Makoto Iwahashi

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.

Surgical management of small gastrointestinal stromal tumors of the stomach.

Small gastrointestinal stromal tumors (GISTs) (<3 cm) occasionally are found in the stomach during endoscopy. There is no consensus about the surgical management of these small tumors, although this clinical issue is crucial because some of the tumors show unexpected malignant behavior. In this study, we evaluated the clinical management of patients with gastric GISTs who underwent surgical resection. Altogether, 31 patients with gastric GISTs were examined retrospectively. Surgical resection was fundamentally indicated for the patients with gastric GISTs suspected to be malignant by endoscopy or endoscopic ultrasonography (EUS). The malignant grade of the GISTs was evaluated by the mitotic rate, tumor size, and MIB-1 index. EUS was useful for differentiating benign from malignant GISTs; but by limiting the study to patients with small tumors (<3 cm), the diagnostic value of EUS was not satisfactory for defining the surgical indication. Tumors that were <50 mm were successfully treated by laparoscopic surgery. Of the 31 patients, 4 had a relapse of the disease, and 1 of those 4 patients had a small tumor (30 mm). All of the recurrences were classified in the high risk category. Surgery is indicated for gastric GISTs that are ≥20 mm or are suspected to be malignant based on EUS findings. Laparoscopic resection is feasible and is recommended as the treatment of choice for patients with tumors < 50 mm. Risk assessment can be most useful for predicting recurrence.

Influence of Overweight on Patients With Gastric Cancer After Undergoing Curative Gastrectomy An Analysis of 689 Consecutive Cases Managed by a Single Center

HYPOTHESIS Overweight (body mass index [calculated as weight in kilograms divided by height in meters squared], > or =25.0) has an effect on surgical results, postoperative complications, and long-term survival in patients with gastric cancer who underwent curative gastrectomy. DESIGN Retrospective study from January 1, 1992, through December 31, 2002. SETTING Wakayama Medical University Hospital. PATIENTS This study included 689 patients who underwent curative gastrectomy (R0). Patients who underwent laparoscopic gastrectomy, gastrectomy with pancreaticoduodenectomy, gastrectomy with another organ resection (liver, colon, or ovary), or gastrectomy with thoracotomy were not included. MAIN OUTCOME MEASURES Duration of operation, amount of blood loss, incidence of postoperative complications, and survival analysis. RESULTS The mean (SD) duration of the operation was longer in the overweight group (315 [75] minutes) than in the normal-weight group (277 [85] minutes) (P < .001). The mean (SD) intraoperative blood loss was larger in the overweight group (882 [764] mL) than in the normal-weight group (536 [410] mL) (P < .001). The rates of postoperative complications (anastomotic leakage, pancreatic fistula, and intra-abdominal abscess) were significantly higher in the overweight group (P < .05). Multivariate logistic regression analysis identified that postoperative complications were significantly associated with being overweight (P = .01) and with undergoing pancreatectomy (P = .03). Disease-specific and overall survival did not show any significant difference between the 2 groups. CONCLUSIONS Being overweight is not a poor risk factor for survival in patients with gastric cancer, although it is independently predictive of postoperative complications.

Analysis of the prognostic factors and evaluation of surgical treatment for synchronous liver metastases from gastric cancer.

Background and aimsWhether or not a synchronous resection of liver metastases from gastric cancer provides a survival benefit has been a key issue. We identify the significant prognostic factors and clarify the beneficial effect on the survival of liver surgical treatment.Materials and methodsWe reviewed 72 patients who underwent a gastrectomy for gastric cancer with synchronous liver metastases and classified the liver metastases into three grades, such as H1: metastases were limited to one of the lobes, H2: there were a few scattered metastases in both lobes, and H3: there were numerous scattered metastases.ResultsH1, 2 metastases, and an absence of peritoneal dissemination (P0) were significantly independent prognostic factors for liver metastases of gastric cancer. In addition, the cumulative 1 and 5-year survival rates of liver surgical treatment (hepatic resection and/or microwave coagulation therapy) were 80.0% and 60.0%, whereas the survival rates for non-hepatic surgical treatment were 36.4% and 0% in 26 patients with H1, 2, and P0. In those patients, the radical operation, the solitary metastatic liver tumor, and no-distant lymph node metastases were independent prognostic determinants of survival.ConclusionThe radical operation including the surgical treatment for metastatic liver tumors should be performed to improve the prognosis in gastric cancer patients with synchronous H1, 2, and P0.

