RET Fluorescence In Situ Hybridization Analysis Is a Sensitive but Highly Unspecific Screening Method for RET Fusions in Lung Cancer

Teodora Radonic,W.R.R Geurts-Giele,Kris G Samsom,Guido M.J M Roemen,Jan H Von Der Thüsen,Erik Thunnissen,Isabelle C Meijssen,Hein F.B M Sleddens,Winand N.M Dinjens,Mirjam C Boelens,Karin Weijers,Ernst Jan M Speel,Stephen P Finn,Cathal O’Brien,Tom Van Wezel,Danielle Cohen,Kim Monkhorst,Paul Roepman,H.J Dubbink

doi:10.1016/j.jtho.2021.01.1619

Abstract

IntroductionRET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions. MethodsBetween January 2016 and November 2019, a total of 4873 patients with NSCLC were routinely screened for RET fusions using either FISH (n = 2858) or targeted RNA next-generation sequencing (NGS) (n = 2015). If sufficient material was available, positive cases were analyzed by both methods (n = 39) and multiple FISH assays (n = 17). In an independent cohort of 520 patients with NSCLC, whole-genome sequencing data were investigated for disruptive structural variations and functional fusions in the RET and compared with ALK and ROS1 loci. ResultsFISH analysis revealed RET rearrangement in 48 of 2858 cases; of 30 rearranged cases double tested with NGS, only nine had a functional RET fusion. RNA NGS yielded RET fusions in 14 of 2015 cases; all nine cases double tested by FISH had RET locus rearrangement. Of these 18 verified RET fusion cases, 16 had a split signal and two a complex rearrangement by FISH. By whole-genome sequencing, the prevalence of functional fusions compared with all disruptive events was lower in the RET (4 of 9, 44%) than the ALK (27 of 34, 79%) and ROS1 (9 of 12, 75%) loci. ConclusionsFISH is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC.

Highlights

RET gene fusions are established oncogenic drivers in 1% of NSCLC
fluorescence in situ hybridization (FISH) is a sensitive but unspecific technique for RET screening, always requiring a confirmation using an orthogonal technique, owing to frequently occurring RET rearrangements not resulting in functional fusions in NSCLC
4873 NSCLC were screened for the presence of RET fusions using either FISH or targeted RNA next-generation sequencing (NGS) (Fig. 1)

Summary

Introduction

RET gene fusions are established oncogenic drivers in 1% of NSCLC. Accurate detection of advanced patients with RET fusions is essential to ensure optimal therapy choice. We investigated the performance of fluorescence in situ hybridization (FISH) as a diagnostic test for detecting functional RET fusions. RET fusions are predominantly found in younger patients with no smoking history, who generally have a good prognosis when treated with conventional cancer therapy,[7] and in particular have a good response when treated with pemetrexed-based chemotherapy.[8] The incidence of RET gene fusions in the literature varies approximately 1% of NSCLC and depends on age, smoking history, histologic subtype, and racial origin (0.7% in non-Asian population[9] and 1.3% in Asian population[10])

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Results

Conclusion