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

Abstract

Neurotrophic tyrosine receptor kinase (NTRK1/2/3) gene fusions are oncogenic drivers in ∼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 (OPA) and Oncomine Focus Assay (OFA) were evaluated. Experiments were conducted using contrived samples (formalin-fixed paraffin-embedded [FFPE] cell lines [n=3] and SeraSeq FFPE 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: 30-620 fusion copies for AFL (in cell lines), versus ∼30-290 copies for TSO500 and ∼1-28 copies for OFA and OPA. All assays showed 100% specificity for NTRK fusions detection, but QC pass rate was variable (AFL, 43%; TSO500, 77%; OFA, 83%). The NTRK fusion detection rate in QC-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.