Comprehensive assessment of the prognosis of pancreatic cancer: peripheral blood neutrophil-lymphocyte ratio and immunohistochemical analyses of the tumour site.

OBJECTIVE: Several studies have suggested that an elevated neutrophil-lymphocyte ratio (NLR) is associated with a poorer prognosis in patients with pancreatic cancer (PC). The correlations between the NLR and immunohistochemical (IHC) analysis with regard to the prognosis of patients with PC remain to be elucidated. By using IHC findings, we determined the value of the NLR as a prognostic factor in patients with PC. MATERIAL AND METHODS: We collected the clinico-pathological data of 28 consecutive patients who underwent surgical resection for PC between January 2008 and December 2012 at The Jikei University Kashiwa Hospital. We investigated whether the NLR and IHC results were related and ensured the consistency of the prognosis of patients with PC. RESULTS: The Kaplan-Meier curves for the disease-free survival (DFS) and the overall survival (OS) revealed that an NLR ≥ 5 is an implicit factor for decreased DFS and OS in patients with PC (p = 0.003, p < 0.001, log-rank test). The density of CD163(+) macrophages and CD66b(+) neutrophils was significantly higher in the high NLR group; on the contrary, the density of CD20(+) lymphocytes was significantly higher in the low NLR group. Moreover, a Mann-Whitney U test showed that the NLR was significantly correlated with a high density of CD20(+) lymphocytes (p = 0.031) and CD163(+) macrophages (p = 0.023), while the NLR was not significantly correlated with CD66b(+) neutrophils (p = 0.397). CONCLUSIONS: Our results demonstrated the validity of the NLR by IHC analyses and we determined that a higher value of NLR is a trustworthy prognostic factor for patients with PC.

Involvement of MAP3K8 and miR-17-5p in poor virologic response to interferon-based combination therapy for chronic hepatitis C.

Despite advances in chronic hepatitis C treatment, a proportion of patients respond poorly to treatment. This study aimed to explore hepatic mRNA and microRNA signatures involved in hepatitis C treatment resistance. Global hepatic mRNA and microRNA expression profiles were compared using microarray data between treatment responses. Quantitative real-time polymerase chain reaction validated the gene signatures from 130 patients who were infected with hepatitis C virus genotype 1b and treated with pegylated interferon-alpha and ribavirin combination therapy. The correlation between mRNA and microRNA was evaluated using in silico analysis and in vitro siRNA and microRNA inhibition/overexpression experiments. Multivariate regression analysis identified that the independent variables IL28B SNP rs8099917, hsa-miR-122-5p, hsa-miR-17-5p, and MAP3K8 were significantly associated with a poor virologic response. MAP3K8 and miR-17-5p expression were inversely correlated with treatment response. Furthermore, miR-17-5p repressed HCV production by targeting MAP3K8. Collectively, the data suggest that several molecules and the inverse correlation between mRNA and microRNA contributed to a host genetic refractory hepatitis C treatment response.

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

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.

Augmentation of antitumor immunity by fusions of ethanol-treated tumor cells and dendritic cells stimulated via dual TLRs through TGF-ß1 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)-ß1, 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-ß1 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.

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-ß1 derived from DC/tumor on the induction of MUC1-specific CTLs. Combined TLR2- and TLR4-activated DC/tumor overcame immune-suppressive effect of TGF-ß1 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-ß1 reduced the efficacy of DC/tumor vaccine in vitro. Incorporating combined TLRs-activation and TGF-ß1-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.

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.

Target chemotherapy of anti-CD147 antibody-labeled liposome encapsulated GSH-DXR conjugate on CD147 highly expressed carcinoma cells.

It was confirmed that CD147 (Emmprin) was expressed on the cell surface of carcinoma cells. For the purpose of studying the efficacy of a CD147-targeting agent on CD147-expressing carcinoma cells, we investigated the effect of a conjugate of glutathione-doxorubicin (GSH-DXR) encapsulated in an anti-CD147 antibody-labeled liposome (aCD147ab-liposome) in terms of specific accumulation and cytotoxicity in CD147-expressing human carcinoma cells. Expression of CD147 was not observed in many normal human tissues. However, slight expression of CD147 in kidney, prostate and breast tissues was observed. By contrast, high-level expression of CD147 in all carcinoma cells such as A431, PC3 and Ishikawa cell lines was confirmed by fluorescent microscopy and Western blot analysis. Specific accumulation of the aCD147ab-liposome in the above-described CD147-expressing cells was observed. GSH-DXR encapsulated in an aCD147ab-liposome expressed specific cytotoxicity against these carcinoma cells. These results suggested that target chemotherapy of GSH-DXR encapsulated in an aCD147ab-liposome on CD147-expressing carcinoma cells was effective.

