Abstract 3728: Two novel EML4-ALK fusion variants involving EML4 exon 17 identified in lung cancer
Authors: Li Hai-Rong , Sanders Heather R. , Sferruzza Anthony D. , Novic Connie , Meloni-Ehrig Aurelia M. , Albitar Maher 
Published: February 2011

Abstract
Introduction: Recently, anaplastic lymphoma kinase (ALK) inhibitors have been used successfully in patients harboring gene fusions between echinoderm microtubule-associated protein-like 4 (EML4) and ALK. These fusions result from a paracentric inversion on chromosome 2 inversion [inv(2)(p21;p23)] and have been identified in 3-7% of all non-small cell lung cancer (NSCLC) cases. To date, 11 variants have been published involving 7 different EML4 exons and invariably involving exon 20 of ALK. To screen for EML4-ALK abnormalities, we developed a multiplex RT-PCR exon screening approach that can detect variants initiating at any of the first 22 EML4 exons. Using this approach we detected both known and novel EML4-ALK fusion variants. We identified 2 novel EML4-ALK fusion variants involving exon 17 of EML4 from a single patient.
Methods: Sixty-one samples were analyzed with the multiplex assay: 56 formalin-fixed paraffin-embedded (FFPE) NSCLC samples, 4 cell line samples (3 NSCLCs; 1 Prostate carcinoma), and 1 control RNA (Human Total RNA, Applied Biosystems). After RNA extraction, RT-PCR was performed using the RNA UltraSense™ One-Step qRT-PCR System (Invitrogen). Twenty-three primers (22 unlabeled EML4 forward; 1 FAM- labeled ALK reverse) were included in 4 master mixes to amplify EML4-ALK fusions initiating within the first 22 EML4 exons to ALK exon 20; 1 endogenous control (beta-2-microglobulin) primer set was included in a separate reaction. After RT-PCR, the PCR products were separated by capillary electrophoresis on a genetic analyzer (ABI 3730, Applied Biosystems) and the fusions identified based on size (bp). Samples with positive results were further analyzed by singleplex RT-PCR to confirm exon involvement and novel fusions were confirmed by sequencing.
Results: One NSCLC cell line was positive for EML4-ALK variant 3a and 3b and 2 NSCLC cell lines were negative, consistent with literature findings. The prostate cancer and control RNA were negative. Two unexpected peaks resulted from multiplex and singleplex RT-PCR reactions containing EML4 exon 17 forward and ALK exon 20 reverse primers. Sequencing revealed 2 previously undescribed variants. One variant (8a) consisted of a complete EML4 exon 17 fused to a partial intron 19-20 and complete exon 20 of ALK. The second variant (8b) consisted of a complete exon 17 with partial intron 17-18 of EML4 fused to the same ALK region as in variant 8a. Overall, 9% (5/56) of lung cancer tumor tissue (FFPE) were positive for EML4-ALK fusions (variants 3a and 3b; 2/56); variant 3a only (1/56), variant 1 (1/56), and novel variants 8a and 8b (1/56)).
Conclusions: This study demonstrates that proper testing for EML4-ALK fusion in lung cancer should encompass at least most of the first 22 exons of the EML4 gene. Encompassing multiplex RT-PCR assays may increase the detection prevalence of EML4-ALK fusion with the detection of new fusion variants as demonstrated in our small series of patients.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3728.