The emerging role of cell-free DNA as a molecular marker for cancer management

Abstract

An increasing number of studies demonstrate the potential use of cell-free DNA (cfDNA) as a surrogate marker for multiple indications in cancer, including diagnosis, prognosis, and monitoring. However, harnessing the full potential of cfDNA requires (i) the optimization and standardization of preanalytical steps, (ii) refinement of current analysis strategies, and, perhaps most importantly, (iii) significant improvements in our understanding of its origin, physical properties, and dynamics in circulation. The latter knowledge is crucial for interpreting the associations between changes in the baseline characteristics of cfDNA and the clinical manifestations of cancer. In this review we explore recent advancements and highlight the current gaps in our knowledge concerning each point of contact between cfDNA analysis and the different stages of cancer management.

Keywords: Biomarker; Cancer; Cell-free DNA; Circulating tumor DNA; Liquid biopsy; Precision oncology.

Fig. 1

Potential applications of cell-free DNA in cancer management. Profiling of cancer-associated genetic alterations in cell-free DNA (cfDNA) may enable the large-scale screening of healthy or at-risk population groups for the early detection of multiple cancers. Furthermore, accumulating data demonstrates the potential clinical value of cfDNA analysis for the care of cancer patients at various stages of the disease. The level of cancer-associated mutations detected in cfDNA generally corresponds with tumor burden, and quantification thereof can serve as an indicator of disease stage and outcome. Tissue-specific nucleosome-spacing patterns and methylation signatures are encoded in cfDNA, and this information is useful for diagnosis, but can also be harnessed to pinpoint the location of tumors, especially those of unknown primary. Novel targeted therapies are only effective when specific pathways are altered in cancer cells. Detection of these mutations in cfDNA can, therefore, guide the selection of matched therapies. Compared to tumor biopsies, cfDNA has been shown to provide a better representation of the complete genetic landscape of a tumor. In addition, cfDNA offers the additional benefit of serial sampling, which allows the longitudinal assessment of dynamic changes in the concentration of cfDNA, the identification of acquired resistance-conferring mutations, and the tracking of clonal evolution. This makes it possible to monitor response of the cancer to therapy, detect the emergence of acquired resistance, as well as monitor and predict minimal residual disease and recurrence following surgery or therapy.

Fig. 2

Characteristics of cell-free DNA in the human body. (a) In cancer patients, cell-free DNA (cfDNA) originates from multiple sources, including cancer cells, cells from the tumor microenvironment, and non-cancer cells from other regions of the body (e.g., hematopoietic stem cells, muscle cells, and epithelial cells). (b) DNA can be liberated from these cells via different mechanisms, most prominently apoptosis, necrosis, and active secretion, although other forms of cell death and clearance may contribute. The physical characteristics of the cfDNA produced by these different mechanisms vary considerably. Apoptosis causes the systematic cleavage of chromosomal DNA into multiples of 160–180 bp stretches, resulting in the extracellular presence of mono- (˜166 bp) and poly-nucleosomes (332 bp, 498 bp). Necrosis results in nuclear chromatin clumping and non-specific digestion, producing cfDNA fragments that are typically larger than 10,000 bp. Mitochondrial DNA can also be released by these mechanisms and typically range between 40 and 300 bp. CfDNA derived from active cellular secretions have been shown to range between 1000 and 3000 bp, while cfDNA originating from extrachromosomal circular DNA ranges between 30 and 20,000 bp. Exosomes, which also enter circulation via regulated release, carry DNA ranging between 150 and 6000 bp. In addition, recent evidence has demonstrated the presence of DNA fragments smaller than 166 bp in blood, which might represent degraded products of any of the aforementioned cfDNA types. Extracellular levels of cfDNA is, therefore, highly dependent on the rate of its release from cells. However, once present in circulation, cfDNA levels are further influenced by its (c) dynamic association and disassociation with extracellular vesicles and several serum proteins, (d) rate of binding, dissociation and internalization by cells, which is dependent on pH, temperature, and can be inhibited by certain substances (such as heparin), and (e) the rate of digestion or clearance, including the activity of DNAse I, renal excretion into urine, and uptake by the liver and spleen.

Fig. 3

A robust preanalytical workflow for tumor-derived cell-free DNA analysis. This workflow was carefully formulated by selecting the most optimal preanalytical steps (indicated by the asterisks) from a wide range of alternative steps that are reported in the literature.

Fig. 4

The value distributions of most cancer biomarkers show an overlap of cancer patients and healthy individuals. The diagnostic performance of a biomarker is best illustrated by ROC curves. To establish this graph, the portion of correctly identified negative controls (specificity) and correctly identified positive cancer patients (sensitivity) are identified for all possible cut-off points (decreasing stepwise from 100% specificity). The area under the curve (AUC) and the sensitivity at a fixed specificity (e.g. 95%) are most informative for the comparison of diagnostic markers. As control groups, healthy individuals and patients with differential diagnostically relevant benign diseases are considered.

Fig. 5

The combination of pre-therapeutic tissue biopsies and serial blood collections for liquid profiling during and after therapy may improve the guidance of cancer patients considerably. The mutational status in tissue is currently required to stratify patients for certain targeted therapies although it allows only a spatially and temporally restricted “snapshot”. Genetic heterogeneity, undetectable dormant and resistant cell clones, and adverse patient conditions limit this approach. CfDNA based liquid profiling, however, can reveal the entire mutational landscape of the cancer, and can be applied serially due to the non-invasiveness of blood collection. Furthermore, it provides essential information on the dynamics of tumor biology that can be used at various time points during the course of the disease for (i) therapy stratification, (ii) assessing prognosis, (iii) monitoring therapy response, (iv) early detection of disease progression, (v) recurrence detection, and (vi) identification of acquired resistances. Together, this may provide accurate individual patient guidance and become the cornerstone of personalized cancer medicine in the future.

Fig. 6

Strategies for improving the specificity and sensitivity of cell-free DNA analyses. The signal-to-noise ratio (mutant allele fraction) of samples can be increased by: (a) optimizing preanalytical steps, especially by using tube types (e.g., Streck BCT), processing procedures (immediate processing and two rounds of centrifugation), and storage conditions (−80 °C) that minimize the release of germline DNA from peripheral blood cells; (b) sampling from body fluids that are in closer proximity to the region of interest; (c) enriching tumor-derived cell-free DNA (cfDNA) by experimental or in silico selection of short cfDNA fragments (<166 bp); and (d) performing independent assays on aliquoted replicates of isolated cfDNA. While dPCR methods demonstrate high sensitivity, multiplexing capacity is limited. (e) Targeted sequencing approaches based on hyprid capture or PCR amplicons can be used to query a larger number of loci. (f) In addition, the sensitivity of targeted sequencing can be improved by molecular barcoding, i.e., assigning unique identifiers (UIDs) to each DNA template during library preparation. This enables post-sequencing stratification of UID families, which allows the construction of a consensus sequence for a single molecule, thereby eliminating random errors that may have been introduced by PCR and sequencing. (g) Finally, the sensitivity and specificity of cfDNA tests could be improved significantly by the combination of multiple biomarkers in a single parallel assessment. Markers of particular interest include miRNAs, proteins, exosomes, metabolites, circulating tumor cells (CTCs), or mitochondrial DNA (mtDNA). In addition, it may be highly relevant to interrogate other features of cfDNA, such as DNA methylation changes and histone post-translational modifications (PTMs).