Abstract 857: Diagnostic test system for sensitive, specific and reproducible detection of EML4-ALK RNA fusion transcripts in the blood of patients with NSCLC

Abstract

Abstract Clinical testing for the detection of RNA fusions in tissue currently include FISH, IHC, PCR and NGS. However, approximately 30% of patients with advanced non-small cell lung cancer (NSCLC) are not candidates for tissue biopsies and in some cases where tissue is obtained, it is not always of sufficient quantity for molecular testing. For these reasons, the detection of nucleic acids in circulation has become increasingly relevant to clinical testing. In this study, we have focused on the development of an EML4-ALK diagnostic test system that includes the prospective collection of whole blood and the reproducible detection of mRNA fusion transcripts by a PCR-based technology. The focus was on the detection of EML4-ALK transcripts from donors with and without previously diagnosed NSCLC. Pre-analytic complexity was reduced by restricting the handling of samples to 72 hours from time of sample receipt to test result. Recovery methods for RNA extraction from donor plasma were then optimized to enrich for RNA recovered from both circulating-free, and RNA within blood vesicles, including platelets and exosomes. A two-step reverse transcription and Droplet Digital™ PCR (RT-ddPCR) method was evaluated in detection testing using a multiplexed EML4-ALK assay that includes variants 1, 2 and 3. Analytic assay specificity and sensitivity was examined for RT-ddPCR efficiency using a cell-line positive for EML4-ALK variant 1 and in vitro RNA designed to mimic variants 1, 2, and 3. Analytic sensitivity was determined to be 0.2% of fusion RNA spiked into a background of normal plasma. Precision studies were conducted with two different amounts of input RNA (high and low), over three consecutive days, three runs in one day and with two operators. The SD of detected fusion transcripts in this study did not exceed 25%. Finally, normal (n = 10) and FISH positive NSCLC donor (n = 9) plasma samples were processed for the recovery of RNA and tested for EML4-ALK fusions. All FISH positive cases were accurately detected by the RT-ddPCR test with EML4-ALK variant copies ranging from 13 to 150 copies. EML4-ALK fusion transcripts were not identified in normal donor plasma. We conclude that the developed test system is highly suited for a 72 hour test to result turnaround, reproducible and sensitive detection of diagnostic fusion RNA variants, including EML4-ALK in blood in the clinical laboratory. Citation Format: Hestia Mellert, Leisa Jackson, Dianna Maar, Dawne Shelton, Gary Pestano. Diagnostic test system for sensitive, specific and reproducible detection of EML4-ALK RNA fusion transcripts in the blood of patients with 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 857.