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Testing NTRK testing: Wet-lab and in silico comparison of RNA-based targeted sequencing assays
Nicole Pfarr1, Martina Kirchner2, Ulrich Lehmann3
1Institute of Pathology, Technical University Munich (TUM), Munich, Germany.
Abstract:
NTRK fusions involving three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, and NTRK3 and a variety of fusion partners were identified as oncogenic drivers across many cancer types. Drugs that target the chimeric protein product require the identification of the underlying gene fusion. This advocates the diagnostic use of molecular assays ranging from fluorescence in situ hybridization (FISH) and reverse transcription polymerase chain reaction (RT-PCR)/Sanger approaches to targeted next-generation sequencing (NGS). Immunohistochemistry may be used as a screening tool and adjunct diagnostic assay in this context. Although FISH and RT-PCR/Sanger approaches are widely adopted in routine diagnostics, current experience with targeted RNA-based NGS is limited. Here, we report on the analysis of major assays (TruSight TST170 and TruSight RNA Fusion [Illumina]; Archer FusionPlex Solid Tumor, Archer FusionPlex Lung, and Archer FusionPlex Oncology [Archer]; Oncomine Comprehensive Assay v3 RNA and Oncomine Focus RNA [Thermo Fisher Scientific]) that are commercially available. The data set includes performance results of a multicentric comparative wet-lab study as well as an in silico analysis on the ability to detect the broad range of NTRK fusions reported until now. A test algorithm that reflects assay methodology is provided. This data will support implementation of targeted RNA sequencing in routine diagnostics and inform screening and testing strategies that have been brought forward.
Insights
Identifying NTRK gene fusions is crucial for targeted cancer therapy. This study evaluates commercially available RNA-based next-generation sequencing assays for accurate and efficient detection of these oncogenic drivers.
Area of Science:
- Oncology
- Molecular Diagnostics
- Genetics
Background:
- NTRK gene fusions (NTRK1, NTRK2, NTRK3) are oncogenic drivers in various cancers.
- Targeted therapies require precise identification of these fusions.
- Current diagnostic methods include FISH, RT-PCR/Sanger, and NGS, with limited experience in RNA-based NGS.
Purpose of the Study:
- To analyze and compare the performance of commercially available targeted RNA-based NGS assays for NTRK fusion detection.
- To provide data supporting the implementation of RNA-based NGS in routine diagnostics.
- To inform screening and testing strategies for NTRK fusions.
Main Methods:
- Comparative wet-lab study of major commercial RNA-based NGS assays (Illumina, Archer, Thermo Fisher Scientific).
- In silico analysis to assess detection capabilities for known NTRK fusions.
- Development of a test algorithm based on assay methodology.
Main Results:
- Performance data from a multicentric comparative study.
- In silico evaluation of assay sensitivity and specificity for a wide range of NTRK fusions.
- A proposed test algorithm to guide assay selection and implementation.
Conclusions:
- Targeted RNA-based NGS assays show promise for NTRK fusion detection.
- Comparative data and a test algorithm can facilitate the adoption of NGS in clinical practice.
- Improved diagnostic strategies are essential for effective targeted cancer therapy.
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