Measuring KRAS Mutations in Circulating Tumor DNA by Droplet Digital PCR and Next-Generation Sequencing

Abstract

Measuring total cell-free DNA (cfDNA) or cancer-specific mutations herein has presented as new tools in aiding the treatment of cancer patients. Studies show that total cfDNA bears prognostic value in metastatic colorectal cancer (mCRC) and that measuring cancer-specific mutations could supplement biopsies. However, limited information is available on the performance of different methods. Blood samples from 28 patients with mCRC and known KRAS mutation status were included. cfDNA was extracted and quantified with droplet digital polymerase chain reaction (ddPCR) measuring Beta-2 Microglobulin. KRAS mutation detection was performed using ddPCR (Bio-Rad) and next-generation sequencing (NGS, Ion Torrent PGM). Comparing KRAS mutation status in plasma and tissue revealed concordance rates of 79% and 89% for NGS and ddPCR. Strong correlation between the methods was observed. Most KRAS mutations were also detectable in 10-fold diluted samples using the ddPCR. We find that for detection of KRAS mutations in ctDNA ddPCR was superior to NGS both in analysis success rate and concordance to tissue. We further present results indicating that lower amount of plasma may be used for detection of KRAS mutations in mCRC.