In 1977 Stroun et al. went on to give what has become a classical definition of cell-free and circulating DNA [5]. In the same year Leon et al. proposed a radioimmunoassay for cfDNA quantification based on the use of labeled DNA as antigens and the serum of patients with systemic lupus erythematosus as a source of antibodies [6]. In 93 % of healthy participants, cfDNA concentrations in the blood serum ranged from 0 to 50 ng/ml. Those values were taken as reference for healthy individuals, which was perfectly legitimate considering the evidence obtained later using state-of-the-art techniques. In Leon’s study concentrations of cfDNA in the blood serum of half of cancer patients (the main group) were considerably higher, ranging from 50 to 5000 ng/ml. After radiation therapy cfDNA concentrations went down in 66 to 90 % of patients with lymphoma, lung, ovarian, uterine and cervical cancers and in 16 to 33 % of patients with glioma, breast tumors and colorectal cancer. Patients with increased or unchanged post- radiation cfDNA levels showed no response to treatment. Later, detection of tumor-specific mutations, microsatellite instability and methylation of DNA circulating in the bloodstream confirmed its tumor-derived origin [7, 8, 9].

The end of the 20th century saw the invention of quantitative real-time polymerase chain reaction, a universal and well- reproducible technique for cfDNA quantification [10, 11], which also made it possible to determine ratios of differently sized cfDNA fragments in the sample [12]. Another milestone was marked in the history of cfDNA exploration by the employment of high-throughput sequencing for whole-genome analysis of specificity and quantification of cfDNA [13, 14].

How do nucleic acids get into the systemic circulation?

In their pioneer study Leon et al. concluded that tumors are not the only source of cfDNA circulating in the blood, as only half of their cancer patients had elevated plasma levels of cfDNA while the other half did not. Recently there has been evidence that concentrations of free DNA in the cells increase within the first few hours or days following cytotoxic chemotherapy administration; the DNA then disappears within a week. In pathology associated with increased cell death or tissue damage (hepatitis, sepsis or trauma), blood plasma concentrations of cfDNA also go up. These facts support the assumption that necrosis, apoptosis and perhaps a few other types of cell death largely determine concentrations of nucleic acids in the blood plasma, especially in cancer patients. Mechanisms of nucleic acid release from the cells might vary in different cancer types. In 1989 Stroun et al. hypothesized that in cancer patients cfDNA is released into the bloodstream mainly by tumor cells [15]; this hypothesis was corroborated by the discovery of oncogenic mutations in cfDNA isolated from the blood plasma of patients with leukemia [16] and pancreatic cancer [7].

Studies of fetal cfDNA demonstrated that its half-life is 16.3 min [17]. These findings were extrapolated onto all types of cfDNA and confirmed by further experiments conducted in animals who received DNA injections [18]. Analysis by gel electrophoresis revealed that cfDNA is fragmented and has characteristics similar to those of DNA isolated from apoptotic cells; it was also found that a small proportion of cfDNA is represented by high-molecular-weight fragments [19]. Jahr et al. believed that the presence of such fragments should be associated with necrosis of tumor cells. It should be noted though that this conclusion has been seriously questioned recently: while analyzing cfDNA obtained from cancer patients, Diehl et al. discovered that its high-molecular-weight fragments do not contain tumor-specific mutations often found in short (less than 200 b. p.) cfDNA fragments [20]. The researchers hypothesized that high-molecular-weight cfDNA could originate from phagocytosed necrotic cells.

According to the commonly shared belief, necrosis triggers release of high-molecular-weight cfDNA into the bloodstream. However, this type of cell death also induces production of large amounts of DNA packaged into typically sized nucleosomes [21, 22]. Normally nucleosomes are released from the cells 24 to 48 hours after apoptosis induction or 12 hours after its early signs begin to show [23]. Although the majority of nucleosomes released into the circulatory system are effectively eliminated by the liver, some of them can still be found in the blood or other bodily fluids. Elevated levels of nucleosomes in the blood can result from increased cell death induced by degenerative, autoimmune, inflammatory, ischemic, or toxin-mediated conditions and traumas, or from the presence of malignant tumors, when elimination mechanismsareoverloadedorcompromised.Thus,circulating nucleosomes can originate from the cells dying from apoptosis or necrosis or a combination of various forms of cell death, depending on the type and intensity of the stimuli and the energy state of the cell.

Another source of cfDNA in the systemic circulation is high- molecular-weight DNA of neutrophils. Neutrophil extracellular traps (NETs) are generated by activated neutrophils during NETosis [24], when nuclear constituents, including DNA molecules, citrullinated histones and granule enzymes, such as elastase, are released into the extracellular environment. Once released, they form web-like structures capable of capturing bacteria, ensuring high concentrations of antibacterial substances. It has become clear in the recent years that NETs can be “built” under sterile proinflammatory conditions, such as thrombosis, cancer, systemic lupus erythematosus, atherosclerosis, and diabetes. Formation of neutrophil traps can be stimulated by chemokines, such as interleukin 8 (CXCL8) [25, 26], and growth factors, such as granulocyte colony-stimulating factor (G-CSF) [27] and transforming growth factor-β. Depending on the microenvironment, expression of the aforementioned factors in the tumor can be stimulated above normal levels, i. e., NETosis may indicate the presence of a malignant tumor. In turn, NETs can stimulate tumor growth and angiogenesis, promote metastases and tumor-induced thromboembolism [28].

It is possible that cfDNA is excreted by exosomes. This mechanism is described in detail in the works by Peters and Reclusa [29, 30].

Finally, cfDNA can be a product of active metabolic secretion [31, 32, 33], coming from a complex it forms with glycolipoproteins and RNA. In their in vitro cell culture experiment, Aucamp et al. demonstrated a statistically significant correlation between the rates of glycolysis and cfDNA release. Fragments of released cfDNA were about 2,000 b. p. long, which ruled out the possibility of their apoptotic or necrotic origin. So far mechanisms and contribution of active secretion into the general pool of cfDNA remain understudied.