Yoshitaro Shindo

A phase I clinical trial of vaccination with KIF20A-derived peptide in combination with gemcitabine for patients with advanced pancreatic cancer.

KIF20A (RAB6KIFL) belongs to the kinesin superfamily of motor proteins, which play critical roles in the trafficking of molecules and organelles during the growth of pancreatic cancer. Immunotherapy using a previously identified epitope peptide for KIF20A is expected to improve clinical outcomes. A phase I clinical trial combining KIF20A-derived peptide with gemcitabine (GEM) was therefore conducted among patients with advanced pancreatic cancer who had received prior therapy such as chemotherapy and/or radiotherapy. GEM was administered at a dose of 1000 mg/m2 on days 1, 8, and 15 in a 28-day cycle. The KIF20A-derived peptide was injected subcutaneously on a weekly basis in a dose-escalation manner (doses of 0.5, 1, and 3 mg/body; 3 patients/cohort). Safety and immunologic parameters were assessed. No severe adverse effects of grade 3 or higher related to KIF20A-derived peptide were observed. Of the 9 patients who completed at least one course of treatment, interferon-&ggr; (IFN-&ggr;)-producing cells were induced in 4 of 9 patients (P2, P3, P6, and P7), and IFN-&ggr;-producing cells were increased in 4 of the 9 patients (P1, P5, P8, and P9). Four of the 9 patients achieved stable disease. The disease control rate was 44%. The median survival time after first vaccination was 173 days and 1-year survival rate was 11.1%. IFN-&ggr;-producing cells were induced by the KIF20A-derived peptide vaccine at a high rate, even in combination with GEM. These results suggest that this combination therapy will be feasible and promising for the treatment of advanced pancreatic cancer.

Phase II clinical trial of peptide cocktail therapy for patients with advanced pancreatic cancer: VENUS-PC study

We previously conducted a phase I clinical trial combining the HLA‐A*2402‐restricted KIF20A‐derived peptide vaccine with gemcitabine for advanced pancreatic cancer (PC) and confirmed its safety and immunogenicity in cancer patients. In this study, we conducted a multicenter, single‐armed, phase II trial using two antiangiogenic cancer vaccines targeting VEGFR1 and VEGFR2 in addition to the KIF20A peptide. We attempted to evaluate the clinical benefit of the cancer vaccination in combination with gemcitabine. Chemotherapy naïve PC patients were enrolled to evaluate primarily the 1‐year survival rate, and secondarily overall survival (OS), progression free survival (PFS), response rate (RR), disease control rate (DCR) and the peptide‐specific immune responses. All enrolled patients received therapy without the HLA‐A information, and the HLA genotypes were used for classification of the patients. Between June 2012 and May 2013, a total of 68 patients were enrolled. No severe systemic adverse effects of Grade 3 or higher related to these three peptides were observed. The 1‐year survival rates between the HLA‐A*2402‐matched and ‐unmatched groups were not significantly different. In the HLA‐A*2402 matched group, patients showing peptide‐specific CTL induction for KIF20A or VEGFR1 showed a better prognosis compared to those without such induction (P = 0.023, P = 0.009, respectively). In the HLA‐A*2402‐matched group, the patients who showed a strong injection site reaction had a better survival rate (P = 0.017) compared to those with a weak or no injection site reaction. This phase II study demonstrated that this therapeutic peptide cocktail might be effective in patients who demonstrate peptide‐specific immune reactions although predictive biomarkers are needed for patient selection in its further clinical application.

Characterization of the T cell repertoire by deep T cell receptor sequencing in tissues and blood from patients with advanced colorectal cancer

