Analytical and clinical validation of a novel amplicon-based NGS assay for evaluation of circulating tumor DNA in metastatic colorectal cancer patients.

e15084 Background: Evaluating the tumor RAS/BRAF status is important for treatment selection and prognosis assessment in metastatic colorectal cancer (mCRC) patients. Correction of artifacts from library preparation and sequencing is essential for accurately analyzing circulating tumor DNA (ctDNA) mutations. Here, we assessed the analytical and clinical performance of a novel amplicon-based next-generation sequencing (NGS) assay, Firefly, which employs a concatemer-based error correction strategy. Methods: Firefly assay targeting KRAS/NRAS/BRAF/PIK3CA was evaluated using cfDNA reference standards and cfDNA samples from 184 mCRC patients. Plasma results were compared to the mutation status determined by ARMS-based PCR from matched tissue. For samples with mutation abundance below the limit of detection (LOD), were retested again by droplet digital polymerase chain reaction (ddPCR) or NGS. Results: Firefly assay demonstrated superior sensitivity and specificity with 98.89% detection rate at allele frequency of 0.2% for 20ng cfDNA. Generally, 40.76% and 48.37% of the patients were reported to be positive by NGS of plasma cfDNA and ARMS of FFPE tissue, respectively. The concordance rate between the two platforms was 80.11%. In pre-treatment cohort, the concordance rate between plasma and tissue was 93.33%, based on the 17 common exons that Firefly and ARMS genotyped, positive percent agreement (PPA), and negative percent agreement (NPA) for KRAS/NRAS/BRAF/PIK3CA was 100% and 99.60%, respectively. Conclusions: Total plasma cfDNA detected by Firefly offers a viable complement for mutation profiling in CRC patients, given highly agreement with matched tumor samples. Together, these data demonstrate that Firefly could be routinely applied for clinical applications in mCRC patients.