Cell-free DNA in the blood provides a non-invasive diagnostic avenue for patients with cancer1. However, characteristics of the origins and molecular features of cell-free DNA are poorly understood. Here we developed an approach to evaluate fragmentation patterns of cell-free DNA across the genome, and found that profiles of healthy individuals reflected nucleosomal patterns of white blood cells, whereas patients with cancer had altered fragmentation profiles. We used this method to analyse the fragmentation profiles of 236 patients with breast, colorectal, lung, ovarian, pancreatic, gastric or bile duct cancer and 245 healthy individuals. A machine learning model that incorporated genome-wide fragmentation features had sensitivities of detection ranging from 57% to more than 99% among the seven cancer types at 98% specificity, with an overall area under the curve value of 0.94. Fragmentation profiles could be used to identify the tissue of origin of the cancers to a limited number of sites in 75% of cases. Combining our approach with mutation-based cell-free DNA analyses detected 91% of patients with cancer. The results of these analyses highlight important properties of cell-free DNA and provide a proof-of-principle approach for the screening, early detection and monitoring of human cancer.
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Sequence data used in this study have been deposited at the database of Genotypes and Phenotypes (dbGaP, study ID 34536).
Code for analyses is available at http:github.com/Cancer-Genomics/delfi_scripts.
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We thank members of our laboratories for critical review of the manuscript. This work was supported, in part, by the Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, the Stand Up to Cancer–Dutch Cancer Society International Translational Cancer Research Dream Team Grant (SU2C-AACR-DT1415), the Commonwealth Foundation, the Cigarette Restitution Fund, the Burroughs Wellcome Fund and the Maryland Genetics, Epidemiology and Medicine Training Program, the AACR-Janssen Cancer Interception Research Fellowship, the Mark Foundation for Cancer Research, US NIH (grants CA121113, CA006973, and CA180950), the Danish Council for Independent Research (11-105240), the Danish Council for Strategic Research (1309-00006B), the Novo Nordisk Foundation (NNF14OC0012747 and NNF17OC0025052), and the Danish Cancer Society (R133-A8520-00-S41 and R146-A9466-16-S2). Stand Up To Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research.
Nature thanks Daniel De Carvalho, Ellen Heitzer and the other anonymous reviewer(s) for their contribution to the peer review of this work.
S.C., A.L., J.P., J.F., V. Adleff, R.B.S. and V.E.V. are inventors on patent applications (62/673,516 and 62/795,900) submitted by Johns Hopkins University related to cell-free DNA for cancer detection. V.E.V. is a founder of Delfi Diagnostics and Personal Genome Diagnostics, a member of their Scientific Advisory Boards and Boards of Directors, and owns Delfi Diagnostics and Personal Genome Diagnostics stock, which are subject to certain restrictions under university policy. Within the last five years, V.E.V. has been an advisor to Daiichi Sankyo, Janssen Diagnostics, Ignyta, and Takeda Pharmaceuticals. The terms of these arrangements are managed by Johns Hopkins University in accordance with its conflict of interest policies.
Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended data figures and tables
Extended Data Fig. 1 Simulations of non-invasive cancer detection based on number of alterations analysed and tumour-derived cfDNA fragment distributions.
a, Monte Carlo simulations were performed using different numbers of tumour-specific alterations to evaluate the probability of detecting cancer alterations in cfDNA at the indicated fraction of tumour-derived molecules. The simulations were performed assuming an average of 2,000 genome equivalents of cfDNA and the requirement of five or more observations of any alteration. These analyses indicate that increasing the number of tumour-specific alterations improves the sensitivity of detection of circulating tumour DNA. b, Cumulative density functions of cfDNA fragment lengths of 42 loci containing tumour-specific alterations from 30 patients with breast, colorectal, lung, or ovarian cancer are shown with 95% confidence bands (orange). Lengths of mutant cfDNA fragments were significantly different in size from wild-type cfDNA fragments (blue) at these loci. c, GC content was similar for mutated and non-mutated fragments. d, GC content was not correlated to fragment length.
