Nucleic acid markers in blood plasma of patients with colorectal cancer

Kondratov A.G.1, Nekrasov K.A.2, Lototska L.V.2, Panasenko G.V.2, Stoliar L.A.2, Kolesnyk O.O.3, Shchepotin I.B.3, Rynditch A.V.1, Kashuba V.I.1

A.G. Kondratov1, K.A. Nekrasov1, L.V. Lototska1, G.V. Panasenko1, L.A. Stoliar1, O.O. Kolesnyk2, I.B. Shchepotin2, A.V. Rynditch1, V.I. Kashuba1

1Institute of Molecular Biology and Genetics, NAS of Ukraine, Kyiv, Ukraine

2National Cancer Institute, Kyiv, Ukraine

o.g.kondratov@imbg.org.ua

Introduction: 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 a painful procedure and can not be recommended for patients with altered topography of colon. Aim: Development of less invasive tools for CRC screening based on detection of nucleic acids in blood plasma. Methods: Concentration of cell-free circulating DNA in blood plasma was analyzed by qPCR. Methylation status of LRRC3B, APC, FHIT and HIC1 genes in cell-free circulating DNA of samples was determined by using methyl-specific PCR (MSP) with subsequent melting curve analysis. Results: It was shown that mean-value of cell-free blood plasma DNA is statistically higher in CRC patients than in healthy donors (p < 0.01). Thus the mean-value of concentration of cell-free circulating DNA in blood plasma of CRC patients was 17.57 ± 3.43 ng/mL and 7.07 ± 0.84 ng/mL — in healthy donors. We have revealed hypermethylation of APC, FHIT and LRRC3B genes in 45% (10/22), 73% (16/22) and 68% (15/22) of tumor samples, respectively. Altogether hypermethylation of at least one of the selected genes was detected in 95% (21/22) of samples. Using MSP with subsequent melting curve analysis we have detected methylated fragments of APC, FHIT and LRRC3B genes in blood plasma of 29% (6/21), 19% (4/21) 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 blood plasma samples of CRC patients. Additionally we have registered high frequency of HIC1 hypermethylation in blood plasma of CRC patients (8/10). We have suggested that two stage verification might be applied for CRC screening, which includes measurement of cell-free circulating DNA concentration with following detection of methylated fragments of APC, FHIT and LRRC3B genes in blood plasma of CRC patients. Overlapping of the above mentioned approaches allowed to increase sensitivity of studied panel up to 71% (15/21) in CRC detection. Conclusion: We have deve­loped approach for screening of CRC which is based on determination of cell-free DNA and methylated fragment of well-known tumor related genes APC, FHIT, LRRC3B in blood plasma. The sensitivity of CRC detection might be increased by using of additional perspective genes like HIC1.

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