Clinical utility and performance of an ultra-rapid multiplex RNA-based assay for detection of ALK, ROS1, RET and NTRK1/2/3 rearrangements and MET exon 14 skipping alterations

Several kinase fusions are established targetable drivers in lung cancers. However, rapid and comprehensive detection remains challenging due to diverse partner genes and breakpoints. We assess the clinical utility and performance of a rapid microfluidic multiplex reverse transcription PCR-based assay for simultaneous query of fusions involving ALK, ROS1, RETand NTRK1/2/3,as well as METexon 14 skipping, using a three-hour automated process. Dual analytic strategies were utilized: fusion-specific amplification (FS) and 3’-5’ expression imbalance (EI). We analyzed 143 independent, formalin-fixed paraffin-embedded tumor samples (112 surgical specimens, 31 cytologic cell blocks); 133 with known kinase gene alterations and 10 negative samples based on clinically validated next-generation sequencing (NGS). Testing was successful in 142(99%) cases. The assay demonstrated a sensitivity of 97% (28/29), 100% (31/31), 92% (22/24), 81% (22/27) and 100% (20/20) for ALK, RET, ROS1, NTRK1/2/3rearrangements and METexon 14 skipping alterations, respectively, with 100% specificity for all. Concordant results were achieved in specimens aged up to 5 years, with >10% tumor, and inputs of at least 9 mm2(surgical specimens) and 9000 cells (cytologic cell blocks). The assay enables rapid screening for clinically actionable kinase alterations with quicker turnaround and lower tissue requirements compared to IHC and molecular methods, while also circumventing the infrastructure dependencies associated with NGS and FISH.