The aim of the present study was to characterize infiltrated T cell clones that define the tumor immune environment and are important in the response to treatment in patients with advanced colorectal cancer (CRC). In order to explore predictive biomarkers for the efficacy of immunochemotherapies, T cell receptor (TCR) repertoire analysis was performed using blood samples and tumor tissues obtained from patients with advanced CRC that had been treated with a combination of five-cancer peptide vaccines and oxaliplatin-based chemotherapy. The TCR-α/β complementary DNAs (cDNAs), prepared from the messenger RNAs (mRNAs) obtained from 17 tumor tissues and 39 peripheral blood mononuclear cells of 9 CRC patients at various time points, were sequenced. The oligoclonal enrichment of certain TCR sequences was identified in tumor tissues and blood samples; however, only a few TCR sequences with a frequency of >0.1% were commonly detected in pre- and post-treatment tumor tissues, or in post-treatment blood and tissue samples. The average correlation coefficients of the TCR-α and TCR-β clonotype frequencies between the post-treatment tumor tissues and blood samples were 0.023 and 0.035, respectively, and were much lower compared with the correlation coefficients of the TCR-α and TCR-β clonotype frequencies between pre- and post-treatment blood samples (0.430 and 0.370, respectively), suggesting that T cell populations in tumor tissues vary from those in blood. Although the sample size was small, a tendency for the TCR diversity in tumor tissues to drastically decrease during the treatment was indicated in two patients, who exhibited a longer progression-free survival time. The results of the present study suggest that TCR diversity scores in tissues may be a useful predictive biomarker for the therapeutic effect of immunochemotherapy for patients with advanced CRC.

A stem cell medium containing neural stimulating factor induces a pancreatic cancer stem-like cell-enriched population

Cancer stem cells (CSCs) have been studied for their self-renewal capacity and pluripotency, as well as their resistance to anticancer therapy and their ability to metastasize to distant organs. CSCs are difficult to study because their population is quite low in tumor specimens. To overcome this problem, we established a culture method to induce a pancreatic cancer stem-like cell (P-CSLC)-enriched population from human pancreatic cancer cell lines. Human pancreatic cancer cell lines established at our department were cultured in CSC-inducing media containing epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), leukemia inhibitory factor (LIF), neural cell survivor factor-1 (NSF-1), and N-acetylcysteine. Sphere cells were obtained and then transferred to a laminin-coated dish and cultured for approximately two months. The surface markers, gene expression, aldehyde dehydrogenase (ALDH) activity, cell cycle, and tumorigenicity of these induced cells were examined for their stem cell-like characteristics. The population of these induced cells expanded within a few months. The ratio of CD24high, CD44high, epithelial specific antigen (ESA) high, and CD44variant (CD44v) high cells in the induced cells was greatly enriched. The induced cells stayed in the G0/G1 phase and demonstrated mesenchymal and stemness properties. The induced cells had high tumorigenic potential. Thus, we established a culture method to induce a P-CSLCenriched population from human pancreatic cancer cell lines. The CSLC population was enriched approximately 100-fold with this method. Our culture method may contribute to the precise analysis of CSCs and thus support the establishment of CSC-targeting therapy.

Highly sensitive stool DNA testing of Fusobacterium nucleatum as a marker for detection of colorectal tumours in a Japanese population.

Background Accumulating evidence shows an over-abundance of Fusobacterium nucleatum in colorectal tumour tissues. Although stool DNA testing of Fusobacterium nucleatum might be a potential marker for the detection of colorectal tumours, the difficulty in detecting Fusobacterium nucleatum in stool by conventional methods prevented further explorations. Therefore, we developed a droplet digital polymerase chain reaction (PCR) assay for detecting Fusobacterium nucleatum in stool and investigated its clinical utility in the management of colorectal tumours in a Japanese population. Methods Feces were collected from 60 healthy subjects (control group) and from 11 patients with colorectal non-advanced adenomas (non-advanced adenoma group), 19 patients with colorectal advanced adenoma/carcinoma in situ (advanced adenoma/carcinoma in situ (CIS) group) and 158 patients with colorectal cancer of stages I to IV (colorectal cancer group). Absolute copy numbers of Fusobacterium nucleatum were measured by droplet digital PCR. Results The median copy number of Fusobacterium nucleatum was 17.5 in the control group, 311 in the non-advanced adenoma group, 122 in the advanced adenoma/CIS group, and 317 in the colorectal cancer group. In comparison with that in the control group, the Fusobacterium nucleatum level was significantly higher in the non-advanced adenoma group, the advanced adenoma/CIS group and the colorectal cancer group. Conclusions This study illustrates the potential of stool DNA testing of Fusobacterium nucleatum by droplet digital PCR to detect individuals with colorectal tumours in a Japanese population.

