Analytical and Clinical Validation of a Highly Sensitive NGS-Based ctDNA Assay with Real-World Concordance in Non-Small Cell Lung Cancer

Abstract

Purpose 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. Materials and Methods 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 AlphaLiquid100 to the tissue-based results. Results The LODs with 30 ng input DNA were 0.11%, 0.11%, 0.06%, 0.21%, and 2.13 copies for detecting single nucleotide variants, insertions, deletions, fusions, and copy number alterations (CNA), respectively. Quantitatively, single nucleotide variants/insertions and deletions, fusions, and CNAs showed a good correlation (R 2 =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 epidermal growth factor receptor ( EGFR ) mutations and 83.3% for ALK translocations. AlphaLiquid100 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. Conclusion The AlphaLiquid100 ctDNA assay demonstrates robust analytical validity, offering clinically important information for NSCLC patients.