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

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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Sub-nucleosomal organization in urine cell-free DNA

10.1101/696633 ◽

2019 ◽

Author(s):

Havell Markus ◽

Jun Zhao ◽

Tania Contente-Cuomo ◽

Elizabeth Raupach ◽

Ahuva Odenheimer-Bergman ◽

...

Keyword(s):

Nucleosome Occupancy ◽

Area Under The Curve ◽

Wide Distribution ◽

Cancer Diagnostics ◽

Cell Of Origin ◽

Cell Free Dna ◽

Noninvasive Diagnostics ◽

Free Dna ◽

Genome Wide ◽

Fragmentation Patterns

AbstractCell-free DNA (cfDNA) in urine is a promising analyte for noninvasive diagnostics. However, urine cfDNA is highly fragmented and whether characteristics of these fragments reflect underlying genomic architecture is unknown. Here, we perform comprehensive characterization of fragmentation patterns in urine cfDNA. We show modal size and genome-wide distribution of urine cfDNA fragments are consistent with transient protection from degradation by stable intermediates of nucleosome disassembly. Genome-wide nucleosome occupancy and fragment sizes in urine cfDNA are informative of cell of origin and renal epithelial cells are amongst the highest contributors in urine. Compared to a nucleosome occupancy map based on control urine samples, we observe a higher fraction of fragments with aberrant ends in cancer patients, distinguishing cancer samples with an area under the curve of 0.89. Our results demonstrate sub-nucleosomal organization in urine cfDNA and are proof-of-principle that genome-wide fragmentation analysis of urine cfDNA can enable cancer diagnostics.

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The use of plasma donor-derived, cell-free DNA to monitor acute rejection after kidney transplantation

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfz091 ◽

2019 ◽

Vol 35 (4) ◽

pp. 714-721 ◽

Cited By ~ 10

Author(s):

Els M Gielis ◽

Kristien J Ledeganck ◽

Amélie Dendooven ◽

Pieter Meysman ◽

Charlie Beirnaert ◽

...

Keyword(s):

Acute Rejection ◽

Serum Creatinine ◽

Characteristic Curve ◽

Area Under The Curve ◽

Threshold Value ◽

Nucleotide Polymorphisms ◽

Kidney Transplant Recipients ◽

Cell Free Dna ◽

Free Dna ◽

Set Up

Abstract Background After transplantation, cell-free deoxyribonucleic acid (DNA) derived from the donor organ (ddcfDNA) can be detected in the recipient’s circulation. We aimed to investigate the role of plasma ddcfDNA as biomarker for acute kidney rejection. Methods From 107 kidney transplant recipients, plasma samples were collected longitudinally after transplantation (Day 1 to 3 months) within a multicentre set-up. Cell-free DNA from the donor was quantified in plasma as a fraction of the total cell-free DNA by next generation sequencing using a targeted, multiplex polymerase chain reaction-based method for the analysis of single nucleotide polymorphisms. Results Increases of the ddcfDNA% above a threshold value of 0.88% were significantly associated with the occurrence of episodes of acute rejection (P = 0.017), acute tubular necrosis (P = 0.011) and acute pyelonephritis (P = 0.032). A receiver operating characteristic curve analysis revealed an equal area under the curve of the ddcfDNA% and serum creatinine of 0.64 for the diagnosis of acute rejection. Conclusions Although increases in plasma ddcfDNA% are associated with graft injury, plasma ddcfDNA does not outperform the diagnostic capacity of the serum creatinine in the diagnosis of acute rejection.

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Modeling cell-free DNA fragment size densities for non-invasive detection of cancer.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.3058 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 3058-3058

Author(s):

Jacob Carey ◽

Bryan Chesnick ◽

Denise Butler ◽

Michael Rongione ◽

Giovanni Parmigiani ◽

...

Keyword(s):

Fragment Size ◽

Length Distribution ◽

Mixture Component ◽

Base Pairs ◽

Machine Model ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Genome Wide ◽

Low Coverage

3058 Background: Circulating cell-free DNA (cfDNA) is largely nucleosomal in origin with typical fragment lengths of 167 base-pairs reflecting the length of DNA wrapped around-the histone and H1 linker. Given the nucleosomal origin of cfDNA, we have previously used low coverage whole genome sequencing to evaluate DNA fragmentation profiles to sensitively and specifically detect tumor-derived DNA with altered fragment lengths or coverage. Methods: Here we evaluate the use of Bayesian finite mixtures to model the fragment length distribution and demonstrate how the parameters from these models can be useful to distinguish between individuals with and without cancer. We examined the number of cfDNA fragments by size ranging from 100-220bp and approximated the mixture component location, scale, and weight using Markov Chain Monte Carlo. The performance of the method was determined using a ten-fold, ten repeat cross-validation of Gradient Boosted Machine model using 1) our previously described genome-wide fragmentation profile approach, 2) the parameters from the mixture model and 3) a combination of approaches 1) and 2) as features. Results: In this study of 215 cancer patients and 208 cancer-free individuals, we observed cross-validated AUCs of 1) 0.94, 2) 0.95, and 3) 0.97 among the three approaches. Conclusions: Our findings indicate that parsimonious mixture models may improve detection of cancer in conjunction with fragmentation profile analyses across the genome.

