Clinical Validation of a Targeted RNA-Sequencing Assay for Driver Gene Alteration Detection in Non-Small Cell Lung Cancer.

Background and objective: With the increasing number of diagnostic biomarkers associated with tumor diagnosis, targeted therapy, and immunotherapy, access to clinical pathological specimens of an appropriate size for analysis is becoming a problem. Conventional high-throughput sequencing assays for non-small cell lung cancer (NSCLC) often necessitate the extraction of separate DNA and RNA samples to achieve precise detection of various mutation types. This study aimed to employ RNA-next-generation sequencing (NGS) technology to simultaneously detect different types of mutations in NSCLC samples, including single nucleotide variations, insertions and deletions, fusions/rearrangements, and exon skipping, thereby addressing the issue of limited sample availability.

Methods: Two hundred and twenty cases of formalin-fixed paraffin-embedded NSCLC clinical specimens were retrospectively included for targeted RNA sequencing based on the principle of probe hybridization capture. Lung cancer tissue samples with different storage times were compared for success in DNA-NGS and RNA-NGS assays. The clinical detection performance of RNA-NGS was evaluated by comparing its results to those of DNA-NGS and clinical assays. Samples with inconsistent results were further verified by immunohistochemistry, amplification refractory mutation system-polymerase chain reaction, or droplet digital polymerase chain reaction.

Results: DNA-NGS exhibited an overall success rate of 91.82% in all samples, while RNA-NGS achieved an overall success rate of 92.73%. However, the success rate declined with longer storage times. Compared with DNA-NGS, targeted RNA sequencing for single nucleotide variation/insertion and deletion detection achieved a sensitivity of 93.75%, a specificity of 100%, and an overall concordance of 97.86%. Compared with the validated results, it achieved a sensitivity of 97.96%, a specificity of 99.28%, an and overall concordance of 98.93% in fusion/rearrangement and Met exon skipping detection, which was superior to DNA-NGS. Compared to clinical testing, this assay demonstrated a sensitivity of 93.33%, a specificity of 100%, and an overall concordance rate of 97.93%.

Conclusions: This study substantiates that the targeted RNA-sequencing assay, based on probe hybridization capture, represents a superior detection technology platform for the application of drug targeting. It expeditiously and reliably provides all the requisite biomarkers for current NSCLC targeted therapies in a single-sample testing workflow, facilitating rapid clinical diagnosis and the formulation of rational treatment plans by clinicians.