Abstract 7030: Reference materials for analysis of DNA methylation in cell-free circulating tumor DNA
Authors: Higgs Alysha , Ryan Angelique , Ramprakash Jayanthi , Lowe Dana Ruminski , Konigshofer Yves , Huang Catherine , Anfora Andrew , Garlick Russell , Anekella Bharathi 
Published: March 2024

Abstract
Aberrant DNA methylation is associated with many cancers including breast, liver, bone, and colon and has been identified as a biomarker for early cancer screening. Liquid biopsies are beginning to screen for DNA methylation in cancer-derived DNA, but accurate assessment of methylation is not trivial, and methods like bisulfite sequencing can result in biased data. Reference materials for establishing the analytical validity of measurements of CpG methylation are not widely available. In order to prepare such reference materials, genomic DNA (gDNA) was extracted from the well characterized human reference cell line GM24385 and fragmented to a circulating tumor DNA (ctDNA)-like size distribution. This DNA was processed further to prepare CpG-methylated and unmethylated versions. The size distribution was determined by using the Bioanalyzer High Sensitivity DNA Kit (Agilent, Santa Clara, CA). Methylation status in the reference materials was confirmed by both digital PCR (dPCR) and NGS based methods. A digital PCR method was developed to analyze heterozygous single nucleotide polymorphisms (SNPs) using the Bio-Rad QX200™ Droplet Digital PCR (ddPCR) System. To determine methylation levels by NGS, the NEBNext™ Enzymatic Methyl-seq Kit was used (EM-seq). Paired-end sequencing of the libraries was performed on a NextSeq™ 2000 (Illumina, San Diego, CA). Data alignment to hg19 was done with the bwa-meth (Pedersen, BS., et al.) aligner, and assessment of methylation in the aligned data was done with MethylDackel (Ryan, D.). Each reference material had an average peak size of ~160 bp in length, similar to patient cfDNA. By dPCR, the unmethylated reference material had an average of 0.73% DNA methylation, while the methylated reference material had an average of 100.10% DNA methylation. Values can be above 100% by dPCR due to unequal representation of both alleles. By NGS, the unmethylated reference material had an average of 0.93 % CpG methylation, which was similar to the background of 1.13 % and 1.31 % for CHG and CHH methylation, respectively. The methylated reference material had an average of 93.95 % CpG methylation, with a background of 0.69 % and 0.74 % for CHG and CHH methylation, respectively. Because EM-seq relies on enzymatic protection and conversion steps, it may be that these reactions did not allow for full protection and full conversion, which led to values above 0 % for unmethylated and below 100 % for methylated materials. We have developed reference materials for the assessment of DNA methylation in ctDNA. One unmethylated and one methylated reference material was created. These reference materials can be mixed together to the level of methylation required to help aid in assay design, optimization, and validation.
Citation Format: Alysha Higgs, Angelique Ryan, Jayanthi Ramprakash, Dana Ruminski Lowe, Yves Konigshofer, Catherine Huang, Andrew Anfora, Russell Garlick, Bharathi Anekella. Reference materials for analysis of DNA methylation in cell-free circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7030.