Pre-diagnosis plasma cell-free DNA methylome profiling up to seven years prior to clinical detection reveals early signatures of breast cancer

Profiling of cell-free DNA (cfDNA) has been well demonstrated to be a potential non-invasive screening tool for early cancer detection. However, limited studies have investigated the detectability of cfDNA methylation markers that are predictive of cancers in asymptomatic individuals. We performed cfDNA methylation profiling using cell-free DNA methylation immunoprecipitation sequencing (cfMeDIP-Seq) in blood collected from individuals up to seven years before a breast cancer diagnosis in addition to matched cancer-free controls. We identified differentially methylated cfDNA signatures that discriminated cancer-free controls from pre-diagnosis breast cancer cases in a discovery cohort that is used to build a classification model. We show that predictive models built from pre-diagnosis cfDNA hypermethylated regions can accurately predict early breast cancers in an independent test set (AUC=0.930) and are generalizable to late-stage breast cancers cases at the time of diagnosis (AUC=0.912). Characterizing the top hypermethylated cfDNA regions revealed significant enrichment for hypermethylation in external bulk breast cancer tissues compared to peripheral blood leukocytes and breast normal tissues. Our findings demonstrate that cfDNA methylation markers predictive of breast cancers can be detected in blood among asymptomatic individuals up to six years prior to clinical detection. ### Competing Interest Statement DDC and SVB are listed as co-inventors on patents filed related to the cfMeDIP-seq technology. SVB is co-inventor of a patent related to mutation-based ctDNA detection that is licensed to Roche. DDC received research funds from Pfizer and Nektar therapeutics. DDC and SVB are co-founders of, have ownership in, and serve in leadership roles at Adela. DWC has acted in a consulting/advisory role for AstraZeneca, Exact Sciences, Eisai, Gilead, GlaxoSmithKline, Inivata, Merck, Novartis, Pfizer and Roche, received research funding (to institution) from AstraZeneca, Gilead, GlaxoSmithKline, Inivata, Merck, Pfizer, and Roche and holds a patent (US62/675,228) for methods of treating cancers characterized by a high expression level of spindle and kinetochore associated complex subunit 3 (ska3) gene. All the other authors declare no conflict of interest. ### Funding Statement OHS biological materials were stored at the Ontario Health Study Biobank, which is supported by the Ontario Institute for Cancer Research through funding provided by the Government of Ontario, the Princess Margaret Cancer Foundation, and a Genome Canada grant (OGI-136) to PA. The OCTANE study was conducted with the support of the Ontario Institute for Cancer Research through funding provided by the Government of Ontario and by the Princess Margaret Cancer Foundation. ### 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: Patient plasma samples were obtained from the Ontario Health Study (OHS) with protocols approved by the University of Toronto Health Sciences research ethics board (protocol #34088). All participants gave written informed consent prior to participation. 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 datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. * ABRNM : Adjacent Breast Normal AUC : Area Under the Receiver Operating Characteristic Curve AUC(t) : Time-dependent Area Under the Receiver Operating Characteristic Curve HBRNM : Healthy Breast Normal BRCA : Breast Cancer cfDNA : Cell-free DNA C-index : Concordance Index CoxPH : Cox Proportional Hazard CV : Cross-validation DMR : Differentially Methylated Regions GEO : Geo Expression Omnibus IC : Input Control Library IHEC : International Human Epigenetics Consortium IP : Input Library KM : Kaplan-Meier LogFC : Log Fold-Change OCTANE : Ontario-wide Cancer TArgeted Nucleic Acid Evaluation OHS : Ontario Healthy Study TCGA : The Cancer Genome Atlas