miR-196b, miR-378a and miR-486 are predictive biomarkers for the efficacy of vaccine treatment in colorectal cancer

MicroRNAs (miRNAs/miRs) regulate the levels of transcripts and serve a critical function in the regulation of tumor microenvironments. Therefore, miRNA levels in cancer tissues are thought to be potential biomarkers for immunotherapy. From a phase I trial of a vaccine treatment using 5 human leukocyte antigen (HLA)-A*2402-restricted peptides (registration no. UMIN000004948), colorectal cancer (CRC) tissues were obtained from 8 patients and normal colorectal tissues from 5 patients via surgery. From a phase II trial using the same peptides (registration no. UMIN000001791), CRC tissues were obtained from 16 patients from the HLA-A*2402-matched group and 10 patients from the HLA-A*2402-unmatched group. These tissues were used for miRNA microarray analysis. As the first step, cancer tissues from the phase I study were used and 10 candidate miRNAs were selected by comparing the miRNA expression between two groups; one with improved prognosis and the other with poor prognosis. The miRNAs were subsequently validated using the cases enrolled in the phase II study. Significantly improved prognoses were identified in 16 patients in the HLA-A*2402-matched group with high expression of miR-196b-5p and low expression of miR-378a-3p and miR-486-5p. There was no difference in prognosis in the 10 patients in the HLA-A*2402-unmatched group. Therefore, high miR-196b expression and low miR-378a-3p and miR-486-5p expression were indicated as useful biomarkers for prediction of the efficacy of vaccine treatment for patients with metastatic CRC. In a planned phase III study, expression levels of these 3 miRNAs (miR-196b-5p, miR-378a-3p and miR-486-5p) may be useful biomarkers for assessing patients who are likely to have an improved outcome following vaccination.

Predictive biomarkers for the efficacy of peptide vaccine treatment: based on the results of a phase II study on advanced pancreatic cancer

BackgroundThe purpose of the present study was to explore novel biomarkers that can predict the clinical outcome of patients before treatment or during vaccination. These would be useful for the selection of appropriate patients who would be expected to exhibit better treatment outcomes from vaccination, and for facilitating the development of cancer vaccine treatments.MethodsFrom a single-arm, non-randomized, human leukocyte antigen (HLA)-A-status-blind phase II trial of a vaccine treatment using three HLA-A*2402-restricted peptides for advanced pancreatic cancer (PC), we obtained peripheral blood samples from 36 patients of an HLA-A*2402-matched group and 27 patients of an HLA-A*2402-unmatched group.ResultsMultivariate analysis (HR = 2.546; 95% CI = 1.138 to 5.765; p = 0.0231) and log-rank test (p = 0.0036) showed that a high expression level of programmed death-1 (PD-1) on CD4+ T cells was a negative predictive biomarker of overall survival in the HLA-A*2402-matched group . Moreover, a high expression level of PD-1 on CD4+ T cells was a negative predictor for the induction of cytotoxic T lymphocytes (p = 0.0007). After treatment, we found that the upregulation of PD-1 and T cell immunoglobulin mucin-3 (Tim-3) expression on CD4+ and CD8+ T cells was significantly associated with a poor clinical outcome in the HLA-A*2402-matched group (p = 0.0330, 0.0282, 0.0046, and 0.0068, respectively). In contrast, there was no significant difference for these factors in the HLA-A*2402-unmatched group.ConclusionsOur results indicate that the upregulation of PD-1 and Tim-3 expression on CD4+ and CD8+ T cells may restrict T cell responses in advanced PC patients; therefore, combination immunotherapy with blockade of PD-1 and Tim-3 to restore T cell responses may be a potential therapeutic approach for advanced PC patients.Trial registrationClinical-Trail-Registration: UMIN000008082.

Novel Indications for Surgical Resection of Metachronous Lung Metastases From Pancreatic Cancer After Curative Resection.

