[Current methods of extracellular DNA methylation analysis].

The discovery of the enormous role methylated cytosine plays in regulating gene expression has led to the development of a variety of techniques for detecting cytosine modification. A majority of these techniques are geared towards analyzing genomic DNA, which is typically available in large quantities. The concentration of cell-free DNAs (cfDNA) extracted from biological fluids including plasma, saliva, tears, or urine is relatively low and their degree of the fragmentation is high. Moreover, for noninvasive diagnostics of cancer, methylation patterns must be studied in minor cancer-specific fractions of DNA molecules substantially diluted by excess unmethylated molecules. The above limitations complicate the application of traditional techniques for cfDNA methylation analysis. In this manuscript, we review the state-of-art analysis of cfDNA methylation, hydroxymethylation, and noncanonical methylation (outside of CpG islands). The review covers methodological approaches to studying individual CpGs and genomic loci, as well as techniques for the large-scale analysis of methylation.

Influence of radical prostatectomy on miRNA dynamics in urine extracellular vesicles.

PURPOSE: Cancer statistics demonstrate leading growth of prostate cancer. As a rule, radical prostatectomy (RP) is a mandatory option in the treatment of localized prostate cancer (PCa). Over 30% of patients develop biochemical resistance after the surgery and over 30% of these patients experience prostate cancer recurrence and metastasis. Currently used PCa patient's diagnostic features fail to identify PCa recurrence. To identify the risk group of PCa patients after RP we attempt to apply miRNAs which were shown as promising liquid biopsy markers for PCa diagnosis and prognosis. MATERIALS AND METHODS: Expression of 14 miRNAs closely involved in the development of prostate cancer from urine extracellular vesicles (uEV) of PCa patients before as well as 3, 6 and 12 months after radical prostatectomy was assessed using RT PCR and compared with their expression from uEV of healthy donors in the current study. RESULTS: It was shown that 22 miRNA pairs prognostic ratios (MPPRs) significantly changed after radical prostatectomy. MPPRs the most promising in terms of evaluating the effectiveness of radical prostatectomy have been identified. These include two groups: MPPRs significantly changed after surgery towards that in healthy donors; and MPPRs, which divided PCa patients into two significantly different subgroups 3 or 6 months after radical prostatectomy. CONCLUSIONS: The obtained data indicate that urine EVs represent a valuable source of both MPDR and MPPR for prostate cancer.

Concentration of cell-free DNA in different tumor types.

Introduction: Cell-free DNA (cfDNA) circulates in the blood for a long time. The levels of cfDNA in the blood are assayed in cancer diagnostics because they are closely related to the tumor burden of patients.Areas covered: cfDNA escapes the action of DNA-hydrolyzing enzymes, being a part of supramolecular complexes or interacting with the plasma membrane of blood cells. cfDNA has heterogeneous size and composition, which impose various restrictions on both isolation methods and subsequent analysis. cfDNA concentration and structural changes with the development of diseases highlight the high potential of cfDNA as a diagnostic and prognostic marker. The concentration of cfDNA released in the blood by tumor cells determines the specificity of such diagnostics and the required blood volume. The present review aimed to synthesize the available data on cfDNA concentration in the cancer patient's blood as well as pre-analytical, analytical, and biological factors, which interfere with cfDNA concentration.Expert opinion: The concentration of cfDNA and tumor cell DNA (ctDNA), and the over-presentation of DNA loci in cfDNA must be considered when looking for tumor markers. Some inconsistent data on cfDNA concentrations (like those obtained by different methods) suggest that the study of cfDNA should be continued.

Data analysis algorithm for the development of extracellular miRNA-based diagnostic systems for prostate cancer.

Urine of prostate cancer (PCa) carries miRNAs originated from prostate cancer cells as a part of both nucleoprotein complexes and cell-secreted extracellular vesicles. The analysis of such miRNA-markers in urine can be a convenient option for PCa screening. The aims of this study were to reveal miRNA-markers of PCa in urine and design a robust and precise diagnostic test, based on miRNA expression analysis. The expression analysis of the 84 miRNAs in paired urine extracellular vesicles (EVs) and cell free urine supernatant samples from healthy donors, patients with benign and malignant prostate tumours was done using miRCURY LNA miRNA qPCR Panels (Exiqon, Denmark). Sets of miRNAs differentially expressed between the donor groups were found in urine EVs and urine supernatant. Diagnostically significant miRNAs were selected and algorithm of data analysis, based on expression data on 24-miRNA in urine and obtained using 17 analytical systems, was designed. The developed algorithm of data analysis describes a series of steps necessary to define cut-off values and sequentially analyze miRNA expression data according to the cut-offs to facilitate classification of subjects in case/control groups and allows to detect PCa patients with 97.5% accuracy.

