5' Rapid Amplification of cDNA Ends (5' RACE)-Based Targeted RNA Sequencing Assay for the Analysis of Clinically...

Next-generation sequencing (NGS) is currently regarded as a preferable method for detection of actionable alterations in non-small cell lung carcinomas (NSCLCs). RNA-based NGS is particularly efficient in detection of gene fusions and allows for mRNA expression analysis of relevant genes. This study describes a novel library preparation pipeline for targeted RNA sequencing, which is based on the 5' rapid amplification of the cDNA ends (5' RACE) and anchored multiplex PCR. The NGS panel was designed for the analysis of 15 genes relevant to NSCLC therapeutic decisions (ALK, BRAF, CD274 (PD-L1), EGFR, ERBB2 (HER2), KRAS, MET, NRAS, NRG1-2, NTRK1-3, RET and ROS1). The validation against PCR was performed for 168 NSCLC samples. The NGS assay successfully detected all 85 mutations previously identified by PCR. It also confirmed the absence of tested mutations in 82 out of 83 PCR-negative samples. The only discordant case was subsequently analyzed by digital droplet PCR and demonstrated low fraction of the mutated allele. The newly designed NGS panel was subsequently used for the analysis of 272 carcinomas from young (≤50 years old) patients with no driver mutations identified by PCR tests or with failed PCR analysis. NGS was successful in 240 (88.2%) cases, including 35 (64.8%) PCR-failed samples. Driver mutations were detected in 55 tumors, including three previously unreported fusions (PAPLN::ALK, CD55::ROS1 and FKBP15::RET). We conclude that 5' RACE-based RNA sequencing is a viable approach for NSCLC molecular testing.