Analytical and Clinical Validation of a Highly Sensitive NGS-Based ctDNA Assay with Real-World Concordance in NSCLC.

Abstract

There have been needs to improve the sensitivity of liquid biopsy. This report aims to report the analytical and clinical validation of a next generation sequencing (NGS)-based circulating tumor DNA (ctDNA) assay. Analytical validation was conducted in vitro by evaluating the limit of detection (LOD), precision, and specificity for various genomic aberrations. The real-world performance in non-small cell lung cancer (NSCLC) was assessed by comparing the results of AlphaLiquid®100 to the tissue-based results. The LODs with 30 ng input DNA were 0.11%, 0.11%, 0.06%, 0.21%, and 2.13 copies for detecting SNVs, insertions, deletions, fusions, and copy number alterations (CNA), respectively. Quantitatively, SNV/INDELs, fusions, and CNAs showed a good correlation (R2=0.91, 0.40, and 0.65; y=0.95, 1.06, and 1.19) to the manufacturer's values, and per-base specificities for all types of variants were near 100%. In real-world NSCLC (n=122), key actionable mutations in NSCLC were detected in 60.7% (74/122) with the ctDNA assay. Comparative analysis against the NGS-based tissue results for all key mutations showed positive percent agreement (PPA) of 85.3%. For individual genes, the PPA was as high as 95.7% for EGFR mutations and 83.3% for ALK translocations. AlphaLiquid 100 detected drug-sensitive EGFR mutation at a variant allele frequency as low as 0.02% and also identified an EGFR mutation in a case where tissue sample missed. Blood samples collected post-targeted therapies revealed additional acquired mutations. The AlphaLiquid®100 ctDNA assay demonstrates robust analytical validity, offering clinically important information for NSCLC patients.