Methylation-Based Markers of Aging and Lifestyle-Related Factors and Risk of Breast Cancer

A Pooled Analysis of Four Prospective Studies

Pierre-Antoine Dugue; Clara Bodelon; Felicia F. Chung; Hannah R. Brewer; Srikant Ambatipudi; Joshua N. Sampson; Cyrille Cuenin; Veronique Chajes; Isabelle Romieu; Giovanni Fiorito; Carlotta Sacerdote; Vittorio Krogh; Salvatore Panico; Rosario Tumino; Paolo Vineis; Silvia Polidoro; Laura Baglietto; Dallas English; Gianluca Severi; Graham G. Giles; Roger L. Milne; Zdenko Herceg; Montserrat Garcia-Closas; James M. Flanagan; Melissa C. Southey


Breast Cancer Res. 2022;24(59) 

In This Article

Abstract and Introduction


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.


Numerous studies have investigated the association of blood DNA methylation and breast cancer risk, for example, at breast cancer-specific genes,[1–3] and overall found mixed results.[4] Lower global levels of DNA methylation are thought to reflect genomic instability and have been hypothesised to increase the risk of cancer,[5] but while several studies were conducted in the context of breast cancer[6–9] they together suggested that there is no substantial association.[10] At individual cytosine-guanine (CpG) sites, our meta-analysis of individual-participant data (1,663 incident cases and matched controls) from the Melbourne Collaborative Cohort Study (MCCS), the European Prospective Investigation into Cancer and Nutrition (EPIC) (EPIC-Italy and EPIC-IARC), and the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) did not find evidence of associations with breast cancer risk.[10] In contrast, a case-cohort analysis within the Sister Study (1,566 breast cancer cases), a US cohort of women with a sister diagnosed with breast cancer, revealed associations at over 2,000 CpGs.[11] Another study with a large sample size found that genetically predicted methylation levels were associated with breast cancer risk,[12] but it is unclear how predicted methylation relates to measured methylation, given that methylation varies with age and exposures accumulated over the life course.[13–16]

Methylation-based markers of aging, such as Horvath-based,[17] Hannum-based,[18] PhenoAge[19] and GrimAge,[20] have become popular tools to evaluate the association between biological aging and risk of disease. While the 'first-generation' measures (Horvath-based and Hannum-based) were developed to predict age accurately, PhenoAge and GrimAge are methylation-based predictors of composite measures (using clinical and physiological data) that are predictive of mortality. The residual of each of these measures on chronological age, named 'age acceleration', best reflects the concept of biological aging. A positive association between epigenetic aging (Horvath first-generation measure) and risk of breast cancer was first reported in an EPIC-IARC study,[8] and later confirmed in the Sister Study for Horvath, Hannum and PhenoAge measures,[21] but not for GrimAge.[22] Only the age acceleration based on Horvath methylation age[17] was therefore studied in relation to breast cancer risk in both previous published studies, so there is a need to accumulate evidence, particularly in women unselected for family history. Associations of epigenetic aging measures with risk of several other types of cancer were also observed in the MCCS, and these tended to be stronger for PhenoAge and GrimAge than for the first-generation measures.[23,24]

Factors other than age, mainly tobacco smoking,[14,25] alcohol consumption[15,26] and body mass index[13,27,28] strongly influence blood DNA methylation and may also increase the risk of breast cancer. Similar to epigenetic aging, methylation marks of lifestyle could be useful markers to increase the precision with which we measure their association with cancer risk. These could reflect unmeasured past and cumulative exposures, imperfect assessments provided by questionnaires, or different individual responses to exposure; epigenetic predictors of lifestyle may therefore have potential to improve the prediction of breast cancer risk.

The aim of this study was to examine the association of previously derived 1) seven methylation-based measures of aging, and 2) methylation-based measures of body mass index, alcohol consumption and tobacco smoking, with breast cancer risk in a meta-analysis of individual-participant data including 1,655 breast cancer cases sampled from the MCCS, EPIC and PLCO.