A novel RT-PCR approach to detecting EML4-ALK fusion genes in archival NSCLC tissue.

Abstract

10535 Background: The echinoderm microtubule-associated protein-like 4–anaplastic lymphoma kinase (EML4-ALK) fusion gene can be detected in subsets of NSCLC, especially never-smoking patients with adenocarcinoma, and appears to be mutually exclusive of EGFR mutation. PF-1066, a potent and selective c-MET/ALK inhibitor, yields high response rates in ALK-positive patients, as detected by labor-intensive fluorescent in situ hybridization (FISH) methodology. Existing RT-PCR assays for these gene variants are designed to amplify large cDNA fragments (> 450 bases), while formalin-fixed paraffin-embedded (FFPE) specimens yield mostly RNA fragments of < 150 bases. Thus, our goal was to design a robust RT-PCR assay for EML4-ALK fusion gene transcripts suitable for use with commonly available FFPE tissues of limited size. Methods: Synthetic fragments representing the nine EML4-ALK fusion genes variants 1, 2, 3a, 3b, 4, 5a, 5b, 6 and 7 were generated by recursive PCR technology. The approach consisted of amplifying over-lapping fragments containing the fusion variant sequences with outside 5′ and 3′ primers. Primer probes were designed to detect specific EML4-ALK fusion gene fragments with a maximum amplicon of 170 bases by RT-PCR. Results: Functional RT-PCR assay were developed for each specific EML4-ALK variant, meeting criteria specified above for optimal clinical testing. Assays detecting multiple fusion variants without distinguishing specific variants were also designed. All nine known EML4-ALK variants can be detected using 6 RT-PCR assays. Screening of NSCLC cDNAs from the Response Genetics database is underway (molecular and clinical correlations to be presented). Conclusions: We developed RT-PCR assays capable of detecting EML4-ALK fusion gene variants from FFPE tissues. Two assays were successfully established: one that can detect and identify each individual variant, and one capable of detecting the presence of any variant as a single assay. We expect this methodology to provide a useful tool for large-scale screening of NSCLC or other FFPE tissues for the EML4-ALK fusion gene. Author Disclosure Employment or Leadership Position Consultant or Advisory Role Stock Ownership Honoraria Research Funding Expert Testimony Other Remuneration Response Genetics Amgen, AstraZeneca, Biodesix, Boehringer Ingelheim, Bristol-Myers Squibb, Genentech, GlaxoSmithKline, Lilly, Merck, Novartis, Pfizer, sanofi-aventis Response Genetics DxS Abbott Laboratories, Bristol-Myers Squibb, Genentech, Lilly, Merck, Novartis, Pfizer