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

Christina Bormann Chung,Jeeyun Lee,Marc Barritault,Pierre-Paul Bringuier,Zhaolin Xu,Weei-Yuarn Huang,Andrea Beharry,Joseph Castillo,Jason Christiansen,Jennifer C Lin,Brandon S Sheffield

doi:10.1016/j.jmoldx.2021.09.008

Abstract

Neurotrophic tyrosine receptor kinase (NTRK1/2/3) fusions are oncogenic drivers in approximately 0.3% of solid tumors. High-quality testing to identify patients with NTRK fusion-positive tumors who could benefit from tropomyosin receptor kinase 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), Thermo Fisher's Oncomine Precision Assay and Oncomine Focus Assay (OFA), were evaluated. Experiments were conducted using contrived samples [formalin-fixed, paraffin-embedded cell lines and SeraSeq formalin-fixed, paraffin-embedded reference material], NTRK fusion-negative clinical samples, and NTRK fusion-positive clinical samples, according to local assays. Estimated limit of detection varied across the four assays: 30 to 620 fusion copies for AFL (cell lines), versus approximately 30 to 290 copies for TSO500 and approximately 1 to 28 copies for OFA and Oncomine Precision Assay. All assays showed 100% specificity for NTRK fusions detection, but quality control pass rate was variable (AFL, 43%; TSO500, 77%; and OFA, 83%). The NTRK fusion detection rate in quality control-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 identify patients with NTRK fusion-positive tumors.

Highlights

Fusions of the neurotrophic tyrosine receptor kinase genes (NTRK1/2/3) are important oncogenic drivers across a large range of tumor types.1, 2 While their prevalence is generally low across all solid tumors, they are found at high frequency (>90%) in rare tumor types such as mammary analogue secretory carcinoma (MASC) and secretory breast carcinoma.2-4 NTRK fusions are Journal Pre-proof promising molecular targets for new therapies and a number of TRK inhibitors have recently been approved, which provide effective targeted treatment options for patients presenting with these genomic rearrangements.Larotrectinib and entrectinib were the first two TRK inhibitors approved in the US, Europe, and other countries for the treatment of NTRK fusion-positive advanced solid tumors;5 a companion diagnostic to larotrectinib (FoundationOne CDx; Foundation Medicine, Cambridge, MA) was subsequently approved
Fusion Copy Number Determination Besides giving different RNA yields, the different extraction methods used in this study yielded variable fusion copies/ng RNA (Table 3). The ReliaPrep (AFL) and RecoverAll (OFA/Oncomine Precision Assay (OPA)) extraction kits yielded similar results, whereas the AllPrep (TSO500) and MagMAX (OPA) kits yielded about one343 third of the fusion copies/ng RNA achieved with the other methods
[RET], ROS1 proto-oncogene 1 [ROS1]) means that testing for oncogenic gene 415 fusions is recommended in the diagnostic process

Summary

Introduction

Larotrectinib and entrectinib were the first two TRK inhibitors approved in the US, Europe, and other countries for the treatment of NTRK fusion-positive advanced solid tumors; a companion diagnostic to larotrectinib (FoundationOne CDx; Foundation Medicine, Cambridge, MA) was subsequently approved (https://www.fda.gov/drugs/fda-approves-companion-diagnostic-identify-ntrk-fusionssolid-tumors-vitrakvi, last accessed July 15, 2021). Both entrectinib and larotrectinib demonstrated strong clinical efficacy in patients with NTRK fusion-positive tumors enrolled in basket trials (response rates of 64% and 78%, respectively), supporting the relevance of NTRK fusions as treatment targets and, the importance of 100 identifying patients harboring these gene rearrangements. . Both entrectinib and larotrectinib demonstrated strong clinical efficacy in patients with NTRK fusion-positive tumors enrolled in basket trials (response rates of 64% and 78%, respectively), supporting the relevance of NTRK fusions as treatment targets and, the importance of identifying patients harboring these gene rearrangements. a third inhibitor, repotrectinib, has shown promising early efficacy; and recently received a

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