Circadian Rhythm of Methylated Septin 9, Cell-Free DNA Amount and Tumor Markers in Colorectal Cancer Patients.

ABSTRACT

To determine the level of cell-free DNA (cfDNA), Septin 9 (SEPT9) and tumor markers (CEA, AFP, CA19-9, TPA, CA72-4). Plasma samples were collected four times a day (0600, 1200, 1800, 2400) from 9 patients with CRC (5 stage I-II, 4 stage III-IV), from one with colorectal adenoma and from one healthy control. CfDNA was isolated, quantified and bisulfite-converted. CfDNA and methylated SEPT9 were determined by RT-PCR. Plasma levels of conventional tumor markers were also measured. The lowest cfDNA concentrations were observed at 2400 and 1800 in stage I-III patients. In stage IV samples low cfDNA level (mean 48.2 ng/ml) were observed at several time points (600, 1200, 1800). The highest cfDNA levels were measured at 600 and 1200 in CRC I-III stages and at 2400 in stage IV samples (78.65 ng/ml). Higher in-day differences were found in stage II (43-48%) than in stage I samples (22%). Interestingly, the highest SEPT9 methylation level was found at 2400 in most CRC cases, in contrast to the cfDNA levels. At 2400, all cancer and adenoma cases were positive for SEPT9 methylation. At other time points (600, 1200, 1800) only 77.7% of CRC samples showed SEPT9 positivity. Stage I samples were SEPT9 positive only at 2400. CEA and CA19-9 levels displayed correlation with the amount of cfDNA in case of late stage cases. Daytime activity can influence SEPT9 positivity in cases with low concentration of cfDNA. Thus, it may improve screening sensitivity by collecting samples earlier in the morning.