Usefulness of an RNA extraction-free test for the multiplexed detection of ALK, ROS1, and RET Gene Fusions in Real Life FFPE Specimens of Non-Small Cell Lung Cancers

Background: ALK, ROS1 and RET rearrangements occur, respectively, in 5%, 2%, and 1% non-small cell lung cancers (NSCLC). ALK and ROS1 fusion proteins detection by immunohistochemistry (IHC) has been validated for rapid patient screening, but ROS1 fusions need to be confirmed by another technique and no RET IHC test is available for clinical use. Research design and methods: We report herein the usefulness of the HTG EdgeSeq Assay, an RNA extraction-free test combining a quantitative nuclease protection assay with NGS, for the detection of ALK, ROS1 and RET fusions from ‘real-life’ small NSCLC samples. A total of 203 FFPE samples were collected from 11 centers. They included 143 rearranged NSCLC (87 ALK, 39 ROS1, 17 RET) and 60 ALK-ROS1-RET negative controls. Results: The assay had a specificity of 98% and a sensitivity for ALK, ROS1 and RET fusions of 80%, 94% and 100% respectively. Among the 19 HTG-assay false negative samples, the preanalytical conditions were identified as the major factors impacting the assay efficiency. Conclusions: Overall, the HTG EdgeSeq assay offers comparable sensitivities and specificity than other RNA sequencing techniques, with the advantage that it can be used on very small and old samples collected multicentrically.