Abstract 4540: Development of well characterized breast, lung, and brain cancer copy number variation reference materials

Abstract

Abstract Introduction: Copy number variation (CNV), including gene amplification and deletion, can be a key driver of oncogenesis. Pathogenic CNVs are often associated with unfavorable prognosis and drug resistance, therefore detection of these types of genetic changes will be important for personalized treatment. Many tumor profiling workflows can detect CNVs in addition to somatic mutations, but well-characterized reference materials for CNVs are not widely available, which makes it difficult to assess accuracy and sensitivity. CNV content of cell lines can be variable and may change with passages, the genomic background may not be diploid, and germline and somatic variants may not be well characterized. Remnant patient samples often lack characterization and the volumes needed for such characterization and larger validation studies. Therefore, we developed and evaluated Seraseq CNV reference materials to serve as accuracy, precision, and limit-of-detection (LOD) controls for tumor profiling assay development and validation. By providing a consistent source of purified DNA with stable CNV content, these reference materials allow for repeatable assessment of clinical diagnostic assays targeting CNVs. Methods: Seraseq CNV reference materials were prepared by mixing genomic DNA with additional copies of target genes from the well-characterized cell line GM24385. Copies of ERBB2, FGFR3, and MYC were combined with genomic DNA to simulate CNVs found in breast cancer, while sequences for EGFR, MET, and MYCN were used to mimic lung and brain cancer CNVs. Three levels of amplification were targeted: +3, +6, and +12 additional copies. The copy number of each gene was precisely quantified using digital PCR and confirmed on several NGS-based assays internally and externally. Results & Conclusions: Testing revealed CNVs of each target gene were close to the expected copy number. Good correlation was observed between digital PCR data and NGS data generated internally with the Archer VariantPlex Solid Tumor assay. In addition, internal and external NGS data showed correlation and broad assay compatibility. While CNVs observed clinically can be upwards of 50 additional copies and more easily detectable, LOD controls such as those described here are imperative in evaluating assay performance. Collectively, the data demonstrate the utility of biosynthetic Seraseq CNV reference materials, which serve as superior alternatives to DNA from cell lines or patient-derived material. Citation Format: Dana Ruminski Lowe, Jessica Dickens, Catherine Huang, Yves Konigshofer, Bharathi Anekella. Development of well characterized breast, lung, and brain cancer copy number variation reference materials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4540.