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Aim. The work is devoted to the development of less invasive tools for the colorectal cancer (CRC) screening. Methods. Q-PCR and methylation-specific PCR techniques were used in the current work. Results. We have shown that the levels of cell-free plasma DNA are higher in the CRC patients compared with the healthy donors (p < 0.01). Hypermethylation of APC, FHIT, LRRC3B and HIC1 genes was studied in the tumor and plasma samples of CRC patients. Two-stage verification for CRC screening was proposed. Conclusions. We proposed and tested a novel approach for CRC screening based on the determination of cell-free DNA and methylated DNA fragments in the plasma. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) is the third commonly diagnosed cancer that causes 400 000 deaths worldwide. The most sensitive modern diagnostic tool of CRC is colonoscopy which is painful procedure and can not be recommended for petient with altered topography of colon. Thus, development of less invasive tools for screening of CRC is actual problem of the modern oncology. To detect concentration of cell free circulating DNA in plasma the qRT-PCR was used. Methylation status of LRRC3B, APC, and FHIT genes in cell free circulating DNA of samples was determined by using methyl-specific PCR with subsequent melting curve analysis. It was shown that mean-value of plasma DNA is statistically higher in CRC patients than healthy donors (p < 0.01). Thus the mean-value concentration of cell free circulating DNA in plasma of CRC patients was 17.57 ± 3.43 ng/mL whereas 7.07 ± 0.84 ng/mL for healthy donors. Accordingly with it the upper cut-off value of the free circulating DNA concentration was 17.74 ng/mL of plasma of healthy donors. Therefore there were 8 out of 22 samples of CRC which fell into criteria as samples with abnormally increased DNA level. We have revealed hypermethylation of APC, FHIT and LRRC3B genes in in 48 % (10/21), 71 % (15/21) and 67 % (14/21) of tumor samples, correspondingly. Altogether hypermethylation at least one of the selected genes was detected in 95 % (20/21) of samples. Using MSP with subsequent melting curve analysis we have previously detected methylated fragments of APC, FHIT and LRRC3B genes in plasma of 19 % (4/21), 29 % (6/21) and 14 % (3/21) of CRC patients, respectively. We have identified hypermethylation at least in one of the selected genes in 48 % (10/21) of plasma samples for CRC patients. We have proposed that two stage verification might be applied for CRC screening. These stages include measurement of cell-free circulating DNA with following detection of methylated fragments of APC, FHIT and LRRC3B genes in plasma of CRC patients. Overlapping of the mentioned approaches allowed increasing sensitivity of studied panel up to 95 % in CRC detection. An approach based on APC, FHIT and LRRC3B genes did not show of 100 % sensitivity in registration of CRC which is essential for prevention of this death disease. Therefore we suggested that higher sensitivity could be achieved by further extension of gene panel for identification of methylated DNA in plasma of CRC patient. [ABSTRACT FROM AUTHOR]