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

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 ...