Michiaki Watanabe

Dendritic Cells Fused with Allogeneic Colorectal Cancer Cell Line Present Multiple Colorectal Cancer–Specific Antigens and Induce Antitumor Immunity against Autologous Tumor Cells

The aim of antitumor immunotherapy is to induce CTL responses against autologous tumors. Previous work has shown that fusion of human dendritic cells and autologous tumor cells induce CTL responses against autologous tumor cells in vitro. However, in the clinical setting of patients with colorectal carcinoma, a major difficulty is the preparation of sufficient amounts of autologous tumor cells. In the present study, autologous dendritic cells from patients with colorectal carcinoma were fused to allogeneic colorectal tumor cell line, COLM-6 (HLA-A2−/HLA-24−), carcinoembryonic antigen (CEA)+, and MUC1+ as an alternative strategy to deliver shared colorectal carcinoma antigens to dendritic cells. Stimulation of autologous T cells by the fusion cells generated with autologous dendritic cells (HLA-A2+ and/or HLA-A24+) and allogeneic COLM-6 resulted in MHC class I– and MHC class II–restricted proliferation of CD4+ and CD8+ T cells, high levels of IFN-γ production in both CD4+ and CD8+ T cells, and the simultaneous induction of CEA- and MUC1-specific CTL responses restricted by HLA-A2 and/or HLA-A24. Finally, CTL induced by dendritic cell/allogeneic COLM-6 fusion cells were able to kill autologous colorectal carcinoma by HLA-A2- and/or HLA-A24-restricted mechanisms. The demonstration of CTL activity against shared tumor-associated antigens using an allogeneic tumor cell line, COLM-6, provides that the presence of alloantigens does not prevent the development of CTL with activity against autologous colorectal carcinoma cells. The fusion of allogeneic colorectal carcinoma cell line and autologous dendritic cells could have potential applicability to the field of antitumor immunotherapy through the cross-priming against shared tumor antigens and provides a platform for adoptive immunotherapy.

Serum vitamin D levels and survival of patients with colorectal cancer: Post-hoc analysis of a prospective cohort study

BackgroundRecently, serum 25-hydroxyvitamin D (25OHD) levels were shown to be associated with the survival of patients with colorectal cancer. However, 25OHD levels were measured a median of 6 years before diagnosis or were predicted levels. In this study, we directly measured serum 25OHD levels at surgery and examined the association with survival among patients with colorectal cancer.MethodsWe started a prospective cohort study to find prognostic factors in patients with colorectal cancer from 2003 to 2008 and stored serum samples and clinical data. As part of a post-hoc analysis, serum 25OHD levels were measured by radioimmunoassay. Association between overall survival and serum 25OHD levels were computed using the Cox proportional hazard model adjusted for month of serum sampling as well as age at diagnosis, gender, cancer stage, residual tumor after surgery, time period of surgery, location of tumor, adjuvant chemotherapy and number of lymph nodes with metastasis at surgery. Unadjusted and adjusted hazard ratios (HR) and 95% confidence intervals (95% CI) were determined.ResultsSerum 25OHD levels were measured in 257 patients. Only 3% had sufficient levels (30 ng/ml and greater). Based on month of blood sampling, an annual oscillation of 25OHD levels was seen, with levels being lower in spring and higher in late summer. Higher 25OHD levels were associated with better overall survival under multi-variate analysis (HR, 0.91: 95% CI, 0.84 to 0.99, P = 0.027).ConclusionsThese results suggest that higher 25OHD levels at surgery may be associated with a better survival rate of patients with colorectal cancer.

Streptococcal Preparation OK-432 Promotes Fusion Efficiency and Enhances Induction of Antigen-Specific CTL by Fusions of Dendritic Cells and Colorectal Cancer Cells

