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Jul 20, 2011

Investigators Find that GNAS Mutations Distinguish IPMNs from Other Invasive and Benign Pancreatic Cysts

  • Investigators claim mutations in a gene known as GNAS can help distinguish between nonmalignant pancreatic cysts and intraductal papillary mucinous neoplasm (IPMN) cysts, which are a known precursor to invasive adenocarcinoma. The team analyzed 169 genes in fluid from IPMN cysts taken from 19 patients. They found that GNAS mutations were present in 66% of IPMNS, while either KRAS or GNAS mutations were present in 96% of samples.

    The researchers, from Johns Hopkins Kimmel Cancer Center, Johns Hopkins Medical Institutions, and Memorial Sloan-Kettering Cancer Center, also evaluated a number of invasive adenocarcinomas that developed as a result of IPMNs harboring GNAS mutations. In 7 of 8 cases tested, the GNAS mutations present in the IPMNs were also found in the invasive lesion. In contrast, GNAS mutations weren’t found in other types of cystic neoplasms of the pancreas or in invasive adenocarcinomas not associated with IPMNs.

    Jian Wu, M.D., Bert Vogelstein, M.D., and colleagues, report their findings in Science Translational Medicine in a paper titled “Recurrent GNAS Mutations Define an Unexpected Pathway for Pancreatic Cyst Development.”

    To try and identify genes that may be mutated specifically in IPMNs, the researchers used massively parallel sequencing to sequence 169 oncogenes and tumor suppressor genes in cyst fluids from 19 IPMNs, each taken from a different patient. In addition, they sequenced the same genes in normal pancreatic, splenic, or intestinal tissues of the same patients to determine which of the alterations identified was the result of somatic mutation.

    The results found only two genes that were mutated in more than one IPMN. KRAS was mutated in 14 of the 19 IPMNs, and the oncogene GNAS was mutated in six IPMNs. The mutations in GNAS all occurred at codon 201 and resulted in either an R201H or an R201C substitution. All 12 KRAS mutations identified were at codon 12.

    The authors note that GNAS mutations have previously been found in pituitary and other uncommon tumor types, but only rarely been identified in common epithelial tumors and, to date, not in pancreatic cysts or in PDAs. In contrast, KRAS mutations at codon 12 have previously been identified in most PDAs as well as in 40% to 60% of IPMNs.

    The team then set out to determine the frequency of three KRAS codon 12 mutations and two GNAS codon 201 mutations in a larger set of IPMNs that included cyst fluid samples from an independent cohort of 65 patients plus the 19 patients whose fluids had been studied by massively parallel sequencing.

    A PCR/ligation method that had the capacity to detect one mutant template molecule among 200 normal (wild-type) templates was used for these analyses. GNAS and KRAS mutations were found in 61% and 82% of the IPMN fluids, respectively.

    Similarly, analysis of macro- and microdissected frozen or paraffin-embedded cyst walls from an independent collection of 48 surgically resected IPMNs identified a high prevalence of GNAS (75%) and KRAS (79%) mutations. “In aggregate, 66% of 132 IPMNs harbored a GNAS mutation, 81% harbored a KRAS mutation, slightly more than half (51%) harbored both GNAS and KRAS mutations, whereas at least one of the two genes was mutated in 96.2%,” the researchers write. “Given background mutation rates in tumors or normal tissues, the probability that either GNAS or KRAS mutations occurred by chance alone was less than 10−30.”

    There was no significant correlation found between the prevalence of KRAS or GNAS mutations and age, sex or smoking history, size or location of cyst, or whether the IPMN was low grade or high grade.

    Interestingly, the authors report, while GNAS and KRAS mutations were present in each of the three major histologic types of IPMNs (intestinal, pancreatobiliary, and gastric), GNAS mutations were most prevalent in the intestinal subtype, whereas KRAS mutations were most frequently found in the pancreatobiliary subtypte, and had the lowest frequency in the intestinal subtype.

    Importantly, no GNAS or KRAS mutations were found in benign serous cystadenomas (SCAs) that had been surgically removed from 44 different patients. And while KRAS mutations were found in 33% of mucinous cystic neoplasms (MCNs), a type of cyst that can progress to invasive carcinoma but less often than IPMNs, none of the MCN cysts harbored GNAS mutations. Alterations in the GNAS gene were also not found in five examples of an uncommon type of cyst, called intraductal oncocytic papillary neoplasm (IOPN), with characteristic oncocytic features.

    A previous whole-exome sequencing study suggested that GNAS mutations also aren’t present in typical PDAs that occurred in the absence of an associated IPM. To extend these finding further Dr. Vogelstein’s team looked for the two GNAS mutations in an additional 95 surgically resected PDAs without evidence of IPMNs. These results confirmed that PDAs developing in the absence of IPMNs don’t carry GNAS mutations.

    Conversely, seven of eight samples from invasive pancreatic adenocarcinomas that developed in association with IPMNs did harbor GNAS mutations. The KRAS mutational status of the PDA was also consistent with that of the associated IPMN in the same seven cases.

    The overall findings suggest that GNAS mutations are specific to IPMN cysts, which means that if a cyst carries a GNAS mutation, it indicates the lesion is potentially neoplastic. “There is no doubt that GNAS mutations play a driving role in this IPMN-specific pathway,” the authors conclude.

    “The mutations are remarkably common and they occur at a single codon (201), mutations of which are known to endow cells with extremely high adenyl cyclase activity and adenosine 3′,5′-monophosphate (cAMP) levels.”

    Apart from providing new clues about the mechanisms involved in IPMN development, the data have obvious practical applications, in terms of distinguishing IPMNs from SCAs and MCNs, the researchers note. “Astute clinicians and pathologists have long suspected that adenocarcinomas of the pancreas arising in IPMNs are a different disease from those arising locally distant or in the absence of an IPMN. We here provide evidence in support of this hypothesis and identify a key molecular component that underlies this difference.”


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