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. Currently available assays for detection of the different variants of EML4-ALK have limitations, however.Methods
We developed an assay system for the detection of EML4-ALK variant 1, 2, 3a, 3b, 4, 5a, 5b, 6, or 7 transcripts in total RNA obtained from formalin-fixed, paraffin-embedded (FFPE) specimens of NSCLC tissue. The assay is based on region-specific polymerase chain reaction (PCR) amplification of EML4-ALK cDNA followed by specific single-base primer extension and analysis of the extension products by MALDI-TOF mass spectrometry. The assay was validated by fluorescence in situ hybridization (FISH) and the results confirmed by subcloning and sequencing of PCR products.Results
Evaluation of the analytic sensitivity of the assay with serial dilutions of plasmids containing EML4-ALK cDNA sequences revealed it to be capable of the reliable detection of one copy of each plasmid per reaction. The assay also detected EML4-ALK variants 1 or 3 in three FFPE samples of surgically resected NSCLC shown to be positive for ALK rearrangement by FISH. Furthermore, the assay identified variant 1 of EML4-ALK in 3 of 20 FFPE biopsy samples from patients with advanced NSCLC. All positive samples were confirmed by subcloning and sequencing.Conclusions
Our novel assay is highly sensitive and effective for the detection of EML4-ALK in FFPE specimens.