Keiji Hayata

Tumor-infiltrating CD4+ Th17 cells produce IL-17 in tumor microenvironment and promote tumor progression in human gastric cancer

Recently, a subset of IL-17 producing T cells distinct from Th1 or Th2 cells has been described as key players in inflammation and autoimmune diseases as well as cancer development. In this study, we investigated the expression level of IL-17 and T helper 17 (Th17)-related cytokines in gastric cancer tissues and assessed the association of their expression with angiogenesis and their clinicopathological parameters. Tumor and adjacent normal tissues were obtained from 82 patients with gastric cancer. IL-17, IL-21 and IL-23 mRNA expression levels were quantified by real-time RT-PCR. Th17 infiltration, microvessel density and neutrophil infiltration in tumor tissues were examined by immunohistochemistry and double immunofluorescence histochemistry. Expression of IL-17, IL-21 and IL-23 mRNA was found to be significantly up-regulated in tumor tissues compared with adjacent normal tissues. The expression level of IL-17 mRNA strongly and positively correlated with that of IL-21 mRNA in tumor tissue. The number of vascular endothelial cells and infiltrating neutrophils was significantly larger in tumors expressing a high level of IL-17 mRNA than in tumors expressing a low level of IL-17 mRNA. In tumor tissues most CD4+ cells were stained with anti-IL-17 antibody. The expression level of IL-17 mRNA in gastric tumors was associated with the depth of the tumors, lymph-vascular invasion and lymph node involvement, suggesting that IL-17 obviously was related to tumor progression. IL-17 and IL-21, which regulates IL-17, would be potential therapeutic targets for the treatment of gastric cancer.

Reconstruction after proximal gastrectomy for early gastric cancer in the upper third of the stomach: An analysis of our 13-year experience

BACKGROUND Fundoplication during esophagogastrostomy (EG) after proximal gastrectomy (PG) is a useful procedure to prevent reflux esophagitis, but it is unclear how much of the remnant stomach should be wrapped around the esophagus. METHODS We analyzed data from 101 patients who underwent PG for upper third early gastric cancer between 1999 and 2011. In all, 64 patients underwent EG, 25 underwent jejunal interposition (JI), and 12 underwent jejunal pouch interposition (JPI). We compared intraoperative details and postoperative outcomes, and investigated the relationships between the degree of the fundoplication during EG and endoscopic findings. RESULTS The length of the operation was significantly shorter in the EG group than in the other 2 groups (P < .05), and the intraoperative blood loss was significantly less in the EG group (P < .05). The JI and the JPI groups had significantly greater rates of early complications than did the EG group (P = .01). Reflux esophagitis was present in 22% of patients in the EG group, 8% in the JPI group, and none in the JI group. In the EG group, reflux esophagitis was significantly less common in patients with a >180° wrap of the remnant stomach around the esophagus than in patients with a smaller wrap (P = .0008). The rate of body weight loss was significantly less in the EG group compared with the other 2 groups (P < .05). CONCLUSION Considering the low invasiveness of the procedure and postoperative outcomes, we consider that EG with a >180° wrap as the optimal reconstructive procedure.

Inhibition of IL-17A in Tumor Microenvironment Augments Cytotoxicity of Tumor-Infiltrating Lymphocytes in Tumor-Bearing Mice

