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

Abstract

Gene fusions involving the three neurotrophic tyrosine receptor kinase genes NTRK1, NTRK2, or NTRK3 were identified as oncogenic drivers in many cancer types. Two small molecule inhibitors have been tested in clinical trials recently and require the detection of a NTRK fusion gene prior to therapeutic application. Fluorescence in situ hybridization (FISH) and targeted next-generation sequencing (tNGS) assays are commonly used for diagnostic profiling of gene fusions. In the presented study we applied an external quality assessment (EQA) scheme in order to investigate the suitability of FISH and RNA-/DNA-based tNGS for detection of NTRK fusions in a multinational and multicentric ring trial. In total 27 participants registered for this study.Nine institutions took part in the FISH-based and 18 in the NGS-based round robin test, the latter additionally subdivided into low-input and high-input NGS methods (regarding nucleic acid input). Regardless of the testing method applied, all participants received tumor sections of 10 formalin-fixed and paraffin-embedded (FFPE) tissue blocks for in situ hybridization or RNA/DNA extraction, and the results were submitted via an online questionnaire. For FISH testing, eight of nine (88.8%) participants, and for NGS-based testing 15 of 18 (83.3%) participants accomplished the round robin test successfully. The overall high success rate demonstrates that FISHand tNGS-based NTRK testing can be well established in a routine diagnostic setting.Complementing this dataset, we provide an updated in silico analysis on the coverage of more than 150 NTRK fusion variants by several commercially available RNA-based tNGS panels. K E Y W O R D S FISH, NGS, NTRK, NTRK fusion variants, RNA sequencing Albrecht Stenzinger and Nicole Pfarr are co-senior authors.

Reproducibility of mRNA-Based Testing of ESR1, PGR, ERBB2, and MKI67 Expression in Invasive Breast Cancer—A Europe-Wide External Quality Assessment

“…Our EQA study included only ten tumor samples, as is often the case in EQA studies [35,36]. It would be interesting to assess the reliability, i.e., the performance relative to reference tests, as well as the predictive and prognostic value of the assay in extensive clinical cohorts.…”

What Is New in Biomarker Testing at Diagnosis of Advanced Non-Squamous Non-Small Cell Lung Carcinoma? Implications for Cytology and Liquid Biopsy

“…IHC must also evaluate the status of NTRK on cytological samples, but in daily practice this may not be adequate. The molecular techniques include fluorescence in situ hybridization, targeted or multiplex RT-PCR, DNA-or RNA-based NGS, and analyses using NanoString technology on tissue sections [109,119,121]. Depending on the quality and quantity of the nucleic acids extracted from the sample, false negative results can be obtained [78,81,109,119,121].…”

“…The molecular techniques include fluorescence in situ hybridization, targeted or multiplex RT-PCR, DNA-or RNA-based NGS, and analyses using NanoString technology on tissue sections [109,119,121]. Depending on the quality and quantity of the nucleic acids extracted from the sample, false negative results can be obtained [78,81,109,119,121]. Depending on the case, these approaches could be performed with cytological samples, but only a few studies in this area have been performed to date and studies comparing cytological and tissue samples are strongly needed [109,[112][113][114][115].…”

Developing Drugs for Tissue‐Agnostic Indications: A Paradigm Shift in Leveraging Cancer Biology for Precision Medicine

“…Instead, a majority of patient were tested using either NGS or fluorescence in situ hybridization. Current testing algorithms suggest that a combination of fluorescence in situ hybridization, NGS, and immunohistochemistry are effective in identifying patients with NTRK fusions 25–29 . While no one diagnostic test was developed alongside either larotrectinib or entrectinib, development of an FDA‐approved companion diagnostic is currently underway.…”