Naoki Hama

MicroRNA-1246 expression associated with CCNG2-mediated chemoresistance and stemness in pancreatic cancer

Background:Pancreatic cancer has a poor prognosis because of its high refractoriness to chemotherapy and tumour recurrence, and these properties have been attributed to cancer stem cells (CSCs). MicroRNA (miRNA) regulates various molecular mechanisms of cancer progression associated with CSCs. This study aimed to identify the candidate miRNA and to characterise the clinical significance.Methods:We established gemcitabine-resistant Panc1 cells, and induced CSC-like properties through sphere formation. Candidate miRNAs were selected through microarray analysis. The overexpression and knockdown experiments were performed by evaluating the in vitro cell growth and in vivo tumourigenicity. The expression was studied in 24 pancreatic cancer samples after laser captured microdissection and by immunohistochemical staining.Results:The in vitro drug sensitivity of pancreatic cancer cells was altered according to the miR-1246 expression via CCNG2. In vivo, we found that miR-1246 could increase tumour-initiating potential and induced drug resistance. A high expression level of miR-1246 was correlated with a worse prognosis and CCNG2 expression was significantly lower in those patients.Conclusions:miR-1246 expression was associated with chemoresistance and CSC-like properties via CCNG2, and could predict worse prognosis in pancreatic cancer patients.

miR-320c regulates gemcitabine-resistance in pancreatic cancer via SMARCC1

Background:Gemcitabine-based chemotherapy is the standard treatment for pancreatic cancer. However, the issue of resistance remains unresolved. The aim of this study was to identify microRNAs (miRNAs) that govern the resistance to gemcitabine in pancreatic cancer.Methods:miRNA microarray analysis using gemcitabine-resistant clones of MiaPaCa2 (MiaPaCa2-RGs), PSN1 (PSN1-RGs), and their parental cells (MiaPaCa2-P, PSN1-P) was conducted. Changes in the anti-cancer effects of gemcitabine were studied after gain/loss-of-function analysis of the candidate miRNA. Further assessment of the putative target gene was performed in vitro and in 66 pancreatic cancer clinical samples.Results:miR-320c expression was significantly higher in MiaPaCa2-RGs and PSN1-RGs than in their parental cells. miR-320c induced resistance to gemcitabine in MiaPaCa2. Further experiments showed that miR-320c-related resistance to gemcitabine was mediated through SMARCC1, a core subunit of the switch/sucrose nonfermentable (SWI/SNF) chromatin remodeling complex. In addition, clinical examination revealed that only SMARCC1-positive patients benefited from gemcitabine therapy with regard to survival after recurrence (P=0.0463).Conclusion:The results indicate that miR-320c regulates the resistance of pancreatic cancer cells to gemcitabine through SMARCC1, suggesting that miR-320c/SMARCC1 could be suitable for prediction of the clinical response and potential therapeutic target in pancreatic cancer patients on gemcitabine-based therapy.

MicroRNA-29a induces resistance to gemcitabine through the Wnt/β-catenin signaling pathway in pancreatic cancer cells

Although we studied previously the mechanisms of resistance of pancreatic cancer cells to gemcitabine (GEM), prediction of the response to GEM remains unsatisfactory. The aim of this study was to investigate the relationship between miR-29a expression and the response to GEM in pancreatic cancer cells. Changes in the growth-inhibitory effect of pancreatic cancer cells (MIAPaCa-2, PSN-1, BxPC-3 and Panc-1) to GEM were examined after overexpression or suppression of miR-29a. We also examined the effect of miR-29a on the Wnt/β-catenin signaling pathway and investigated whether the altered growth-inhibitory effect by miR-29a suppression was weakened after the addition of Wnt3a, a Wnt/β-catenin signaling activator. MIAPaCa-2 and PSN-1 cells transfected with anti-miR-29a showed significantly lower resistance to GEM. In the anti-miR-29a-transfected cells, GEM induced significantly larger numbers of apoptotic cells and S phase accumulation compared to control cells, demonstrated by Annexin V assay and flow cytometric analysis of the cell cycle, respectively. The transfected cells showed overexpression of putative target molecules including Dkk1, Kremen2 and sFRP2 and lower activation of the Wnt/β-catenin signaling pathway. The addition of Wnt3a weakened the augmented growth-inhibitory effect of anti-miR-29a transfection. Our findings suggest that miR-29a expression correlates significantly with the growth-inhibitory effect of GEM and that activation of the Wnt/β-catenin signaling pathway mediated the miR-29a-induced resistance to GEM in pancreatic cancer cell lines.

