Rapid detection and genotyping of ALK fusion variants by adapter multiplex PCR and high-resolution melting analysis

Anaplastic lymphoma kinase (ALK) fusion is a promising predictive biomarker of ALK-tyrosine kinase inhibitor (ALK-TKI) treatment. Furthermore, different fusion variants correlate to different ALK-TKIs responses. Although variant identification assists in treatment direction, most ALK detection assays do not genotype different fusion variants. We developed a high-resolution melting (HRM) assay to rapidly detect ALK fusions and automatically distinguish at least 20 fusion variants in one tube. Adapter multiplex PCR was designed to amplify ALK fusion variants and the reference gene GAPDH. After HRM, negative derivative curves showed a low temperature GAPDH peak, and if an ALK fusion was present, a high temperature peak from the ALK segment and variably a middle temperature part associated with the fusion partner. Selected regions of the second derivative curves were analyzed to extract features (∆T_m, PTS/ITS, H₁/H₂) that define two curve types (monotonic and non-monotonic). Synthetic samples of 20 ALK fusion variants were used to train a quadratic discriminate analysis model, and the accuracy was 97.06% (66/68) and 85.71% (144/162) for monotonic and non-monotonic variants, respectively. The limit of detection of the assay was 1%. The analytical sensitivity of genotyping was 1 and 5% for monotonic and non-monotonic variants, respectively. In a blinded study, we detected ALK fusion from formalin-fixed paraffin-embedded lung cancer samples with a 100% 47) and genotyping /47) and genotyping (7/7). Multiplex adapter HRM is a simple, fast, and sensitive way of ALK fusion detection and genotyping. Automatic genotyping with parameters extracted from second derivative curves is a promising method that may be applicable to other types of gene variants detected by HRM.