A digital PCR based assay to detect all ALK fusion species

Accurate detection of Anaplastic lymphoma kinase (ALK) fusion in lung cancer cells is essential to screen for patients suitable for targeted drug treatments such as crizotinib. ALK fusion involves multiple genes and different exon junctions. When an ALK fusion results in overexpression of the ALK protein, a malignant transformation can occur. The challenge is to develop a single test that will detect ALK overexpression from all fusion combinations including novel/unidentified fusion partner(s). In this study, we evaluated two strategies for detecting ALK fusion events using digital PCR: 5′/3′ imbalance and ALK overexpression relative to a reference gene. Our data shows that the determination of ALK RNA expression levels is a better option when a reference gene is included in the assay. We further determined the threshold to call for positive or negative samples and evaluated the analytical specifications of the assay in 28 FFPE samples from Non-small cell lung cancer (NSCLC) patients with know ALK fusion status. We validated this threshold with 36 clinical samples with ALK status determined by IHC. Digital PCR ALK assay we developed had a concordance of 97.2% (35/36). Testing the assay on clinical samples demonstrated consistency with reference assays, suggesting a great potential for the dPCR assay to service the clinical detection needs.