Comparison of RNA-Based Next-Generation Sequencing Assays for the Detection of NTRK Gene Fusions

Recently, the US Food and Drug Administration approved several targeted therapies directed against oncogenic fusions. One of the most effective such targeted therapies is Vitrakvi (larotrectinib), highly specific oral tropomyosin receptor kinase inhibitor indicated for the treatment of patients with any solid tumor harboring a fusion involving one of the neurotrophic receptor tyrosine kinase (NTRK) genes. Although several diagnostic approaches can be used to detect these NTRK fusions, RNA-based next-generation sequencing remains one of the most sensitive methods, as it can directly detect the transcribed end product of gene fusion at the mRNA level. In this study, performance characteristics of three RNA-based next-generation sequencing assays with distinct mechanisms and chemistries were investigated: anchored multiplex PCR, amplicon-based multiplex PCR, and hybrid capture-based enrichment method. Analytical sensitivity analysis shows that the amplicon-based multiplex PCR method has the lowest limit of detection. However, both hybrid-capture and anchored multiplex PCR methods can detect NTRK fusions with uncommon or novel fusion partners, which is challenging for the amplicon-based multiplex method. As for clinical sensitivity, all three methods were highly concordant in detecting NTRK fusions in patient samples. Additionally, they all presented equivalent high-level performance in specificity, suggesting that all three platforms can detect NTRK fusions in clinical samples with similar performance characteristics.