Early Detection of Metastatic Relapse and Monitoring of Therapeutic Efficacy by a Five Circulating Tumor DNA Methylation Signature in Colorectal Cancer

Purpose: To develop a circulating tumor DNA (ctDNA) methylation signature for early detection of metastatic relapse and surveillance of therapeutic efficacy in stage III CRC. Perimental design: The deferentially methylated locations (DMLs) between 52 normal intestinal mucosa and 134 stage III CRC tissue and between plasma from 96 stage I/II CRC patients and 73 stage IV CRC patients were detected by high-throughput targeted methylation sequencing. CtDNA methylation signature was established by random forest with DMLs shared by plasma and tissue samples. This signature was then applied to predict the risk of metastatic disease recurrence in blood samples from 50 stage III CRC patients and dynamic monitor the recurrence risk in serial blood samples from 4 CRC patients. Results: 2237 and 1185 hypermethylation markers were identified in CRC tissue and plasma from stage IV CRC patients, respectively. Our signature achieved the sensitivity of 84.21% and specificity of 81.81% in distinguishing late CRC from early CRC in the validation cohort. Moreover, this signature yielded high sensitivity (87.5%) and specificity (94.12%) for predicting metastatic disease relapse in a cohort of 50 stage III CRC patients, and could be an independent risk factor for relapse in those patients (HR, 18.7; 95% CI, 5.3-66.2; P = 9.86e-6). The analyses of serial blood samples of CRC showed that this model was more sensitive to monitor treatment response than imaging examination and serum carcinoembryonic antigen (CEA). Conclusions: We identify a ctDNA methylation signature for stratifying risk of recurrence in stage III CRC and non-invasively monitoring treatment response.Statement of translational relevanceWe successfully developed a ctDNA methylation signature for risk assessment of metastatic disease recurrence in stage III CRC patients and surveillance of therapeutic efficacy of CRC. Funding: This work was supported by the National Key R&D program of China (2017YFC1309002), National Natural Science Foundation of China (81672821, 81872041, 81773101, 81802306, 81903002), Natural Science Foundation Key program of Guangdong Province of China (2018B0303110017), Natural Science Foundation of Guangdong Province of China (2018A030310457, 2019A1515011436), Science and Technology Planning Project of Guangdong Province, China (2017B020226005). Declaration of Interest: The authors Jianbing Fan, Xia Li are employees of AnchorDx Medical Co., Ltd., a company that focuses on the development of next generation sequencing diagnostic products for cancer detection using liquid biopsy. All other authors declare no competing financial interest. Ethical Approval: It was approved by the Human Research Ethical Review Board in Southern Hospital of Southern Medical University and Southern Theater General Hospital of Chinese people's Liberation Army.