Preoperative next-generation sequencing of pancreatic cyst fluid is highly accurate in cyst classification and detection of advanced neoplasia

Abstract

Objective: DNA-based testing of pancreatic cyst fluid (PCF) is a useful adjunct to the evaluation of pancreatic cysts (PCs). Mutations in KRAS/GNAS are highly specific for intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs), while TP53/PIK3CA/PTEN alterations are associated with advanced neoplasia. A prospective study was performed to evaluate preoperative PCF DNA testing.

Design: Over 43-months, 626 PCF specimens from 595 patients were obtained by endoscopic ultrasound (EUS)-fine needle aspiration and assessed by targeted next-generation sequencing (NGS). Molecular results were correlated with EUS findings, ancillary studies and follow-up. A separate cohort of 159 PCF specimens was also evaluated for KRAS/GNAS mutations by Sanger sequencing.

Results: KRAS/GNAS mutations were identified in 308 (49%) PCs, while alterations in TP53/PIK3CA/PTEN were present in 35 (6%) cases. Based on 102 (17%) patients with surgical follow-up, KRAS/GNAS mutations were detected in 56 (100%) IPMNs and 3 (30%) MCNs, and associated with 89% sensitivity and 100% specificity for a mucinous PC. In comparison, KRAS/GNAS mutations by Sanger sequencing had a 65% sensitivity and 100% specificity. By NGS, the combination of KRAS/GNAS mutations and alterations in TP53/PIK3CA/PTEN had an 89% sensitivity and 100% specificity for advanced neoplasia. Ductal dilatation, a mural nodule and malignant cytopathology had lower sensitivities (42%, 32% and 32%, respectively) and specificities (74%, 94% and 98%, respectively).

Conclusions: In contrast to Sanger sequencing, preoperative NGS of PCF for KRAS/GNAS mutations is highly sensitive for IPMNs and specific for mucinous PCs. In addition, the combination of TP53/PIK3CA/PTEN alterations is a useful preoperative marker for advanced neoplasia.

Keywords: pancreatic cancer; pancreatic epidemiology; pancreatic pathology; pancreato-biliary disorders.

Figure 1

Study cohort. In total, 642 patients with 648 PCs underwent an EUS-FNA and PCF) molecular testing. Forty-seven PCF specimens from 47 patients were unsatisfactory for molecular testing; however, the remaining 601 PCF specimens from 595 patients were satisfactory. Repeat EUS-FNA and molecular testing was performed for 25 PCs from 25 (of 595) patients. (*) Of note, 1 of 47 PCs with a VHL alteration also harboured a TP53 mutation. EUS-FNA, endoscopic ultrasound–fine needle aspiration; PC, pancreatic cyst; PCF, pancreatic cyst fluid.

Figure 2

DNA-based molecular testing of a pancreatic head cyst. An incidental 3 cm pancreatic head cyst with no associated main duct dilatation or mural nodule by both (A) CT (red arrow) and (B) endoscopic ultrasound. Fine-needle aspiration and subsequent (C) cytopathology showed atypical cells with no definitive mucin. However, DNA analysis identified mutations in GNAS and PIK3CA with MAFs of 51% and 50%, respectively. Follow-up surgical resection revealed an (D) invasive moderately differentiated adenocarcinoma (black arrows) arising in an intraductal papillary mucinous neoplasm. MAFs, mutant allele frequencies.