Abstract 4344: Comparison of nCounter, immunohistochemistry, RT-PCR and FISH to detect ALK, ROS1 and RET rearrangements in advanced non-small cell lung cancer (NSCLC)
Authors: Teixido Cristina , Reguart Noemí , Giménez-Capitán Ana , Molina-Vila Miguel Ángel , Galván Patricia , Rodriguez Sonia , Paré Laia , Viteri Santiago , Peg Vicente , Yeste Zaira , Viñolas Núria , Rosell Rafael , Prat Aleix 
Published: July 2016

Abstract
Background: Targetable rearrangements in anaplastic lymphoma kinase (ALK), ROS1, and RET genes are present in approximately 7% of patients (p) with advanced NSCLC. Current methods for detecting gene fusions are based on FISH (FDA-approved companion diagnostic test for ALK), immunohistochemistry (IHC) or RT-PCR. However, these tests have disadvantages in terms of sensitivity, cost and throughput, and often show discrepancies. The nCounter platform allows simultaneous detection with no enzymatic reaction, within 72 hours, of several fusion genes in formalin-fixed paraffin-embedded (FFPE) samples using a transcript-based method.
Methods: A custom set of 5′and 3′ probes and fusion-specific probes to detect ALK, ROS1 and RET fusion transcripts was designed and evaluated. A panel of ALK-ROS1-RET positive cell lines (H2228, H3122 [EML4-ALK], SUDHL-1 [NPM-ALK], HCC78 [SLC34A2-ROS1], BaF3 pBABE [CD74-ROS1], LC2/ad [RET]) and negative cell lines (PC9, H1975 [EGFR mut], H460, H23 [KRAS mut]) was used to validate the technique. Then, a total of 70 FFPE samples was analyzed, 49 of them positive by FISH, IHC and/or qRT-PCR for ALK (n = 30), ROS1 (n = 17) and RET (n = 2). Total RNA was isolated from cell lines and FFPE and &lt; 225 ng were used for hybridization. Raw counts were normalized using positive controls, negative controls and four house-keeping genes (GAPDH, GUSB, OAZ1 and POLR2A) as described in Lira et al. J Mol Diagn 2013. Positive and negative translocations were defined by two criteria: (1) a 3’/5’ ratio score of &gt; 2.0 and ≤ 2.0 respectively or/and (2) a signal for a fusion-specific probe above background. Response to crizotinib by RECIST criteria was retrospectively collected in p with ALK-positive NSCLC by any technique.
Results: nCounter sensitivity to detect fusion transcripts ALK, ROS1 and RET in cell lines was 100% using the two criteria (3’/5’ and direct probes) and specificity was also 100%. Among 20 ALK-FISH-positive p, ALK 3’-5’ scoring was positive in 18 (95%). One p was non-evaluable (NE) by ALK 3’-5’ scoring. Among 48 ALK-FISH-negative p, nCounter score was positive in 13 (27%). All p positive for ALK by nCounter were either positive or NE for ALK by IHC. A total of 17 p were treated with crizotinib, 16 of whom responded to treatment and were positive by nCounter. Regarding FISH, five p responding to crizotinib were negative and one was NE. Finally, one p not responding to crizotinib was positive by RT-PCR but negative by nCounter.
Conclusion: The ALK/ROS1/RET nCounter-based assay is a highly sensitive screening assay that identifies ALK-FISH-negative/NE NSCLC patients who could benefit from treatment with ALK inhibitors.
Citation Format: Cristina Teixido, Noemí Reguart, Ana Giménez-Capitán, Miguel Ángel Molina-Vila, Patricia Galván, Sonia Rodriguez, Laia Paré, Santiago Viteri, Vicente Peg, Zaira Yeste, Núria Viñolas, Rafael Rosell, Aleix Prat. Comparison of nCounter, immunohistochemistry, RT-PCR and FISH to detect ALK, ROS1 and RET rearrangements in advanced non-small cell lung cancer (NSCLC). [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4344.