Frequent GNAS mutations in low-grade appendiceal mucinous neoplasms.
ABSTRACT: The molecular basis for the development of appendiceal mucinous tumours, which can be a cause of pseudomyxoma peritonei, remains largely unknown.Thirty-five appendiceal mucinous neoplasms were analysed for GNAS and KRAS mutations. A functional analysis of mutant GNAS was performed using a colorectal cancer cell line.A mutational analysis identified activating GNAS mutations in 16 of 32 low-grade appendiceal mucinous neoplasms (LAMNs) but in none of three mucinous adenocarcinomas (MACs). KRAS mutations were found in 30 LAMNs and in all MACs. We additionally analysed a total of 186 extra-appendiceal mucinous tumours and found that GNAS mutations were highly prevalent in intraductal papillary mucinous tumours of the pancreas (88%) but were rare or absent in mucinous tumours of the colorectum, ovary, lung and breast (0-9%). The prevalence of KRAS mutations was quite variable among the tumours. The introduction of the mutant GNAS into a colorectal cancer cell line markedly induced MUC2 and MUC5AC expression, but did not promote cell growth either in vitro or in vivo.Activating GNAS mutations are a frequent and characteristic genetic abnormality of LAMN. Mutant GNAS might play a direct role in the prominent mucin production that is a hallmark of LAMN.
Project description:Mucinous neoplasms of the appendix (MNA) are rare tumors which may progress from benign to malignant disease with an aggressive biological behavior. MNA is often diagnosed after metastasis to the peritoneal surfaces resulting in mucinous carcinomatosis peritonei (MCP). Genetic alterations in MNA are poorly characterized due to its low incidence, the hypo-cellularity of MCPs, and a lack of relevant pre-clinical models. As such, application of targeted therapies to this disease is limited to those developed for colorectal cancer and not based on molecular rationale.We sequenced the whole exomes of 10 MCPs of appendiceal origin to identify genome-wide somatic mutations and copy number aberrations and validated significant findings in 19 additional cases.Our study demonstrates that MNA has a different molecular makeup than colorectal cancer. Most tumors have co-existing oncogenic mutations in KRAS (26/29) and GNAS (20/29) and are characterized by downstream PKA activation. High-grade tumors are GNAS wild-type (5/6), suggesting they do not progress from low-grade tumors. MNAs do share some genetic alterations with colorectal cancer including gain of 1q (5/10), Wnt, and TGF? pathway alterations. In contrast, mutations in TP53 (1/10) and APC (0/10), common in colorectal cancer, are rare in MNA. Concurrent activation of the KRAS and GNAS mediated signaling pathways appears to be shared with pancreatic intraductal papillary mucinous neoplasm.MNA genome-wide mutational analysis reveals genetic alterations distinct from colorectal cancer, in support of its unique pathophysiology and suggests new targeted therapeutic opportunities.
Project description:In 2010, the World Health Organization reclassified the entity originally described as intraductal oncocytic papillary neoplasm as the 'oncocytic subtype' of intraductal papillary mucinous neoplasm. Although several key molecular alterations of other intraductal papillary mucinous neoplasm subtypes have been discovered, including common mutations in KRAS, GNAS, and RNF3, those of oncocytic subtype have not been well characterized. We analyzed 11 pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms. Nine pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms uniformly exhibited typical entity-defining morphology of arborizing papillae lined by layers of cells with oncocytic cytoplasm, prominent, nucleoli, and intraepithelial lumina. The remaining two were atypical. One lacked the arborizing papilla and had flat oncocytic epithelium only; the other one had focal oncocytic epithelium in a background of predominantly intestinal subtype intraductal papillary mucinous neoplasm. Different components of this case were analyzed separately. Formalin-fixed, paraffin-embedded specimens of all cases were microdissected and subjected to high-depth-targeted next-generation sequencing for a panel of 300 key cancer-associated genes in a platform that enabled the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Fresh frozen specimens of two cases were also subjected to whole-genome sequencing. For the nine typical pancreatic 'oncocytic subtype' of intraductal papillary mucinous neoplasms, the number of mutations per case, identified by next-generation sequencing, ranged from 1 to 10 (median=4). None of these cases had KRAS or GNAS mutations and only one had both RNF43 and PIK3R1 mutations. ARHGAP26, ASXL1, EPHA8, and ERBB4 genes were somatically altered in more than one of these typical 'oncocytic subtype' of intraductal papillary mucinous neoplasms but not in the other two atypical ones. In the neoplasm with flat oncocytic epithelium, the only mutated gene was KRAS. All components of the intestinal subtype intraductal papillary mucinous neoplasms with focal oncocytic epithelium manifested TP53, GNAS, and RNF43 mutations. In conclusion, this study elucidates that 'oncocytic subtype' of intraductal papillary mucinous neoplasm is not only morphologically distinct but also genetically distinct from other intraductal papillary mucinous neoplasm subtypes. Considering that now its biologic behavior is also being found to be different than other intraductal papillary mucinous neoplasm subtypes, 'oncocytic subtype' of intraductal papillary mucinous neoplasm warrants being recognized separately.
