Abstract 2290: Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies

Abstract Multiple studies have shown that the plasma cell-free DNA (cfDNA) from cancer patients differs from healthy individuals in both fragment length and fragment end motifs (FEMs). Yet, there is a lack of understanding regarding how the two factors combined can be associated with cancer and gene transcription. In this study, we evaluated cfDNA fragmentomics in plasma from lung cancer patients (n = 17) and healthy individuals (n = 7) using targeted sequencing (lung cancer: n = 12, healthy: n = 7) and whole-genome sequencing (WGS) (lung cancer: n = 5). A personal gene expression profile was established with H3K36me3 cell-free chromatin immunoprecipitation sequencing (cfChIP-seq). cfChIP-seq isolates nucleosomes with histone marks of active transcription directly from plasma, meaning cfDNA enrichment with cfChIP reflects gene activity in the cells of origin. Short cfDNA fragments were isolated using in vitro size-selection. Somatic mutations were identified from input cfDNA through the AVENIO pipeline. The findings were validated in an external public dataset comprising of WGS-cfDNA data from healthy individuals. Cancer patients contain a larger fraction of short (<150bp) cfDNA fragments compared to healthy individuals. Cancer patients also demonstrate an enrichment of distinct FEMs compared to healthy individuals. Investigating the association between gene activity and fragment lengths revealed that genes with high expression display an enrichment of short cfDNA fragments (median = 19.99%, IQR: 16.94% - 27.13%, P < 0.0001) compared to genes with low expression. Furthermore, distinct GC-rich FEMs are enriched in active genes. Combining the frequency of short cfDNA fragments with the presence of distinct FEMs results in a further enrichment of the highest expressed genes (median = 37.85%, IQR: 30.10% - 39.49%, P < 0.0001). In vitro size selection of short cfDNA also results in enrichment of GC-rich FEMs. In vitro size-selection can isolate cfDNA representing active genes and in vitro size-selection enrichment correlates with the cfChIP-seq enrichment (Spearman’s ρ range: 0.499-0.882). This study expands the knowledge regarding cfDNA fragmentomics. We document how cancer patients and healthy individuals have different fragmentomic features. In addition, we shed new light on how gene activity in the cells of origin is associated with cfDNA fragment lengths and FEMs. Together, the results can increase the utility of liquid biopsies by determining gene activity in cancer patients. Citation Format: Christoffer Trier Maansson, Louise Skov Thomsen, Peter Meldgaard, Anders Lade Nielsen, Boe Sandahl Sorensen. Integration of cell-free DNA end motifs and fragment lengths can improve identification of active genes in liquid biopsies [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 2290.