Tumor Cell-Free DNA Assay Analytical Performance in 101 Clinical Laboratories Participating in a College of American Pathologists Proficiency Testing Program

Abstract

Tumor cell-free DNA (cfDNA) assays are increasingly used for mutation detection in therapy selection and disease monitoring. This study evaluates the laboratory performance in College of American Pathologists (CAP) Molecular Oncology Committee tumor cfDNA proficiency testing (PT) during a 2-year period. To assess the analytical performance of clinical laboratories offering tumor cfDNA assays using well-characterized PT specimens and compare performance across analytical methods. This study examined laboratory performance in CAP cfDNA PT programs from 2018 and 2019, with laboratories receiving multiple mailings longitudinally. Testing encompassed 12 specimens targeting 16 clinically significant canonical single-nucleotide variants in 5 oncogenes: EGFR (epidermal growth factor receptor), KRAS (KRAS proto-oncogene, GTPase), BRAF (B-Raf proto-oncogene, serine/threonine kinase), NRAS , (NRAS proto-oncogene, GTPase), and IDH1 (isocitrate dehydrogenase 1). A total of 5088 PT responses from 101 laboratories were analyzed. Results were classified as true positive, false negative, true negative, or false positive, and stratified by next-generation sequencing (NGS) versus non-NGS methods and US Food and Drug Administration–approval status. Gene-specific sensitivities ranged from 87.1% (176 of 202) to 100.0% (80 of 80), with specificities from 98.7% (1284 of 1301) to 100.0% (827 of 827). For EGFR mutations, which had samples at lower variant allele fractions (VAFs), NGS-based assays demonstrated higher sensitivity than non-NGS methods (95.7% [88 of 92] versus 81.7% [85 of 104]; P = .002). Within the 0.1% to 0.3% VAF range, NGS-based approaches achieved significantly higher sensitivity (96.1% [73 of 76]) than non-NGS methods (78.2% [61 of 78]) ( P = .01). Clinical laboratories achieved high analytical sensitivity and specificity for tumor cfDNA PT across 5 oncogenes. NGS-based assays demonstrated superior analytical performance for EGFR mutation detection at low VAFs, with performance differences most pronounced in the 0.1% to 0.3% VAF range.