P1.02-043 Multiplexed FISH (ALK/ROS1, RET, NTRK1) in Lung Adenocarcinomas: Novel Dual ALK/ROS1 Probe and Automated Scanning System

Although the use of NGS (next-generation sequencing) is indeed feasible for the study of druggable rearrangements, the sensitivity and specificity of targeted NGS has been challenged on several grounds: use of fixed tissue, poor quality/insufficient sample, RNA-contamination risk or long turn-around time. Our relatively high rate of failures (7.6%) with the RNA workflow of targeted NGS (data not shown), prompted us to investigate possible alternatives. The objective of this study is to demonstrate an efficient solution based on multiplexed fluorescence in situ hybridization (FISH) for the comprehensive characterization of fusions (ALK, ROS1, RET and NTRK1) in lung adenocarcinomas (ACs). We have implemented for the first time in the literature a novel four-color ALK/ROS1 probe and used an automated scanning system for scoring these four translocations. A total of 64 patients with early stage lung ACs who underwent surgery at HM Sanchinarro University Hospital were considered. All slides were carefully reviewed to identify the different histological patterns. Afterwards we performed FISH to study ALK, ROS1, RET and NTRK1 in each pattern. We used both commercially (RET and NTRK1) and non-commercially (ALK/ROS1 dual) available Vysis break-apart probes (Abbott Molecular, USA). Slides were captured and scored with the BioView (Rehovot, Israel) automated imaging and analysis system, using dedicated applications for each probe. Positive cases were all confirmed with targeted NGS (Oncomine Focus AssayTM, Life Technologies, USA). Seven out of 420 slides did not hybridize (1.6%). We found two ALK (2.8%) and two ROS1 (2.8%) positive tumors, while no translocations were identified in RET or NTRK1. The rearrangements were present in all the different histological patterns within a given tumor, with a similar proportion of positive cells. The two major patterns of positivity were represented. The mean percentage of positive cells was 65% (range 46 to 84%). The NGS results confirmed the FISH findings. Simultaneous FISH testing for ALK and ROS1 is feasible on a single slide. The strategy reported herein provided reduced overall scoring time and ensured sensitive counting. This model might be easier to reconcile (lower cost, shorter turn-around time, and less failure rate) with subsequent comprehensive genotyping. Therefore, it could be used prospectively in advanced lung ACs to align the use of several technologies.