Toyokazu Yukawa

Current Immunotherapeutic Approaches in Pancreatic Cancer

Pancreatic cancer is a highly aggressive and notoriously difficult to treat. As the vast majority of patients are diagnosed at advanced stage of the disease, only a small population is curative by surgical resection. Although gemcitabine-based chemotherapy is typically offered as standard of care, most patients do not survive longer than 6 months. Thus, new therapeutic approaches are needed. Pancreatic cancer cells that develop gemcitabine resistance would still be suitable targets for immunotherapy. Therefore, one promising treatment approach may be immunotherapy that is designed to target pancreatic-cancer-associated antigens. In this paper, we detail recent work in immunotherapy and the advances in concept of combination therapy of immunotherapy and chemotherapy. We offer our perspective on how to increase the clinical efficacy of immunotherapies for pancreatic cancer.

Combined TLR2/4-Activated Dendritic/Tumor Cell Fusions Induce Augmented Cytotoxic T Lymphocytes

Induction of antitumor immunity by dendritic cell (DC)-tumor fusion cells (DC/tumor) can be modulated by their activation status. In this study, to address optimal status of DC/tumor to induce efficient antigen-specific cytotoxic T lymphocytes (CTLs), we have created various types of DC/tumor: 1) un-activated DC/tumor; 2) penicillin-killed Streptococcus pyogenes (OK-432; TLR4 agonist)-activated DC/tumor; 3) protein-bound polysaccharides isolated from Coriolus versicolor (PSK; TLR2 agonist)-activated DC/tumor; and 4) Combined OK-432- and PSK-activated DC/tumor. Moreover, we assessed the effects of TGF-β1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-β1 in comparison to those single activated or un-activated DC/tumor as demonstrated by: 1) up-regulation of MHC class II and CD86 expression on DC/tumor; 2) increased fusion efficiency; 3) increased production of fusions derived IL-12p70; 4) activation of CD4+ and CD8+ T cells that produce high levels of IFN-γ; 5) augmented induction of CTL activity specific for MUC1; and 6) superior efficacy in inhibiting CD4+CD25+Foxp3+ T cell generation. However, DC/tumor-derived TGF-β1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-β1-blockade of DC/tumor may enhance the effectiveness of DC/tumor-based cancer vaccines and have the potential applicability to the field of adoptive immunotherapy.

A protocol for immunoaffinity separation of the accumulated ubiquitin-protein conjugates solubilized with sodium dodecyl sulfate.

Certain proteins insoluble in aqueous salt solutions are difficult to separate from impurities by immunoaffinity techniques, even when the proteins are solubilized with denaturants due to interference of the antigen-antibody reaction. Representative examples of such proteins are the ubiquitin-protein conjugates that accumulate in neuronal tissues of neurodegenerative diseases, the hallmark of such disorders. In this study, we developed a novel sample preparation method comprising two successive steps: Sodium dodecyl sulfate (SDS) removal from the SDS-containing extracts and renaturation of the denatured proteins. The application of this method was tested on ubiquitin-protein conjugates in the brains of Niemann-Pick type C disease mouse and in heat-shocked K562 erythroleukemia cells. The ubiquitin-protein conjugates in both cases are insoluble in Tris-buffered saline but soluble in 2% SDS. The SDS-solubilized fractions prepared from each of the samples were further pretreated by the method mentioned above, and the ubiquitin-protein conjugates were efficiently immunoprecipitated with the anti-ubiquitin antibody from them. This method was also applied successfully to the immunoprecipitation of flotillin-1, a lipid raft protein, from mouse brain extract prepared with 2% SDS. These results indicate that this simple protocol has potential applications for excellent immunoaffinity separation of the less-soluble proteins in diverse cells and tissues.

Long-Term Alteration of Intestinal Microbiota in Patients with Ulcerative Colitis by Antibiotic Combination Therapy

