Cancer Res Treat.  2023 Jan;55(1):28-40. 10.4143/crt.2021.1572.

Interlaboratory Comparison Study (Ring Test) of Next-Generation Sequencing–Based NTRK Fusion Detection in South Korea

• Lee SE
• Lee MS
• Jeon YK
• Shim HS
• Kang J
• Kim J
• Choi YL

• KMID: 2537989
• DOI: http://doi.org/10.4143/crt.2021.1572

Abstract

Purpose
Tropomyosin receptor kinase (TRK) inhibitors are approved for the treatment of neurotrophic receptor tyrosine kinase (NTRK) fusion-positive tumors. The detection of NTRK fusion using a validated method is required before therapeutic application. An interlaboratory comparison study of next-generation sequencing (NGS)–based NTRK gene fusion detection with validated clinical samples was conducted at six major hospitals in South Korea.
Materials and Methods
A total of 18 samples, including a positive standard reference and eight positive and nine negative clinical samples, were validated using the VENTANA pan-TRK (EPR17341) and TruSight Oncology 500 assays. These samples were then tested using four different NGS panels currently being used at the six participating institutions.
Results
NTRK fusions were not detected in any of the nine negative clinical samples, demonstrating 100% specificity in all six participating institutions. All assays showed 100% analytical sensitivity to identify the NTRK fusion status in formalin-fixed paraffin-embedded (FFPE) samples, although with variable clinical sensitivity. False-negative results were due to low tumor purity, poor RNA quality, and DNA-based sequencing panel. The RNA-based targeted NGS assay showed an overall high success rate of identifying NTRK fusion status in FFPE samples.
Conclusion
This study is the first to test the proficiency of NGS-based NTRK detection in South Korea with the largest participating institutions. RNA-based NGS assays to detect NTRK fusions can accurately characterize fusion transcripts if sufficient RNA of adequate quality is available. The comparative performance data will support the implementation of targeted NGS-based sequencing assays for NTRK fusion detection in routine diagnostics.

Keyword

fusion; Ring test; RNA-based NGS assay

Figure

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  Fig. 1 Overview cutting of the tumor tissue slices with formalin-fixed paraffin-embedded (FFPE) blocks. Seventeen FFPE tissue samples were cut with a 5 μm curl for next-generation sequencing analysis and curls were numbered according to the order of cut followed by placed in a microcentrifuge tube. Two vials were assigned to each institution. IHC, immunohistochemistry; TRK, tropomyosin receptor kinase.

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  Fig. 2 Tumor histology and pan–tyrosine receptor kinase (TRK) staining of ring trial samples. Resected formalin-fixed paraffin-embedded (FFPE) samples were stained with VENTANA pan-TRK (EPR17341) assay kits; cases determined to be positive for staining were further validated for neurotrophic receptor tyrosine kinase (NTRK) fusion gene detection via additional next-generation sequencing (NGS) analysis. Samples BN01, BN03, BN04, BN07, BN10, BN11, BN13, and BN17 were confirmed as positive by NGS analysis; these are marked in red. BN16 is a reference obtained from Seraseq FFPE NTRK Fusion RNA and is not an experimental sample.

Reference

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