Cell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented. Whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we characterized fragmentation patterns in urine cfDNA using whole-genome sequencing. Size distribution of urine cfDNA fragments showed multiple strong peaks between 40 and 120 base pairs (bp) with a modal size of 81- and sharp 10-bp periodicity, suggesting transient protection from complete degradation. These properties were robust to preanalytical perturbations, such as at-home collection and delay in processing. Genome-wide sequencing coverage of urine cfDNA fragments revealed recurrently protected regions (RPRs) conserved across individuals, with partial overlap with nucleosome positioning maps inferred from plasma cfDNA. The ends of cfDNA fragments clustered upstream and downstream of RPRs, and nucleotide frequencies of fragment ends indicated enzymatic digestion of urine cfDNA. Compared to plasma, fragmentation patterns in urine cfDNA showed greater correlation with gene expression and chromatin accessibility in epithelial cells of the urinary tract. We determined that tumor-derived urine cfDNA exhibits a higher frequency of aberrant fragments that end within RPRs. By comparing the fraction of aberrant fragments and nucleotide frequencies of fragment ends, we identified urine samples from cancer patients with an area under the curve of 0.89. Our results revealed nonrandom genomic positioning of urine cfDNA fragments and suggested that analysis of fragmentation patterns across recurrently protected genomic loci may serve as a cancer diagnostic.

A 10-year-old boy presented with spontaneous bruising and was found to have extreme thrombocytosis without neutrophilia/shift to immaturity, basophilia or eosinophilia. While the peripheral blood and bone marrow findings initially suggested essential thrombocythemia, BCR-ABL1 translocation was detected and chronic myeloid leukemia, chronic phase, was diagnosed. Apheresis for platelet depletion was performed as a bridge given the delayed effects of medical therapy.

Cell-free DNA (cfDNA) in plasma has been shown to be a promising analyte for noninvasive diagnostics. The collection of blood plasma requires venipuncture, and plasma volume obtainable at a single point is limited. In contrast, cfDNA in urine can be collected noninvasively, with minimal assistance and in larger volumes. However, so far there has been limited success in diagnostic development using urine cfDNA as urine cfDNA is highly fragmented, and whether the characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we perform a comprehensive characterization of fragmentation patterns in urine cfDNA using high-depth whole-genome sequencing from 30 healthy volunteers. We show the distribution of fragment sizes and genome-wide distribution of urine cfDNA fragments are consistent with transient protection from complete degradation by stable intermediates of nucleosome disassembly. Genome-wide nucleosome occupancy and fragment sizes in urine cfDNA are informative of the cell of origin and renal epithelial cells are among the highest contributors in urine. Based on a reference nucleosome map for urine cfDNA positioning, we developed a computational method to measure the fraction of urine cfDNA fragments with aberrant ends at unexpected genomic loci. We observe a higher fraction of fragments with aberrant ends in pediatric and adult cancer patients, distinguishing cancer samples with an area under the curve of 0.89. Our results demonstrate genomic architecture is preserved to an unexpected degree in urine cfDNA and are proof of principle that genome-wide fragmentation analysis of urine cfDNA can enable cancer diagnostics. This abstract is also being presented as Poster B12. Citation Format: Havell Markus, Jun Zhao, Tania Contente-Cuomo, Elizabeth Raupach, Ahuva Odenheimer-Bergman, Sydney Connor, Bradon McDonald, Elizabeth Hutchins, Marissa McGilvrey, Michelina C. de la Maza, Kendall Van Keuren-Jensen Van Jensen, Patrick Pirrotte Pirrotte, Ajay Goel, Carlos Becerra, Daniel D. Von Hoff, Scott Celinski, Pooja Hingorani, Muhammed Murtaza. Sub-nucleosomal fragmentation in urine cell-free DNA [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr PR14.