a, Cumulative density functions of fragment lengths at 44 loci containing germline alterations (non-tumour derived) from 38 patients with breast, colorectal, lung or ovarian cancer are shown with 95% confidence bands. Fragments with germline mutations (orange) were comparable in length to wild-type cfDNA fragment lengths (blue). b, Cumulative density functions of fragment lengths at 41 loci containing haematopoietic alterations (non-tumour derived) from 28 patients with breast, colorectal, lung or ovarian cancer are shown with 95% confidence bands. After correction for multiple testing, there were no significant differences (α = 0.05) in the size distributions of mutated haematopoietic cfDNA fragments (orange) and wild-type cfDNA fragments (blue).
a, cfDNA fragment lengths are shown for healthy individuals (n = 30, grey) and patients with lung cancer (n = 8, blue). b–d, cfDNA fragmentation profiles from healthy individuals (n = 30) had high correlations, whereas patients with lung cancer (n = 8) had lower correlations to median fragmentation profiles of lymphocytes (b), lymphocyte nucleosome distances (c) and healthy cfDNA (d). Pearson correlations are shown with box plots depicting minimum, 25th percentile, median, 75th percentile, and maximum values. e, High coverage (9×) WGS data were subsampled to 2×, 1×, 0.5×, 0.2× and 0.1×-fold coverage. Mean centred genome-wide fragmentation profiles in 5-Mb bins for 30 healthy individuals and 8 patients with lung cancer are depicted for each subsampled fold coverage with median profiles shown in blue. f, Pearson correlation of subsampled profiles to initial profile at 9× coverage for healthy individuals and patients with lung cancer.
Detection and monitoring of cancer in serial blood draws from patients with non-small cell lung cancer (n = 19) undergoing treatment with targeted tyrosine kinase inhibitors (black arrows) was performed using targeted sequencing (top) as previously reported29, and genome-wide fragmentation profiles (bottom). For each case, the vertical axis of the bottom panel displays −1 times the Pearson correlation of each sample to the median healthy cfDNA fragmentation profile. Error bars depict confidence intervals from binomial tests for mutant allele fractions, and confidence intervals calculated using Fisher transformation for genome-wide fragmentation profiles. Although the approaches analyse different aspects of cfDNA (whole genome compared with specific alterations), the targeted sequencing and fragmentation profiles were similar for patients responding to therapy as well as those with stable or progressive disease. As fragmentation profiles reflect both genomic and epigenomic alterations (whereas mutant allele fractions only reflect individual mutations), mutant allele fractions alone may not reflect the absolute level of correlation of fragmentation profiles to healthy individuals.
Extended Data Fig. 5 Profiles of cfDNA fragment lengths in copy neutral regions in healthy individuals and one patient with colorectal cancer.
a, The fragmentation profiles in 211 copy neutral windows in chromosomes 1–6 are shown for 25 randomly selected healthy individuals (grey). For a patient with colorectal cancer (CGCRC291) with an estimated mutant allele fraction of 20%, we diluted the cancer fragment length profile to an approximate 10% tumour contribution (blue). a, b, Although the marginal densities of the fragment profiles for the healthy samples and patient with cancer show substantial overlap (a, right), the fragmentation profiles are different as can be seen through visualization of the fragmentation profiles (a, left) and by the separation of the patient with colorectal cancer from the healthy samples (n = 25) in a principal component analysis (b).
