Validation of CRC and NSCLC somatic mutations detected by NGS using ddPCR

Abstract

Next Generation Sequencing (NGS) has become powerful tool in molecular oncology. It allows multiparallel targeted sequencing that enables comprehensive assessment of tumor heterogeneity. Detection of mutations in colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) defines patients diagnosis, therapy and prognosis. Multiple genes, their somatic mutations to be precise, carry different degrees of importance for any of these stages. Ion AmpliSeq™ Colon and Lung Cancer Research Panel v2, which was used in this study, allows detection of hotspot mutations on 22 genes in a single reaction. Droplet digital PCR (ddPCR) has a unique advantage in low frequency mutation detection and it has been used as a validation method for mutations that were detected with NGS. It has high sensitivity and enables accurate detection of mutant allele in a background of abundant wild type alleles. For this study 35 samples of CRC and NSCLC were sequenced and same samples were analysed on ddPCR for KRAS, NRAS, EGFR and BRAF genes. All processed samples were successfully sequenced and had average base coverage >500X. NGS sequencing proved itself to be cost effective, has shorter turnaround time and is highly sensitive. Out of 35 samples, 25 had genetic alterations, while 10 samples are reported as wild type but were still tested on ddPCR as controls. In three samples low frequency somatic mutations were detected by NGS and verified using ddPCR, which leads us to conclusion that ddPCR is a good tool for verification of somatic mutations in CRC and NSCLC.

Droplet Digital PCR as a Molecular Tool for the Detection of the EGFR T790M Mutation in NSCLC Patients with the EGFR Activating Mutations

“…In 2019, Salihefendić et al [ 5 ] studied whether ddPCR technology could be used as a confirmatory method for the detection and quantification of somatic mutations, previously detected by NGS. A total of 35 samples from CRC and NSCLC patients were analyzed, and the results showed that there were no statistically significant differences between the results obtained by NGS and ddPCR methods.…”

“…A total of 35 samples from CRC and NSCLC patients were analyzed, and the results showed that there were no statistically significant differences between the results obtained by NGS and ddPCR methods. Therefore, high sensitivity and resolution of ddPCR, make it an adequate method for validating low-frequency somatic mutations [ 5 ] .…”

“…TKIs target the EGFR receptor in NSCLC patients and thus harbor activating EGFR mutations [ 3 , 4 ]. The presence of mutations in NSCLC is crucial for a patient’s diagnosis, therapy assessment, and prognosis [ 5 ] .…”

“…Droplet digital PCR has several unique advantages, especially regarding rare mutation detection and precise DNA quantification [ 5 ] . Droplet digital PCR utilizes water-oil emulsion technology to partition each sample into 20 000 nanoliter-sized droplets, where target and background DNA are distributed randomly into the droplets.…”