepatocellular carcinoma.

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.

Protocol For An Adjuvant Alpha-Fetoprotein-Derived Peptide After Transarterial Chemoembolization in Patients With Hepatocellular Carcinoma: Safety Study.

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. TRIAL REGISTRATION: Japan Registry of Clinical Trials jRCTs041180155; https://jrct.niph.go.jp/latest-detail/jRCTs041180155. INTERNATIONAL REGISTERED REPORT IDENTIFIER (IRRID): DERR1-10.2196/17082.

Development of novel diagnostic system for pancreatic cancer, including early stages, measuring mRNA of whole blood cells.

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.

Characteristics of Immune Response to Tumor-Associated Antigens and Immune Cell Profile in Patients With Hepatocellular Carcinoma.

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.

Immune responses of human T lymphocytes to novel hepatitis B virus-derived peptides.

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.

Immune responses against tumour-associated antigen-derived cytotoxic T lymphocyte epitopes in cholangiocarcinoma 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 , ß-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.

Immune response to human telomerase reverse transcriptase-derived helper T cell epitopes in hepatocellular carcinoma patients.

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.

Association Between High-Avidity T-Cell Receptors, Induced by α-Fetoprotein-Derived Peptides, and Anti-Tumor Effects in Patients With Hepatocellular Carcinoma.

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 (AFP357 and AFP403) 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 <120% of the baseline measurement, without new lesions. 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 AFP357-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.

Cellular Immune Responses for Squamous Cell Carcinoma Antigen Recognized by T Cells 3 in Patients with Hepatocellular Carcinoma.

BACKGROUND & AIMS: Squamous cell carcinoma antigen recognized by T cells 3 (SART3), a tumor-associated antigen expressed in many cancers, functions in tumor rejection. In this study, we investigated its usefulness as an immunotherapeutic target in hepatocellular carcinoma (HCC). METHODS: The expression of SART3 in hepatoma cell lines and HCC tissues was investigated by immunofluorescence and immunohistochemical analyses. Two peptides derived from SART3 (SART3109 and SART3315) were used for immunological analysis. T-cell responses were investigated by interferon-gamma (IFN-γ) enzyme-linked immunospot and cytotoxic T lymphocyte (CTL) assays using peripheral blood mononuclear cells (PBMCs) in 47 patients, and tumor-infiltrating lymphocytes in 8 of 47 patients with HCC. The safety of immunotherapy using a SART3-derived peptide was investigated by vaccinations of SART3109 in 12 patients with HCC (trial registration: UMIN000005677). RESULTS: The immunofluorescence and immunohistochemical analyses showed that SART3 was expressed in six HCC cell lines, and in HCC tissues including of alpha-fetoprotein-negative individuals. SART3-specific CTLs were generated by stimulating PBMCs with the peptides, and they showed cytotoxicity against HCC cells expressing the protein. Of the 47 HCC patients, 25.5% and 10.6% showed significant responses to SART3109 and SART3315, respectively. The infiltration of SART3109-specific IFN-γ-producing CTLs into the tumor site was confirmed. In the vaccination study, no severe adverse events were observed, and the peptide-specific CTLs were newly induced in four of five patients tested. CONCLUSIONS: SART3 is an immunotherapeutic candidate, and peptides from this antigen may be applied in HCC immunotherapy. TRIAL REGISTRATION: UMIN000005677.

Myeloid-derived suppressor cells correlate with patient outcomes in hepatic arterial infusion chemotherapy for hepatocellular carcinoma.

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 (<3.0 cm), absence of major portal vein invasion, absence of distant metastasis, Union Internationale Contre Le Cancer tumor lymph node metastasis stage (I or II), neutrophil lymphocytic ratio (<2.1) and the frequency of MDSCs (<30.5 %) as factors that prolonged overall survival time after HAIC. Even in the group adjusted with progressive levels of tumors, patients with a low frequency of MDSCs had a significantly longer overall survival time. In conclusion, the frequency of MDSCs before the treatment is a prognostic factor in HAIC against HCC.

Clinical characteristics and prognosis of non-B non-C hepatocellular carcinoma patients with modest alcohol consumption.

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.