Abstract 4648: Development of a rapid, precise, and sensitive molecular assay for ALK fusion detection
Authors: Shahbazian Mona D. , Shang Yulei , Jamba Maidar , Powell Michael J. 
Published: July 2017

Abstract
Introduction In non-small cell lung cancer (NSCLC), chromosomal translocation events that result in overactivation of the anaplastic lymphoma kinase (ALK) have been identified and shown to play a key role in tumorigenesis. Inhibitors of the ALK kinase, such as the FDA-approved crizotinib, are effective in treating ALK-fusion-positive non-small cell lung cancer. Molecular studies have identified many ALK fusion variants in various cancer types, encompassing a combination of 22 different gene partners as well as various break points within the partner genes. The most common fusion events in non-small cell lung cancer are between the echinoderm microtubule associated protein like 4 (EML4) gene and the ALK gene. Fusion events between the Kinesin Family Member 5B (KIF5B) gene and ALK have also been identified. Existing assays for ALK fusion detection include the time-consuming, costly, and challenging fluorescence in situ hybridization (FISH) method, which is the current gold standard. Next generation sequencing approaches are also available, which are also costly to implement, difficult to interpret, and have lengthy workflows. Reverse-transcriptase PCR methods are ideal, as they are cost-effective, sensitive, rapid, and require little interpretation expertise. Methodology We designed a multiplexed real-time RT-PCR method to enable indiscriminate detection of ten EML4-ALK and three KIF5B-ALK fusion variants (encompassing approximately 95% of EML4-ALK and 90% of KIF5B-ALK characterized fusion variants, based on the Catalogue of Somatic Mutations in Cancer (COSMIC) database). The assay uses standard real-time PCR instrumentation and was characterized using the Bio-Rad CFX384™, Roche LightCycler® 96, and Roche LightCycler® 480 II instruments. Assay preparation and run time requires 2 hours or less. Sensitivity testing showed that for the Roche LightCycler® 480 II and Bio-Rad CFX384™, 100% detection was achieved for 50 copies of fusion template for each of the 13 targeted variants. The Roche LightCycler® 96 showed slightly poorer sensitivity, with 100% detection observed at 50 copies for 9 fusion variants and 100 copies for the remaining four variants. Inter- and intra-run precision testing demonstrated good reproducibility and repeatability, with a coefficient of variation of less than 10% for all targeted fusion variants. Using ALK-positive and ALK-negative FFPE reference materials, it was demonstrated that 50 ng of total FFPE RNA was sufficient and up to approximately 200 ng showed good performance. Conclusion In summary, we have developed a rapid, precise, and sensitive assay for the detection of thirteen EML4-ALK and KIF5B-ALK fusion events to enable molecular characterization of non-small cell lung cancer tumors.
Citation Format: Mona D. Shahbazian, Yulei Shang, Maidar Jamba, Michael J. Powell. Development of a rapid, precise, and sensitive molecular assay for ALK fusion detection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4648. doi:10.1158/1538-7445.AM2017-4648