Dendritic/tumor fusion cell (FC) vaccine is an effective approach for various types of cancer but has not yet been standardized. Antitumor activity can be modulated by different mechanisms such as dendritic cell (DC) maturation state. This study addressed optimal strategies for FC preparations to enhance Ag-specific CTL activity. We have created three types of FC preparations by alternating fusion cell partners: 1) immature DCs fused with autologous colorectal carcinoma cells (Imm-FCs); 2) Imm-FCs followed by stimulation with penicillin-inactivated Streptococcus pyogenes (OK-432) (Imm-FCs/OK); and 3) OK-432-stimulated DCs directly fused to autologous colorectal carcinoma cells (OK-FCs). Both OK-FCs and Imm-FCs/OK coexpressed the CEA, MUC1, and significantly higher levels of CD86, CD83, and IL-12 than those obtained with Imm-FCs. Short-term culture of fusion cell preparations promoted the fusion efficiency. Interestingly, OK-FCs were more efficient in stimulating CD4+ and CD8+ T cells capable of high levels of IFN-γ production and cytolysis of autologous tumor or semiallogeneic targets. Moreover, OK-FCs are more effective inducer of CTL activation compared with Imm-FCs/OK on a per fusion cell basis. The pentameric assay confirmed that CEA- and MUC1-specific CTL was induced simultaneously by OK-FCs at high frequency. Furthermore, the cryopreserved OK-FCs retained stimulatory capacity for inducing antitumor immunity. These results suggest that OK-432 promotes fusion efficiency and induction of Ag-specific CTL by fusion cells. We conclude that DCs fused after stimulation by OK-432 may have the potential applicability to the field of antitumor immunotherapy and may provide a platform for adoptive immunotherapy in the clinical setting.

Induction of antigen-specific CD4- and CD8-mediated T-cell responses by fusions of autologous dendritic cells and metastatic colorectal cancer cells.

Human metastatic colorectal carcinomas (CRCAs) express carcinoembryonic antigen (CEA) and/or MUC1 tumor‐associated antigens as potential targets for the induction of active specific immunity. In the present study, freshly isolated metastatic CRCA cells were successfully fused with immature autologous human monocyte‐derived dendritic cells (DCs). The created heterokaryons (DC/CRCA) coexpress the CRCA‐derived CEA and MUC1 antigens and DC‐derived MHC class II and costimulatory molecules. The fusion cells were functional in stimulating the proliferation of autologous T cells. In addition, both CD4+ and CD8+ T cells were activated by fusion cells, as demonstrated by the production of high levels of IFN‐γ. More importantly, coculture of fusion cells with patient‐derived peripheral blood mononuclear cells (PBMCs) resulted in the induction of antigen‐specific cytotoxic T lymphocytes (CTLs). CTLs were effective at lysis of not only autologous CRCA cells but also the CEA and/or MUC1‐positive and HLA partially matched target cells. Antigen‐specific CTL responses were confirmed by tetrameric analysis. Coculture of PBMCs with fusion cells resulted in increased frequency of CEA‐ and MUC1‐specific CTLs simultaneously. Taken together, these results indicate that freshly isolated human metastatic CRCA cells expressing the CEA and/or MUC1 may represent a potential partner for the creation of DC/tumor fusion cells targeting induction of antigen‐specific CTL responses. Our report demonstrates the simultaneous induction of CRCA‐specific CTL responses restricted by HLA‐A2 and ‐A24.

Synergistic Induction of Antigen-Specific CTL by Fusions of TLR-Stimulated Dendritic Cells and Heat-Stressed Tumor Cells

Dendritic cell (DC)/tumor cell fusion cells (FCs) can induce potent CTL responses. The therapeutic efficacy of a vaccine requires the improved immunogenicity of both DCs and tumor cells. The DCs stimulated with the TLR agonist penicillin-killed Streptococcus pyogenes (OK-432; OK-DCs) showed higher expression levels of MHC class I and II, CD80, CD86, CD83, IL-12, and heat shock proteins (HSPs) than did immature DCs. Moreover, heat-treated autologous tumor cells displayed a characteristic phenotype with increased expression of HSPs, carcinoembryonic Ag (CEA), MUC1, and MHC class I (HLA-A2 and/or A24). In this study, we have created four types of FC preparation by alternating fusion cell partners: 1) immature DCs fused with unheated tumor cells; 2) immature DCs fused with heat-treated tumor cells; 3) OK-DCs fused with unheated tumor cells; and 4) OK-DCs fused with heat-treated tumor cells. Although OK-DCs fused with unheated tumor cells efficiently enhanced CTL induction, OK-DCs fused with heat-treated tumor cells were most active, as demonstrated by: 1) up-regulation of multiple HSPs, MHC class I and II, CEA, CD80, CD86, CD83, and IL-12; 2) activation of CD4+ and CD8+ T cells able to produce IFN- γ at higher levels; 3) efficient induction of CTL activity specific for CEA or MUC1 or both against autologous tumor; and 4) superior abilities to induce CD107+IFN-γ+CD8+ T cells and CD154+ IFN-γ+CD4+ T cells. These results strongly suggest that synergism between OK-DCs and heat-treated tumor cells enhances the immunogenicity of FCs and provides a promising means of inducing therapeutic antitumor immunity.