Project description:G?protein ?s (GNAS) mediates receptor-stimulated cAMP signalling, which integrates diverse environmental cues with intracellular responses. GNAS is mutationally activated in multiple tumour types, although its oncogenic mechanisms remain elusive. We explored this question in pancreatic tumourigenesis where concurrent GNAS and KRAS mutations characterize pancreatic ductal adenocarcinomas (PDAs) arising from intraductal papillary mucinous neoplasms (IPMNs). By developing genetically engineered mouse models, we show that GnasR201C cooperates with KrasG12D to promote initiation of IPMN, which progress to invasive PDA following Tp53 loss. Mutant Gnas remains critical for tumour maintenance in vivo. This is driven by protein-kinase-A-mediated suppression of salt-inducible kinases (Sik1-3), associated with induction of lipid remodelling and fatty acid oxidation. Comparison of Kras-mutant pancreatic cancer cells with and without Gnas mutations reveals striking differences in the functions of this network. Thus, we uncover Gnas-driven oncogenic mechanisms, identify Siks as potent tumour suppressors, and demonstrate unanticipated metabolic heterogeneity among Kras-mutant pancreatic neoplasms.
Project description:PURPOSE:The natural history and prognosis of appendiceal adenocarcinomas differ from those of adenocarcinomas arising in other large bowel sites. We aimed to compare the molecular profiles exhibited by appendiceal adenocarcinomas and colorectal cancers, or between the histopathologic subtypes of appendiceal adenocarcinoma. EXPERIMENTAL DESIGN:A total of 183 samples from appendiceal adenocarcinoma [46 adenocarcinoma, not otherwise specified (NOS), 66 pseudomyxoma peritonei (PMP), 44 mucinous adenocarcinoma (MU), and 27 signet ring cell carcinoma (SR)], 994 from right-sided colorectal cancer (R-CRC), and 1,080 from left-sided CRC (L-CRC) were analyzed by next-generation sequencing (NGS) and IHC markers. Microsatellite instability (MSI) and tumor mutational burden (TMB) were tested by NGS, and programmed death ligand 1 (PD-L1) by IHC. RESULTS:We observed high mutation rates in appendiceal adenocarcinoma samples for KRAS (55%), TP53 (40%), GNAS (31%), SMAD4 (16%), and APC (10%). Appendiceal adenocarcinoma exhibited higher mutation rates in KRAS and GNAS, and lower mutation rates in TP53, APC, and PIK3CA (6%) than colorectal cancers. PMP exhibited much higher mutation rates in KRAS (74%) and GNAS (63%), and much lower mutation rates in TP53 (23%), APC (2%), and PIK3CA (2%) than NOS. Alterations associated with immune checkpoint inhibitor response (MSI-high, TMB-high, PD-L1 expression) showed similar frequency in appendiceal adenocarcinoma compared with L-CRC, but not R-CRC, and those of NOS were higher than other subtypes of appendiceal adenocarcinoma and L-CRC. CONCLUSIONS:Molecular profiling of appendiceal adenocarcinoma revealed different molecular characteristics than noted in R-CRC and L-CRC, and molecular heterogeneity among the histopathologic subtypes of appendiceal adenocarcinoma. Our findings may be critical to developing an individualized approach to appendiceal adenocarcinoma treatment.
