Testing NTRK testing: Wet‐lab and in silico comparison of RNA‐based targeted sequencing assays

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.

NTRKtesting: First results of theQuiP‐EQAscheme and a comprehensive map ofNTRKfusion variants and their diagnostic coverage by targetedRNA‐basedNGSassays

“…All of these six participants used panels of the same composition, namely the Oncomine Focus Assay (Thermo Fisher Scientific) or the equivalent AmpliSeq Focus for Illumina Assay. As shown in our previous in silico analysis (21), this was not unexpected since these two panels are not capable of detecting this special NTRK fusion. One of the participants also rated a TPM3::NTRK1 fusion (case 2.4) as negative.…”

“…While current guidelines (16) suggest the use of RNA-based tNGS panels for NTRK testing, less is known about the influence of the assay design on the ability to detect specific NTRK fusions. Filling this gap, we recently performed a preliminary in silico analysis by analyzing bed files of several assays and their coverage of major NTRK fusion variants (21). Complementing these results as well as the ring trial data described above, we extended our previous analysis by analyzing additional RNA-based tNGS assays as well as additional NTRK gene fusion variants.…”

“…All cases had been pretested thoroughly by the institute that provided the samples and were retested centrally at the Institute of Pathology-University Hospital Heidelberg, yielding concordant results for FISH and RNA sequencing for all 24 tumor samples. 21 Of these, samples with sufficient tumor material for RNA-based NGS and FISH were selected comprising six unequivocally NTRK fusion-negative and four unequivocally NTRK fusion-positive cases each. A detailed overview on the cohort is provided in Table S1.…”

Predictive molecular pathology of lung cancer in Germany with focus on gene fusion testing: Methods and quality assurance

“…The detection of fusions on RNA level has proven to be a robust technology due to its reliability irrespective of the method used for RNA extraction. 71 In addition, RNA-based assays are able to determine whether a fusion event is in-frame, which might be essential for functional interpretation. Detection of additional activating aberrations like the skipping of exon 14 of the MET gene and the duplication of the EGFR kinase domain is also feasible using such assays.…”

“…Their major advantage is that only a small amount of input RNA is needed, thus the targeted assays work with as little as 10 ng of RNA; in contrast, other approaches require up to 250 ng of RNA, which can be challenging in biopsy samples. 71 Anchored multiplex PCR (AMP-PCR) allows the detection of yet unknown gene fusions. Amplicons are generated using the technology of rapid amplification of complementary DNA ends PCR, which employs a gene-specific primer and a universal primer for the potentially unknown fusion partner.…”