Three-dimensional culture using a radial flow bioreactor induces matrix metalloprotease 7-mediated EMT-like process in tumor cells via TGFbeta1/Smad pathway.

To confirm the usefulness of the radial flow type bioreactor (RFB) for a three-dimensional (3D) culture system, which provides a tissue architecture and molecular function mimicking the in vivo environment, molecular expression in the A431 human squamous carcinoma cell line during culture were analyzed under the physically different environments of 3D culture in the RFB, 2D culture in a monolayer as well as in nude mice. Time-dependent accumulation of autocrine transforming growth factor (TGF) beta1 was found in spent culture media obtained only from 3D cultured A431 cancer cells, which grew well with a stratified-sheet morphology. Cells in the RFB overexpressed matrix metalloproteinase 7 (MMP7) and showed an increased release of soluble 80-kDa fragments of E-cadherin into the media time-dependently, resulting in the reduction of E-cadherin protein at the cell surface without down-regulation of the mRNA. beta-Catenin and its nuclear partner, LEF1, were up-regulated and Wnt protein secretion was also accelerated. Additional up-regulation of the transcriptional factors, HMGA2 and down-stream Slug, was noted. TGFbeta1-dependent, MMP7-mediated up-regulation of beta-catenin/LEF1 signaling and TGFbeta1-activated HMGA2 pathways consequently converged with Slug overexpression, due to disassembly and further repression of E-cadherin expression, which was reproducible in the epithelial mesenchymal transition process without any manipulation. Other transcriptional factors, Notch/HEY1 and NF-kappaB, were also up-regulated in 3D-cultured cells. These signals recruited molecules related to extracellular matrix-cell remodeling and angiogenesis. Expression of several representative molecules in the 3D cultured cells was parallel with that in xenotransplanted A431 tumor tissues in nude mice. 3D culture of tumor cells in the RFB is a useful tool for cancer experimental biology and evaluation of cancer therapeutic-like systems in nude mice.

The association of deamidation of Bcl-xL and translocation of Bax to the mitochondria through activation of JNK in the induction of apoptosis by treatment with GSH-conjugated DXR.

We investigated the induction of apoptosis via deamidation of Bcl-xL and translocation of Bax to the mitochondria by treatment with GSH-DXR. GSH-DXR treatment of HepG2 cells, which did not express GST P1-1, exhibited deamidation of Bcl-xL, and the degree of deamidation was related to the activation of caspase-3. Overexpression of GST P1-1 in HepG2 cells decreased both the Bcl-xL deamidation and caspase-3 activation induced by treatment with GSH-DXR. Bcl-xL deamidation and caspase-3 activation were also suppressed by co-treatment with SP600125, a specific inhibitor of JNK activity. Overexpression of wild-type Bcl-xL in HepG2 decreased GSH-DXR-induced apoptosis although deamidation was observed. However, expression of the deamidated mutant of Bcl-xL, in which aspartic acid was substituted for both arginine 52 and 66 (N52,66D-Bcl-xL), exhibited high sensitivity for the induction of apoptosis. Expression of the Bcl-xL mutant, in which alanine was substituted for both arginine 52 and 66 (N52,66A-Bcl-xL), suppressed deamidation and showed resistance to the induction of apoptosis by treatment with GSH-DXR. On the other hand, endogenous Bax and overexpressed Flag-Bax were localized in the cytosolic fraction of HepG2 cells. Treatment of the cells with GSH-DXR caused translocation of Flag-Bax to the mitochondrial fraction following the induction of apoptosis. The induced apoptosis was enhanced by the expression of Flag-Bax. Moreover, Flag-Bax was partly located in the mitochondrial fraction in N52,66D-Bcl-xL-expressed cells without the induction of apoptosis. Therefore, the induction of apoptosis by treatment of HepG2 with GSH-DXR was enhanced, thereby facilitating the release of cytochrome c by both deamidated inactivation of Bcl-xL and functional translocation of Bax to the mitochondria via JNK activation. Deamidation of Bcl-xL might be induced in order to translocate Bax to the mitochondria.