Detection of KRAS mutations in colon adenocarcinoma by droplet digital PCR.

Abstract

e15080 Background: The screening of mutations in the KRAS gene is a traditional marker of the effectiveness of targeted therapy for colorectal cancer. Mutations in the 12 codon of the second exon of the KRAS gene are present in 40% of colorectal tumors, and monitoring of the mutational status together with the level of mutant DNA is of particular clinical interest. However, the sensitivity level of the traditionally used real-time polymerase chain reaction (RT-PCR) method is insufficient in some cases. Recently, Droplet Digital PCR (DD-PCR) has been considered as an alternative method, which is more sensitive. The purpose of this study was to compare the methods of detection of somatic mutations in the second exon of the KRAS gene using DD-PCR versus RT-PCR. Methods: This study included 134 patients with colon adenocarcinoma. The presence or absence of activating mutations in the second exon of the KRAS gene in samples of FFPE blocks was detected by RT-PCR using the “Real-time-PCR-KRAS-7M reagent kit” (Biolink, Russia) and Digital Droplet PCR using the “KRAS Screening Multiplex kit” (Bio-Rad, USA). Results: For all 134 samples, selected for the study, the WT status of the KRAS gene was identified by the RT-PCR method. According to the results of DD-PCR for 131 samples (96.2%), a positive amplification response of mutant DNA with more than 200 events was obtained. The number of amplicons varied from 312 to 117917 per sample, the median was 2940 copies. According to recent trials, a clinically significant level of mutational events must exceed 5% of the total number of amplicons in a sample. In our study, 12.9% of cases (17 patients) met this criterion. Conclusions: The DD-PCR method demonstrated a much higher analytical sensitivity for the detection of SNP mutations in comparison with RT-PCR that may be of critical importance in the therapeutic decision.