It remains controversial whether IL-17A promotes or inhibits cancer progression. We hypothesized that IL-17A that is locally produced in the tumor microenvironment has an important role in angiogenesis and tumor immunity. We investigated the effect of inhibiting IL-17A at tumor sites on tumor growth and on local and systemic anti-tumor immunity. MC38 or B16 cells were inoculated subcutaneously into mice, and intratumoral injection of an adenovirus vector expressing siRNA against the mouse IL-17A gene (Ad-si-IL-17) significantly inhibited tumor growth in both tumor models compared with control mice. Inhibition of IL-17A at tumor sites significantly suppressed CD31, MMP9, and VEGF expression in tumor tissue. The cytotoxic activity of CD8+ T cells from tumor-infiltrating lymphocytes in mice treated with Ad-si-IL-17 was significantly higher than in control mice; however, CD8+ T cells from splenocytes had similar activity levels. Suppression of IL-17A at tumor sites led to a Th1-dominant environment, and moreover, eliminated myeloid-derived suppressor cells and regulatory T cells at tumor sites but not in splenocytes. In conclusion, blockade of IL-17A at tumor sites helped suppress tumor growth by inhibiting angiogenesis as well as cytotoxic T lymphocytes activation at tumor sites.

Clinicopathological characteristics of remnant gastric cancer after a distal gastrectomy.

IntroductionThe survival rate of patients with remnant gastric cancer (RGC) is unfavorable in comparison to that of cancer in the nonresected stomach. However, when RGC is curatively resected, no significant differences have been reported between both groups in regard to survival. The aim of this study is to analyze the clinicopathological factors influencing a curative resection of RGC.MethodsThirty-eight consecutive patients with RGC from January 1, 1994 through March 31, 2009 were enrolled in this retrospective study.ResultsTheir primary diseases were gastric cancers (21; 55.3%) and benign diseases (17; 44.7%). The type of the reconstruction methods of first gastrectomy were Billroth I (28; 73.7%) and Billroth II (10; 26.3%). A total of 31 patients underwent a laparotomy. Twenty patients underwent a curative resection, four patients underwent a palliative resection, and seven underwent a nonresective operation. A total of seven patients underwent an endoscopic resection for early gastric cancer, and all patients received a curative resection. Univariate and multivariate logistic regression analyses were performed to identify the clinicopathological and background factors influencing a curative resection of RGC. A multivariate analysis revealed only an annual follow-up endoscopic examination after the initial gastrectomy to be an independent factor for a curative resection (p = 0.016; odds ratio, 35.3).ConclusionsAn annual follow-up endoscopic examination an after initial gastrectomy may be related to improving the prognosis of patients with RGC.

Dendritic cells adenovirally-transduced with full-length mesothelin cDNA elicit mesothelin-specific cytotoxicity against pancreatic cancer cell lines in vitro

Mesothelin (MSLN) is an attractive candidate as a molecular target for pancreatic cancer immunotherapy. The purpose of this study was to demonstrate that cytotoxic T lymphocytes (CTLs) generated from peripheral blood mononuclear cells (PBMCs) by stimulation with genetically-modified dendritic cells (DCs) expressing MSLN could produce specific anti-tumor immunity against pancreatic cancer cells endogenously expressing MSLN. MSLN-specific CTLs were generated from PBMCs of healthy donors by in vitro stimulation with DCs adenovirally-transduced with the full-length MSLN gene (DC-AxCAMSLN). The cytotoxic activity was tested using a 4-h (51)Cr-release assay. The pancreatic cancer cell lines (PK1, CfPAC1, AsPC1), a lymphoblastoid cell lines (LCL) transduced with the MSLN gene, and LCL pulsed with MSLN-epitope peptides were used as target cells. MSLN-specific CTLs induced by in vitro stimulation with DC-AxCAMSLN killed pancreatic cancer cell lines expressing MSLN in an HLA-restricted fashion. These CTLs also showed cytotoxic activity against autologous LCL pulsed with multiple MSLN-derived epitope peptides. In addition, CD8(+) T cells, as well as CD4(+) T cells, sorted from these CTLs showed significant production of interferon-γ when stimulated with DC-AxCAMSLN. The in vitro stimulation of PBMCs with DCs transduced with the full-length MSLN gene elicited a potent MSLN-specific cytotoxic activity against pancreatic cancer cell lines endogenously expressing MSLN by recognizing multiple MSLN epitopes and activating both CD8(+) T cells and CD4(+) helper T cells. These results therefore suggest the potential of developing future clinical applications of the vaccines using genetically-modified DCs expressing MSLN.

