Analysis of KRAS Mutation Subtype in Tissue DNA and Cell-Free DNA Using Droplet Digital PCR and the Function of Cell-Free DNA as a Recurrence Predictive Marker in Pancreatic Cancer

Abstract

KRAS mutation is a major regulator in the tumor progression of pancreatic cancer. Here, we compared the frequency and mutation burden of KRAS mutation subtypes with paired tumor tissue and blood in patients and examined their clinical significance. DNA from tumor tissues and cell-free DNA (cfDNA) from preoperative blood were obtained from 70 patients with pancreatic cancer. Subtypes and mutation burdens of KRAS G12D and G12V mutations were evaluated using droplet digital PCR. Comparing the presence of mutations in tissue, accumulative and simultaneous mutations of G12D or G12V were identified of 67 (95.7%), and 48 patients (68.6%). Conversely, in blood, they were only identified in 18 (25.7%) and four (5.7%) patients; respectively. Next, comparing the mutation burden in tissue, the mutation burden varied from less than 0.1 to more than five, whereas that of cfDNA in blood was mostly between one and five, as cases with a mutation burden lower than 0.1 and higher than five were rare. Finally, the presence of the G12V mutation alone in cfDNA and the combination of the G12V mutation with elevated CA 19-9 levels were associated with poor recurrence-free survival. These fundamental data on the KRAS mutation subtypes and their clinical significance could support their potential as predictive markers for postoperative recurrence.