MEASURING KRAS MUTATIONS IN PANCREATIC CYST FLUID BY DROPLET DIGITAL PCR AND NEXT-GENERATION SEQUENCING

Aims:

DNA-based testing of pancreatic cyst fluid, obtained with endoscopic ultrasound-fine needle aspiration (EUS-FNA), may be useful in the evaluation of pancreatic cystic neoplasms (PCN). Mutations in KRAS/GNAS are highly specific for mucinous pancreatic cystic neoplasm and can be detected by next generation sequencing (NGS; multiple genes simultaneously) or by droplet digital PCR (ddPCR; one gene at a time, but presumed higher sensitivity). The aim of this pilot study was to evaluate and compare NGS and ddPCR for measuring KRAS mutations in cyst fluid.

Methods:

In this pilot study, pancreatic cyst fluid from patients who underwent EUS-FNA were included. KRAS mutation analysis was performed using ddPCR (Bio-Rad) and next-generation sequencing (Ion Torrent PGM). The detection of mutant alleles in cyst fluid by the different techniques was compared and correlated with histopathology.

Results:

Twenty-four EUS-FNA obtained cyst fluid specimens of patients who subsequently underwent surgery were included for analysis (2007 – 2014). Surgical pathology showed 13 IPMNs, 7 MCNs, 3 SCNs and 1 cNET. Among these cases, 14 specimens were satisfactory for molecular testing by NGS (6 IPMNs, 5 MCNs, 1 cNET, 2 SCNs), whereas 17 specimens by ddPCR (9 IPMNs, 6 MCNs, 1 cNET, 1 SCN). Mutations in KRAS were detected in 3/6 and 9/9 IPMNs by NGS and ddPCR, respectively. In addition, KRAS mutations were identified in 0/5 and 2/6 MCNs by NGS and ddPCR. One mutation was identified in cNET by ddPCR. No mutations were identified in SCNs.

Conclusions:

In this pilot study, ddPCR was superior to NGS for the detection of mutations in cyst fluid in concordance with surgical pathology. Although the sensitivity of both ddPCR and NGS assays is often limited by the amount of DNA that can be evaluated, less DNA is required for mutation analysis by ddPCR. The lower limits of detection of ddPCR should still be determined.