Detection of EML4-ALK in serum RNA from lung cancer patients using MassARRAY platform.

Abstract

10569 Background: The presence of the transforming fusion gene EML4-ALK in non–small cell lung cancer (NSCLC) is a predictive marker for the efficacy of ALK kinase inhibitors. The break apart fluorescence in situ hybridization (FISH) assay has served as the standard test for identification of NSCLC patients with ALK gene rearrangement. Circulating RNA in plasma/serum is an emerging field for noninvasive molecular diagnosis. We previously developed a MassARRAY assay to detect nine types of EML4-ALK fusion gene (variants 1, 2, 3a, 3b, 4, 5a, 5b, 6, and 7). The assay is based upon region-specific PCR followed by specific single-base primer extension reactions; the extended products are resolved using MALDI-TOF mass spectrometry. In this study, we evaluated whether EML4-ALK fusion RNA could be detected in serum obtained from NSCLC patients. Methods: The RNA was extracted from serum obtained from 12 lung cancer patients with ALK rearrangements by FISH analysis. The extracted RNA was reverse transcribed into cDNA, and the resultant cDNA was amplified by nested PCR. The PCR product was subjected to MassARRAY assay to identify EML4-ALK variants. Results: The EML4-ALK RNA (variant 1 and 3) was detected in serum from 2 of 12 patients. These are confirmed by subcloning and sequencing. Conclusions: The detection of EML4-ALK in serum RNA samples by MassARRAY assay is feasible. This noninvasive assay will be useful in patients with insufficient or unavailable tumor specimens. Additional experiments will reveal the sensitivity and specificity of this detection system.