[Representation analysis of miRNA from clarified urine and urine microvesicles in prostate malignancies and non-malignant neoplasms]. / Analiz predstavlennosti mikroRNK v mikrovezikulakh mochi i beskletochnoi moche pri zabolevaniiakh predstatel'noi zhelezy.

Urine of prostate cancer patients contains tumor-specific biopolymers, including protein- and microvesiclesassociated miRNAs that can potentially be used as oncomarkers. Previously we have characterized urine extracellular vesicles and demonstrated diagnostic potential of their miRNA cargo. In this study, we have performed a comparative analysis of the expression of 84 miRNA in paired samples of urine microvesicles and clarified urine from healthy men, patients with benign hyperplasia and cancer of the prostate using miRCURY LNA miRNA qPCR Panels. Subsets of miRNAs with differences in expression between the fractions of the urine were found in all three groups. Two groups of miRNA were identified based on the patterns of their differential expression. They regulate several key signaling pathways associated with prostate cancer development.

[Contamination of exosome preparations, isolated from biological fluids]. / Kontaminatsiia preparatov ékzosom, vydelennykh iz biologicheskikh zhidkostei.

The aim of our study was to attract the attention of researchers at the problem of contamination of exosome preparations. Using a transmission electron microscope JEM-1400 ("JEOL", Japan) we have examined exosome preparations, isolated according to the conventional scheme of sequential centrifugation from different biological fluids: plasma and urine of healthy persons and patients with oncologic diseases, bovine serum, and culture fluid (MDCK, MDA-MB и MCF-7 cells). All exosome preparations (over 200) contained exosomes, which were identified by immuno-electron microscopy using antibodies to tetraspanins CD63 or CD9. Besides exosomes, all the studied preparations contained contaminating structures: distinct particles of low electron density without limiting membrane ("non-vesicles"). Two main kinds of the "non-vesicles" species were found in exosome preparations: 20-40 nm in size, representing 10-40% of all structures in the preparations; and 40-100 nm in size (identical to exosomes by size). Morphology of the "non-vesicles" allowed to identify them as lipoproteins of intermediate and low density (20-40 nm), and very low density (40-100 nm). The highest level of the contamination was detected in exosome preparations, isolated from blood samples. The results of our study indicate the need to control the composition of exosome preparation by electron microscopy and take into account the presence of contaminating structures in analysis of experimental data.

[Generation of blood circulating DNA: the sources, peculiarities of circulation and structure].

Extracellular nucleic acids (exNA) were described in blood of both healthy and illness people as early as in 1948, but staied overlooked until middle 60th. Starting from the beginning of new millennium and mainly in the last 5 years exNA are intensively studied. Main attention is directed to investigation of exNA as the source of diagnostic material whereas the mechanisms of their generation, as well as mechanisms to providing long-term circulation of exNA in the bloodstream are not established unambiguously. According to some authors, the main source of circulating nucleic acids in blood are the processes of apoptosis and necrosis, while others refer to the possible nucleic acid secretion by healthy and tumor cells. Circulating DNA were found to be stable in the blood for a long time, escaping from the action of DNA hydrolyzing enzymes and are apparently packed in different supramolecular complexes. This review presents the opinions of various authors and evidence in favor of all the theories describingappearance of extracellular DNA, the features of the circulation and structure of the extracellular DNA and factors affecting the time of DNA circulation in blood.

Extracellular Nucleic Acids in Urine: Sources, Structure, Diagnostic Potential.

Cell-free nucleic acids (cfNA) may reach the urine through cell necrosis or apoptosis, active secretion of nucleic acids by healthy and tumor cells of the urinary tract, and transport of circulating nucleic acids (cir- NA) from the blood into primary urine. Even though urinary DNA and RNA are fragmented, they can be used to detect marker sequences. MicroRNAs are also of interest as diagnostic probes. The stability of cfNA in the urine is determined by their structure and packaging into supramolecular complexes and by nuclease activity in the urine. This review summarizes current data on the sources of urinary cfNA, their structural features, diagnostic potential and factors affecting their stability.

Redistribution of Free- and Cell-Surface-Bound DNA in Blood of Benign and Malignant Prostate Tumor Patients.

