Abstract The properties of cancer-associated genetic changes in cell-free DNA (cfDNA) are not fully understood. We performed whole-genome sequencing (WGS) of cfDNA as well as tumor tissue and white blood cells (WBCs) from 1,807 samples of 1,064 patients across eight common cancer types. Characterization of single base substitutions, small insertions and deletions, structural variants (SVs), and phased variants in single cfDNA molecules revealed unique properties of tumor-derived alterations as well as differences in error rates that spanned orders of magnitude. Given the low error rate associated with detection of tumor-specific rearrangement junctions in cfDNA, we hypothesized that these types of changes could enable detection of circulating tumor DNA (ctDNA) without prior knowledge of the alterations in the tumor tissue. As an example of this approach, we scanned each sequenced fragment genome-wide in cfDNA samples from the CheckPAC trial of patients with metastatic pancreatic cancer treated with radiation and immunotherapy to identify putative rearrangement junctions. We identified 22,010,911 such fragments but only 1,572 (0.007%) and 58,339 (0.27%) of these were present in the matched tumor or WBC samples, respectively, with the remaining identified only in cfDNA. We characterized each cfDNA fragment by the SV type, SV size, microhomology and insertion at the breakpoint junction, fragment size, and the location of the breakpoint with respect to the nearest fragment end, identifying differences depending on the origin of the SV. Machine learning analyses of SVs from cfDNA resulted in a high cross-validated performance for detection of tumor-specific SVs with an area under the curve (AUC) of 0.97 (95% CI: 0.97-0.98). After enriching for fragments most likely to be tumor-derived, we found that the number of cfDNA fragments containing SVs was highly correlated with the number obtained using a tumor-informed approach (Pearson correlation coefficient = 0.87, p0.001), and could recapitulate longitudinal ctDNA levels and clinical outcomes using only low-coverage (∼4x) plasma WGS. The universal nature of tumor-associated sequence and structural alterations in cfDNA may be broadly useful for cancer detection. Citation Format: Daniel C. Bruhm, Carolyn Hruban, Adrianna L. Bartolomucci, Akshaya V. Annapragada, Sarah Short, Shashikant Koul, Kaui P. Lebarbenchon, Julia S. Johansen, Inna M. Chen, Andrei Sorop, Razvan Iacob, Speranta Iacob, Liana Gheorghe, Simona Dima, Katherine A. McGlynn, Manuel Ramírez-Zea, John Groopman, PLCRC-MEDOCC group, Remond J. Fijneman, Gerrit A. Meijer, Zachariah H. Foda, Jillian Phallen, Robert B. Scharpf, Victor E. Velculescu. Sequence and structural DNA alterations in the circulation of patients with cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 2591.
Bruhm et al. (Fri,) studied this question.