Epigenetics, fragmentomics, and topology of cell-free DNA in liquid biopsies – Science


Enhancing liquid biopsies

Liquid biopsies, analyses of cell-free DNA that circulates in the blood, can be used in prenatal testing, oncology, and to monitor organ transplant recipients. Lo et al. review the nongenetic information that can be gleaned from analyses of cell-free DNA, which offers additional promise for the applications of this procedure. These analyses include DNA methylation patterns, fragmentation profiles, and topology, which can be informative about the health and origin of the tissue from which they are derived.

Science, this issue p. eaaw3616

Structured Abstract


Liquid biopsies that are based on analysis of cell-free DNA from plasma offer diagnostic information that is otherwise accessible conventionally through invasive biopsies. Noninvasive prenatal testing has been used globally for the screening of fetal chromosomal aneuploidies and has led to a considerable reduction in invasive prenatal testing, such as use of amniocentesis. Cancer liquid biopsies have been used for the selection of targeted therapies and monitoring of disease progression. Liquid biopsies for organ transplant patients have been used to monitor graft dysfunction. The first applications of liquid biopsies are based on the detection of genetic markers in cell-free DNA, such as sex differences, genetic polymorphisms, or mutations. By studying nongenetic features of cell-free DNA molecules—including DNA methylation, fragmentation, and topology—understanding of cell-free DNA biology has expanded the spectrum and utilities of liquid biopsies.


Cell-free DNA in plasma consists of a mixture of fragmented DNA molecules released from various tissues within the body. Each cell-free DNA fragment bears molecular signatures of its cell of origin, such as DNA methylation status. The methylation profile of circulating fetal DNA in the mother’s plasma correlates with that of the placenta and has been exploited as a means to develop noninvasive fetus-specific biomarkers that are not dependent on fetal sex or genotype. Circulating tumor-derived DNA bear methylation states that resemble the tumor tissue and have enabled the development of tests for the screening and localization of cancer. The fragmentation of plasma DNA is related to the nucleosomal organization, chromatin structure, gene expression, and nuclease content of the tissue of origin, resulting in characteristic signatures in the form of fragment size, nucleotide motifs at the fragment ends, single-stranded jagged ends, and the genomic locations of the fragmentation endpoints. For mitochondrial DNA that is originally in a circular form, fragmentation will also change its topology into a linear form. By noting these features of cell-free DNA fragments, the anatomical site of pathology could potentially be deduced, providing additional information than just quantifying mitochondrial DNA without regard to its form. The study of such characteristics has also enhanced our understanding of the biology and generation of cell-free DNA. The roles of nucleases in plasma DNA biology, such as deoxyribonuclease 1–like-3, have been explored by using gene-deletion mouse models and confirmed in humans bearing nuclease gene mutations, with potential implications for the pathogenesis of autoimmune diseases.


The use of DNA methylation, fragmentomic, and topologic analyses of circulating DNA, either in a targeted fashion or in a genomewide manner, will be expected to impact clinical practice. Clinical specimens covering more disease entities will need to be investigated to identify tissue-specific and disease-relevant signatures. During the discovery phase, to better delineate these signatures, mining is performed on high volumes of DNA data pooled within and across samples by using customized bioinformatics algorithms. Once these putative sets of signatures have been identified, signature- and target-specific assays could be developed, and large-scale clinical trials will be needed to validate these approaches. One application is in the development of plasma DNA–based cancer screening. One advantage of these approaches is the potentially large number of markers that can be developed to differentiate cancer and noncancer cells and in the ability to locate the tissue of origin of the detected cancer, possibly including cancers of unknown primary. In the area of noninvasive prenatal testing, the correlation of DNA methylation, fragmentomic, and topologic aberrations in circulating DNA to clinical outcomes would expand the spectrum of diagnostic applications beyond current ones. Fragmentomic approaches have the potential for enriching the cell-free DNA species of interest, such as through the use of automated platforms that allow the size separation of circulating DNA. In the area of organ transplant monitoring, the development of DNA methylation, fragmentomic, and topologic markers would provide an alternative to genetic markers for detecting rejection. These nongenetic markers may enable further differentiation of the donor’s contribution to the circulating DNA pool into its constituent tissue components. The understanding between circulating DNA and nucleases is in its infancy. Circulating DNA signatures attributable to changes in nuclease expression in health and disease need to be elucidated and may have emerging diagnostic applications.

Cell-free DNA contains molecular features characteristic of their tissues of origin.

Different tissues, including cancer cells and trophoblasts in pregnancy, contribute cell-free DNA to the circulation. The cell-free DNA molecules may bear methylation states reflective of the cell of origin. The DNA molecule size, fragment end locations, and end motifs are influenced by the nucleosome organization, chromatin structure, nuclease content, and gene expression of the tissue of origin. Parameters that can be measured to quantify these characteristics are shown on the right.


Liquid biopsies that analyze cell-free DNA in blood plasma are used for noninvasive prenatal testing, oncology, and monitoring of organ transplant recipients. DNA molecules are released into the plasma from various bodily tissues. Physical and molecular features of cell-free DNA fragments and their distribution over the genome bear information about their tissues of origin. Moreover, patterns of DNA methylation of these molecules reflect those of their tissue sources. The nucleosomal organization and nuclease content of the tissue of origin affect the fragmentation profile of plasma DNA molecules, such as fragment size and end motifs. Besides double-stranded linear fragments, other topological forms of cell-free DNA also exist—namely circular and single-stranded molecules. Enhanced by these features, liquid biopsies hold promise for the noninvasive detection of tissue-specific pathologies with a range of clinical applications.