Evaluating Targeted Next-Generation Sequencing (NGS) Assays and Reference Materials for NTRK Fusion Detection

Neurotrophic tyrosine receptor kinase (NTRK1/2/3) gene fusions are oncogenic drivers in approximately 0.3% of solid tumors. High-quality testing to identify patients with NTRK fusion-positive tumors who could benefit from TRK inhibitors is recommended, but the current NTRK testing landscape, including next-generation sequencing (NGS), is fragmented and availability of assays varies widely. The analytical and clinical performance of four commonly available RNA-based NGS assays, Archer's FusionPlex Lung panel (AFL), Illumina's TruSight Oncology 500 (TSO500), as well as Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA), were evaluated. Experiments were conducted using contrived samples [formalin-fixed, paraffin-embedded cell lines (n = 3) and SeraSeq formalin-fixed, paraffin-embedded reference material (three lots)], NTRK fusion-negative clinical samples (n = 30), and NTRK fusion-positive clinical samples (n = 14), according to local assays. Estimated limit of detection varied across the four assays: 30 to 620 fusion copies for AFL (in cell lines), versus approximately 30 to 290 copies for TSO500 and approximately 1 to 28 copies for OFA and Oncomine Precision Assay. All assays showed 100% specificity for NTRK fusions detection, but quality control pass rate was variable (AFL, 43%; TSO500, 77%; and OFA, 83%). The NTRK fusion detection rate in quality control-validated clinical samples was 100% for all assays. This comparison of the strengths and limitations of four RNA-based NGS assays will inform physicians and pathologists regarding optimal assay selection to support identification of patients with NTRK fusion-positive tumors.