Few reports exist regarding surgical resection of metachronous lung metastases (MLM) from pancreatic ductal adenocarcinoma (PDA) after curative resection. To elucidate the indications for surgical resection of MLM and long-term survival, we analyzed Japanese case reports of MLM from PDA. Between 1983 and 2014, 17 Japanese case reports concerning surgical resection of MLM from PDA were published. We determined long-term survival in 16 patients (considering the published data of 15 patients and 1 of our own) by using a questionnaire survey and analyzing the relationships between background factors and long-term survival. In 16 patients with long-term survival, 4 patients were still alive without recurrence. The remaining 12 patients died of disease after recurrence. The median survival after the initial lobectomy was 37 months and the 3- and 5-year survival for all patients after lobectomy was 50% and 41%, respectively. Fourteen patients had a disease-free interval after initial resection of the primary pancreatic tumor of >20 months. These patients had a longer median survival period after lobectomy (46 vs. 25.5 mo, P=0.19). Seven patients had MLM of <16 mm. These patients had a statistically longer overall survival after lobectomy (83 vs. 16 mo, P=0.04). Three of 4 patients with primary stage I tumors were still alive without recurrence. We found that the conventional criteria for surgical resection of MLM from PDA (first disease-free interval of >20 mo with no other metastatic lesions) were appropriate. In addition, it is possible that MLM of <16 mm or primary stage I tumors are novel criteria.

Low invasiveness of thoracoscopic esophagectomy in the prone position for esophageal cancer: a propensity score-matched comparison of operative approaches between thoracoscopic and open esophagectomy

BackgroundIn this study, cytokine levels, outcome, and survival rates after esophagectomy for esophageal cancer were retrospectively investigated in a propensity score-matched comparison of operative approaches between the thoracoscopic esophagectomy (TE) in the prone position and open esophagectomy (OE).Patients and MethodsBetween 2005 and 2014, TE was performed on a group of 85 patients, which was compared with a group of 104 OE cases. Eventually, 65 paired cases were matched using propensity score matching.ResultsAlthough the TE group underwent a significantly longer operation time than the OE group (P < 0.001), the TE group exhibited less blood loss (P < 0.001) and had a shorter postoperative hospital stay (P = 0.038) than the OE group. The serum interleukin-6 levels on ICU admission (P < 0.001) and on POD 1 (P < 0.001) were significantly lower in the TE group. The interleukin-10 levels on ICU admission (P < 0.001), POD 1 (P = 0.016), and POD 3 (P < 0.001) were also significantly lower in the TE group. Pulmonary complication was significantly lower in the TE group (P = 0.043). The 5-year PFS rates in the TE and OE groups were 70.6 and 58.7% (P = 0.328), respectively, and OS rates were 64.9 and 50.2% (P = 0.101), respectively.ConclusionTE compared to OE is a less invasive procedure with lower surgical stress and less pulmonary complication for the treatment of esophageal squamous cell carcinoma.

Postoperative Adjuvant Therapy for Resectable Pancreatic Cancer With Gemcitabine and Adoptive Immunotherapy

Objectives We previously described adoptive immunotherapy (AIT) with cytotoxic T lymphocytes (CTLs) stimulated by the mucin 1 (MUC1)–expressing human pancreatic cancer cell line YPK-1 (MUC1-CTLs) and demonstrated that MUC1-CTLs might prevent liver metastasis. In the present study, we combined gemcitabine (GEM) and AIT for the treatment of pancreatic cancer. Methods A total of 43 patients who underwent radical pancreatectomy received treatment with MUC1-CTLs and GEM. After surgery, MUC1-CTLs were induced and administered intravenously 3 times, and GEM administered according to the standard regimen for 6 months. The patients whose relative dose intensity of GEM was 50% or more and who received 2 or more MUC1-CTL treatments were used as the adequate treatment group (n = 21). Results In the adequate treatment group, disease-free survival was 15.8 months, and overall survival was 24.7 months. Liver metastasis was found only in 7 patients (33%), and local recurrence occurred in 4 patients (19%). The independent prognostic factor of long-term disease-free survival on multivariate analysis was the average number of CTLs administered (P = 0.0133). Conclusions The combination therapy with AIT and GEM prevented liver metastasis and local recurrence. Moreover, the disease free-survival was improved in patients who received sufficient CTLs.