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

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

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

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

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

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

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