Automated nCounter-based assay for identifying clinically relevant ALK, ROS1 and RET rearrangements in advanced non-small cell lung cancer (NSCLC)

Abstract

Background: Targetable rearrangements in anaplastic lymphoma kinase (ALK), ROS1, and RET genes can be detected in ∼5-7% of patients with advanced NSCLC. Fluorescent in situ hybridization (FISH) and immunohistochemistry (IHC) are currently used for screening but present disadvantages in terms of sensitivity, reproducibility, cost and throughput. The Elements nCounter multiplexed platform has the potential for quick, sensitive and simultaneous detection of several clinically relevant fusion transcripts, but it needs validation in the clinical setting. Methods: A set of probes for detection of ALK, ROS1 and RET fusion transcripts were designed and initially assessed in a panel of cell lines. Subsequently, a total of 108 FFPE samples from advanced NSCLC patients were analyzed with the nCounter-multiplexed platform. Results were compared with FISH, IHC and, in the case of ALK, RT-PCR. Response to crizotinib was retrospectively collected in a subset of patients. Results: All patients categorized as positive for ALK by nCounter were concordant with IHC (100% sensitivity [CI = 88.3-100], 97.2% specificity [CI = 85.8-99.5]) while 10/31 were negative by FISH (95.5% sensitivity [CI = 78.2-99.2], 84.9% specificity [CI = 74.3-91.6]). Twenty patients received crizotinib based on ALK results and 18 derived clinical benefit. Of those, all were positive by nCounter while 3 were negative or not evaluable by FISH. In the case of ROS, 19/21 positive patients by nCounter were also positive by FISH (96.1% specificity [CI = 86.8-98.9]) but 8 samples were found positive by FISH and negative by nCounter (70.4% sensitivity [CI = 51.5-84.2]). Six of them were also negative by IHC, indicating lack of protein expression. Ten patients received crizotinib based on ROS results. Of the seven patients deriving clinical benefit, six were positive by nCounter. Two patients were nCounter positive for RET, one of them was FISH positive. Conclusions: We have validated an ALK/ROS1/RET nCounter multiplexed assay that allows for effective screening of FFPE samples and identifies advanced NSCLC patients who will benefit from targeted therapies.