Abstract 5109: Development of cfDNA reference standards for methylation-sequencing tests

Abstract Introduction: Recently, methylation sequencing has drawn significant attention as it has shown great promise for cancer detection and relapse surveillance. However, as with any low-input sequencing method, quality control is critical for implementation into the clinic, and it is difficult to obtain a sustainable source of patient samples for validation activities. To address these unmet needs, here we describe a design of fit-for-purpose cfDNA Methylation Reference Standards (MRS) to evaluate the performance of cfDNA-based methylation tests for oncology. Methods: We analyzed the genome-wide methylation landscape of 6 human cancer cell lines, 4 normal human lymphocyte cell lines, and 24 healthy donor cfDNA samples via whole genome bisulfite sequencing (WGBS), the gold standard of methylation analysis. Using cancer cell line genomic DNA cleaved by double-stranded DNA endonuclease (Zymo), we developed a range of cfDNA MRS spanning clinically relevant tumor fractions. DNA fragmentation were validated using the Labchip system (Perkin Elmer) and tumor fractions were independently verified by droplet digital PCR (Biorad). Results: The MRS series represent surrogates for lung, colorectal, liver, ovarian, pancreatic, and esophageal cancers. They display a size distribution comparable to cfDNA extracted from plasma, and represent a broad range of tumor fractions from 10% to 0.01%. In addition, the assembly accuracy was confirmed by digital droplet PCR (ddPCR) and ultra-deep mutation sequencing. Conclusions: These results demonstrated that the analytical feature and clinical relevance of MRS provides a versatile tool for assay optimization, validation, and cross-platform comparison. Citation Format: Bingsi Li, Jing Su, Jiayue Xu, Guangliang Zhang, Xiaoling Li, Ya Zhou, Hao Wang, Shuai Fang, Zhihong Zhang. Development of cfDNA reference standards for methylation-sequencing tests [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5109.