**3. Circulating cell-free and tumour DNA**

Circulating cell-free DNA (cfDNA) is present in the blood even in healthy individuals although higher levels are observed in patients diagnosed with autoimmune diseases and especially malignant tumours. Nevertheless, cfDNA seems to represent an essential biological regulatory mechanism. cfDNA is either released passively from dying—apoptotic or necrotic—cells or secreted actively from viable cells. cfDNA can be destroyed by DNAse, but at least part of cfDNA poll follows another way of further biological turnover entering healthy cells. The subsequent genomic integration hypothetically can have a myriad of significant outcomes in health and disease ranging from senescence to autoimmune diseases or transfer of the cfDNA to germ cells.

In cancer patients, a fraction of circulating cell-free DNA burden is attributable to the tumour and consequently is designated as circulating tumour DNA (ctDNA). These DNA fragments are released from neoplastic cells and therefore can reflect the tumour-specific events in DNA, including somatic mutations, methylation patterns and degree of microsatellite instability. Thus, the presence of a malignant tumour can manifest by multiple quantitative and qualitative changes in circulating DNA. First, the concentration and features, e.g. fragment length of cfDNA in cancer patients, differ from healthy individuals. Second, the ctDNA reflects the specific dynamic genetic landscape of the cancer.

Evaluation of circulating DNA in certain situations can be diagnostically useful, e.g. to disclose an occult tumour. However, pitfalls exist, e.g. mutations can be present in cfDNA of healthy volunteers who do not develop cancer at least during the follow-up period. Thus, overdiagnosis of cancer by liquid biopsy must be avoided, especially when screening asymptomatic individuals. In contrast, qualitative or quantitative dynamic changes in the ctDNA of a known oncological patient bring reliable, biologically justified information. In patients with

already confirmed cancer, ctDNA can identify either minimal residual disease after surgery with curative intent or tumour relapse. Similarly, the response to treatment can be monitored. Molecular alterations can be assessed to select personalised treatment.

In addition to the diagnostic, prognostic and predictive role in oncology, cfDNA analysis might be useful in other medical situations. Thus, circulating cell-free DNA can be derived from transplanted organs or from the foetus during pregnancy, serving as an early manifestation of graft rejection or reflecting genetic features on the foetus, respectively. In addition, cfDNA levels can be valuable also as a nonspecific biomarker of tissue damage in critical care medicine and related clinical situations, e.g. sepsis, haemodialysis and others [4].
