Non-invasive Early Diagnosis Of Colorectal Cancer By Detecting DNA Methylation Based On Second Generation Sequencing

Background:Second-generation sequencing is a technology for studying biological genes.Its cost has been gradually reduced from $1 per bp at the beginning to affordable range in various fields.Colorectal cancer(CRC)is one of the most common malignancies.About 2/3 of colorectal cancer patients in China have lymph node or distant metastasis at the time of diagnosis.Only two thirds of patients can receive radical surgery,and the recurrence rate is 30 to 50 percent.Most colorectal cancers are sporadic and develop as adenomaneoplasms over several years.Therefore,it is very important to improve the early screening and diagnosis rate of CRC and precancerous polyps for timely treatment and improvement of prognosis.Early diagnosis and removal of precancerous lesions remain the most effective means of reducing CRC mortality.At present,it have their limitations for the commonly used primary screening methods for high-risk groups such as colonoscopy,fecal occult blood test,CEA and other tumor markers.It is urgent to search for reliable non-invasive biomarkers for early diagnosis of CRC.Abnormal DNA methylation is closely related to the occurrence and development of CRC and other tumors.In the early stage of CRC,the level of free DNA methylation in the plasma of cancer patients is generally higher than that of normal people,which indicates that the detection of DNA methylation may become a second-generation marker molecule for the diagnosis of colorectal polyps and colorectal cancer.Objective:(1)To identify DNA methylation markers in tissues and plasma of patients with colorectal cancer based on second-generation sequencing,and to screen DNA methylation markers in peripheral blood.(2)In order to reduce the cost of labtest,a technology platform for detecting peripheral blood DNA methylation markers was established for non-invasive early diagnosis of colorectal cancer based on multiplex PCR.Methods:(1)Blood cell and plasma samples were collected from 73 cases of colorectal cancer,38 cases of colorectal adenoma and plasma,and 69 cases of normal volunteers.Tissue and plasma DNA were extracted respectively and captured by flow microarray.Methylated sequencing was used to obtain single-base resolution data.Machine learning was used to establish a discrimination model to screen out alternative marker sites and verify them.According to p<0.05&2 times significant difference,the final DNA methylation differential region(DMR)was obtained by filtering the difference between cancer and benign+difference between cancer and normal-difference between benign and normal.(2)Difference of free DNA methylation in peripheral blood plasma was detected by multiple TaqMan real-time quantitative PCR assay.It was verified for the accuracy of the diagnosis of colorectal cancer at the three sites of Septin9,AXL4 and SDC2.978 cases from 5 centers were recruited for this test.Results:(1)315 potential loci were found in CRC tissues.These 315 loci provided a high accuracy of more than 95%,158 of which loci provided 6343 combinations for the differentiation of CRC.In the peripheral blood plasma,15 DMRs were found,7 of which were located in the promoter region of the gene.DMRs of cancer showed a good distinction,not appear in the benign group and the normal group.There were 9 DMRs between the benign and normal groups.(2)940 cases participated in the establishment of the final PCR system and the performance evaluation test.The accuracy,sensitivity and specificity of the PCR system were 87.8%(95%CI 82.9%-91.5%),82.7%(95%CI 71.8%-90.1%),and 90.1%(95%CI 84.3%-93.9%).It is worth mentioning that the detection rate of precancerous lesions was 55.0%(95%CI 38.7%-70.4%)by this method.Conclusion:(1)Colorectal cancer has a rich combination of DNA methylation marker sites,and three differential methylation sites can well distinguish tumors.The gene of HBX20 with hypermethylation has tumor suppressive effect.Hypermethylation may inhibit the tumor suppressive function of hbx20.Seven new blood derived methylation markers were found in plasma.(2).A platform was established for noninvasive detection of colorectal cancer using multiple PCR to detect the combination of three sites of plasma DNA methylation,which can reduce the detection cost and verification cost.The multiplex PCR design tool ultiplex can assist in multiplex primer design.