Abstract 3243: Fragmentomic features of plasma cell-free DNA in patients with osteosarcoma

The rapid advancement of liquid biopsy technologies represents a major breakthrough in cancer management, allowing for sensitive detection of tumor-derived molecules and providing real-time insights into disease progression. Osteosarcoma (OS), the most common primary bone malignancy, currently lacks robust biomarkers for therapy response. We hypothesize that a multi-omic and multi-modal approach to identifying OS-specific markers in circulating tumor DNA (ctDNA) will facilitate precise patient stratification and improve treatment outcomes. It has been demonstrated by others that plasma ctDNA in OS patients carry specific genomic markers, such as point mutations and copy number alterations. However, the fragmentomic features of cell-free DNA (e.g., fragment length and nucleotide composition) remain poorly characterized in OS patients. To provide the rationale for studying fragmentomic features of plasma DNA in OS, we sought to demonstrate the feasibility of detecting distinct fragmentomic patterns in the publicly available whole genome sequencing data from cfDNA of 3 OS patients (NCBI SRA accession number PRJNA917431) together with our previously published shallow whole genome sequencing data from cell-free DNA of 7 patients with leiomyosarcoma, 12 patients with leiomyoma, 3 patients with dedifferentiated liposarcoma and 3 patients with well-differentiated liposarcoma (PMIDs: 29463554, 32232185, 34986184). We determined the first 4 nucleotide sequence (i.e., a 4-mer motif) on each 5′ fragment end of plasma DNA molecules. We calculated the frequency of each of the 256 possible motifs and normalized it to the total number of reads in each specimen. We applied multiclass differential analysis of the frequency of these motifs in 5 different types of tumors, and identified 64 motifs significantly enriched in OS (mean frequency difference between groups > 2.5-fold, false discovery rate < 0.05). In OS cell-free DNA, we observed decrease of motifs associated with DNASE1L3 nuclease (e.g., CCCA, CCAG, CCTG) and increase of motifs such as TAAA, AAAA, and TTTT. In the next steps, we will examine the expression of different DNA nucleases in patient tissue specimens by immunohistochemistry and will analyze plasma DNA from a new cohort of 50 OS patients to validate our preliminary findings. We will investigate whether these fragmentomic patterns of cell-free DNA have the predictive and prognostic value in OS patients. Our preliminary results warrant further studies of the frequency and diversity of the 4-mer motifs and expression of different DNA nucleases in OS. Citation Format: Anastasia E. Mackeracher, Joanna Przybyl. Fragmentomic features of plasma cell-free DNA in patients with osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 3243.