Optimal Dose of Preoperative Enteral Immunonutrition for Patients with Esophageal Cancer

PurposeA preoperative immunonutrition pharmaceutics diet (IMPACT) significantly reduced the incidence of postoperative infectious complications, but the optimal regimen still remains unclear. We evaluated the optimal dose of a preoperative IMPACT for patients with esophageal carcinoma and the incidence of postoperative complications based on the dose of IMPACT.MethodsThis study design was a prospective nonrandomized study. Twenty patients with thoracic esophageal carcinoma who underwent a right transthoracic subtotal esophagectomy were divided into two groups. These patients were administered immunonutrition of 500 ml/day (IMP500) or 1000 ml/day (IMP1000) for 7 days before the operation.ResultsThe incidence of postoperative mortality and morbidity was not different between the IMP500 group and the IMP1000 group. No difference was observed in the perioperative changes in inflammatory, immunological and nutritional variables between the two groups. There were no adverse effects in the IMP500 group, but four patients (40%) had diarrhea and four patients (40%) had appetite loss in the IMP1000 group. In the IMP1000 group, only four patients (40%) could take 1000 ml, but others reduced the quantity of IMPACT because of diarrhea and discomfort.ConclusionThis study suggests that 500 ml of IMPACT is recommended as an optimal dose for patients with esophageal cancer.

Complications of endoscopic submucosal dissection for gastric noninvasive neoplasia: an analysis of 647 lesions.

Purpose: This study aimed to determine risk factors for postoperative complications of gastric endoscopic submucosal dissection (ESD). Methods: This retrospective study included 647 lesions in 580 consecutive patients who underwent ESD for gastric noninvasive neoplasia from January 1, 2002 through December 31, 2011. Results: The overall perforation rate was 5.1%. Multivariate logistic regression analysis indicated that perforation was significantly associated with tumors in the greater curvature of the stomach (P<0.0001), scars in tumor lesions (P=0.002), long operative time (P=0.007), and tumors in the remnant stomach (P=0.036). The bleeding rate after gastric ESD was 3.9%. Multivariate logistic regression analysis indicated a statistically significant association between postoperative bleeding and oral anticoagulant or antiplatelet drugs (P<0.0001), dialysis (P=0.009), and use of antihypertensive drugs (P=0.015). Conclusion: It is important to perform gastric ESD with particular care in patients with risk factors.

Prognostic significance of IL-17 mRNA expression in peritoneal lavage in gastric cancer patients who underwent curative resection

Peritoneal dissemination is frequently detected in patients with advanced gastric cancer. The peritoneal cavity is a compartment in which an immunologic host-tumor interaction can occur. There are no reports on the relationship between IL-17 expression in peritoneal lavage and prognosis in gastric cancer patients. Therefore, we investigated the expression of IL-17 mRNA in peritoneal lavage from gastric cancer patients and assessed the association of its expression with clinicopathological parameters and prognosis. Peritoneal lavage was obtained from 114 patients with gastric cancer at initial surgery. Seventy-nine patients underwent curative resection. Among these 79 patients, IL-17 mRNA expression was associated with the depth of tumor invasion (p<0.05). Twelve of the 79 patients who underwent curative resection died, and 9 of those 12 developed peritoneal metastasis. Notably, among the 79 patients who underwent curative resection, those with high expression of IL-17 mRNA in peritoneal lavage had significantly prolonged survival when compared to these patients with low expression of IL-17 mRNA in peritoneal lavage (p<0.05) as evidence by the survival curves. In a multivariate analysis, low expression of IL-17 mRNA in peritoneal lavage and tumor size were found to be independent significant predictive factors for prognosis (HR, 7.91; 95% CI, 1.65-38.03) in the patients who underwent curative resection. IL-17 mRNA expression in peritoneal lavage is a reliable prognostic factor for patients undergoing curative resection for gastric cancer. Low IL-17 expression in the peritoneal cavity may correlate with cancer development in the peritoneal cavity in patients with gastric cancer.

