br Materials and Methods br Results A

Materials and Methods
Results A total of 28 NSCLC tumor samples from 26 patients (2 patients with paired tumor samples) were included. According to the initial FISH (Abbott Molecular probes) and IHC results, 13 samples were ALK IHC+FISH+, 5 samples were ALK IHC+FISH−, 2 samples (from the same patient, patient 18) were ALK IHC−FISH+, 3 samples were ROS1 IHC+FISH+, 4 samples were ROS1 IHC+ FISH−, and 1 sample was ALK and ROS1 IHC−FISH− (patient 19). Among the samples lacking FISH positivity criteria, 2 samples (from patients 8 and 19) presented with > 50% of tumor nuclei with isolated 5′-ALK signals, 1 sample (patient 20) had a polysomic/amplified ROS1 status (ie, > 6 ROS1 copies per tumor nuclei), and 4 samples had other known oncogenic molecular alterations (2 with EGFR L858R and 2 with KRAS G12C mutations). The dual ALK and ROS1 FISH test (ZytoVision probe) results were fully concordant with the results of the single ALK and ROS1 FISH tests (Abbott Molecular probes) and detected ALK rearrangements in 15 samples and ROS1 rearrangements in 3 samples (examples shown in Figure 1B-M). No false-positive result was observed using the dual probe in GSK1016790A sale to the results with the single probes. The dual ALK and ROS1 FISH test also confirmed the atypical FISH patterns previously obtained. For 1 patient (patient 8) with ALK IHC+FISH− status showing a partial response to crizotinib, the dual probe FISH test also showed a negative FISH result, with > 50% single 5′-ALK signals (Figure 1N-P). This single 5′-ALK signal pattern was also obtained in another sample and was concordant with the previously concluded ALK IHC− FISH− status (patient 19). High percentages of ALK nuclei were also confirmed in the paired samples from patient 18 with the dual probe FISH test, despite the IHC-negative status of these 2 samples and in the samples from patient 4 (1 sample) and patient 9 (2 samples) with no response to crizotinib therapy despite concordant ALK IHC+FISH+ status. No patient with ROS1 rearrangement was treated by crizotinib in our study. The detailed results are listed in Table 1.
Discussion With the increase in the biomarkers of interest to test for in patients with NSCLC, the tissue handling, processing, and sectioning must be optimal to minimize tumor wastage and to permit the analysis of every potentially relevant targets, especially in the case of small biopsy samples. New molecular genetics technologies such as NGS, RNA sequencing, or NanoString gene expression profiling technologies offer opportunities to analyze concurrently many oncogenic targets, including subject-to-rearrangement targets, such as the ALK and ROS1 rearrangements considered in our study.22, 23 Nevertheless, the sensitivity of targeted NGS has been challenged because of several factors, such as poor quality/insufficient sample, RNA contamination risk, and the long turnaround time with ∼7% of failures with the RNA workflow of targeted NGS reported by some investigators. The NanoString system has shown promising performance in the detection of oncogenic rearrangements, especially in small poor cell samples, and will probably constitute a very interesting tool to minimize tumor tissue wastage and to maximize the capacity of analyses in the near future. Nevertheless, to date, it is not yet very widespread; thus, other methods could be considered to minimize tumor tissue wastage and concurrently analyze several oncogenes to search for the activating and treatable rearrangements using multiplexed FISH methods.22, 24, 25 In our study, the FlexISH ALK/ROS1 DistinguISH Probe (ZytoVision) focused on the 2 oncogenic rearrangements that enable the use of approved tyrosine kinase inhibitor therapies for NSCLC. The results were in absolute concordance with those from the single classically used ALK FISH and ROS1 FISH tests without any false-positive or false-negative results. As a consequence, with the now-recommended NSCLC-dedicated guidelines for first-line IHC screening of ALK and ROS1 expression, this dual ALK and ROS1 FISH test could become a valuable CE-IVD test to confirm druggable oncogenic alterations in NSCLC in the case of a score of 1+ or 2+ for ALK IHC or in the case of a score of 1+, 2+, or 3+ for ROS1 IHC.5, 6, 9, 10