To estimate and control for the effects of GC content on sequencing coverage, we calculated coverage in non-overlapping 100-kb genomic windows across the autosomes. For each window, we calculated the average GC of the aligned fragments. a, LOESS smoothing of raw coverage (top row) for two randomly selected healthy subjects (CGPLH189 and CGPLH380) and two patients with cancer (CGPLLU161 and CGPLBR24) with undetectable aneuploidy (PA score < 2.35). After subtracting the average coverage predicted by the LOESS model, the residuals were rescaled to the median autosomal coverage (bottom row). As fragment length may also result in coverage biases, we performed this GC correction procedure separately for short (≤150 bp) and long (>150 bp) fragments. Although the 100-kb bins on chromosome 19 (blue points) consistently have less coverage than predicted by the LOESS model, we did not implement a chromosome-specific correction as such an approach would remove the effects of chromosomal copy number on coverage. b, Overall, we found a limited correlation between short or long fragment coverage and GC content after correction among healthy individuals (n = 211, interquartile range: −0.03–0.03) and patients with cancer (n = 128, interquartile range: −0.06–0.02) with a PA score < 3. Box plots depict 25th percentile, median, and 75th percentile values.
a, We used gradient tree boosting machine learning to examine whether cfDNA can be categorized as having characteristics of a patient with cancer or a healthy individual. The machine learning model included fragmentation size and coverage characteristics in windows throughout the genome, as well as chromosomal arm and mitochondrial DNA copy numbers. We used a tenfold cross-validation approach in which each sample is randomly assigned to a fold, and nine of the folds (90% of the data) are used for training and one fold (10% of the data) is used for testing. The prediction accuracy from a single cross-validation is an average over the ten possible combinations of test and training sets. As this prediction accuracy can reflect bias from the initial randomization of patients, we repeat the entire procedure, including the randomization of patients to folds, ten times. For all cases, feature selection and model estimation were performed on training data and were validated on test data, and the test data were never used for feature selection. Ultimately, we obtained a DELFI score that could be used to classify individuals as likely to be healthy or having cancer. b, Distribution of AUCs across the repeated tenfold cross-validation. The 25th, 50th and 75th percentiles of the 100 AUCs for the cohort of 215 healthy individuals and 208 patients with cancer are indicated by dashed lines.
Extended Data Fig. 8 Whole-genome analyses of chromosomal arm copy number changes and mitochondrial genome representation.
a, Z-scores for each autosome arm are depicted for healthy individuals (n = 215) and patients with cancer (n = 208). The vertical axis depicts normal copy at zero with positive and negative values indicating arm gains and losses, respectively. Z-scores greater than 50 or less than −50 are thresholded at the indicated values. b, The fraction of reads mapping to the mitochondrial genome is depicted for healthy individuals (n = 215) and patients with cancer (n = 208). Box plots depict the minimum, 25th percentile, median, 75th percentile, and maximum values.
a, Analyses of individual cancer types using DELFI had AUCs ranging from 0.86 to >0.99. b, Receiver operator characteristics for detection of cancer using cfDNA fragmentation profiles and other genome-wide features in a machine learning approach are depicted for a cohort of 215 healthy individuals and each stage of 208 patients with cancer with ≥95% specificity shaded in blue. c, Receiver operator characteristics for DELFI tissue prediction of bile duct, breast, colorectal, gastric, lung, ovarian or pancreatic cancer are depicted. To increase sample sizes within cancer type classes, we included cases detected with a 90% specificity, and the lung cancer cohort was supplemented with the addition of baseline cfDNA data from 18 patients with lung cancer with prior treatment36. d, DELFI tissue of origin prediction.
DELFI (green) and targeted sequencing10 for mutation identification (blue) were performed independently in a cohort of 126 patients with breast, bile duct, colorectal, gastric, lung or ovarian cancer. The number of individuals detected by each approach and in combination are indicated for DELFI detection with a specificity of 98%, targeted sequencing specificity at >99%, and a combined specificity of 98%. ND, not detected.
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Cristiano, S., Leal, A., Phallen, J. et al. Genome-wide cell-free DNA fragmentation in patients with cancer. Nature 570, 385–389 (2019). https://doi.org/10.1038/s41586-019-1272-6
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