Performance Evaluation And Application Development Of SEPT9 Gene Methylation In Colorectal Cancer

The SEPT9 gene methylation assay was approved by the US FDA,the EU CE mark and the Chinese CFDA as a peripheral blood gene methylation assay for colorectal cancer(CRC)screening(FDA)and/or early detection(CE and CFDA),and it is the first FDA-approved epigenetic assay for tumor liquid biopsy.Although the performance of the assay has been widely validated and assessed in CRC screening and early detection,the effects of many factors on its performance have not been systematically investigated.In addition,few studies have been reported on its performance on colorectal precancerous lesions.In this study,we aim to investigate the effects of sex,age,cancer stage,cancer location and algorithms on the performance of the SEPT9 assay,and to study its performance on colorectal precancerous lesions,including various types of adenomas and hyperplastic polyps.A total of 428 subjects were recruited in this opportunistic screening study,including 98 CRC patients,86 adenoma patients,67 patients with hyperplastic polyps and 177 subjects with no evidence of diseases(NED).The quantitative fluorescent real-time polymerase chain reaction(qRT-PCR)was performed to measure the methylation level of the promoter region of the SEPT9 gene,and the ABI 7500 fast DX equipment and Epi proColon 2.0 CE assay were used in this study.All procedures,including blood collection,sample transportation,processing,DNA extraction,storage,bisulfite conversion,PCR,and subsequent data analysis and interpretation,were performed based on the instructions for use of the Epi proColon 2.0 CE assay.Statistical analysis was performed and the figures were made using the GraphPad PRISM 5.0 software.Our study shows that the 1/3 algorithm exhibited higher positive detection rate(PDR)than the 2/3 algorithm in the detection of CRC,adenoma and hyperplastic polyps,while the false positive rate in NED subjects was also higher with the 1/3 algorithm than the 2/3 algorithm.The PDR for stage 0 and I CRC was significantly lower than that of the stage II-IV CRC,regardless of what algorithm was chosen.However,the PDR for stage 0 and I CRC were all above 50%,supporting its use in early-stage CRC screening.The PDR of NED subjects over 60 years old was significantly higher than those younger than 60,while gradual increase in PDR was observed with the increase of age in NED subjects younger than 60.In contrast,no significant differences were identified between the age groups in CRC patients.Further studies revealed that no difference in PDR was found between male and female in every CRC stage,and no difference in PDR was observed among all locations of colorectal tract,including ileocecus,ascending colon,transverse colon,descending colon,sigmoid colon and rectum.Assays using 1/3 algorithm found that the PDR for serrated adenoma(SA),tubular adenoma(TA),tubulovillous adenoma(TVA)and villous adenoma was 27.8%,28.7%,53.7% and 83.3%,respectively,suggesting the positive correlation between the PDR and the amount of villous components.The high PDR in TVA and VA indicates that the SEPT9 assay can be used in the screening of precancerous advanced adenoma.In this study,we found that algorithms,cancer staging and subject age were factors affecting the performance of the SEPT9 assay,while sex and cancer location appeared to have no effect on its performance.The SEPT9 assay exhibited adequate sensitivity to adenomas with villous components or LGD/HGD,while showed low sensitivity to hyperplastic polyps.Thus,it can be used as a blood assay for screening of advanced adenoma and early-stage CRC.