Deep Sequencing of Cancer-Related Genes Revealed <i>GNAS</i> Mutations to Be Associated with Intraductal Papillary Mucinous Neoplasms and Its Main Pancreatic Duct Dilation

<div><p>Background</p><p>To clarify the genetic mutations associated with intraductal papillary mucinous neoplasms (IPMN) and IPMN-related pancreatic tumours, we conducted cancer-related gene profiling analyses using pure pancreatic juice and resected pancreatic tissues.</p><p>Methods</p><p>Pure pancreatic juice was collected from 152 patients [nine with a normal pancreas, 22 with chronic pancreatitis (CP), 39 with pancreatic ductal adenocarcinoma (PDAC), and 82 with IPMN], and resected tissues from the pancreas were collected from 48 patients (six IPMNs and 42 PDACs). The extracted DNA was amplified by multiplexed polymerase chain reaction (PCR) targeting 46 cancer-related genes containing 739 mutational hotspots. The mutations were analysed using a semiconductor-based DNA sequencer.</p><p>Results</p><p>Among the 46 cancer-related genes, <i>KRAS</i> and <i>GNAS</i> mutations were most frequently detected in both PDAC and IPMN cases. In pure pancreatic juice, <i>GNAS</i> mutations were detected in 7.7% of PDAC cases and 41.5% of IPMN cases (<i>p</i><0.001 vs. others). All PDAC cases with <i>GNAS</i> mutations (n = 3) were accompanied by IPMN. Multivariate analysis revealed that <i>GNAS</i> mutations in IPMN cases were associated with dilated main pancreatic ducts (MPD, <i>p</i> = 0.016), while no statistically independent associations with clinical variables were observed for <i>KRAS</i> mutations. In the resected pancreatic tissues, <i>GNAS</i> mutations were detected in 50% of PDAC cases concomitant with IPMN, 33.3% of PDAC cases derived from IPMN, and 66.7% of IPMN cases, while no <i>GNAS</i> mutations were detected in cases of PDAC without IPMN.</p><p>Conclusions</p><p>The <i>GNAS</i> mutation was specifically found in the cases with IPMN and it was speculated that some PDACs might be influenced by the concomitant but separately-located IPMN in their pathogenic mechanism. Furthermore, the <i>GNAS</i> mutation was significantly associated with MPD dilatation in IPMN cases, suggesting its role in mucus hypersecretion.</p></div

Results of univariate and multivariate analyses of <i>GNAS</i> status in the pancreatic juice of cases with IPMN.

<p>WT, wild type; IPMN, intraductal papillary mucinous neoplasm;</p><p>MPD, main pancreatic duct (normal MPD diameter ≤2 mm);</p><p>Ph, pancreas head; Pbt, pancreas body and tail; ^†<i>p</i><0.05.</p

Characteristics of the cases from whom the pancreatic juice was obtained.

<p>CP, chronic pancreatitis; PDAC, pancreatic ductal adenocarcinoma; IPMN, intraductal papillary mucinous neoplasm; MPD, main pancreatic duct.</p

Genetic mutations in pure pancreatic juice.

<p>The y-axis of the figure represents the percentage of cases with mutations in each gene. A. No mutation was detected in the pure pancreatic juice from cases with normal pancreas tissue. B. <i>GNAS</i> mutation was detected in one case (4.5%) with CP (chronic pancreatitis) and a small cystic lesion. C. <i>GNAS</i> and <i>KRAS</i> mutations were detected in 7.7% (three of 39 cases) and 20.5% (eight of 39 cases), respectively, that had pancreatic ductal adenocarcinoma (PDAC). The <i>GNAS</i> mutation was detected in all cases with intraductal papillary mucinous neoplasm (IPMN, n = 3). D. <i>GNAS</i> and <i>KRAS</i> mutations were detected in 41.5% (34 of 82 cases) and 39.0% (32 of 82 cases), respectively, with IPMN.</p

Comparison of detected mutations between the pancreatic juice and resected tissue.

<p>Upper lines of each case indicate the results for tissue samples and lower lines indicate the results for pancreatic juice.</p><p>VF, Variant frequency; WT, wild type;</p><p>PDAC, pancreatic ductal adenocarcinoma; IPMN, intraductal papillary mucinous neoplasm.</p

Schema of pancreatic ductal adenocarcinoma (PDAC) with associated intraductal papillary mucinous neoplasm (IPMN).

<p>A. The <i>KRAS</i> mutation was detected in ordinary PDAC without IPMN. B. <i>KRAS</i> and <i>GNAS</i> mutations were detected in IPMN. C. <i>KRAS</i> and <i>GNAS</i> mutations were detected in PDAC derived from primary IPMN. D. <i>KRAS</i> and <i>GNAS</i> mutations were detected not only in IPMN but also in PDAC that developed separately from IPMN.</p

Cancer-related gene profiling in resected pancreatic tumours.