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Cell-Free DNA Screening for Fetal Aneuploidy

50 Studies Every Obstetrician-Gynecologist Should Know ◽

10.1093/med/9780190947088.003.0021 ◽

2021 ◽

pp. 115-120

Author(s):

Ashley N. Battarbee ◽

Neeta L. Vora

Keyword(s):

Trisomy 21 ◽

Dna Analysis ◽

Chromosomal Abnormalities ◽

Area Under The Curve ◽

First Trimester ◽

Outcome Data ◽

Cell Free Dna ◽

High Risk Women ◽

Free Dna ◽

Trimester Screening

In a prospective, multicenter blinded study at 35 international centers, the Noninvasive Examination of Trisomy (NEXT) study evaluated the performance of cell-free DNA screening for fetal trisomy compared to standard first trimester screening with nuchal translucency and serum analytes in a routine prenatal population. Among the 15,841 women who had standard screening and cell-free DNA analysis with neonatal outcome data, there were 68 chromosomal abnormalities (1 in 236). Of these, 38 were Trisomy 21 (1 in 417). Cell-free DNA analysis had a higher area under the curve (AUC) for trisomy 21, compared to standard screening (0.999 vs. 0.958, p = 0.001). Cell-free DNA analysis also had greater sensitivity, specificity, and positive predictive value compared to standard screening for trisomy 21, 18, and 13. While cell-free DNA analysis cannot detect all chromosome abnormalities, it performed better than standard screening for detection of trisomies 21, 18, and 13 in a routine population including low- and high-risk women.

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Fragmentation patterns of circulating cell-free DNA demonstrate biomarker potential for human cancers

Biotarget ◽

10.21037/biotarget.2019.08.02 ◽

2019 ◽

Vol 3 ◽

pp. 16-16 ◽

Cited By ~ 1

Author(s):

Zhou Zhang ◽

Wei Zhang

Keyword(s):

Cell Free Dna ◽

Human Cancers ◽

Free Dna ◽

Fragmentation Patterns ◽

Circulating Cell Free Dna

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Cell-Free DNA Analysis of Targeted Genomic Regions in Maternal Plasma for Non-Invasive Prenatal Testing of Trisomy 21, Trisomy 18, Trisomy 13, and Fetal Sex

Clinical Chemistry ◽

10.1373/clinchem.2015.252502 ◽

2016 ◽

Vol 62 (6) ◽

pp. 848-855 ◽

Cited By ~ 29

Author(s):

George Koumbaris ◽

Elena Kypri ◽

Kyriakos Tsangaras ◽

Achilleas Achilleos ◽

Petros Mina ◽

...

Keyword(s):

Trisomy 21 ◽

Dna Analysis ◽

Prenatal Testing ◽

Cost Effective ◽

Maternal Plasma ◽

Trisomy 13 ◽

Cell Free Dna ◽

Free Dna ◽

Genomic Regions ◽

Fetal Fraction

Abstract BACKGROUND There is great need for the development of highly accurate cost effective technologies that could facilitate the widespread adoption of noninvasive prenatal testing (NIPT). METHODS We developed an assay based on the targeted analysis of cell-free DNA for the detection of fetal aneuploidies of chromosomes 21, 18, and 13. This method enabled the capture and analysis of selected genomic regions of interest. An advanced fetal fraction estimation and aneuploidy determination algorithm was also developed. This assay allowed for accurate counting and assessment of chromosomal regions of interest. The analytical performance of the assay was evaluated in a blind study of 631 samples derived from pregnancies of at least 10 weeks of gestation that had also undergone invasive testing. RESULTS Our blind study exhibited 100% diagnostic sensitivity and specificity and correctly classified 52/52 (95% CI, 93.2%–100%) cases of trisomy 21, 16/16 (95% CI, 79.4%–100%) cases of trisomy 18, 5/5 (95% CI, 47.8%–100%) cases of trisomy 13, and 538/538 (95% CI, 99.3%–100%) normal cases. The test also correctly identified fetal sex in all cases (95% CI, 99.4%–100%). One sample failed prespecified assay quality control criteria, and 19 samples were nonreportable because of low fetal fraction. CONCLUSIONS The extent to which free fetal DNA testing can be applied as a universal screening tool for trisomy 21, 18, and 13 depends mainly on assay accuracy and cost. Cell-free DNA analysis of targeted genomic regions in maternal plasma enables accurate and cost-effective noninvasive fetal aneuploidy detection, which is critical for widespread adoption of NIPT.

