Multiplexed, Digital Gene Expression And Fusion Transcript Analysis To Screen For Eml4-Alk Positive Lung Cancer

Purpose: EML4-ALK fusions occur in approximately 5% of non-small cell lung carcinoma and define a subpopulation of lung cancer patients highly responsive to ALK kinase inhibitors. Current methodologies for detecting presence of ALK rearrangements are labor-intensive, costly, and not ideally suited for screening large number of patient samples. To allow for a sensitive, facile, and inexpensive methodology to detect EML4-ALK fusions, we developed a direct transcript profiling to detect common variants of EML4-ALK fusions. Experimental Design: Using a single multiplex assay, we simultaneously interrogated presence of EML4-ALK transcripts and ALK 3′ over-expression in 8 ALK-positive and 22 ALK-negative NSCLC samples previously tested by fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC). For controls, we also tested ALK-positive cancer cells lines NCI-H3122 and NCI-H2228, and an ALK-negative cell line A549. Purified RNA from formalin-fixed paraffin-embedded (FFPE) tissue sections was hybridized in duplicates to a biotinylated capture probe and a color-coded reporter probe designed to hybridize to sequences spanning EML4-ALK junction. In addition, multiple probes were created to hybridize within 5′ and 3′ regions of ALK transcript to detect discordant levels between the breakpoint, indicative of fusion event. Purified hybridization complexes are immobilized and aligned on a cartridge where a microscope CCD camera images and counts the molecular color-coded tags attached to reporter probes. The number of reporter counts corresponds to the number of transcripts being interrogated. Results: With this assay, we obtained 100% concordance in EML4-ALK fusion calls to results generated by FISH and IHC analyses. Similar findings were obtained for control cancer cell lines. The assay is highly reproducible and sensitive, detecting ALK-fusion transcripts even in samples with low tumor content. Using the combined strategy of fusion detection and ALK 5′ and 3′ transcript quantification, we were able to validate the status of one patient negative for ALK rearrangement by FISH but positive for ALK protein expression by IHC. Conclusions: We have developed a novel and sensitive methodology to screen for common EML4-ALK fusions in NSCLC. The assay is inexpensive, easy to perform, high-throughput and compatible with FFPE tissue samples. This is a promising technology highly suitable for screening large numbers of tumor samples without the need for cDNA synthesis and PCR amplification. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4553. doi:1538-7445.AM2012-4553