Methylation changes at the GNAS imprinted locus in pancreatic cystic neoplasms are important for the diagnosis of malignant cysts

BACKGROUND: Guanine nucleotide-binding protein, alpha stimulating (GNAS) mutations are characteristic of intraductal papillary mucinous neoplasms (IPMNs). Pancreatic ductal adenocarcinomas (PDACs) harboring GNAS mutations originate in IPMNs. GNAS is a complex imprinted locus that produces five transcripts regulated by differential methylated regions, NESP55, GNASAS, GNASXL, GNAS1A, and GNAS. AIM: To evaluate if methylation changes in the differential methylated regions of GNAS locus contributed to malignant progression of pancreatic cysts. METHODS: GNAS locus methylation was analyzed in archival pancreatic cyst fluid (PCF) obtained by endoscopic ultrasound with fine-needle aspiration by methylation specific–multiplex ligation dependent probe amplification. Results were normalized and analyzed using Coffalyser.Net software. RESULTS: Fifty-two PCF samples obtained by endoscopic ultrasound with fine-needle aspiration and previously characterized for KRAS and GNAS mutations were studied. The final diagnoses were surgical (11) and clinicopathological (41), including 30 benign cysts, 14 pre-malignant cyst, and eight malignant cysts. Methylation changes at NESP55, GNASAS, GNAS1A, and especially GNASXL were more frequent in malignant cysts, and NESP55 and GNASAS were useful for diagnosis. A combined variable defined as “GNAS locus methylation changes” was significantly associated with malignancy (6/8 malignant cysts and only 2/20 benign cysts) and improved classification. Hypermethylation in both maternally (NESP55) and paternally (GNASXL) derived promoters was found in 3/3 PDACs. CONCLUSION: This is the first study to identify methylation changes in the GNAS locus, improving the diagnosis of malignant pancreatic cysts and suggesting a role in progression to PDAC.