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The fragmentation patterns of maternal plasma cell‐free DNA and its applications in non‐invasive prenatal testing

Prenatal Diagnosis ◽

10.1002/pd.5680 ◽

2020 ◽

Vol 40 (8) ◽

pp. 911-917 ◽

Cited By ~ 1

Author(s):

Min Pan ◽

Pingsheng Chen ◽

Jiafeng Lu ◽

Zhiyu Liu ◽

Erteng Jia ◽

...

Keyword(s):

Plasma Cell ◽

Prenatal Testing ◽

Maternal Plasma ◽

Cell Free Dna ◽

Non Invasive ◽

Free Dna ◽

Fragmentation Patterns

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Comparative Analysis of Urine Fractions for Optimal Bladder Cancer Detection Using DNA Methylation Markers

Cancers ◽

10.3390/cancers12040859 ◽

2020 ◽

Vol 12 (4) ◽

pp. 859 ◽

Cited By ~ 5

Author(s):

Anouk E. Hentschel ◽

Jakko A. Nieuwenhuijzen ◽

Judith Bosschieter ◽

Annina P. van Splunter ◽

Birgit I. Lissenberg-Witte ◽

...

Keyword(s):

Dna Methylation ◽

Bladder Cancer ◽

Comparative Analysis ◽

Area Under The Curve ◽

Practical Reasons ◽

Methylation Analysis ◽

Cell Free Dna ◽

Molecular Tests ◽

Free Dna ◽

Tumor Tissues

DNA methylation analysis of full void urine and urine pellet seems promising for bladder cancer (BC) detection and surveillance. Urinary cell-free DNA from urine supernatant is now gaining interest for other molecular tests in BC. This study aims to evaluate which urine fraction is preferred for BC diagnosis using methylation markers: full void urine, urine pellet or supernatant. Methylation levels of nine markers were determined in the three urine fractions and correlated with their respective tumor tissues in BC patients and compared to controls. For all markers and marker panel GHSR/MAL, diagnostic performance was determined by calculating the area under the curve (AUC) of the respective receiver operating characteristic curves. For most of the markers, there was a significant correlation between the methylation levels in each of the urine fractions and the matched tumor tissues. Urine pellet was the most representative fraction. Generally, AUCs for BC diagnosis were comparable among the fractions. The highest AUC was obtained for GHSR/MAL in urine pellet: AUC 0.87 (95% confidence interval: 0.73–1.00), corresponding to a sensitivity of 78.6% and a specificity of 91.7%. Our results demonstrate that cellular and cell-free DNA in urine can be used for BC diagnosis by urinary methylation analysis. Based on our comparative analysis and for practical reasons, we recommend the use of urine pellet.

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Increased plasma concentration of cell-free DNA precedes disease recurrence in children with high-risk neuroblastoma

10.21203/rs.2.13924/v3 ◽

2020 ◽

Author(s):

Yan Su ◽

Lijun Wang ◽

Chiyi Jiang ◽

Zhixia Yue ◽

Hongjun Fan ◽

...

Keyword(s):

High Risk ◽

Maintenance Treatment ◽

Multidisciplinary Treatment ◽

Area Under The Curve ◽

Predictive Biomarkers ◽

Disease Recurrence ◽

High Rate ◽

Roc Curve Analysis ◽

Cell Free Dna ◽

Free Dna

Abstract Background: Neuroblastoma is the most common extracranial solid tumor of childhood. The high rate of recurrence is associated with a low survival rate for patients with high-risk neuroblastoma. There is thus an urgent need to identify effective predictive biomarkers of disease recurrence. Methods: A total of 116 patients with high-risk neuroblastoma were recruited at Beijing Children’s Hospital between February 2015 and December 2017. All patients received multidisciplinary treatment, were evaluated for the therapeutic response, and then initiated on maintenance treatment. Blood samples were collected at the beginning of maintenance treatment, every 3 months thereafter, and at the time of disease recurrence. Plasma levels of cell-free DNA (cfDNA) were quantified by qPCR. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the ability of plasma cfDNA concentration to predict recurrence. Results: Of the 116 patients, 36 (31.0%) developed recurrence during maintenance treatment. The median time to recurrence was 19.00, 9.00, and 8.00 months for patients who had achieved complete response (n = 6), partial response (n = 25), and stable disease (n = 5), respectively, after multidisciplinary treatment. The median plasma cfDNA concentration at the time of recurrence was significantly higher than the concentration in recurrence-free patients throughout maintenance treatment (29.34 ng/mL vs 10.32 ng/mL). Patients recorded a plasma cfDNA level ≥29 ng/mL an average of 0.55 months before diagnosis of disease recurrence. ROC analysis of the power of plasma cfDNA to distinguish between patients with or without recurrence yielded an area under the curve of 0.825, with optimal sensitivity and specificity of 80.6% and 71.3%, respectively, at a cfDNA level of 12.93 ng/mL. Conclusions: High plasma cfDNA concentration is a potential molecular marker to signal disease recurrence in patients with high-risk neuroblastoma.

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