Analytical Validation of a Plasma-Based Rapid Liquid Biopsy Assay Using Next-Generation Sequencing.

Abstract

Cell-free total nucleic acid (cfTNA)-based liquid biopsy (LBx) offers a minimally invasive alternative to tissue-based next generation sequencing (NGS). Most NGS-based LBx assays require several days for results. This study validated a rapid NGS-based LBx assay with a two-day turnaround time (TAT). Patient plasma samples were used to validate the Oncomine Precision Assay for single nucleotide variants (SNVs), insertion deletions (indels) and fusions across 50 genes on the Genexus Sequencer. Manual extraction using the QIAamp cfTNA Kit was performed to optimize fusion detection. Analytical sensitivity in synthetic controls (N=20) using the automated workflow was 99.2% for SNVs and 95% for indels, with 100% specificity (allele frequency ≥0.5%). In clinical samples (N=107), sensitivity was 99.4% for SNVs and 100% for indels; specificity was 100% for both, compared with orthogonal assays. Manual extraction improved overall performance compared to the automated extraction and was used for the final clinical workflow. Analytical sensitivity of fusions in synthetic controls (N=22) was 98.9%, with 100% specificity (≥7 copies). In clinical samples (N=38) sensitivity and specificity for both SNVs and indels were 100% and 99.9%, respectively; sensitivity for fusions was 72.2%; specificity was 100%. Overall precision was >99% and average TAT was ≤2 days. This study reports the feasibility and validation of a two-day TAT NGS-based cfTNA assay that can potentially reduce time-to-treatment.