Abstract Background Cell-free circulating tumor DNA (ctDNA)-based next-generation sequencing (NGS) has demonstrated the potential to guide the personalized treatment of non-small cell lung cancer (NSCLC). Furthermore, clinical treatment-response data guided by ctDNA remain scarce. This study systematically evaluates an NGS platform’s detection accuracy and clinical utility in a large Chinese NSCLC cohort, establishing therapeutic-response evidence. Methods We first established the cutoff and quality control parameters for the NGS test using clinical plasma samples from NSCLC patients, with ddPCR serving as the reference standard. The performance of the assay was then robustly validated in an independent cohort of 522 samples. Finally, we analyzed the driver and resistance mutations for their distribution, concordance with tissue samples, and therapeutic relevance. Results Through analysis of plasma samples using ROC and downsample methods, we established a 0.2% detection threshold and identified > 1400x mean effective depth as the critical QC metric; under these parameters, ddPCR validation of 17 specific sites in the 522-sample cohort demonstrated > 80% positive percentage agreement (PPA) and > 95% negative percentage agreement (NPA), confirming strong NGS-ddPCR concordance. The 21-gene panel detected mutations in 73.75% of patients, with 45.59% harboring NCCN-recommended targetable mutations. In the tissue-plasma concordance analysis, stage-specific performance was observed: Stage III showed 28.57% PPA (2/7) versus 99.20% NPA (124/125), while Stage IV demonstrated 99.20% PPA (124/125) and 99.46% NPA (183/184). Clinically relevant plasma-specific mutations were identified throughout, and pooled data demonstrated equivalent targeted therapy response rates between this ctDNA-based NGS and National Medical Products Administration (NMPA)-approved tissue-based assays. Conclusions This study highlights the feasibility of ctDNA-based NGS in clinical practice, offering valuable and clinically relevant mutational genomic profiling for stage III/IV NSCLC in Chinese patients. Trial registration This study is registered with Chinese Clinical Trial Registry, ChiCTR2000041034, on December 16, 2020.
Yang et al. (2025) studied this question.