**4. Circulating microRNAs**

Considering the pathogenesis of malignant tumours, the burden of CTCs should appear and increase in parallel with advancing cancer course. Indeed, higher numbers of circulating tumour cells were observed in patients having tumours with higher pT or pN or higher pTNM stage [10]. However, CTCs were found in more than 80% of patients presenting with pT1 and/or pN0 gastric cancer [2]—an interesting finding that has a practical value regarding the possibilities of

Thus, CTC detection has manifold roles in oncology. A high number of CTCs is an adverse prognostic factor, shown in breast, prostatic, colorectal, gastric, pancreatic and neuroendocrine carcinomas and sarcomas as well as non-small cell lung carcinoma [5]. However, a diagnostic role has also been confirmed, e.g. in the case of breast, prostate and colorectal carcinoma [5]. Early diagnostics by CTCs has been verified in non-small cell lung carcinoma [5] and gastric cancer [2]. CTCs can provide the information on key genetic features of cancer cells and on the epigenetic changes. Hence, prediction of the treatment response by CTC has been demonstrated in breast, prostate and colorectal cancer as well as in melanoma and non-small cell lung carcinoma. Further, changes of CTC count during treatment dynamically

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

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

early diagnostics but also an impact on the theoretical considerations of carcinogenesis.

reflects the response to treatment paralleling the residual tumour burden [5].

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

6 Liquid Biopsy

specific dynamic genetic landscape of the cancer.

MicroRNAs (miRNAs) are small, evolutionary conserved, single-stranded, non-coding RNA molecules (approximately 22 nucleotides in length) that bind target mRNA to regulate gene expression [11, 12] at the posttranscriptional level [13]. These molecules act as large-scale molecular switches. MicroRNAs are involved in different physiological and pathological events, including apoptosis, cell proliferation and differentiation; therefore, it is not surprising to see miRNAs participating in carcinogenesis as either tumour suppressors [14, 15] or oncogenes [16]. The cardinal tumour features include cell proliferation, invasion and metastasis as well as activated angiogenesis. miRNAs regulate all the steps. In addition, up- or down regulation of certain miRNAs is associated with the biological potential of cancer, e.g. proliferation, invasivity and epithelial-mesenchymal transition or grade. miRNAs can be assessed either in tissues or in biological liquids, in the last case becoming a target for liquid biopsy.

Again, the diagnostic and regulatory roles of miRNAs are not limited to oncology. Women who develop pre-eclampsia and spontaneous preterm birth are characterised by specific exosomal miRNA profile at early gestation. Considering the interplay between exosomal secretion, oxygen tension and endothelial proliferation, aberrant exosomal signalling by placental cells is suggested to have a pathogenetic role in pregnancy complications [17].
