New Perspectives on the Importance of Cell-Free DNA Biology

Abstract

Body fluids are constantly replenished with a population of genetically diverse cell-free DNA (cfDNA) fragments, representing a vast reservoir of information reflecting real-time changes in the host and metagenome. As many body fluids can be collected non-invasively in a one-off and serial fashion, this reservoir can be tapped to develop assays for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as solid tumors, fetal genetic abnormalities, rejected organ transplants, infections, and potentially many others. The translation of cfDNA research into useful clinical tests is gaining momentum, with recent progress being driven by rapidly evolving preanalytical and analytical procedures, integrated bioinformatics, and machine learning algorithms. Yet, despite these spectacular advances, cfDNA remains a very challenging analyte due to its immense heterogeneity and fluctuation in vivo. It is increasingly recognized that high-fidelity reconstruction of the information stored in cfDNA, and in turn the development of tests that are fit for clinical roll-out, requires a much deeper understanding of both the physico-chemical features of cfDNA and the biological, physiological, lifestyle, and environmental factors that modulate it. This is a daunting task, but with significant upsides. In this review we showed how expanded knowledge on cfDNA biology and faithful reverse-engineering of cfDNA samples promises to (i) augment the sensitivity and specificity of existing cfDNA assays; (ii) expand the repertoire of disease-specific cfDNA markers, thereby leading to the development of increasingly powerful assays; (iii) reshape personal molecular medicine; and (iv) have an unprecedented impact on genetics research.

Keywords: cell-free DNA; cfDNA; circulating tumor DNA; ctDNA; liquid biopsy.

Figure 1

The diverse possible origins of cfDNA in humans. Through various pathways of cell death, clearance, and regulated release, whole or partial genomes of diverse origins are constantly shed into human body fluids in the form of fragmented cfDNA.

Figure 2

Clinical applications and potential roles of cfDNA in human biology. Human body fluids are constantly replenished by cfDNA fragments of various origins. CfDNA profiling thus offers the unique opportunity to reconstruct major portions of the host- and metagenome, and this information can be harnessed to develop tests for the diagnosis, prognosis, and monitoring of wide-ranging pathologies, such as (A) various cancer indications, (B) fetal genetic abnormalities and pregnancy complications, (C) organ transplant complications, (D) infections, (E) chronic illnesses, and (F) acute illnesses. CfDNA profiling may also be used to characterize (G) the gut microbiome and (H) assimilated environmental DNA. (I) Beyond its use as a clinical biomarker, cfDNA may have many other potential uses and roles in human biology and pathology.

Figure 3

Factors that can potentially affect total cfDNA levels. Here we summarize the wide-ranging factors that have been experimentally shown to modulate total cfDNA levels. We also show several other factors that possibly affect total cfDNA levels, but which have not yet been conclusively demonstrated or have not yet been investigated. This makes it very difficult to correlate total cfDNA levels with a specific factor and limits the clinical use of total cfDNA measurements.

Figure 4

Features of cfDNA that could serve as clinical biomarkers. In addition to DNA hotspot mutations, various disease- and tissue-specific genetic, epigenetic, and structural features are encoded into cfDNA. Much of this information can be leveraged for the detection and monitoring of a wide range of diseases, physiological states, and other clinical scenarios.

Figure 5

Determining the tissue-of-origin of cfDNA molecules. The cfDNA population in a typical biospecimen is highly complex and derives from numerous different cell and tissue types. However, cfDNA molecules contain multiple layers of cell-type specific epigenetic signatures, such as differentially methylated DNA regions, post-translational histone modifications, nucleosome occupancy, as well as various fragmentation features. Through the use of increasingly sophisticated molecular analysis methods coupled with machine learning algorithms, the epigenetic information carried by cfDNA molecules can be decoded to determine the contribution of different tissue types.

Figure 6

Mechanisms of NET formation. (A) Suicidal NETosis occurs over several hours and involves the decondensation of chromatin in activated neutrophils leading to the release of chromatin into the cytoplasm. Subsequently, the cell membrane is ruptured and chromatin is released from the lysed neutrophil into extracellular space. (B) Vital NET formation/extrusion is a rapid process occurring within 60 min of cell stimulation where vesicles containing nuclear DNA are fused with the plasma membrane and chromatin is released entirely, leaving anuclear neutrophils. These cells maintain their viability; therefore, this type of NET formation is best categorized as NET extrusion rather than NETosis, which implies that the NET-forming cells are lysed. (C) Mitochondrial vital NET formation/extrusion is a secondary form of vital NET formation in which mitochondrial DNA is released as NETs after the rupture of mitochondrial membranes, leaving viable neutrophils lacking mitochondria.

Figure 7

Biological factors that modulate the characteristics of cfDNA. The composition and fluctuation of the cfDNA population in the circulatory system is influenced by numerous, often mutually non-exclusive determinants. The major factors include (A) the relative contribution that different cell types make toward the total cfDNA pool; (B) the mechanisms by which cfDNA is released from the various contributing cells; (C) factors that affect the movement of cfDNA from tissues or cells into circulation; (D) modifications and rate of degradation by extracellular nucleases and proteolytic enzymes; (E) the rate of uptake and digestion by the liver, spleen or kidneys; (F) binding and detachment to circulating or epithelial cells, and (G) association with DNA-binding proteins, other macromolecules, or extracellular vesicles. (H) All of the former factors are amendable by a web of factors, many of which may interact in known and unknown ways, including (i) a variety of diseases, physiological states, and other clinical scenarios, (ii) phenomena that cause cell death or constitutive DNA release, such as mechanical stress, oxidative stress, hypoxia, inflammation, DNA damage, genomic instability, lesions, and (iii) other factors such as, e.g., time of day, body mass index, diet, medication, fitness, exercise, and metabolic rate, most of which are liable to significant intra- and interindividual variation.

Figure 8

Factors that contribute to the complexity and fluctuation of cfDNA composition.

Figure 9

Factors that need to be addressed in order to increase the fidelity of cfDNA analysis. In order to (i) use cfDNA for studying temporal genomic changes, (ii) investigate the role of cfDNA in human health and disease, and (iii) develop increasingly powerful clinical cfDNA assays, the quantitative and qualitative information contained in cfDNA samples needs to be reconstructed with high fidelity. Here we briefly summarize various factors that need to be considered and steps that can be taken towards increasingly accurate cfDNA measurements.