Data collection

Self-reported weight and height in the year prior to diagnosis were assessed as part of the baseline interviewer-administered structured 100-min questionnaire, which was completed, on average, within 3 months of diagnosis. These assessments were used to calculate the body mass index (BMI) for each participant [weight (kg)/height (m²)], as a measure of obesity. Participants were additionally queried on their demographic characteristics (including age, race/ ethnicity, income, and education), medical histories (including family history of BC, exogenous hormone use, and mammography screening), and other potential prognostic factors as previously detailed [15–17]. Medical records were also abstracted for clinically relevant prognostic factors (including treatment and hormone receptor status).

As part of the LIBCSP protocol, medical records were abstracted at baseline and again at the 5-year follow-up to determine tumor characteristics (e.g., ER/PR status, tumor size, and nodal involvement) and treatment regimen of the first primary BC diagnosis.

Archived FFPE tumor tissue of the first primary BC was obtained, and DNA extraction was performed, as previously described [18]. Thirteen genes known to be involved in breast carcinogenesis, and frequently methylated in promoter regions, were selected for assessing interactions with obesity. Promoter methylation of ERa, PR, and BRCA1 was determined by methylation-specific (MSP)-PCR and was dichotomized (i.e., methylated vs. unmethylated) based on the presence or absence of the PCR band [18, 19]. Methylation status of the 10 remaining genes was assessed by the Methyl Light assay [20, 21]. The percentage of methylation was calculated by the 2^−ΔΔC_T method, where ΔΔC_T = (C_T,Target − C_T,Actin)_sample − (C_T,Target − C_T,Actin)_{full methylated DNA} [22] and multiplying by 100. Using a 4 % cut-off, we dichotomized into methylated or unmethylated cases as previously reported [23].

For 73.1 % of women with BC, trained phlebotomists obtained a non-fasting 40 mL blood sample at the baseline interview, and DNA was isolated as previously described [24]. Details of LUMA and LINE-1 assessment in the LIBCSP have been described previously [14]. Briefly, LUMA followed the modified protocol described by Bjornsson et al. [25] and was expressed as a percentage based on the following equation: methylation methylation(%) = [1 − (HpaII ΣG/ΣT)/(MspI ΣG/ΣT)]* 100 [25]. Four CpG sites in the promoter region of LINE-1 were assessed using a pre-validated pyrosequencing-based methylation assay [20] and were individually analyzed as a T/C single-nucleotide polymorphism using QCpG software (Qiagen). These data were subsequently averaged to provide an overall percentage 5mC status.

Vital status through the end of 2011 was determined through the NDI as previously reported [26]. Briefly, after approximately 14.7 (0.2–15.4) years of follow-up, among the 1308 patients with any global or gene-specific methylation assessments and complete BMI data, we identified 441 who died from all causes and 194 whose deaths were related to BC. BC-related deaths were determined using the International Classification of Diseases (codes 174.9 or C-50.9).