The Value Of Circulating DNA In Serum In Diagnosis And Therapy Evaluation For Colorectal Cancer Patients

Background Colorectal cancer(CRC) is the third most commonly diagnosed cancer in males and the second in females, with an estimated 1.4 million cases and 693,900 deaths occurring in 2012. As early stage of CRC usually do not lead to specific symptoms, most CRC patients are in late stages when they are diagnosed, and thus losing the best opportunity for treatment. Therefore, early detection and monitoring recurrence are crucially important for CRC patients.Circulating DNA is defined as a kind of extracellular DNA that exists in plasma,cerebrospinal fluid and synovial fluid.It is generally believed that Circulating DNA released from apoptotic cells and necrotic cells.It is estimated that a 100 g tumor can realease about 3×1010 tumor cells into blood. Tumor cells in the blood circulation and distant metastasis also release circulating DNA. The detection of circulating DNA in plasma or serum is like "biopsy Liquid". We can not only estimate the tumor burden or evaluate the therapeutic effect by quantity of circulating DNA, but also we can distinguish malignant tumors from nonmalignant ones and screen drug targets by detecting the changes of genetics and epigenetics(such as gene mutation,DNA methylation,microsatellite instability(MSI),micro RNAs and so on. foremost, the detection of DNA is convenient, economical and repeatable.Objective The aim of this study was(1) to compare the concentrations and integrity of circulating DNA between CRC patients, patients with benign intestinal diseases and normal controls to see whether circulating DNA in serum could help diagnose CRC and(2) investigate whether the circulating DNA has a role as response biomarker in patients receiving surgery and neoadjuvant chemotherapy.Methods Peripheral blood samples from 148 primary CRC patients, 46 patients with benign intestinal diseases and 41 normal controls were collected at Shandong Cancer Hospital and institute from June 2014 to June 2015. The CRC group consisted of 112 patients who then received operation and 36 patients treated by neoadjuvant chemotherapy. The samples from operative patients were collected at 3 days before operation, 7 days and 14 days after operation. Serum samples of 36 patients undergoing neoadjuvant chemotherapy were taken before onset of the neoadjuvant chemotherapy(cycle 1) and before cycle 3. We assessed the concentration and integrity index of circulating DNA using real-time quantitative PCR by amplifying the LINE1 repeats(LINE1-qPCR). DNA integrity index was calculated as the ratio of LINE1-292 to LINE1-149. The Clinical response to therapy was assessed by the Response Evaluation Criteria in Solid Tumors(RECIST). The software package SPSS17.0 was used to perform the analyses stated above. A P value < 0.05 was considered significant.Results 1.Circulating DNA and DNA integrity index(DII)varied significantly with histologic differentiation, disease stage and distant metastasis(all P<0.05),and no association was found between them and gender, age, tumour site and physical scores(all P>0.05). CEA and CA199 in primary CRC patients were correlated with disease stage and distant metastasis(all P<0.05), while they are not correlated with gender, age, tumour site, histologic differentiation and physical scores.2.The concentrations of circulating DNA, CEA or CA199 was significant different among colorectal cancer patients, benign intestinal diseases patients and normal controls(all P<0.05), while DNA integrity index(DII)was not( P>0.05)?3.The AUC for distinguishing primary CRC patients from normal controls by circulating DNA, CEA and CA199 were 0.71, 0.67 and 0.69, respectively.4.The sensitivity of circulating DNA, CEA and CA199 were 62.21 %, 59.46% and 43.92%, respectively.The specificity were 79.31%, 68.97% and 96.55 %, respectively.5.At 7 days after surgery, the concentration of circulating DNA was significantly higher than that before surgery( P<0.05), while at 14 days after surgery it was lower than that before surgery or 7 days after surgery(all P<0.05). At 7 days after surgery, DII was higher than that before surgery(P <0.05), while at 14 days after surgery DII decreased significantly than that before(all P <0.05).6.The concentration of circulating DNA in remission group(CR+PR) after treatment decreased significantly than that before the treatment(P<0.05),while it in stable group(SD) or progression group(PD) was significantly higher after treatment.(all P <0.05)?There was no significant difference in DII concentrations among the three subgroups before and after chemotherapy(all P>0.05).There was no significant difference in CEA or CA199 in remission group(CR+PR) and stable group(SD) before and after chemotherapy(all P>0.05).For patients in PD group, CEA and CA199 after chemotherapy was significantly increased compared with that before the chemotherapy(all P <0.05)?Conclusions 1. Circulating DNA, DNA integrity index(DII),CEA and CA199 were correlated with disease stage and distant metastasis,which indicated that tumor markers could reflect the stage of disease.2. The concentrations of circulating DNA, CEA or CA199 was significant different among colorectal cancer patients, benign intestinal diseases patients and normal controls, while DNA integrity index(DII)was not.3. The AUC for distinguishing primary CRC patients from normal controls by circulating DNA was biggest.4. Circulating DNA had the highest sensitivity for diagnosis of CRC and CA199 had the highest specificity.5. There was a general trend that Circulating DNA and DNA integrity index(DII)were significantly higher before surgery, and decreased progressively in the follow-up period after surgery. During the observation period, the two markers were still higher than the healthy people.6. Changes of Circulating DNA seemed to be more precise than other tumor markers as the tool for evaluating the effectiveness of chemotherapy.