Recent studies suggest that extracellular vesicles may be the key to timely diagnosis and monitoring of genito-urological malignancies. In this study we investigated the composition and content of extracellular vesicles found in the urine of healthy donors and prostate cancer patients. Urine of 14 PCa patients and 20 healthy volunteers was clarified by low-speed centrifugation and total extracellular vesicles fraction was obtain by high-speed centrifugation. The exosome-enriched fraction was obtained by filtration of total extracellular vesicles through a 0.1 μm pore filter. Transmission electron microscopy showed that cell-free urine in both groups contained vesicles from 20 to 230 nm. Immunogold staining after ultrafiltration demonstrated that 95% and 90% of extracellular vesicles in healthy individuals and cancer patients, respectively, were exosomes. Protein, DNA and RNA concentrations as well as size distribution of extracellular vesicles in both fractions were analyzed. Only 75% of the total protein content of extracellular vesicles was associated with exosomes which amounted to 90–95% of all vesicles. Median DNA concentrations in total extracellular vesicles and exosome-enriched fractions were 18 pg/ml and 2.6 pg/ml urine, correspondingly. Urine extracellular vesicles carried a population of RNA molecules 25 nt to 200 nt in concentration of no more than 290 pg/ml of urine. Additionally, concentrations of miR-19b, miR-25, miR-125b, and miR-205 were quantified by qRT-PCR. MiRNAs were shown to be differently distributed between different fractions of extracellular vesicles. Detection of miR-19b versus miR-16 in total vesicles and exosome-enriched fractions achieved 100%/93% and 95%/79% specificity/sensitivity in distinguishing cancer patients from healthy individuals, respectively, demonstrating the diagnostic value of urine extracellular vesicles.

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.

A vast wealth of recent research has seen attempts of using microRNA (miRNA) found in biological fluids in clinical research and medicine. One of the reasons behind this trend is the apparent their high stability of cell-free miRNA conferred by small size and packaging in supramolecular complexes. However, researchers in both basic and clinical settings often face the problem of selecting adequate methods to extract appropriate quality miRNA preparations for use in specific downstream analysis pipelines. This review outlines the variety of different methods of miRNA isolation from biofluids and examines the key determinants of their efficiency, including, but not limited to, the structural properties of miRNA and factors defining their stability in the extracellular environment.

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.

The aim of this study was to investigate miRNA profiles of clarified urine supernatant and combined urine vesicle fractions of healthy donors and patients with benign prostatic hyperplasia and prostate cancer (PCa). The comparative analysis of miRNA expression was conducted with a custom miRCURY LNA miRNA qPCR panel. Significant combinations of miRNA pairs were selected by the RandomForest-based feature selection algorithm Boruta; the difference of the medians between the groups and a 95% confidence interval was built using the bootstrap approach. The Asymptotic Wilcoxon-Mann-Whitney Test was performed for miRNA combinations to compare different groups of donors. Benjamini-Hochberg correction was used to adjust the statistical significance for multiple comparisons. The most diagnostically significant miRNAs pairs were miR-107-miR-26b.5p and miR-375.3p-miR-26b.5p in the urine supernatant fraction that discriminated the group of healthy patients and PCa patients, as well as miR-31.5p-miR-16.5p, miR-31.5p-miR-200b, miR-31.5p-miR-30e.3p and miR-31.5p-miR-660.5p in the fraction extracellular vesicles that were different between healthy men and benign prostate hyperplasia patients. Such statistical criteria as the occurrence of individual significant miRNA pairs in the total number of comparisons, median ΔC_t difference, and confidence interval can be useful tools for determining reliable markers of PCa.