Genes copy number as a marker of low-invasive assessment of rectal tumors radiotherapy effectiveness.

Abstract

3025 Background: Radiotherapy (RT) is a key component of rectal cancer (RC) treatment, however, nonresponsiveness in patients to preoperative RT is very common, usually due to the tumor cells radioresistance, mediated by their molecular characteristics, such as gene expression. The features of mRNA rapid degradation in extracellular environment make this indicator unsuitable for low invasive diagnostics. The solution to this problem is possible by switching to a more stable marker - the copy number variation (CNV), which can be determined in the extracellular DNA (cfDNA) circulating in the blood plasma. Therefore, the aim of the study was to identify the relationship between the level of genes CNV in the cfDNA of blood plasma with the effectiveness of rectal tumors RT. Methods: We used cfDNA preparations from blood plasma obtained before RT from 200 patients with RC, as well as from blood plasma of 50 apparently healthy donors (AHD, without cancer). RT was carried out on a linear accelerator Novalis TX (SFD = 2.4 Gy, TFD = 54.0 Gy). Blood samples were separated into plasma and cell fraction by centrifugation. Isolation of cfDNA from blood plasma was performed using the phenol-chloroform method. Determination CNV of 5 genes (BRCA2, H2AX, CASP9, RBBP8 and BCL2) was performed using Real-Time qPCR method. Differences were assessed using Mann-Whitney test; the Bonferroni correction was used to correct multiple comparisons. Results: RT results for 200 patients allowed them to be divided into 2 groups. After RT, 120 patients showed complete tumor regression (group 1), 50 patients showed insignificant tumor regression and 30 patients did not regress (group 2). In cfDNA of group 1 patients was found CNV decrease (p < 0.05) of H2AX and RBBP8 genes by 2.5 and 2.0 times, respectively, relative to AHD group. In the cfDNA of group 2patients an increase (p < 0.05) of BRCA2, H2AX, RBBP8 and BCL2 genes CNV was found by 2.0, 2.2, 2.0 and 2.0 times, respectively, relative to AHD group. Only 2 genes CNV differed in group 1 from group 2: the CNV of H2AX and RBBP8 was 5.4 and 4.0 times less respectively (p < 0.005). Conclusions: Thus, it has been found that increased CNV of genes BRCA2, H2AX, BCL2, RBBP8 in blood plasma cfDNA is associated with low efficiency of RT. At the same time, the CNV of H2AX and RBBP8 genes in cfDNA of patients with RC has the greatest potential as a marker of the RT effectiveness.