Clinical validation of a next-generation sequencing assay for 17 cancer related mutations in non-small cell Lung cancer

作者：Bing Xu, Guangyu Shan, Sihang Gao, Yanrong Wang, Weiwei Wang, Xiaoxi Pan, Dongxing Zhang, Lijiao Lin, Jing Gao, Nanying Che, Xiaoyong Ji, Junhua Pan · 发表于：Research Square · 年份：2023 · DOI：10.21203/rs.3.rs-2740545/v1 · 研究领域：Cancer Genomics and Diagnostics、Lung Cancer Treatments and Mutations、RNA modifications and cancer

Abstract Background Circulating tumor DNA (ctDNA) enables rapid and repeat testing of actionable mutations with fewer side effects compared to tissue biopsy. And the use of next-generation sequencing (NGS) based on ctDNA as a diagnostic tool in clinical settings is growing. We developed a hybridization capture massively parallel NGS assay using ctDNA, USCI-CT, across 20 cancer relevant genes of non-small cell lung cancer (NSCLC). Clinical validations of this assay across 17 mutations were presented. Methods Clinical NSCLC samples and simulated negative and positive cell-free DNA (cfDNA) samples were applied to validate the technical performance of this assay. First, 40 simulated negative control cfDNA samples were used to evaluate the systemic error levels to delimit the limit of detection of variant allele frequency (VAF). And, 92 clinical NSCLC samples were genotyped by USCI-CT and ddPCR to decide the proper limit of VAF, depth of coverage, and the average depth of target regions for confidently detecting mutations for USCI-CT. Second, one simulated negative cfDNA sample and two simulated positive cfDNA samples in seven replicates were used to assess the precision of the assay. Finally, 518 clinical NSCLC samples were recruited to evaluate the analytical sensitivity and specificity of USCI-CT. Results The assay-specific systemic error rate was below 0.20% by sequencing sixty-seven simulated negative control cfDNA samples. The proper cutoff of detection of VAF, coverage of d...