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Pancreatic ductal adenocarcinoma (PDAC) is difficult to distinguish from autoimmune pancreatitis (AIP) because of their clinical and radiological similarities, and therefore simple and minimally invasive surrogate markers for differential diagnosis would be useful. In our previous studies, we identified four microRNAs (miRNAs)–miR-7, miR-34a, miR-181d, and miR-193b –as MAPK-associated microRNAs whose expression was altered significantly with upregulation of the MAPK signaling pathway. Recently it has been reported that these miRNAs could be used as biomarkers in serum samples for accurate diagnosis of pancreatic lesions. The aim of the present study was to evaluate whether these MAPK-associated miRNAs in serum could be used as biomarkers for differentiating PDAC from AIP. We enrolled 69 patients with PDAC, 26 with intraductal papillary mucinous neoplasm (IPMN) and 15 with AIP. The expression of MAPK-associated miRNAs in serum was measured by quantitative real-time PCR. The 2-ΔCT method was used to quantify the expression of miRNAs, and the data were normalized using spiked-in synthetic cel-miR-39. Patients with PDAC or IPMN showed significantly higher amounts of serum MAPK-associated miRNAs than those with AIP (p<0.009 for miR-7, p<0.002 for miR-34a, p<0.001 for miR-181d, p<0.002 for miR-193b). ROC curve analysis demonstrated that these miRNAs had an area under the ROC curve (AUC) of 0.723–0.882 for differentiation between PDAC or IPMN from AIP. Furthermore, serum miR-181d was significantly associated with the presence of metastasis in patients with PDA (p = 0.014). Serum MAPK-associated miRNAs could be novel noninvasive biomarkers for differentiation between PDAC or IPMN and AIP.

Background: The incidence of pancreatic cancer is increasing year-by-year in Japan. Among the diseases that complicate pancreatic cancer, diabetes is the most common. Recently, it has become evident that patients suffering from diabetes and obesity show increased expression of osteopontin (OPN). The purpose of this study was to investigate the effect of high glucose and high insulin culture conditions on a human pancreatic duct epithelial cell line (HPDE-6), focusing particularly on OPN expression. Methods: HPDE-6 were cultured under various conditions, employing several combinations of glucose (normal, 6 mM high, 30 mM, and 60 mM) and insulin (0.1 nM, 1 nM) concentration. Results: HPDE-6 cell proliferation was significantly accelerated under high glucose culture conditions in comparison to samples in 6 mM glucose, and was more prominent under high insulin conditions. At the same time, the expression of OPN mRNA was also increased significantly. In comparison with 6 mM glucose, the expression of 8-OHdG DNA was increased in high glucose culture. Conclusion: HPDE-6 cells show accelerated proliferation and increased OPN expression when cultured under high glucose and high insulin conditions. Furthermore, the cells show increased oxidative stress in the presence of high glucose.

Extracellular vesicles (EVs) are released from all cells. Bile directly contacts bile duct tumor; bile-derived EVs may contain high concentrations of cancer biomarkers. We performed a proteomic analysis of human bile-derived EVs and identified a novel biomarker of cholangiocarcinoma (CCA). EVs were isolated using ultracentrifugation, and chelating agents, ethylenediaminetetraacetic acid and ethylene glycol tetraacetic acid (EDEG) and phosphate buffered saline (PBS) were used as dissolution solutions. Bile was collected from 10 CCA and 10 choledocholithiasis (stones) cases. Proteomic analysis was performed; subsequently, ELISA was performed using the candidate biomarkers in a verification cohort. The vesicles isolated from bile had a typical size and morphology. The expression of exosome markers was observed. RNA was more abundant in the EDEG group. The proportion of microRNA was higher in the EDEG group. EDEG use resulted in the removal of more contaminants. Proteomic analysis identified 166 proteins as CCA-specific. ELISA for Claudin-3 revealed statistically significant difference. The diagnostic accuracy was AUC 0.945 and sensitivity and specificity were 87.5%. We report the first use of EDEG in the isolation of EVs from human bile and the proteomic analysis of human bile-derived EV-proteins in CCA. Claudin-3 in bile-derived EVs is a useful biomarker for CCA.

Pancreatic cancer (PC) is a highly lethal malignancy, with a 5-year survival rate of 6%. Cancer gene panel testing is expected to allow selection of suitable therapeutic drugs in individual patients with PC and improve their prognosis. Although somatic mutations can be identified in formalin-fixed, paraffin-embedded samples derived from surgical specimen, the rate of surgical indication among patients with PC is only 20%. To acquire genome information with a less invasive method, we used rapid on-site evaluation (ROSE) specimens from endoscopic ultrasound-guided fine-needle aspiration. The present study aimed to retrospectively evaluate the utility of comprehensive cancer gene panel testing with ROSE specimens. DNA was extracted from preserved ROSE specimens of 26 patients diagnosed with PC between 2011 and 2017. DNA sequences of oncogenes and cancer-related genes were determined using the Ion AmpliSeq Comprehensive Caner Panel. We compared KRAS mutations between cancer gene panel testing by next-generation sequencing (NGS) and KRAS mutation analysis by polymerase chain reaction. The mean yield of DNA per extraction from ROSE specimens was 171 ng (range, 34-478 ng). On cancer gene panel testing, we noted KRAS mutations (92%), TP53 mutations (50%), CDKN2A mutations (15%), and SMAD4 mutations (31%). The concordance rate of KRAS mutations between cancer gene panel testing by NGS using ROSE specimens and KRAS mutation analysis by the companion diagnostics using residual materials was 81%. Among five cases of KRAS discordance, three showed KRAS mutations in cancer gene panel testing but not in KRAS mutation analysis. Cancer gene panel testing with ROSE specimens can help stratify unresectable PC patients without additional invasive approaches, and it can be used for therapeutic drug selection.