Research Of Circulating Cell-free DNA Concentrations And Integrity In Serum Of Colorectal Cancer Patients

Objective To establish a method that quantitative real-time PCR by amplifying the ALU repeats(ALU-q PCR) for detecting concentrations and integrity of circulating cell-free DNA(cf-DNA) and investigate cf-DNA expression in patients with benign and malignant colorectal tumors and healthy controls, also explore its clinical significance of increasing the early complementary diagnosis and monitoring of progression and prognosis of colorectal cancer(CRC). Evaluate the diagnostic efficiency of combination cf-DNA concentrations and integrity with conventional serum marker carcinoembryonic antigen(CEA) in CRC.Methods Serum samples were collected at random from 104 with primary CRC, 85 with operated CRC, 16 with recurrent/metastatic CRC, 63 patients with intestinal polyps who were from the Affiliated Hospital of Nantong University and 110 healthy controls were collected during the same period from the Physical Examination Center of the same hospital. Compare cf-DNA levels between the groups, longer and shorter DNA fragments(247bp, 115bp) in serum were determined by ALU-q PCR. ALU115 represent the absolute amount of serum DNA. DNA integrity index was calculated as the ratio of ALU-q PCR results(ALU247/115). Correlative serum carcinoembryonic antigen(CEA) level was detected by ARCHITECT assay.Results The median absolute serum ALU115 and the ALU247/115 index(1046.0 ng/ml, 0.62) in primary CRC were significantly higher than in intestinal polyps(423.3 ng/ml, 0.41) and healthy controls(385.4 ng/ml, 0.38)(P<0.0001, respectively). The ALU115 and ALU247/115 index of serum DNA in intestinal polyps were no statistical differences compared with healthy controls(P>0.05, respectively). In the relevant indicators of prognosis, ALU115 and ALU247/115 index in primary CRC patients were correlated with age, histological differentiation and tumor stage(P<0.05). With 694.0 ng/ml, 0.52 and 5 ng/ml as the cut-off values of ALU115, ALU247/115 index and CEA level, the area under the ROC curve(AUC) for distinguishing primary CRC patients from healthy controls was 0.85(95% CI 0.81~0.91), 0.89(95% CI 0.85~0.93) and 0.78(95% CI 0.72~0.84), respectively. Compared with the diagnostic efficiency of ALU115, ALU247/115 or CEA alone, combined detection of ALU115, ALU247/115 and CEA improved the diagnostic efficiency of primary CRC to some extent. The median absolute serum ALU115 and the ALU247/115 index(2228.1 ng/ml, 0.68) of serum DNA in recurrent/metastatic CRC were significantly higher compared with primary CRC(1046.0 ng/ml, 0.62)(P=0.0021, P=0.0018) or operated CRC(524.1 ng/ml, 0.48)(P<0.0001, respectively). We found that cf-DNA measurements could be used to monitor tumor dynamics, ALU115 and ALU247/115 in 20 cases of follow-up had a distinct increase at pre-operation and then decrease progressively at post-operation.Conclusions Combined detection of ALU115, ALU247/115 and CEA could improve the diagnostic efficiency for CRC. Serum DNA concentrations and integrity index may be valuable in early complementary diagnosis and monitoring of progression and prognosis of CRC.