Previous work has demonstrated that intestinal bacteria, such as Fusobacterium varium (F. varium), contribute to the clinical activity in ulcerative colitis (UC); thus, an antibiotic combination therapy (amoxicillin, tetracycline, and metronidazole (ATM)) against F. varium can induce and maintain UC remission. Therefore, we investigated whether ATM therapy induces a long-term alteration of intestinal microbiota in patients with UC. Patients with UC were enrolled in a multicenter, randomized, double-blind, placebo-controlled study. Biopsy samples at the beginning of the trial and again at 3 months after treatment completion were randomly obtained from 20 patients. The terminal restriction fragment length polymorphism (T-RFLP) in mucosa-associated bacterial components was examined to assess the alteration of the intestinal microbiota. Profile changes of T-RFLP in mucosa-associated bacterial components were found in 10 of 12 patients in the treatment group and in none of 8 in the placebo group. Dice similarity coefficients using the unweighted pair group method with arithmetic averages (Dice-UPGMA) confirmed that the similarity of mucosal microbiota from the descending colon was significantly decreased after the ATM therapy, and this change was maintained for at least 3 months. Moreover, at 3 months after treatment completion, the F. varium/β-actin ratio, examined by real-time PCR using nested PCR products from biopsy samples, was reduced less than 40% in 8 of 12 treated patients, which was higher, but not significantly, than in 4 of 8 patients in the placebo group. Together, these results suggest that ATM therapy induces long-term alterations in the intestinal microbiota of patients with UC, which may be associated, at least in part, with clinical effects of the therapy.

Augmentation of Antitumor Immunity by Fusions of Ethanol-Treated Tumor Cells and Dendritic Cells Stimulated via Dual TLRs through TGF-β1 Blockade and IL-12p70 Production

The therapeutic efficacy of fusion cell (FC)-based cancer vaccine generated with whole tumor cells and dendritic cells (DCs) requires the improved immunogenicity of both cells. Treatment of whole tumor cells with ethanol resulted in blockade of immune-suppressive soluble factors such as transforming growth factor (TGF)-β1, vascular endothelial growth factor, and IL-10 without decreased expression of major histocompatibility complex (MHC) class I and the MUC1 tumor-associated antigen. Moreover, the ethanol-treated tumor cells expressed “eat-me” signals such as calreticulin (CRT) on the cell surface and released immunostimulatory factors such as heat shock protein (HSP)90α and high-mobility group box 1 (HMGB1). A dual stimulation of protein-bound polysaccharides isolated from Coriolus versicolor (TLR2 agonist) and penicillin-inactivated Streptococcus pyogenes (TLR4 agonist) led human monocyte-derived DCs to produce HSP90α and multiple cytokines such as IL-12p70 and IL-10. Interestingly, incorporating ethanol-treated tumor cells and TLRs-stimulated DCs during the fusion process promoted fusion efficiency and up-regulated MHC class II molecules on a per fusion basis. Moreover, fusions of ethanol-treated tumor cells and dual TLRs-stimulated DCs (E-tumor/FCs) inhibited the production of multiple immune-suppressive soluble factors including TGF-β1 and up-regulated the production of IL-12p70 and HSP90α. Most importantly, E-tumor/FCs activated T cells capable of producing high levels of IFN-γ, resulting in augmented MUC1-specific CTL induction. Collectively, our results illustrate the synergy between ethanol-treated whole tumor cells and dual TLRs-stimulated DCs in inducing augmented CTL responses in vitro by FC preparations. The alternative system is simple and may provide a platform for adoptive immunotherapy.

Hepatic Angiosarcoma Associated with Esophageal Variceal Hemorrhage.

Primary hepatic angiosarcoma is a very rare malignancy with a poor prognosis. Because patients present with no specific symptoms, the cancer can grow undetected and most cases are diagnosed too late for resection. We present the case of a 78-year-old Japanese man admitted to our hospital with massive hematemesis and melena. A total gastrectomy had previously been performed on the patient to treat gastric cancer. Endoscopic injection sclerotherapy was performed to control the bleeding from varices over the anastomosis. Computed tomography revealed the presence of multiple atypical liver nodules in the enhanced image. Histological diagnosis of hepatic angiosarcoma was obtained by percutaneous ultrasound-guided liver biopsy. To our knowledge, this is the first report of a patient with hepatic angiosarcoma and acute variceal hemorrhage.

Long-term management of gemcitabine in a patient with advanced pancreatic cancer undergoing haemodialysis

Abstract Gemcitabine application for patients with impaired renal function or undergoing haemodialysis will increase if the efficacy and safety are proved as the treatment for pancreatic cancer of these patients. However, there is no guideline about the usage of gemcitabine in patients with impaired renal function or haemodialysis. We report the case of a 70-year-old man with advanced pancreatic cancer undergoing haemodialysis. After discontinuation of 100% or 80% dosage, 60% dose of gemcitabine was administered biweekly. Serum carbohydrate antigen 19-9 and carcinoembryonic antigen levels were marked by slight variations and abdominal computed tomography (CT) showed the tumour size hardly changed. We administered gemcitabine for the patient 14 times in total, and he survived over 8 months from the definitive diagnosis. These findings confirm the efficacy and safety of treatment with a biweekly 60% dose of gemcitabine for patients with advanced pancreatic cancer undergoing haemodialysis in the face of dose modification.