Orthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay Discordance

PURPOSEDiscordance between plasma and tumor variant calling has been attributed primarily to tumor heterogeneity, whereas technical variables remain largely unexplored.MATERIALS AND METHODSTo measure these variables, we tested four next-generation sequencing (NGS) gene panel assays for mutations in circulating tumor DNA (ctDNA) using replicate sets of 24 plasma samples and compared the results with matched tumor-normal tissue pairs.RESULTSOur orthogonal approach identified false-negative (FN) and false-positive (FP) variants with high confidence and revealed substantial variability among the ctDNA assays, with a range of sensitivity (38% to 89%) and positive predictive value (36% to 80%). Most discordance in our cross-vendor study was observed below 1% variant allele frequency. FP variants displayed mutational biases and tended to be novel variants not found in somatic databases. Of the 56 unique variants called by all four ctDNA assays, 41 (68%) resulted from technical discordance.CONCLUSIONThese finding...