Serum methylation of GALNT9, UPF3A, WARS, and LDB2 as non-invasive biomarkers for the early detection of colorectal cancer and premalignant adenomas

Background Early detection through screening programs has proven to be the most effective strategy to reduce the incidence and mortality of colorectal cancer. The most widely implemented non-invasive screening test is the fecal immunochemical test, which presents an inadequate sensitivity for the detection of precancerous advanced adenomas. This fact, together with the modest participation rates in screening programs, highlights the need for a blood test that could improve both the adherence to screening and the selection to colonoscopy. Methods In this study, we conducted a serum-based discovery and validation of circulating cell-free DNA (cfDNA) methylation biomarkers for colorectal cancer screening in a multicentre cohort of 433 serum samples including healthy controls, benign pathologies, advanced adenomas, and colorectal cancer. First, we performed an epigenome-wide methylation analysis with the MethylationEPIC array in 280 cfDNA samples using a pooling approach, followed by a robust prioritization of candidate biomarkers for the joint detection of advanced adenomas and colorectal cancer (advanced neoplasia). Then, candidate biomarkers were validated by pyrosequencing in independent individual 153 cfDNA samples. Results We report GALNT9 , UPF3A , WARS , and LDB2 as new non-invasive methylation biomarkers for the early detection of colorectal advanced neoplasia. A model composed of GALNT9 , UPF3A , WARS , and LDB2 reported a sensitivity of 62.1% and a specificity of 97.4% for the detection of advanced neoplasia. On the other hand, the combination of GALNT9 and UPF3A by logistic regression discriminated advanced neoplasia with 78.8% sensitivity and 100% specificity, outperforming the commonly used fecal immunochemical test and the methylated SEPT9 blood test. Conclusions Serum methylation levels of GALNT9 , UPF3A , WARS , and LDB2 represent highly specific and sensitive novel blood-based biomarkers for the detection of colorectal cancer and premalignant advanced adenomas of both distal and proximal locations. The reported results show the feasibility of DNA sample pooling strategies for biomarker discovery. Overall, this study highlights the utility of cfDNA methylation for the early detection of colorectal neoplasia, with the potential to be implemented as a non-invasive test for colorectal cancer screening. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work received funding from Plan Nacional I +D +I 2015-2018 (Accion Estrategica en Salud) Instituto de Salud Carlos III (Spain)-FEDER (PI15/02007), Fundacion Cientifica de la Asociacion Espanola contra el Cancer (GCB13131592CAST), and support from Centro Singular de Investigacion de Galicia (Conselleria de Cultura, Educacion e Ordenacion Universitaria) (ED431G/02, Xunta de Galicia and FEDER-European Union). Maria Gallardo-Gomez was supported by a predoctoral fellowship from Ministerio de Educacion, Cultura y Deporte (Spanish Government) (FPU15/02350). ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: Written informed consent was obtained from all patients with approval by the Ethics Committee for Clinical Research of Galicia (2018/008). The study was conducted according to the clinical and ethical principles of the Spanish Government and the Declaration of Helsinki. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable. Yes The Infinium MethylationEPIC data from all the pooled samples generated and analyzed during this study have been deposited in the NCBI Gene Expression Omnibus (GEO) ([www.ncbi.nlm.nih.gov/geo][1]) and are accessible through GEO Series accession number GSE186381. [1]: https://www.ncbi.nlm.nih.gov/geo