Plasma miR-21 is a novel diagnostic biomarker for biliary tract cancer

Biliary tract cancer (BTC) has a generally poor prognosis. Furthermore, it is difficult to distinguish BTC from benign biliary disease (BBD) with commonly used modalities. Therefore, a novel biomarker to facilitate cancer detection is highly desirable. Recent studies have reported the use of circulating microRNAs (miRNAs) as biomarkers for cancers. The purpose of this study was to evaluate whether circulating miRNA‐21 (miR‐21) could be used as a biomarker for BTC. Plasma samples were obtained from 94 BTC patients, 50 healthy volunteers (HVs), and 23 BBD patients. miR‐21 levels in the samples were measured by qRT‐PCR. Plasma miR‐21 levels in patients with BTC were significantly higher than in HVs or in patients with BBD (P < 0.0001 for both). Receiver–operator curve (ROC) curve analysis in differentiating BTC patients from HVs indicated that area under the curve (AUC), optimal sensitivity and specificity was 0.93, 85.1% and 100%, respectively, and those in differentiating BTC patients from BBD patients was 0.83, 72.3%, 91.3%, respectively. Validation of these results indicated that the negative predictive value, positive predictive value, sensitivity, specificity, and accuracy in differentiating BTC patients from HVs was 76.6%, 98.6%, 84.0%, 98.0%, and 88.9%, respectively, and those in differentiating BTC patients from BBD patients was 42.2%, 93.0%, 71.2%, 82.6%, and 72.6%, respectively. These sets of values were improved by combining miR‐21 and CA19‐9 measurements. Plasma miR‐21 is a novel diagnostic biomarker for BTC, and may be useful in distinguishing between BTC and BBD patients.

Detection of AFP mRNA-expressing cells in the peripheral blood for prediction of HCC recurrence after living donor liver transplantation

The aim of this study was to evaluate the hypothesis that the detection of alpha‐fetoprotein (AFP) mRNA‐expressing cells could be a novel, direct and accurate method for predicting tumor recurrence after living donor liver transplantation (LDLT) in patients with hepatocellular carcinoma (HCC). The test group consisted of 32 patients who underwent LDLT for end‐stage liver disease with HCC. Quantitative real‐time reverse transcription polymerase chain reaction was used for the detection of AFP mRNA‐expressing cells in the peripheral blood. Nine (28.1%) of the 32 patients developed tumor recurrences during the follow‐up period (mean, 27.9 months). The test for the presence of AFP mRNA in the peripheral blood was positive either preoperatively or postoperatively in 11 (34.3%) of the 32 patients, and positive preoperatively in three patients (9.4%). Univariate analysis revealed that a positive preoperative test for peripheral blood AFP mRNA, as well as exceeding Milan criteria and microscopic evidence of vascular invasion were significant predictors for the recurrence of HCC (P = 0.002, 0.049, and 0.001, respectively). Multivariate analysis using Coxs proportional hazards model revealed that a positive preoperative test for peripheral blood AFP mRNA was an independent risk factor for the recurrence of HCC. We concluded that the presence of AFP mRNA‐expressing cells preoperatively could be a useful predictor of the recurrence of HCC in liver transplant patients.

Differential transcriptome patterns for acute cellular rejection in recipients with recurrent hepatitis C after liver transplantation

Histopathological evaluation of the liver via biopsy remains the standard procedure for the diagnosis of both acute cellular rejection (ACR) and recurrent hepatitis C (RHC) after liver transplantation. Nevertheless, it is often difficult to diagnose ACR in hepatitis C virus–positive recipients because of changes in common and overlapping with RHC. The aim of this study was to identify potential target genes for ACR in recipients with RHC. We analyzed 22 liver biopsy samples obtained from 21 hepatitis C virus–positive recipients. The clinicopathological diagnosis based on biopsy examination was ACR‐predominant with superimposed RHC in 9 samples (ACR group) and RHC without ACR (non‐ACR group) in 13. Using oligonucleotide microarrays, we compared the transcriptional changes in the 2 groups and selected 2206 genes that were significantly modulated in ACR. We analyzed the regulatory networks in ACR with Ingenuity Pathway Analysis software, and we confirmed with quantitative real‐time polymerase chain reaction the reproducibility of caspase 8, apoptosis‐related cysteine peptidase and bone morphogenetic protein 2 up‐regulation in another group of validation samples, representing 2 genes from the core network as the target genes for ACR. Our results demonstrated novel transcriptome patterns for ACR with concurrent RHC that were distinct from those of recipients with only RHC, suggesting that gene expression profiling may be useful in the diagnosis of ACR in recipients with hepatitis C. Liver Transpl 15:1738–1749, 2009.