Laparoscopic resection for gastrointestinal stromal tumors of the stomach.

BACKGROUND We reviewed our experience with primary gastrointestinal tumors (GISTs) after surgical treatment. METHODS Between 1998 and 2003, 56 patients who underwent surgical treatment for primary GIST of the stomach were enrolled in this study. Statistical analyses of the risk factors for recurrence were assessed. RESULTS The proportion of cases undergoing laparoscopic surgery was 25 of 56 (44%) in these retrospective data. The site of recurrence was only the liver in all cases. These recurrent cases were defined as high-risk category. Tumors measuring over 2 cm in size tended to recur earlier, namely within 32 months. A statistical analysis showed a statistically significant correlation between the disease progression and the pathological phenotype. CONCLUSIONS This retrospective study has shown that an initial laparoscopic resection of gastric GISTs is feasible even when the tumor size is relatively small (2-5 cm). The pathological phenotype (especially tumor mitosis) directly correlates to the patients survival even if the resected tumor size was relatively small.

Dendritic Cells Transduced with Tumor-Associated Antigen Gene Elicit Potent Therapeutic Antitumor Immunity: Comparison with Immunodominant Peptide-Pulsed DCs

Several studies have shown that vaccine therapy using dendritic cells (DCs) pulsed with specific tumor antigen peptides can effectively induce antitumor immunity. Peptide-pulsed DC therapy is reported to be effective against melanoma, while it is still not sufficient to show the antitumor therapeutic effect against epithelial solid tumors such as gastrointestinal malignancies. Recently, it has been reported that vaccine therapy using DCs transduced with a surrogate tumor antigen gene can elicit a potent therapeutic antitumor immunity. In this study, we investigated the efficacy of vaccine therapy using DCs transduced with the natural tumor antigen in comparison with peptide-pulsed DCs. DCs derived from murine bone marrow were adenovirally transduced with murine endogenous tumor antigen gp70 gene, which is expressed in CT26 cells, or DCs were pulsed with the immunodominant peptide AH-1 derived from gp70. We compared these two cancer vaccines in terms of induction of antigen-specific cytotoxic T lymphocyte (CTL) responses, CD4+ T cell response against tumor cells, migratory capacity of DCs and therapeutic immunity in vivo. The cytotoxic activity of splenocytes against CT26 and Meth-A pulsed with AH-1 in mice immunized with gp70 gene-transduced DCs was higher than that with AH-1-pulsed DCs. CD4+ T cells induced from mice immunized with gp70 gene-transduced DCs produced higher levels of IFN-γ by stimulation with CT26 than those from mice immunized with AH-1-pulsed DCs (p < 0.0001), and it was suggested that DCs transduced with tumor-associated antigen (TAA) gene induced tumor-specific CD4+ T cells, and those CD4+ T cells played a critical role in the priming phase of the CD8+ T cell response for the induction of CD8+ CTL. Furthermore, DCs adenovirally transduced with TAA gene showed an enhancement of expression of CC chemokine receptor 7 and improved the migratory capacity to draining lymph nodes. In subcutaneous models, the vaccination using gp70 gene-transduced DCs provided a remarkably higher therapeutic efficacy than that using AH-1-pulsed DCs. These results suggested that vaccine therapy using DCs adenovirally transduced with TAA gene can elicit potent antitumor immunity, and may be useful for clinical application.

