Endoscopic Ultrasound (EUS) Guided Pancreatic Fluid Aspiration for DNA Analysis of KRAS and GNAS Mutations for the Evaluation of Pancreatic Cystic Neoplasia

Abstract

Purpose: Discriminating benign from premalignant cysts of the pancreas is limited by the performance of cyst fluid aspirate and cytology. The presence of KRAS mutations in cyst fluid has been reported to increase the yield of cyst fluid aspirate for the presence of neoplastic cysts. Recently, mutations in GNAS have been found to have an oncogenic role in intraductal papillary mucinous neoplasms (IPMN) through promotion of adenyl cyclase activity and cAMP levels. Our aim was to evaluate whether analysis of cyst fluid DNA obtained by EUS for GNAS mutations can enhance the preoperative diagnosis of neoplastic pancreatic cysts. Methods: Endoscopic ultrasound-guided pancreatic cyst aspirates were collected during a 24-month period and investigated for cytology, carcinoembryonic antigen (CEA) level, and molecular analysis. KRAS and GNAS point mutations were detected by dideoxy chain termination sequencing (Applied Biosystems) of extracted DNA (qiagen) from cyst fluid. The cyst fluid analysis was compared to the final surgical pathology or cytology. Results: We identified 23 patients (11 male, mean age = 68.2 ± 13.8 [SD] years) with pancreatic cysts who underwent EUS-guided cyst aspiration. The mean cyst diameter was 2.3 cm (range 1-8 cm). Cysts were located in the head (n=10), body (n=8), and tail (n=5) of the pancreas. The cohort consisted of four serous cystadenomas (SCA), three mucinous cyst neoplasms (MCN), one pseudocyst, five solid-cystic adenocarcinoma (PDA), two solid-cystic endocrine neoplasms, one lymphoepithelial cyst, and seven IPMNs. The mean cyst fluid CEA levels in IPMNs was 3,895 ng/mL (range 22-24,747); 3/7 patients with IPMN had CEA levels <192 ng/mL. Mutations in KRAS were present in 5/7 (71%) IPMNs; LOH was demonstrated in three of these. Three of seven (43%) IPMNs carried a mutation in GNAS, all of which harbored degrees of dysplasia. The mutations in GNAS occurred at codon 201. All IPMNs carried a mutation in at least one of the two oncogenes. No mutations were identified in the SCA, in contrast to the IPMNs (p<0.01). GNAS mutations were not identified in any of the MCNs or PDA (p<0.005 when compared to IPMNs), although KRAS mutations were found in 1/3 (33%) of MCNs and 3/5 (60%) of PDAs. Cyst fluid CEA >192 ng/mL had a sensitivity of 57% to predict the presence of an IPMN. The sensitivity of KRAS mutation in cyst fluid was 71% for the diagnosis of IPMN, but with the addition of GNAS mutations, all IPMNs were identified (sensitivity = 100%, p<0.05). Conclusion: The presence of GNAS in combination with KRAS in pancreatic cystic fluid improves the sensitivity for diagnosis of IPMN when compared to CEA or KRAS alone. This demonstrates the feasibility of GNAS analysis from pancreatic cyst fluid obtained by EUS guided aspiration.