Gene expression profiling of acute cellular rejection in rat liver transplantation using DNA microarrays

Acute cellular rejection (ACR) is still a major problem in organ transplantation, and its genetic and molecular mechanisms remain poorly understood. We used DNA microarrays to investigate the gene expression profiles in ACR. We hypothesized that changes of gene expression in grafts could also be detected in peripheral blood leukocytes. We first compared the gene expression profiles in liver isografts (Lewis to Lewis) and allografts (Dark Agouti to Lewis) harvested from rats at days 1, 3, 5, and 7 after transplantation. Hierarchical clustering analysis indicated that gene expression started to change on day 3, and 89 differentially expressed genes were extracted from allografts in comparison with isografts at day 3. Most of the up‐regulated genes were associated with graft‐infiltrating leukocytes. We then confirmed the similarity of gene expression changes in peripheral leukocytes by quantitative real‐time polymerase chain reaction. We also investigated the gene expression changes in other inflammatory and liver dysfunction models. Two interferon‐gamma inducible genes, interferon regulatory factor 1 and guanylate nucleotide binding protein 2, were overexpressed in both the peripheral leukocytes and liver graft during ACR. Although further studies are necessary, these 2 genes in peripheral leukocytes could be potentially useful markers for rejection or immunosuppression. Liver Transpl 15:509–521, 2009.

Preoperative chemoradiotherapy, surgery and adjuvant therapy for resectable pancreatic cancer.

BACKGROUND/AIMS In order to improve the poor prognosis of pancreatic cancer, a combination therapy consisting of preoperative chemoradiotherapy, surgery and postoperative chemotherapy may be an ideal strategy; nevertheless, the influence of preoperative therapy to postoperative therapy is not investigated. METHODOLOGY Thirty patients with resectable pancreatic ductal adenocarcinoma were enrolled. A 40Gy of radiation (2Gy/day x 20 fractions/4 weeks) was administered together with intravenous infusion of gemcitabine (800mg/m2, days 1, 8 and 15) before surgery. Surgery was performed 3-7 weeks after the final fraction of radiation, and postoperative chemotherapy consisting of 1000mg/m2 gemcitabine (days 1, 8 and 15 every 4 weeks for 6 cycles) was started within 8 weeks after surgery. RESULTS All 30 patients successfully completed preoperative therapy. Re-staging after such therapy showed radiologically unresectable disease in 4 patients and 1 patient rejected surgery. Among the 25 patients who underwent laparotomy, 21 underwent curative resection. After curative resection, 4 were inadequate in performance status, thus postoperative therapy could not be started. Ten patients completed postoperative adjuvant therapy. CONCLUSIONS The combination therapy for resectable pancreatic cancer seems a feasible and effective approach, though preoperative therapy may reduce the feasibility of postoperative therapy.

Prognostic impact of preoperative NLR and CA19-9 in pancreatic cancer

BACKGROUND Recently, several preoperative proinflammatory markers and nutritional factors such as neutrophil-to-lymphocyte ratio (NLR) and prognostic nutrition index (PNI) have been reported as significant predictor for poor prognosis of various malignant tumors. In this study, we evaluated the prognostic values of these preoperative parameters in patients with resectable pancreatic head cancer. METHODS We retrospectively reviewed consecutive patients who underwent PD for pancreatic head cancer between 2007 and 2012. A total of 46 patients were enrolled in this analysis. Preoperative parameters such as CRP, CA19-9, NLR and PNI at the time of presentation were recorded as well as overall survival. Cancer specific survival was assessed using Kaplan-Meier method. Univariate and multivariate Cox regression models were applied to evaluate the prognostic relevance of preoperative parameters. The correlations between CA19-9 values, NLR and pathological findings, first recurrence site were respectively reviewed. RESULTS In multivariable analysis preoperative high NLR (≧2.7) and high CA19-9 (≧230) were independent prognostic factors for poor survival (P value: 0.03 and 0.025, respectively). Kaplan-Meier survival analysis demonstrated the overall 2-year survival rate in patients with high NLR or high CA19-9 were 37.5% compared with 89.9% in patients with low NLR and low CA19-9. CONCLUSION Preoperative NLR and serum CA19-9 offer significant prognostic information associated with overall survival following PD in the patients with pancreatic head cancer.

Steroid-free living donor liver transplantation in adults: impact on hepatitis C recurrence

Abstract:  Introduction:  Although steroid‐free immunosuppression has been proven to be safe and feasible for liver transplantation, its impact on hepatitis C virus (HCV) recurrence remains unknown. We aimed to clarify the impact of steroid‐free immunosuppression on post‐operative HCV recurrence after living donor liver transplantation (LDLT).