Successful cancer vaccine therapy for carcinoembryonic antigen (CEA)-expressing colon cancer using genetically modified dendritic cells that express CEA and T helper-type 1 cytokines in CEA transgenic mice

This study was designed to determine whether the vaccination of genetically modified dendritic cells (DCs) simultaneously expressing carcinoembryonic antigen (CEA), granulocyte macrophage colony‐stimulating factor (GM‐CSF) and interleukin 12 (IL‐12) can overcome the peripheral T‐cell tolerance to CEA and thereby elicit a therapeutic response in CEA transgenic mice. CEA transgenic mice were immunized once by subcutaneous injection with DCs adenovirally transduced with CEA and T helper‐type 1 cytokine genes. The cytotoxic activity of spleen cells against CEA‐expressing tumors, MC38‐CEA, in the mice immunized with DCs expressing CEA (DC‐AxCACEA) was higher than that in those immunized with DCs‐AxCALacZ (p < 0.0001), and was augmented by the cotransduction with the GM‐CSF/IL‐12 gene (p < 0.05). The vaccination with DC‐AxCACEA/GM‐CSF/IL‐12 could elicit a more potent therapeutic immunity than the vaccination with DC‐AxCACEA in subcutaneous tumor models (p < 0.0001), and 4 of 5 mice showed a complete eradication of the subcutaneous tumors in these vaccination groups. Even in a large tumor model, this vaccination therapy completely eliminated the subcutaneous tumors in all mice. This antitumor activity mostly vanished with the depletion of CD8+ T cells and NK cells in vivo and was completely abrogated with the depletion of CD4+ T cells. A histopathological examination showed no evidence of an autoimmune reaction. No other adverse effects were observed. This vaccination strategy resulted in the generation of highly efficient therapeutic immune responses against MC38‐CEA in the absence of autoimmune responses and demonstrated no adverse effects, and may therefore be useful for future clinical applications as a cancer vaccine therapy.

Vaccination with peptides derived from cancer-testis antigens in combination with CpG-7909 elicits strong specific CD8+ T cell response in patients with metastatic esophageal squamous cell carcinoma.

Potent helper action is necessary for peptide‐based vaccines to efficiently induce antitumor immune responses against advanced cancer. A phase I trial for advanced esophageal squamous cell carcinoma was carried out for patients with HLA‐A*2402 using epitope peptides derived from novel cancer‐testis antigens, LY6K and TTK, in combination with CpG‐7909 (NCT00669292). This study investigated the feasibility and the toxicity as well as induction of tumor antigen‐specific immune responses. Nine patients were vaccinated on days 1, 8, 15, and 22 of each 28‐day treatment cycle with peptide LY6K‐177, peptide TTK‐567, and CpG‐7909 (level‐1; 0, level‐2; 0.02, level‐3; 0.1 mg/kg) and all were tolerated by this treatment. LY6K‐specific T cell responses in PBMCs were detected in two of the three patients in each level. In particular, two patients in level‐2/3 showed potent LY6K‐specific T cell responses. In contrast, only two patients in level‐2/3 showed TTK‐567‐specific T cell responses. The frequency of LY6K‐177 or TTK‐567‐specific CD8+ T cells increased in patients in level‐2/3 (with CpG). The vaccination with peptides and CpG‐7909 increased and activated both plasmacytoid dentritic cells and natural killer cells, and increased the serum level of α‐interferon. There were no complete response (CR) and partial response (PR), however, one of three patients in level‐1, and four of six patients in level‐2/3 showed stable disease (SD). In conclusion, vaccination with LY6K‐177 and TTK‐567 in combination with CpG‐7909 successfully elicited antigen‐specific CD8+ T cell responses and enhanced the innate immunity of patients with advanced esophageal squamous cell carcinoma. This vaccine protocol is therefore recommended to undergo further phase II trials. (Cancer Sci 2010; 101: 2510–2517)

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.