Increased Lymphatic Vessels in Patients with Encapsulating Peritoneal Sclerosis

♦ Background: The angiogenic response is partly involved in the progression of encapsulating peritoneal sclerosis (EPS). However, the details of the angiogenic response, especially for lymphatic vessels in patients with EPS, remain unclear. In addition, because of technical limitations, morphology studies reported to date have examined only the parietal peritoneum. The morphologies of parietal and visceral lymphatic vessels in patients with EPS both need to be analyzed. ♦ Methods: We examined peritoneal samples from 18 patients with EPS who underwent enterolysis of the visceral peritoneum and compared them with samples from 17 autopsy cases (controls). To examine the angiogenic response, we performed immunohistochemistry for the endothelial markers CD34 (blood vessels) and podoplanin (lymphatic vessels) and for the cell proliferation marker Ki-67. Immunogold electron microscopy analysis for podoplanin was also performed. In 7 of 18 cases, we compared differences in the angiogenic response of the parietal and visceral peritoneal membranes. ♦ Results: Angiogenic responses were more frequent in the compact zone than in regenerated layers. The number of capillaries positive for anti-CD34 and anti-podoplanin monoclonal antibodies per unit area of visceral peritoneal tissue was, respectively, 41.1 ± 29.3/mm2 in EPS patients and 2.7 ± 4.4/mm2 in controls (p ≤ 0.01) and 48.1 ± 43.9/mm2 in EPS patients and 4.1 ± 5.4/mm2 in controls (p ≤ 0.01). The percentage of capillaries positive for anti-Ki-67, CD34, and podoplanin was 4.6% in EPS patients (p ≤ 0.01) and 0.8% in controls (p = 0.09). The immunogold electron microscopy analysis revealed that podoplanin was localized to endothelial cells with anchoring filaments, a specific feature of lymphatic vessels. Furthermore, compared with parietal peritoneal membrane, visceral peritoneal membrane had a more prominent podoplanin-positive capillary profile, but not a prominent CD34-positive capillary profile. In addition, fibroblast-like cells double-positive for podoplanin and smooth muscle actin were markedly increased in the degenerated layer, as previously reported. ♦ Conclusions: Our study demonstrated that lymphatic vessels are increased in the visceral peritoneum of patients with EPS.

Thymidine phosphorylase mRNA expression may be a predictor of response to post‑operative adjuvant chemotherapy with S‑1 in patients with stage III colorectal cancer

The aim of the present study was to investigate markers in surgically resected specimens of colorectal cancer that can be used to predict the response to chemotherapy. The mRNA expression levels of enzymes involved in 5-fluorouracil (5-FU) metabolism and folate metabolism were measured in formalin-fixed, paraffin-embedded tumor sections obtained from the primary tumors of 54 patients with resected stage II or III colorectal cancer who received S-1 for one year. The 5-FU metabolizing enzymes studied were thymidylate synthase, dihydropyrimidine dehydrogenase and thymidine phosphorylase (TP). The folate metabolizing enzymes studied were folypolyglutamate synthetase, γ-glutamyl hydrolase and dihydrofolate reductase. The associations between the mRNA expression levels of these enzymes and clinical variables were investigated. Tumors were classified as exhibiting high or low expression as compared with the median mRNA expression level of each metabolizing enzyme defined as the cutoff value. The associations between the high and low expression levels of each enzyme and disease-free survival (DFS) were analyzed with the use of Kaplan-Meier curves and the log-rank test. DFS was not significantly associated with the relative mRNA expression level of any metabolizing enzyme in the study group as a whole, but there was a trend toward longer DFS in patients with high TP expression (P=0.066). In patients with stage III colorectal cancer, high TP expression was associated with significantly improved outcomes compared with low TP expression (P=0.039). These results indicate that the mRNA expression of TP, a metabolizing enzyme of 5-FU, is a significant predictor of response to post-operative chemotherapy with S-1 in patients with stage III colorectal cancer.

