Abstract 6258: Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free DNA

Abstract Cell-free DNA (cfDNA) circulating in blood plasma arises primarily from cellular chromatin fragmentation and release due to cell death. Studies have demonstrated that the assessment of fragmentomic features of cfDNA enables gene expression inference and tissue-of-origin classification with potential applications for noninvasive cancer detection. However, due to low depth of coverage of sites of interest, current whole genome sequencing (WGS) methods have not been able to infer expression of individual genes or limited gene sets. Therefore, we developed a targeted sequencing approach that enables individual gene expression inference from cfDNA fragmentation patterns based on transcriptional start-site gene activation probability (TSS-GAP). To assess the sensitivity of this method for cfDNA-inferred gene expression profiling, we applied TSS-GAP to blends of micrococcal nuclease (MNase) -digested DNA from a cancer cell line (LS180) and sorted peripheral blood mononuclear cells (PBMC), with LS180 DNA at varying concentrations (0. 1-10%). This enabled the determination of the limit of detection (LoD), relative to healthy donor background levels, on the basis of the genes’ activation probabilities (TSS-GAP scores). We found 936 genes with high inferred expression in the LS180 cancer cell line and low inferred expression in non-cancer samples (i. e. CD4, CD14, and plasma cfDNA from 4 healthy donors) and used those for healthy donor background and LoD assessment. Using these gene-level background values as cutoffs, blends were assessed to determine the lowest blend level at which expression of the gene was able to be differentiated from background. Out of the 936 genes, 326 (34. 8%) were detectable at a blend level of 0. 1%, 59 (6. 3%) at 0. 3%, 31 (3. 3%) at 1%, 19 (2. 0%) at 3%, and 39 (4. 2%) at 10%. In total, 416 genes could be detected by TSS-GAP at levels 1% of or lower above background, highlighting the potential of our method for detecting expression of certain genes even from a small fraction of cells contributing cfDNA to liquid biopsy samples. Further expansion of this work could consider the utility of such a method for circulating tumor DNA (ctDNA) detection, cfDNA tissue-of-origin classification, and deconvolution of cell types contributing to cfDNA found in blood. Citation Format: Emily Leff, Alexander Tseng, Victoria Cheung, Ehsan Tabari, Kimberly Walter, Alex Lovejoy, Cheng-Ho J. Lin. Inference of gene expression using fragmentation patterns from targeted high-depth sequencing of cell-free 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 (6Suppl): Abstract nr 6258.