• Precision Medicine, School of Clinical Sciences at Monash Health, Monash University, Clayton, VIC, Australia. pierre-antoine.dugue@monash.edu.
• Divison of Cancer Epidemiology and Genetics, National Cancer Institute, Bethesda, USA.
• International Agency for Research On Cancer (IARC), Lyon, France.
• Department of Surgery and Cancer, Faculty of Medicine, Imperial College London, London, UK.
• Department of Biomedical Sciences, University of Sassari, Sassari, Italy.
• Unit of Cancer Epidemiology, Città della Salute e Della Scienza University-Hospital, Turin, Italy.
• Department of Research, Epidemiology and Prevention Unit, Fondazione IRCCS Istituto Nazionale dei Tumori di Milano, Milan, MI, Italy.
• Dipartimento di Medicina Clinica e Chirurgia Federico II University, Naples, Italy.
• Hyblean Association for Epidemiological Research AIRE-ONLUS, Ragusa, Italy.
• MRC Centre for Environment and Health, School of Public Health, Imperial College London, London, UK.

BACKGROUND: DNA methylation in blood may reflect adverse exposures accumulated over the lifetime and could therefore provide potential improvements in the prediction of cancer risk. A substantial body of research has shown associations between epigenetic aging and risk of disease, including cancer. Here we aimed to study epigenetic measures of aging and lifestyle-related factors in association with risk of breast cancer.

METHODS: Using data from four prospective case-control studies nested in three cohorts of European ancestry participants, including a total of 1,655 breast cancer cases, we calculated three methylation-based measures of lifestyle factors (body mass index [BMI], tobacco smoking and alcohol consumption) and seven measures of epigenetic aging (Horvath-based, Hannum-based, PhenoAge and GrimAge). All measures were regression-adjusted for their respective risk factors and expressed per standard deviation (SD). Odds ratios (OR) and 95% confidence intervals (CI) were calculated using conditional or unconditional logistic regression and pooled using fixed-effects meta-analysis. Subgroup analyses were conducted by age at blood draw, time from blood sample to diagnosis, oestrogen receptor-positivity status and tumour stage.

RESULTS: None of the measures of epigenetic aging were associated with risk of breast cancer in the pooled analysis: Horvath 'age acceleration' (AA): OR per SD = 1.02, 95%CI: 0.95-1.10; AA-Hannum: OR = 1.03, 95%CI:0.95-1.12; PhenoAge: OR = 1.01, 95%CI: 0.94-1.09 and GrimAge: OR = 1.03, 95%CI: 0.94-1.12, in models adjusting for white blood cell proportions, body mass index, smoking and alcohol consumption. The BMI-adjusted predictor of BMI was associated with breast cancer risk, OR per SD = 1.09, 95%CI: 1.01-1.17. The results for the alcohol and smoking methylation-based predictors were consistent with a null association. Risk did not appear to substantially vary by age at blood draw, time to diagnosis or tumour characteristics.

CONCLUSION: We found no evidence that methylation-based measures of aging, smoking or alcohol consumption were associated with risk of breast cancer. A methylation-based marker of BMI was associated with risk and may provide insights into the underlying associations between BMI and breast cancer.