A high multiplex assay to quantify copy number variation in the FGFR1, 2, 3 genes and chromosome 11q in a single reaction

• Published in: European journal of cancer (1990) 2016-12, Vol.69, p.S134-S134
• Main Authors: Madanahally Divakar, K, Boehme, M, Korn, K
• Format: Article
• Language: English
• Subjects: Amplification; Angiogenesis; Assaying; Biomarkers; Chromosome 11; Chromosome translocations; Chromosomes; Computer simulation; Copy number; Data analysis; Data processing; Deoxyribonucleic acid; Diagnostic systems; DNA; Fibroblast growth factor 3; Fibroblast growth factor receptor 1; Fibroblast growth factor receptors; Genes; Hematology, Oncology and Palliative Medicine; Homeostasis; Mathematical analysis; Metabolism; Multiplexing; Mutation; Nucleic acids; Organogenesis; Polymerase chain reaction; Precision medicine; Primers; Run time (computers); Sensitivity analysis; Studies; Variation
• ISSN: 0959-8049; 1879-0852
• DOI: 10.1016/S0959-8049(16)32998-7

Background: The fibroblast growth factor receptor (FGFR) pathway plays a major role in regulating several basic biologic processes, from organogenesis to metabolism homeostasis, and angiogenesis. Aberrations, including gene amplifications, point mutations, and chromosomal translocations, in FGFR or FGF genes result in an oncological event which has diagnostic value in personalized medicine. Here, we report development of a highly multiplex quantitative assay to detect copy number variation (CNV) in the human FGFR (receptor) oncogenes 1, 2, and 3, and FGF (ligands) 3, 4, and 19 (Chromosome 11q) from FFPE specimen on the fully automated Modaplex platform. Methods: Primers were designed using proprietary technology to amplify FGFR 1. 2. 3. Chromosome 11q, and three reference genes from FFPE extracted DNA. Reaction conditions were optimized using proprietary PCR chemistry. Assay development was carried out using commercially extracted nucleic acid. The CNV was simulated using synthetic targets (gBlocks) spiked-in to wild-type genomic DNA. DNA extracted from FFPE specimen was tested. Results: The Modaplex CNV panel can detect copy number variation in FGFR1, 2, 3 and. chromosome 11q. Assay designed to include two amplicons for each of FGFR1. 2, and 3 genes, and one amplicon each for FGF3, 4, 19 (in total three amplicons for Chromosome 11q) in order to provide reliability in CNV calculation. Reference genes were characterized for their ability to serve as normalization genes to be used in CNV calculation. The total run time including data analysis and setup is less than 4 hours. Analytical sensitivity studies demonstrated the assay is capable of detecting and quantifying 2 fold increase in copy number variation in the genes of interest. Dynamic range and PCR efficiency for each assay target were measured and a quantification algorithm was developed to measure CNV. Finally, the assay was tested on known wild type FFPE DNA to establish cut-off for CNV calculation. Conclusion: The Modaplex FGFR-CNV assay provides a simple and sensitive method of detection of CNV of the FGFR and FGF genes. The assay can be performed on a single platform with a fast turn-around time of 4 hrs. The Modaplex FGFR CNV Panel offers an excellent tool for use in biomarker discovery and screening of prognostic markers from biological specimens.