DNA methylation markers for sensitive detection of circulating tumor DNA in patients with gastroesophageal cancers

Background Patients with gastric and gastroesophageal junction adenocarcinomas (G/GEJ AC) face poor outcomes. Thus, sensitive biomarkers for improved clinical management are highly warranted. Detection of circulating tumor DNA (ctDNA) using DNA methylation biomarkers is a highly sensitive approach for cancer detection and management. Here, we explored the potential of a tumor-agnostic test targeting DNA methylation to detect ctDNA in patients with resectable and advanced G/GEJ AC. Methods Tumor DNA from 29 patients, and plasma cell free DNA from 17 patients with advanced- and 17 patients with resectable G/GEJ AC, and from 50 healthy controls was analyzed. A tumor agnostic, digital PCR test, TriMeth, targeting the gastrointestinal cancer specific methylated genes C9orf50, KCNQ5, and CLIP4, was performed. Results TriMeth detected tumor DNA in 29/29 (100%) of the tumor tissue samples. Furthermore, TriMeth detected ctDNA in plasma from 13/17 (76%) of patients with advanced disease, 7/17 (41%) of patients with resectable disease, and in 0/50 (0%) of healthy controls. Conclusions This study demonstrates that TriMeth may hold potential as a biomarker for identification of ctDNA in patients with G/GEJ AC. The study sets the scene for ongoing larger clinical studies investigating the performance of TriMeth in different clinical settings. ### Competing Interest Statement The authors have declared no competing interest. ### Funding Statement This work was supported by Aage & Johanne Louis-Hansens foundation \[Grant number: j.nr. 18-2B-2525\] (Andersen); NEYE Foundation \[Grant number: 2018-02-23\] (Ogaard); Dansk kraeftforskningsfond \[Grant numbers: DKF-2018-14 - (207) and DKF-2021-104 - (704) (Ogaard and Iden); Knud & Edith Eriksens foundation [Grant number: j.nr. 62786\] (Ogaard); Aarhus University \[No grant number]; DCCC ctDNA Research Center [Project number: 20220926_3\] (Iden); Danish Cancer Society \[Grant no. R257-A14700\] (Clausen); Jubilaeumsfonden af 1986 (Iden); Novo Nordic Foundation \[Grant numbers: NNF17OC0025052 and NFF22OC0074415\] (Andersen); Cancer Research UK \[Grant number: C6199/A26932\] (Andersen); Sejer Persson and Lis Kluwer Perssons scholarship \[No grant number\] (Mau-Sorensen). The authors declare that no funds, grants, or other support were received during the preparation of this manuscript. ### 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: This study was performed in line with the principles of the Declaration of Helsinki. As stated in the Methods section of the manuscript, approvals were granted from The Ethics Committee of the Capital Region Denmark for the collection and use of biological samples (j. nos. H-16046103, H-4-2013-050, H-2-2013-101, H-19076846, H-20033168) and the capture of clinical data from patient journals (j. nos. H-21052982, H-1300530, H-16046103). Informed consent was obtained from all participating patients. The Danish Data Protection Agency (j. nos. HVH-2013-022/2007-58-0015, P-2019-701) approved the study. 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, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes The original data presented in the study are included in the Supplemental Tables S3-S4. Further inquiries can be directed to the corresponding author.