<p>VF, Variant frequency; WT, wild type;</p><p>PDAC, pancreatic ductal adenocarcinoma; IPMN, intraductal papillary mucinous neoplasm.</p>†<p>DNA from formalin-fixed, paraffin-embedded samples.</p

Radiological imaging of pancreatic ductal adenocarcinoma (PDAC) concomitant with intraductal papillary mucinous neoplasm (IPMN).

<p>A. A case of CP with a small cyst (blue arrow) that had the <i>GNAS</i> mutation (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098718#pone-0098718-g001" target="_blank">Fig. 1B</a>) in pure pancreatic juice on magnetic resonance cholangiopancreatography (MRCP) imaging. B–D. PDAC with <i>GNAS</i> mutations distinct from concomitant IPMN. In cases 30 (B) and 31 (C), MRCP imaging revealed stenosis in the MPD in the pancreatic head near the PDAC (yellow arrowhead). The IPMN was located in the pancreatic body (blue arrow). Computed tomographic imaging in case 28 (D) revealed that the PDAC was located in the pancreatic body (yellow arrowhead), whereas the IPMN was located in the pancreatic head (blue arrow). A list of cases B–D is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098718#pone-0098718-t004" target="_blank">Table 4</a>.</p

Inhibitory Effects of Caffeic Acid Phenethyl Ester Derivatives on Replication of Hepatitis C Virus

<div><p>Caffeic acid phenethyl ester (CAPE) has been reported as a multifunctional compound. In this report, we tested the effect of CAPE and its derivatives on hepatitis C virus (HCV) replication in order to develop an effective anti-HCV compound. CAPE and CAPE derivatives exhibited anti-HCV activity against an HCV replicon cell line of genotype 1b with EC₅₀ values in a range from 1.0 to 109.6 µM. Analyses of chemical structure and antiviral activity suggested that the length of the n-alkyl side chain and catechol moiety are responsible for the anti-HCV activity of these compounds. Caffeic acid n-octyl ester exhibited the highest anti-HCV activity among the tested derivatives with an EC₅₀ value of 1.0 µM and an SI value of 63.1 by using the replicon cell line derived from genotype 1b strain Con1. Treatment with caffeic acid n-octyl ester inhibited HCV replication of genotype 2a at a similar level to that of genotype 1b irrespectively of interferon signaling. Caffeic acid n-octyl ester could synergistically enhance the anti-HCV activities of interferon-alpha 2b, daclatasvir, and VX-222, but neither telaprevir nor danoprevir. These results suggest that caffeic acid n-octyl ester is a potential candidate for novel anti-HCV chemotherapy drugs.</p></div

Effect of compound 10 on the viral replication in the replicon cell line and HCVcc.

<p>(A) Molecular structure of compound <b>10</b>. (B) Huh7/Rep-Feo cells were incubated for 72 h in a medium containing various concentrations of compound <b>10</b>. Luciferase and cytotoxicity assays were carried out by the method described in Materials and Methods. Error bars indicate standard deviation. The data represent three independent experiments. (C) Protein extract was prepared from Huh7/Rep-Feo cells treated for 72 h with the indicated concentration of compound <b>10</b> and it was then subjected to Western blotting using antibodies to NS3 and beta-actin. (D) Huh7 cell line was transfected with pEF Fluc IN encoding firefly luciferase or pEF Rluc IN encoding <i>Renilla</i> luciferase. Both transfected cell lines were incubated with DMSO (Control) or 40 µg/ml compound <b>10</b>. Firefly or <i>Renilla</i> luciferase activity was measured 72 h post-treatment. Luciferase activity was normalized with protein concentration. Error bars indicate standard deviation. The data were represented from three independent experiments. (E) Schematic structure of RNA transcribed from the plasmids was shown (Top). The bicistronic gene is transcribed under the control of elongation factor 1α (EF1α) promoter. The upstream cistron encoding <i>Renilla</i> luciferase (RLuc) is translated by a cap-dependent mechanism. The downstream cistron encodes the fusion protein (Feo), which consists of the firefly luciferase (Fluc) and neomycin phosphotransferase (Neo^r), and is translated under the control of the EMCV or HCV IRES. Huh7 cell line was transfected with each plasmid and incubated for 72 h post-treatment with DMSO (control) or 40 µg/ml of compound <b>10</b>. Firefly and <i>Renilla</i> luciferase activities were measured. Relative ratio of Firefly luciferase activity to <i>Renilla</i> luciferase activity was represented as percentage of the control condition. Error bars indicate standard deviation. The data were represented from three independent experiments. (F) Huh7 OK1 cell line was infected with HCVcc derived from JFH-1 strain and then treated with several concentrations of compound <b>10</b> at 24 h post-infection. The resulting cells were harvested 72 h post-infection. The viral RNA of supernatant was purified and estimated by the method described in Materials and Methods. Error bars indicate standard deviation. The data represent three independent experiments. Treatment with DMSO corresponds to ‘0’.</p