Project description:More than 2% of the adult U.S. population harbors a pancreatic cyst. These often pose a difficult management problem because conventional criteria cannot always distinguish cysts with malignant potential from those that are innocuous. One of the most common cystic neoplasms of the pancreas, and a bona fide precursor to invasive adenocarcinoma, is called intraductal papillary mucinous neoplasm (IPMN). To help reveal the pathogenesis of these lesions, we purified the DNA from IPMN cyst fluids from 19 patients and searched for mutations in 169 genes commonly altered in human cancers. In addition to the expected KRAS mutations, we identified recurrent mutations at codon 201 of GNAS. A larger number (113) of additional IPMNs were then analyzed to determine the prevalence of KRAS and GNAS mutations. In total, we found that GNAS mutations were present in 66% of IPMNs and that either KRAS or GNAS mutations could be identified in 96%. In eight cases, we could investigate invasive adenocarcinomas that developed in association with IPMNs containing GNAS mutations. In seven of these eight cases, the GNAS mutations present in the IPMNs were also found in the invasive lesion. GNAS mutations were not found in other types of cystic neoplasms of the pancreas or in invasive adenocarcinomas not associated with IPMNs. In addition to defining a new pathway for pancreatic neoplasia, these data suggest that GNAS mutations can inform the diagnosis and management of patients with cystic pancreatic lesions.
Project description:Activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Intraductal papillary neoplasms of the bile ducts (IPNBs) morphologically resemble pancreatic IPMNs. This study sought to assess the mutational status of GNAS at codon 201 in IPNBs.Thirty-four patients were included. DNA from microdissected IPNBs was subjected to a polymerase chain reaction and ligation method for the detection of GNAS mutations at codon 201 and of KRAS mutations at codon 12. Mutational status was compared with clinical and pathologic data.The IPNBs had a median diameter of 3.5 cm and were located intrahepatically (n= 6), extrahepatically (n= 13), both intra- and extrahepatically (n= 4) or in the gallbladder (intracystic papillary neoplasms, n= 11). Most exhibited pancreatobiliary differentiation (n= 20), high-grade dysplasia (n= 26) and an associated adenocarcinoma (n= 20). Analysis of GNAS codon 201 identified only one mutant sample in a multifocal intestinal subtype intrahepatic IPNB with high-grade dysplasia. Six lesions harboured a KRAS codon 12 mutation.GNAS codon 201 mutations are uncommon in IPNBs, by contrast with pancreatic IPMNs. More comprehensive molecular profiling is needed to uncover the pathways involved in IPNB development.
Project description: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.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.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.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.
Project description:Pancreatic cystic neoplasms (PCNs) are a heterogeneous group with varying risks of malignancy. To explore the clinical utility of liquid biopsy in cyst type classification, we analyzed the GNAS/KRAS mutations in circulating cell-free DNA (cfDNA) obtained from 57 patients with histologically diagnosed PCNs, including 34 with intraductal papillary mucinous neoplasms (IPMNs) and compared the mutant allele prevalence and variant patterns with the paired resected specimens using next-generation sequencing. The positive prevalence of GNAS mutations in cfDNA of patients with IPMN (n?=?11, 32%) was significantly higher than that in those with other PCNs (0%, P?=?0.002). Conversely, KRAS mutations were detected in cfDNA of only 2 (6%) IPMN patients. The paired-sample comparison revealed highly concordance between the GNAS mutation status of cfDNA and resected IPMN specimens. Similar distributions of GNAS mutation positivity in cfDNA were observed across the different histological grades, whereas IPMNs with intestinal subtype showed a significantly higher prevalence of GNAS mutations than other subtypes (P?=?0.030). GNAS mutation positivity in cfDNA was significantly associated with the acellular mucin pool of histological findings in primary IPMN lesions (P?=?0.017). Detection of GNAS mutation in cfDNA can serve as a novel biomarker for cyst type classification and differentiation of intestinal subtype IPMN from the other PCNs.
