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

Area of Science:

• Oncology
• Molecular Diagnostics
• Genomics

Background:

• Neurotrophic tyrosine receptor kinase (NTRK) fusions are rare but actionable oncogenic drivers in solid tumors, occurring in approximately 0.3% of cases.
• Accurate identification of NTRK fusion-positive tumors is essential for patient eligibility for tropomyosin receptor kinase inhibitor therapy.
• The current landscape of NTRK testing, particularly using next-generation sequencing (NGS), is fragmented with variable assay availability and performance.

Purpose of the Study:

• To evaluate and compare the analytical and clinical performance of four commonly available RNA-based NGS assays for NTRK fusion detection.
• To provide insights into the strengths and limitations of these assays to guide physicians and pathologists in selecting optimal testing methods.

Main Methods:

• Evaluation of four RNA-based NGS assays: Archer's FusionPlex Lung panel (AFL), Illumina's TruSight Oncology 500 (TSO500), and Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA).
• Experiments utilized contrived samples (cell lines, reference material), NTRK fusion-negative clinical samples, and NTRK fusion-positive clinical samples.
• Assessment of analytical sensitivity (limit of detection) and specificity, alongside quality control (QC) pass rates.

Main Results:

• The estimated limit of detection varied significantly across assays, ranging from approximately 1-28 copies (OFA, Oncomine Precision) to 30-620 copies (AFL).
• All four assays demonstrated 100% specificity for NTRK fusion detection.
• Quality control pass rates were variable: AFL (43%), TSO500 (77%), and OFA (83%).
• All assays achieved 100% detection rate for NTRK fusions in quality control-validated clinical samples.

Conclusions:

• All evaluated RNA-based NGS assays can accurately detect NTRK fusions in clinical samples, showing 100% specificity.
• Assay performance, particularly in terms of limit of detection and QC pass rates, varies, necessitating careful selection based on clinical needs.
• This comparative analysis aids in informed decision-making for NTRK fusion testing to optimize patient identification for targeted therapies.