Identification of ALK, ROS1, and RET Fusions by a Multiplexed mRNA-Based Assay in Formalin-Fixed, Paraffin-Embedded Samples from Advanced Non–Small-Cell Lung Cancer Patients

Abstract

Abstract BACKGROUND Anaplastic lymphoma receptor tyrosine kinase (ALK), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), and ret proto-oncogene (RET) fusions are present in 5%–7% of patients with advanced non–small-cell lung cancer (NSCLC); their accurate identification is critical to guide targeted therapies. FISH and immunohistochemistry (IHC) are considered the gold standards to determine gene fusions, but they have limitations. The nCounter platform is a potentially useful genomic tool for multiplexed detection of gene fusions, but has not been validated in the clinical setting. METHODS Formalin-fixed, paraffin embedded (FFPE) samples from 108 patients with advanced NSCLC were analyzed with an nCounter-based assay and the results compared with FISH, IHC, and reverse transcription PCR (RT-PCR). Data on response to fusion kinase inhibitors was retrospectively collected in a subset of 29 patients. RESULTS Of 108 FFPE samples, 98 were successfully analyzed by nCounter (91%), which identified 55 fusion-positive cases (32 ALK, 21 ROS1, and 2 RET). nCounter results were highly concordant with IHC for ALK (98.5%, CI = 91.8–99.7), while 11 discrepancies were found compared with FISH (87.5% concordance, CI = 79.0–92.9). For ROS1, nCounter showed similar agreement with IHC and FISH (87.2% and 85.9%), but a substantial number of samples were positive only by 1 or 2 techniques. Of the 25 patients deriving clinical benefit from fusion kinase inhibitors, 24 were positive by nCounter and 22 by FISH. CONCLUSIONS nCounter compares favorably with IHC and FISH and can be used for identifying patients with advanced NSCLC positive for ALK/ROS1/RET fusion genes.