A direct correlation between the concentration of cell-free and cell-surface-bound circulating DNA (cfDNA and csbDNA, respectively) was demonstrated. Based on an inverse correlation between blood plasma DNase activity and the cfDNA concentration, blood DNases are supposed to regulate the cfDNA concentration. However, no correlation was found between the DNase activity in blood plasma and the csbDNA concentration, indicating that blood DNases are not involved in csbDNA dissociation from the cell surface. The possibility of DNA redistribution between cfDNA and csbDNA indicates that the total pool of circulating DNA (cfDNA + csbDNA) should be used for a correct analysis of marker DNA concentrations and data standardization.

Deoxyribonuclease activity in biological fluids of healthy donors and cancer patients.

Integral activity of neutral deoxyribonucleases in the plasma and urine or donors, patients with benign prostatic hyperplasia, and patients with stomach and prostatic cancer was studied by IFA based on hydrolysis of DNA fragment modified with haptene molecules. In donors plasma deoxyribonuclease activity was 0.16+/-0.04, urinary activity 1.49+/-1.41 act. U/ml. In patients with benign prostatic hyperplasia and malignant tumors the integral activity of blood deoxyribonucleases was significantly below the normal, and in tumors it did not correlate with tumor size and disease stage. A significant correlation between blood and urinary deoxyribonuclease activities was detected.

[Breast cancer diagnostics based on extracellular DNA and RNA circulating in blood].

Extracellular DNA and RNA were extracted from blood plasma and cell surface-bound fractions of patients with breast tumors and healthy controls. Frequency of RASSF1A, Cyclin D2 and RARbeta2 methylation was detected using methylation-specific PCR in the extracellular DNA, extracted from plasma and cell-surface bound fractions of patient blood. Methylation of at least one of these genes was found in plasma of 13% patients with benign breast fibroadenoma and in 60% of breast cancer patients. Using cell-surface bound DNA as a substrate for PCR have lead to increase of gene methylation detection frequency up to 87% in fibroadenoma and 95% in breast cancer patients without false positive controls. GAPDH, RASSF8, Ki-67 RNA and 18S RNA were quantified using RT-qPCR of the extracellular RNA circulating in blood of patients with breast tumors and healthy controls. The main part of the extracellular RNA was shown to be cell-surface bound. Results show a higher amount of RASSF8, Ki-67 RNA and 18S RNA in plasma and cell-bound fraction of patients with breast cancer compared with patients with benign tumors and healthy controls. The data indicate that the specific RNA quantification in blood plasma is valuable for discrimination between cancer and benign tumors, which can be detected with high sensitivity using analysis of methylated RASSF1A, Cyclin D2 and RARbeta2 genes in extracellular circulating DNA.

[Modern methods of prostate cancer diagnostics].

The review highlights analytics capacities of immunochemical and molecular-genetic methods used in the non-invasive test for prostate cancer and monitoring of efficacy of anticancer therapy. The perspectives of their applications in clinical practice also have been evaluated.

Cell-free and cell-bound circulating DNA in breast tumours: DNA quantification and analysis of tumour-related gene methylation.

Tumour development is characterised by the increased circulating DNA (cirDNA) concentration and by tumour-related changes in blood plasma DNA. Concentration of cirDNA and methylation of RARbeta2, RASSF1A and HIC-1 gene promoters were investigated in cell-free and cell-surface-bound fractions from healthy donors, patients with breast cancer, and patients with breast fibroadenoma. Tumour development was shown to lead to significant changes in the distribution of cirDNA between cell-free and cell-surface-bound fractions. Analysis of RARbeta2 and RASSF1A methylation in the total cirDNA provides 95% diagnostic coverage in breast cancer patients, 60% in patients with benign lesions, and is without false-positive results in healthy women. Results of the study indicate that methylation-specific PCR of RARbeta2 and RASSF1A genes based on the total cirDNA combined with the quantitative analysis of cirDNA distribution between cell-bound and cell-free fractions in blood provide the sensitive and accurate detection and discrimination of malignant and benign breast tumours.

[Circulating nucleic acids in blood of patients with stomach and colon neoplasms].

Concentrations of extracellular deoxy- and ribonucleic acids in blood plasma and cell-surface-bound of blood cells were investigated in healthy donors and patients with malignant gastrointestinal tumors. Our results indicate that high concentration of extracellular DNA in blood plasma along with decreased level of extracellular RNA on the surface of blood cells correlate with development of gastrointestinal cancer. Ratio of nucleic acids in plasma to total amount of nucleic acids circulated in blood is a characteristic parameter distinguishing cancer patients from healthy persons.