Abstract 3961: Methodological considerations in the preparation of biomimetic reference materials for ctDNA assays

Clinical utilization of cell-free DNA (cfDNA) has become commonplace for NIPStesting and has recently been more widely deployed to quantitate cell-free circulating tumor DNA(ctDNA) for use in cancer patient care longitudinally across the treatment spectrum from subclinical, to diagnosis and through treatment. The scarcity and unstable nature of the cfDNA analyte in human plasma is problematic and thus the lack of robust detection methods has limited clinical validation. In this work we sought to avoid the inherent limitations of plasma-based patient reference materials and developed a strategy to prepare a biomimetic reference material which is sufficiently equivalent to the cfDNA found in patients with malignant cancers. Thus far the scarcity and physical form of ctDNA in human plasma has attenuated the validation of assays for clinical utility. To this end, we have generated a biosynthetic construct with short DNA fragments containing relevant cancer mutation sequences, including single-nucleotide variations, insertions and deletions in BRAF, EGFR, KRAS, NRAS, PIK3CA or HER/ERBB2. The mutant fragments were titrated into normal human reference DNA, GM24385, and ultrasonically sheared to ∼160 bp. Precise allele frequency blends were verified by digital PCR (dPCR) prior to encapsulation to enhance stability and compatibility with commutable plasma. The reference material is formulated to 20 ng/mL of extractable DNA in modified Seracare Matribase™ to confer commutability in pre-analytic processes. These materials have been quantitated by dPCR and/or Next Generation Sequencing (NGS). A dilution panel was prepared at 10%, 5%, 2.5%, 1.25%, 0.1% allele frequency (mutant:normal DNA). Linearity (R 2 = 0.9997) was observed by dPCR (measured vs. expected) and the NGS detected, Swift Accel-Amplicon 56G Oncology Panel (R 2 = 0.997). Due to the low abundance of ctDNA, a characterized reference set is critical to validate assays that are developed with extremely low limit of detection. Data will be presented to show the breadth of this methodology and its utility in validating NGS and dPCR assays. Citation Format: Seth B. Harkins, Farol L. Tomson, Bharathi Anekella, Russell Garlick. Methodological considerations in the preparation of biomimetic reference materials for ctDNA assays. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3961.