Epigenome-wide association study for lifetime estrogen exposure identifies an epigenetic signature associated with breast cancer risk

Abstract

Background: It is well established that estrogens and other hormonal factors influence breast cancer susceptibility. We hypothesized that a woman's total lifetime estrogen exposure accumulates changes in DNA methylation, detectable in the blood, which could be used in risk assessment for breast cancer.

Methods: An estimated lifetime estrogen exposure (ELEE) model was defined using epidemiological data from EPIC-Italy (n = 31,864). An epigenome-wide association study (EWAS) of ELEE was performed using existing Illumina HumanMethylation450K Beadchip (HM450K) methylation data obtained from EPIC-Italy blood DNA samples (n = 216). A methylation index (MI) of ELEE based on 31 CpG sites was developed using HM450K data from EPIC-Italy and the Generations Study and evaluated for association with breast cancer risk in an independent dataset from the Generations Study (n = 440 incident breast cancer cases matched to 440 healthy controls) using targeted bisulfite sequencing. Lastly, a meta-analysis was conducted including three additional cohorts, consisting of 1187 case-control pairs.

Results: We observed an estimated 5% increase in breast cancer risk per 1-year longer ELEE (OR = 1.05, 95% CI 1.04-1.07, P = 3 × 10^-12) in EPIC-Italy. The EWAS identified 694 CpG sites associated with ELEE (FDR Q < 0.05). We report a DNA methylation index (MI) associated with breast cancer risk that is validated in the Generations Study targeted bisulfite sequencing data (OR_{Q4_vs_Q1} = 1.77, 95% CI 1.07-2.93, P = 0.027) and in the meta-analysis (OR_{Q4_vs_Q1} = 1.43, 95% CI 1.05-2.00, P = 0.024); however, the correlation between the MI and ELEE was not validated across study cohorts.

Conclusion: We have identified a blood DNA methylation signature associated with breast cancer risk in this study. Further investigation is required to confirm the interaction between estrogen exposure and DNA methylation in the blood.

Keywords: Biomarker; Breast cancer; Cancer risk; DNA methylation; EWAS; Epigenetics; Estrogen exposure; Hormonal exposures.

Fig. 1

EWAS identifies CpG sites significantly associated with ELEE in EPIC-Italy. An EWAS with DNA methylation (HM450K beta values) as outcome and ELEE as exposure was conducted in EPIC-Italy (n = 216) using a beta regression model adjusted for age, BMI, alcohol consumption, and smoking duration (all variables reported at recruitment), and batch, position on batch, and WBC composition. a Manhattan plot of minus log₁₀Q values for FDR-corrected P values from the EWAS of all 404,596 probes across the genome. Blue line indicates FDR Q value threshold of 0.05. b Volcano plot of the regression coefficients (estimates) from the beta regression model, showing significant hypomethylated (in blue) and hypermethylated probes (in purple)

Fig. 2

The MI is associated with breast cancer risk. The MI was developed in combined HM450K data from EPIC-Italy (dataset 2, n = 237) and the Generations Study (dataset 3, n = 65) using ridge regression. The correlation between the MI and ELEE and the association between the MI and breast cancer risk were evaluated. a The correlations between the MI and ELEE in the development of HM450K data were as follows: r = 0.60 and P = 6 × 10⁻²⁵ for EPIC-Italy and r = 0.27 and P = 0.027 for the Generations Study b The MI and ELEE were not correlated in the Generations Study targeted sequencing data (r =− 0.04, P = 0.340). c Density plot of the MI values in controls and cases in EPIC-Italy HM450K data. The MI was significantly associated with breast cancer risk in EPIC-Italy (n = 162 pairs, OR = 1.51, 95% CI 1.26–1.82, P = 1 × 10⁻⁵). d Density plot of the MI values in controls and cases in the Generations Study targeted sequencing data. The MI was significantly associated with breast cancer risk in the Generations Study (n = 339 pairs, OR = 1.04, 95% CI 1.01–1.08, P = 0.022). ORs were adjusted for age, BMI, alcohol consumption, and smoking duration (all variables reported at recruitment) and WBC composition

Fig. 3

Meta-analysis of the association between MI and breast cancer risk. The association between MI and risk for breast cancer, as a continuous variable (a) or as a categorical variable (b), was estimated in the four studies included in the meta-analysis using conditional logistic regression adjusted for age, BMI, alcohol consumption, and smoking duration (all variables reported at recruitment) and WBC composition. The log odds ratios were combined in a meta-analysis using restricted-maximum likelihood model. The square boxes represent the odds ratios (ORs) and the lines the 95% confidence intervals (CIs). ^aEPIC-Italy corresponds to the new EPIC-Italy samples, not included in the development of the MI

Fig. 4

Time to diagnosis and the association between the MI and breast cancer risk. Matched case-control pairs were stratified on median time to diagnosis in EPIC-Italy HM450K data (dataset 2) and in the four study cohorts included in the meta-analysis. The association between the MI and breast cancer risk was analyzed in the two groups. a The MI was significantly associated with breast cancer risk in EPIC-Italy pairs with a shorter time to diagnosis (n = 81 pairs, OR = 1.47, 95% CI 1.12–1.93, P = 0.005). b The MI was significantly associated with breast cancer risk in EPIC-Italy pairs with a longer time to diagnosis (n = 81 pairs, OR = 1.84, 95% CI 1.30–2.61, P = 0.001). c The combined meta-analysis including pairs with shorter time to diagnosis showed no significant association between the MI and breast cancer risk (OR = 1.03, 95% CI 0.98–1.08, P = 0.241). d The combined meta-analysis including pairs with shorter time to diagnosis showed no significant association between the MI and breast cancer risk (OR = 1.05, 95% CI 1.01–1.10, P = 0.021). The log odds ratios were combined in the meta-analyses using restricted-maximum likelihood model. ORs were adjusted for age, BMI, alcohol consumption, and smoking duration (all variables reported at recruitment) and WBC composition