Introduction: Extracellular nucleic acids are found in human blood and cell culture medium as cell-free or being adsorbed at cell surface. In the last years, the circulating extracellular nucleic acids in blood were shown to be associated with certain diseases. Attempts are made to develop non-invasive methods of early tumor diagnostics based on analysis of circulating DNA and RNA. Areas covered: This article reviews accumulating data regarding cell-free and cell-surface-bound extracellular nucleic acid nature and generation mechanisms. Their existence as a constituent of the naturally occurring complexes with proteins or membrane-bearing particles is discussed with regard to their homeostatic concentration and distribution in healthy donor blood which are significantly altered in cancer patients. Gene-target and whole-genome studies reveal significant differences in gene representation between extracellular DNA and genome DNA. Overrepresentation of regions with high transcription activity has led to proposal that extracellular DNA generation is strongly dependent on the parent genome functionality, which is associated with chromosome packaging and DNA methylation levels. Expert opinion: Recent studies provide evidence of the circulating nucleome organization complexity indicating that discovery of extracellular DNA generation and circulation patterns in healthy condition and cancer is essential to enable the development of proper approaches for the selection of valid diagnostic markers.

Cancer remains one of the main causes of human mortality despite significant progress in its diagnostics and therapy achieved in the past decade. Massive hypomethylation of retrotransposons, in particular LINE-1, is considered a hallmark of most malignant transformations as it results in the reactivation of retroelements and subsequent genomic instability. Accumulating data on LINE-1 aberrant methylation in different tumor types indicates its significant role in cancer initiation and progression. However, direct evidence that LINE-1 activation can be used as a cancer biomarker is still limited. The objective of this review was to critically evaluate the published results regarding the diagnostic/prognostic potential of the LINE-1 methylation status in cancer. Our analysis indicates that LINE-1 hypomethylation is a promising candidate biomarker of cancer development, which, however, needs validation in both clinical and laboratory studies to confirm its applicability to different cancer types and/or stages. As LINE-1 is present in multiple cell-free copies in blood, it has advantages over single-copy genes regarding perspectives of using its methylation status as an epigenetic cancer biomarker for cell-free DNA liquid biopsy.

Lung cancer is a complex disease that often manifests at the point when treatment is not effective. Introduction of blood-based complementary diagnostics using molecular markers may enhance early detection of this disease and help reduce the burden of lung cancer. Here we evaluated the diagnostic potential of seven plasma miRNA biomarkers (miR-21, -19b, -126, -25, -205, -183, -125b) by quantitative reverse transcription PCR. Influence clinical and demographical characteristics, including age, tumor stage and cancer subtype on miRNA levels was investigated. Four miRNAs were significantly dysregulated (miR-19b, -21, -25, -183) in lung cancer patients. Combination of miR-19b and miR-183 provided detection of lung cancer with 94.7% sensitivity and 95.2% specificity (AUC = 0.990). Thus, miRNAs have shown the potential to discriminate histological subtypes of lung cancer and reliably distinguish lung cancer patients from healthy individuals.

Alterations in the patterns of DNA methylation are among the earliest and most common events in tumorigenesis. Epigenetic changes were shown to be detectable in DNA, circulating in blood (cirDNA) of cancer patients, indicating the resources to create the minimally invasive diagnostic tests based on tumor-specific DNA markers. RARβ2 methylation level was significantly increased in plasma cirDNA and cell surface-bound cirDNA (csb-cirDNA) from patients with non-small cell lung cancer compared with healthy individuals (7620 and 1083 copies/ml in the csb fractions, 3589 and 1068 copies/ml in the blood plasma; P=0.003 and 0.001). The cell-bound-to-cell-free RARβ2 methylation ratio was found to be elevated in patients with non-small cell lung cancer compared with control (2.12 and 1.01, respectively; P=0.023). RARβ2 methylation level in csb-cirDNA and plasma cirDNA was higher in stage III patients compared with stage I-II patients (P=0.02 and 0.03). In the subgroup of patients with squamous cell carcinoma, RARβ2 methylation level in the cbs-cirDNA was higher compared with patients with adenocarcinoma (P=0.04). Epigenetic alterations of tumor suppressor gene RARβ2 in the total cirDNA (plasma cirDNA and csb-cirDNA) were found to be associated with lung cancer progression. The data obtained indicate that cirDNA-based testing provides a valuable source for subsequent verification of methylated DNA markers for lung cancer diagnostics and prognosis.