Abstract 5139: Development of novel copy number variation (CNV) reference materials in genome in a Bottle background

Abstract

Abstract Introduction: Advancements in tumor characterization by solid and liquid biopsy are instrumental in the evolution of personalized medicine. Recent improvements in assay sensitivity for profiling DNA variants such as single nucleotide polymorphisms (SNP) and indels have facilitated expansion of both solid and liquid biopsy applications. However, measurement of copy number variations (CNV) is a relatively new application, and few reference materials exist to aid in assay development and optimization. In this study, we demonstrate for the first time the development of 17 whole gene CNV reference standards in a background of the highly characterized NIST Genome in a Bottle GM24385 genomic DNA. Method: DNA molecules containing full genes of MET, ERBB2, ERBB3, PIK3CA, EGFR, BRAF, FGFR1, FGFR2, FGFR4, KIT, KRAS, MYC-N, PDGFRA, MDM2, CD274, MYC-L and MYC were each spiked separately into background GM24385 genomic DNA to generate 2.8 copies of each gene. Copy number for each of the 17 CNV samples was determined using Bio-Rad® Droplet DigitalTM PCR (ddPCRTM). To assess linearity over a range of copy numbers, MET and ERBB2 CNV ladder reference materials with copy gains at 6 different levels 3, 6, 9, 12 and 15 copies were also generated. To mimic circulating tumor DNA, each CNV sample was fragmented and size selected to recover a population containing ~170 bp size fragments. Copy number was also verified post size selection using ddPCRTM and the Ion S5TM XL system. Multiplexed triple CNV controls with MET, EFGR and ERBB2 were also developed at 3, 6, 9, 12 and 15 copies and analyzed by ddPCRTM. Results: Copy number for all 17 genes measured at 2.80 ± 0.28 copies by ddPCR. When tested in a ctDNA copy number ladder format, MET and ERBB2 showed good linearity of R2=0.99 for observed versus expected # of copies. Good correlation between ddPCR and NGS was also observed, with a slope of 1.004 for MET and 0.969 for ERBB2 across increasing copy numbers and standard deviation (SD) of ± 10%. Notably, SD increased at copy numbers higher than 12. The average size of fragmented ctDNA CNV reference standards were observed to be ~170 bp on the Agilent 2100 Bioanalyzer system. Finally, the multiplexed triple CNV control also performed as expected on ddPCR with SD of ± 10%. Conclusion: A novel method for producing CNV and ctDNA CNV controls has been developed that enables preparation of any copy gain level with a known gDNA background. Simpler QC materials mimicking patient samples will enable simpler CNV test method development and analytical validation, which will be critical for laboratories to introduce CNV solid and liquid biopsy testing into the field. Citation Format: Kunal Banjara, Lu Zheng, Samyuktha Dasari, Kara Norman. Development of novel copy number variation (CNV) reference materials in genome in a Bottle background [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5139.