Abstract 974: DNA methylation and gene expression as determinants of genome-wide cell-free DNA fragmentation
Authors: Noë Michaël , Annapragada Akshaya V. , Foda Zachariah H. , Medina Jamie E. , Mathios Dimitrios , Cristiano Stephen , Cherry Christopher , Bruhm Daniel C. , Niknafs Noushin , Adleff Vilmos , Ferreira Leonardo , Easwaran Hari , Baylin Stephen , Phallen Jillian , Scharpf Robert B. , Velculescu Victor E. 
Published: March 2024

Cell-free DNA (cfDNA) in the bloodstream is increasingly gaining attention as a diagnostic tool for the early detection of cancer. Nevertheless, the characteristics and sources of cfDNA fragmentation in the blood remain poorly understood. In this study, we sought to unravel the impact of DNA methylation and gene expression on the naturally occurring genome-wide fragmentation of cfDNA. We performed a comprehensive analysis of plasma samples from 969 individuals, including 182 individuals diagnosed with cancer (pancreatic, colorectal, ovarian, lung and breast cancer). In healthy individual cfDNA fragmentation patterns like DNA motifs at cfDNA fraqgments ends, recurrent cfDNA fragment start sites, cfDNA coverage and cfDNA fragment size were compared to publicly available data on cfDNA methylation and gene expression from myeloid cells. We discovered that the ends of cfDNA fragments, particularly those containing CGs or CCGs, displayed a distinct pattern of enrichment or depletion, respectively, at methylated CpG positions throughout the genome. This phenomenon is consistent with structural models suggesting an increased interaction of CG fragment ends with nucleosomes. cfDNA fragments were more prevalent (up to 3.7 fold) and of larger sizes (up to 4-5 base larger) when derived from regions of methylated CpGs or transcriptional start sites of genes that were not actively expressed. Differences in cfDNA fragmentation reflected specific biological pathways associated with their tissue of origin (like E2f transcription factors and blood cell metabolism genes in cfDNA from healthy individuals). Through analyses of cfDNA from patients with cancer, we identified a connection between tumor-related hypomethylation, increased gene expression, and a global reduction in cfDNA fragment size. This connection may offer an explanation for the generally smaller cfDNA fragments found in individuals with cancer. We found that cancer-specific methylation at CpG sites from pancreatic cancers were associated with widespread changes in cfDNA fragment ends, particularly for patients with pancreatic (test statistic = Welch two sample t-test; p&lt;2.2e-16) and other cancers. These findings establish a direct link between epigenetic modifications and cfDNA fragmentation that may have implications for non-invasive detection of cancer.

Citation Format: Michaël Noë, Akshaya V. Annapragada, Zachariah H. Foda, Jamie E. Medina, Dimitrios Mathios, Stephen Cristiano, Christopher Cherry, Daniel C. Bruhm, Noushin Niknafs, Vilmos Adleff, Leonardo Ferreira, Hari Easwaran, Stephen Baylin, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. DNA methylation and gene expression as determinants of genome-wide cell-free DNA fragmentation [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 974.