Validation of a next-generation sequencing assay for the detection of EGFR mutations in cell-free circulating tumor DNA

Exp Mol Pathol. 2021 Sep 21:104685. doi: 10.1016/j.yexmp.2021.104685. Online ahead of print.ABSTRACTDetection of EGFR mutations from circulating tumor DNA (ctDNA) from blood plasma represents a gentle, non-invasive alternative to rebiopsy and can therefore be used for therapy monitoring of non-small-cell lung cancer (NSCLC) patients. The aim of this project was to investigate whether the Reveal ctDNA™ 28 NGS assay (ArcherDX, Boulder, CO), has a comparable sensitivity and specificity to droplet digital PCR (ddPCR, gold-standard) and is therefore suitable for therapy monitoring of progressing lung cancer patients. First, we validated the NGS assay with commercially available reference material (SeraCare, Massachusetts, US). Using an input of 22 ng reference material, a sensitivity of 96% and a specificity of 100% could be achieved for variant allele frequencies (VAF) of 0.5%. For variants at a VAF of 0.1% the sensitivity was substantial reduced. Next, 28 plasma samples from 16 patients were analyzed and results were compared to existing ddPCR data. This comparative analysis of patient samples revealed a concordance of 91% between NGS and ddPCR. These results confirm that the Reveal ctDNA™ 28 NGS assay can be used for therapy monitoring of patients under TKI therapy. However, due to the slightly superior sensitivity of ddPCR, a combination of NGS (with broad coverage of a large number of genomic loci) and ddPCR (with targeted highly sensitive detection of specific mutations)...
Source: Experimental and Molecular Pathology - Category: Pathology Authors: Source Type: research