Deep Sequencing of Cancer-Related Genes Revealed GNAS Mutations to Be Associated with Intraductal Papillary Mucinous Neoplasms and Its Main Pancreatic Duct Dilation Shinichi Takano*, Mitsuharu Fukasawa, Shinya Maekawa, Makoto Kadokura, Mika Miura, Hiroko Shindo, Ei Takahashi, Tadashi Sato, Nobuyuki Enomoto First Department of Internal Medicine, Faculty of Medicine, University of Yamanashi, Chuo, Yamanashi, Japan Abstract Background: 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. Methods: 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. Results: Among the 46 cancer-related genes, KRAS and GNAS mutations were most frequently detected in both PDAC and IPMN cases. In pure pancreatic juice, GNAS mutations were detected in 7.7% of PDAC cases and 41.5% of IPMN cases (p, 0.001 vs. others). All PDAC cases with GNAS mutations (n = 3) were accompanied by IPMN. Multivariate analysis revealed that GNAS mutations in IPMN cases were associated with dilated main pancreatic ducts (MPD, p = 0.016), while no statistically independent associations with clinical variables were observed for KRAS mutations. In the resected pancreatic tissues, GNAS 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 GNAS mutations were detected in cases of PDAC without IPMN. Conclusions: The GNAS 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 GNAS mutation was significantly associated with MPD dilatation in IPMN cases, suggesting its role in mucus hypersecretion. Citation: Takano S, Fukasawa M, Maekawa S, Kadokura M, Miura M, et al. (2014) Deep Sequencing of Cancer-Related Genes Revealed GNAS Mutations to Be Associated with Intraductal Papillary Mucinous Neoplasms and Its Main Pancreatic Duct Dilation. PLoS ONE 9(6): e98718. doi:10.1371/journal.pone.0098718 Editor: Jo ¨ rg D. Hoheisel, Deutsches Krebsforschungszentrum, Germany Received January 31, 2014; Accepted May 2, 2014; Published June 4, 2014 Copyright: ß 2014 Takano et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan (25860527, http://www.jsps.go.jp/ j-grantsinaid/) and by grants from the Foundation for Advancement of International Science (http://www.fais.or.jp/grant/fy25/01_file/2013_01members.pdf). The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: stakano@yamanashi.ac.jp Introduction Intraductal papillary mucinous neoplasm (IPMN) is a pancre- atic exocrine tumour characterised by cystic dilatation of the main and/or branch pancreatic ducts; these ducts are lined with a mucin-producing atypical epithelium that often proliferates in a papillary fashion [1–3]. IPMN is associated with a spectrum of diseases ranging from adenoma to invasive pancreatic ductal adenocarcinoma (PDAC). PDAC may be derived from IPMN or may concomitantly develop in other regions of a pancreas in which IPMN has developed. The two IPMN-related forms of PDAC are considered to be different disease entities due to their different proximities to the IPMN in the pancreas. However, the prognosis of these IPMN-related forms of PDAC is often better than that of ordinary PDAC if early diagnosis is made [4,5]. In contrast, the genetic characteristics of these two IPMN-related forms of PDAC and the reasons for their differing prognoses are not fully understood. PDAC arises as a result of the accumulation of genetic and epigenetic mutations that confer a selective advantage to cancer cells [6,7]. Mutations in KRAS, CDKN2A, TP53, and SMAD4 have been frequently reported in cases of PDAC using conventional methods such as direct sequencing [8]. Recently, whole-exome analysis using next-generation sequencing has also revealed high- frequency alterations in these few genes [6,7,9]. In contrast, somatic oncogenic mutations in the guanine nucleotide binding protein, alpha stimulating (GNAS)-encoding G PLOS ONE | www.plosone.org 1 June 2014 | Volume 9 | Issue 6 | e98718