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

Abstract

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.

From Somatic Variants Toward Precision Oncology: An Investigation of Reporting Practice for Next-Generation Sequencing-Based Circulating Tumor DNA Analysis

“…These findings highlight a potential source of errors for these laboratory tests, raising concerns of whether analytical validation procedures are as rigorous as claimed . Similar results were also found in several recently published orthogonal comparison studies and the recent European pilot schemes, which also underscores the importance of analytically validated assays with sensitivity below 1% VAF . Thus, in the future, stricter experiments for the confirmation of performance parameters (especially LOD) need to be further developed in all laboratories by using materials with natural ctDNA characteristics and precise VAF rather than clinical samples or plasmids .…”

Completing the Translation

“…Unfortunately, this embarrassment of diagnostic riches in care and treatment of patients with cancer is not without consequences, encumbered by both technical challenges and administrative or reporting difficulties. Multiple sources have previously focused on technical comparisons among ctDNA technologies and laboratories, with ample evidence that significant differences in results exist between labs that question the validity and actionability of a result .…”

UltraPrep is a scalable, cost-effective, bead-based method for purifying cell free DNA

“…Moreover, there is an acute need for "truth samples", comprised of bona fide cfDNA, that can be used for proficiency testing. The current paradigm of comparing cancer patient cfDNA with matched DNA extracted from tumor biopsies invariably generates discrepancies that are most often explained away as biological phenomenon [15]. Similarly, "synthetic cfDNA" spiked with known markers is an uncertain approximation of genuine, physiologically generated DNA [12].…”