Project description:Intraductal neoplasms are important precursors to invasive pancreatic cancer and provide an opportunity to detect and treat pancreatic neoplasia before an invasive carcinoma develops. The diagnostic evaluation of these lesions is challenging, as diagnostic imaging and cytological sampling do not provide accurate information on lesion classification, the grade of dysplasia or the presence of invasion. Moreover, the molecular driver gene mutations of these precursor lesions have yet to be fully characterized. Fifty-two intraductal papillary neoplasms, including 48 intraductal papillary mucinous neoplasms (IPMNs) and four intraductal tubulopapillary neoplasms (ITPNs), were subjected to the mutation assessment in 51 cancer-associated genes, using ion torrent semiconductor-based next-generation sequencing. P16 and Smad4 immunohistochemistry was performed on 34 IPMNs and 17 IPMN-associated carcinomas. At least one somatic mutation was observed in 46/48 (96%) IPMNs; 29 (60%) had multiple gene alterations. GNAS and/or KRAS mutations were found in 44/48 (92%) of IPMNs. GNAS was mutated in 38/48 (79%) IPMNs, KRAS in 24/48 (50%) and these mutations coexisted in 18/48 (37.5%) of IPMNs. RNF43 was the third most commonly mutated gene and was always associated with GNAS and/or KRAS mutations, as were virtually all the low-frequency mutations found in other genes. Mutations in TP53 and BRAF genes (10% and 6%) were only observed in high-grade IPMNs. P16 was lost in 7/34 IPMNs and 9/17 IPMN-associated carcinomas; Smad4 was lost in 1/34 IPMNs and 5/17 IPMN-associated carcinomas. In contrast to IPMNs, only one of four ITPNs had detectable driver gene (GNAS and NRAS) mutations. Deep sequencing DNA from seven cyst fluid aspirates identified 10 of the 13 mutations detected in their associated IPMN. Using next-generation sequencing to detect cyst fluid mutations has the potential to improve the diagnostic and prognostic stratification of pancreatic cystic neoplasms.
Project description:BACKGROUND & AIMS:Mutations at hotspots in GNAS, which encodes stimulatory G-protein, ? subunits, are detected in approximately 60% of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. We generated mice with KRAS-induced IPMNs that also express a constitutively active form of GNAS in pancreas and studied tumor development. METHODS:We generated p48-Cre; LSL-KrasG12D; Rosa26R-LSL-rtTA-TetO-GnasR201C mice (Kras;Gnas mice); pancreatic tissues of these mice express activated KRAS and also express a mutant form of GNAS (GNASR201C) upon doxycycline administration. Mice that were not given doxycycline were used as controls, and survival times were compared by Kaplan-Meier analysis. Pancreata were collected at different time points after doxycycline administration and analyzed by histology. Pancreatic ductal adenocarcinomas (PDACs) were isolated from mice and used to generate cell lines, which were analyzed by reverse transcription polymerase chain reaction, immunoblotting, immunohistochemistry, and colony formation and invasion assays. Full-length and mutant forms of yes-associated protein (YAP) were expressed in PDAC cells. IPMN specimens were obtained from 13 patients with IPMN undergoing surgery and analyzed by immunohistochemistry. RESULTS:All Kras;Gnas mice developed pancreatic cystic lesions that resemble human IPMNs; the grade of epithelial dysplasia increased with time. None of the control mice developed cystic lesions. Approximately one third of Kras;Gnas mice developed PDACs at a median of 30 weeks after doxycycline administration, whereas 33% of control mice developed PDACs. Expression of GNASR201C did not accelerate the development of PDACs compared with control mice. However, the neoplasms observed in Kras;Gnas mice were more differentiated, and expressed more genes associated with ductal phenotypes, than in control mice. PDACs isolated from Kras;Gnas mice had activation of the Hippo pathway; in cells from these tumors, phosphorylated YAP1 was sequestered in the cytoplasm, and this was also observed in human IPMNs with GNAS mutations. Sequestration of YAP1 was not observed in PDAC cells from control mice. CONCLUSIONS:In mice that express activated KRAS in the pancreas, we found expression of GNASR201C to cause development of more differentiated tumors, with gene expression pattern associated with the ductal phenotype. Expression of mutant GNAS caused phosphorylated YAP1 to be sequestered in the cytoplasm, altering tumor progression.