Research ArticleCancer

Analysis of recurrently protected genomic regions in cell-free DNA found in urine

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Science Translational Medicine  17 Feb 2021:
Vol. 13, Issue 581, eaaz3088
DOI: 10.1126/scitranslmed.aaz3088

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Detecting cancer by urine cell-free DNA

Detection of cell-free DNA (cfDNA) in urine could be a noninvasive approach to diagnose cancer. However, urine cfDNA is very fragmented, making it difficult to use. Markus et al. analyzed fragmentation patterns in urine and plasma cfDNA using whole-genome sequencing in healthy individuals and those with cancer. Compared to cfDNA from healthy individuals, tumor-derived fragmentation patterns ending within recurrently protected regions occurred more frequently in urine. By comparing genome-wide differences in urine cfDNA fragmentation patterns, the authors could distinguish cancer samples from controls, suggesting that this approach might complement plasma cfDNA as a cancer diagnostic.


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.

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