Open AccessOrthogonal Comparison of Four Plasma NGS Tests With Tumor Suggests Technical Factors are a Major Source of Assay Discordance
Author(s) -
Daniel Stetson,
Ambar Ahmed,
Xin Xu,
Barrett Nuttall,
Tristan Lubinski,
Justin Johnson,
J. Carl Barrett,
Brian Dougherty
Publication year - 2019
Publication title -
jco precision oncology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.405
H-Index - 22
ISSN - 2473-4284
DOI - 10.1200/po.18.00191
Subject(s) - replicate , biology , somatic cell , circulating tumor dna , predictive value , genetics , gene , computational biology , medicine , cancer , statistics , mathematics
PURPOSE Discordance between plasma and tumor variant calling has been attributed primarily to tumor heterogeneity, whereas technical variables remain largely unexplored.MATERIALS AND METHODS To 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.RESULTS Our 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.CONCLUSION These findings suggest that most NGS assay discordance is a result of technical variations and, to a lesser extent, biologic factors such as clonal hematopoiesis of indeterminate potential and tumor heterogeneity.