An armed oncolytic herpes simplex virus expressing thrombospondin‐1 has an enhanced in vivo antitumor effect against human gastric cancer

Advanced gastric cancer is a common disease, but the conventional treatments are unsatisfactory because of the high recurrence rate. One of the promising new therapies is oncolytic virotherapy, using oncolytic herpes simplex viruses (HSVs). Thrombospondin‐1 (TSP‐1) suppresses tumor progression via multiple mechanisms including antiangiogenesis. Our approach to enhance the effects of oncolytic HSVs is to generate an armed oncolytic HSV that combines the direct viral oncolysis with TSP‐1‐mediated function for gastric cancer treatment. Using the bacterial artificial chromosome (BAC) system, a 3rd generation oncolytic HSV (T‐TSP‐1) expressing human TSP‐1 was constructed for human gastric cancer treatment. The enhanced efficacy of T‐TSP‐1 was determined in both human gastric cancer cell lines in vitro and subcutaneous tumor xenografts of human gastric cancer cells in vivo. In addition, we examined the apoptotic effect of T‐TSP‐1 in vitro, and the antiangiogenic effect of T‐TSP‐1 in vivo compared with a non‐armed 3rd generation oncolytic HSV, T‐01. No apparent apoptotic induction by T‐TSP‐1 was observed for human gastric cancer cell lines TMK‐1 cells but for MKN1 cells in vitro. Arming the viruses with TSP‐1 slightly inhibited their replication in some gastric cancer cell lines, but the viral cytotoxicity was not attenuated. In addition, T‐TSP‐1 exhibited enhanced therapeutic efficacy and inhibition of angiogenesis compared with T‐01 in vivo. In this study, we established a novel armed oncolytic HSV, T‐TSP‐1, which enhanced the antitumor efficacy by providing a combination of direct viral oncolysis with antiangiogenesis. Arming oncolytic HSVs may be a useful therapeutic strategy for gastric cancer therapy.

Antitumor immune response of dendritic cells (DCs) expressing tumor‐associated antigens derived from induced pluripotent stem cells: In comparison to bone marrow‐derived DCs

It is generally accepted that the difficulty in obtaining a sufficient number of functional dendritic cells (DCs) is a serious problem in DC‐based immunotherapy. Therefore, we used the induced pluripotent stem (iPS) cell‐derived DCs (iPSDCs). If the therapeutic efficacy of iPSDCs is equivalent to that of bone marrow‐derived DCs (BMDCs), then the aforementioned problems may be solved. In our study, we induced iPSDCs from iPS cells and examined the capacity for maturation of iPSDCs compared to that of BMDCs in addition to the capacity for migration of iPSDCs to regional lymph nodes. We adenovirally transduced the hgp100 gene, natural tumor antigens, into DCs and immunized mice once with the genetically modified DCs. The cytotoxic activity of CD8 (+) cytotoxic T lymphocytes (CTLs) was assayed using a 51Cr‐release assay. The therapeutic efficacy of the vaccination was examined in a subcutaneous tumor model. Our results showed that iPSDCs have an equal capacity to BMDCs in terms of maturation and migration. Furthermore, hgp100‐specific CTLs were generated in mice immunized with genetically modified iPSDCs. These CTLs exhibited as high a level of cytotoxicity against B16 cells as BMDCs. Moreover, vaccination with the genetically modified iPSDCs achieved as high a level of therapeutic efficacy as vaccination with BMDCs. Our study clarified experimentally that genetically modified iPSDCs have an equal capacity to BMDCs in terms of tumor‐associated antigen‐specific therapeutic antitumor immunity. This vaccination strategy may therefore be useful for future clinical application as a cancer vaccine.