Expression of CXCR‑4 and IDO in human colorectal cancer: An immunohistochemical approach

C-X-C chemokine receptor type 4 (CXCR4), the receptor for the chemokine stromal cell-derived factor (SDF)-1 [also known as C-X-C motif chemokine 12 (CXCL12)], is involved in lymphocyte trafficking. Recent studies have demonstrated that, during pregnancy, a placental enzyme called indoleamine 2, 3-dioxygenase (IDO) exerts a key role in suppressing the maternal T-cell response against the fetus. In the present study, the significance of CXCR4 and IDO expression in human colorectal cancer (CRC) has been investigated by immunohistochemical assay, and their association with survival was analyzed. Tumor specimens (n=60) from patients with different American Joint Committee on Cancer (AJCC) stages of CRC (I or IV) were assessed. In the stage IV group, 23 of 30 cases (77%) stained positive for CXCR4, and 9 of 30 (30%) were positive for IDO. By contrast, in the stage I group, 7 of 30 cases (23%) stained positive for CXCR4, and 15 of 30 cases (50%) were positive for IDO. The 5-year survival rate of those with high CXCR4 expression in tumor specimens (n=30) was significantly worse compared with those with negative CXCR4 expression (16.3 vs. 60.7%, P=0.02). By contrast, the 5-year survival rate of those with high IDO expression in tumor specimens (n=24) was not significantly different compared with those with negative IDO expression (36.4 vs. 56.8%). In the stage I group, 4 patients in the high IDO expression group (n=15) had distant metastases (2 in the liver 1 in the brain, and 1 in the lung). Taken together, CXCR4 appears to be a novel predictive indicator of survival, and IDO expression in the early stage may be a predictor of distant metastasis.

Arterial Infusion Chemotherapy in Patient with Repeated Recurrent Tumor of Cecal Cancer: Report of a Case

Purpose: We report a patient with a repeated recurrent tumor after Right-hemicolectomy for advanced cecal cancer who was treated by intra-arterial infusions of 5-fluorouracil (5-FU). Methods: A computed tomography scan revealed a pelvic mass involving the psoas major muscle and quadratos lumborum muscle, in contact with the widely projecting toward L2-S2. The fluorodeoxyglucose-positron emission tomography (FDG-PET) revealed an accumulation spot in the same place. This case was deemed in operable, and one-shot bolus of 5-FU was administered through the tumor feeding arteries: the left 3rd, 4th lumbar, and ilio - lumbar arteries at a dosage of 250 mg/body from each artery. Results: A partial regression of the tumor was observed by computed tomography. The serum level of carbohydrate antigen 19–9 returned normal in 8 months. During chemotherapy, the side effect and complications were tolerable, and she experienced only grade-1 nausea caused by 5-fluorouracil. Conclusion: A long–time, intra-arterial 5-fluorouracil infusion could control effectively and safely.

Surgical Approach for Recurrent Encapsulating Peritoneal Sclerosis A Case Report

症例は55歳の男性で, 慢性腎不全に対して1993年9月に腹膜透析が導入された. 腹膜炎のため, 2002年5月に血液透析に移行した. 保存的治療に抵抗性の被. 性腹膜硬化症 (以下, EPS)と診断され同年11月に初回の手術 (被嚢剥離術) が施行された. その後, EPS再発によるイレウスとなり保存的治療では改善せず, 2004年4月に再手術(被嚢剥離術) が施行された. 初回と同様に小腸全体が硬い厚い膜で被われ癒着していた. さらに, 初回術後の癒着もあったが被嚢をすべて剥離した. 術中2か所で小腸を損傷したためにその口側で人工肛門を造設した. 手術時間6時間30分, 出血量728g.術後11日目で全粥摂取可能となり内科に転科転床した. EPSに対する手術治療は成績不良かつ致命的合併症の発生率が高いとされていた. 術前の全身状態, 感染を確実にコントロールし, 慎重な手技を徹底することで, EPSに対する再手術は可能と考える.