Blood DNA methylation and breast cancer risk: a prospective nested case-control study

Juan Rodriguez; Felix Grassmann; Damien Kaukonen; Sara Hägg; Keith Humphreys; Mikael Eriksson; Yuqi Zhang; Katharina Morawitz; Marike Gabrielson; Per Hall; Kamila Czene

doi:10.1016/j.ebiom.2026.106352

Blood DNA methylation and breast cancer risk: a prospective nested case-control study

EBioMedicine. 2026 Jul:129:106352. doi: 10.1016/j.ebiom.2026.106352. Epub 2026 Jun 25.

Authors

Affiliations

¹ Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden. Electronic address: juan.rodriguez@ki.se.
² Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden; Health and Medical University, Potsdam, Germany.
³ Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden; InFLAMES Research Flagship Center, University of Turku, Turku, Finland.
⁴ Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden.
⁵ Health and Medical University, Potsdam, Germany.
⁶ Department of Medical Epidemiology and Biostatistics, Karolinska Institutet, Stockholm, Sweden; Department of Oncology, Södersjukhuset, Stockholm, Sweden.

Abstract

Background: Blood tests that predict breast cancer (BC) risk before diagnosis could complement mammography screening. Whole-blood DNA methylation is stable and may capture early systemic changes related to tumour development.

Methods: We conducted a prospective matched case-control study nested within Sweden's mammography screening programme, profiling whole-blood DNA methylation on the Illumina MethylationEPIC array in 377 female participants who later developed BC and 378 age-matched female controls. We identified methylation sites (CpG sites) in Stockholm and validated them in Skåne, after stringent quality control and adjustment for technical and epidemiological confounders. We then constructed a methylation risk score (MRS).

Findings: We identified 87 CpG sites in Stockholm (FDR < 0.05; |β| > 0.015), of which 22 validated in Skåne (P < 0.05; concordant direction) and formed the MRS. The MRS demonstrated significant associations with BC risk, was consistent across different BC subtypes, and independent of established risk factors. The association of MRS with BC remained relatively stable over a pre-diagnostic window of up to five years. MRS exhibited predictive performance comparable to a polygenic risk score, and it significantly improved the 3-5-year prediagnosis discrimination of established BC risk models (P < 0.05, DeLong's test). Several CpG sites correlated with immune-related genes, suggesting mechanisms involving immune modulation and tumour progression.

Interpretation: Our methylation signature was significantly associated with BC risk and may complement existing risk models, particularly for estimating 3-5-year risk. These findings suggest that blood DNA methylation may support BC risk stratification, although further validation and assessment of clinical utility are needed.

Funding: Swedish Research Council; Swedish Cancer Society; Stockholm County Council; Cancerföreningen i Stockholm; Erling-Persson Foundation; Sjöberg Foundation; and EU funds via ILB.

Keywords: Blood-based biomarkers; Breast cancer; DNA methylation; MRS; Risk prediction.

MeSH terms

Aged
Biomarkers, Tumor*
Breast Neoplasms* / blood
Breast Neoplasms* / diagnosis
Breast Neoplasms* / epidemiology
Breast Neoplasms* / genetics
Case-Control Studies
CpG Islands
DNA Methylation*
Female
Genetic Predisposition to Disease
Genetic Risk Score
Humans
Middle Aged
Prospective Studies
Risk Factors
Sweden / epidemiology

Substances

Biomarkers, Tumor