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Background Environmental and genetic factors play an important role in the etiology of breast cancer. Several small blood-based DNA methylation studies have reported risk associations with methylation at individual CpGs and average methylation levels; however, these findings require validation in larger prospective cohort studies. To investigate the role of blood DNA methylation on breast cancer risk, we conducted a meta-analysis of four prospective cohort studies, including a total of 1663 incident cases and 1885 controls, the largest study of blood DNA methylation and breast cancer risk to date. Methods We assessed associations with methylation at 365,145 CpGs present in the HumanMethylation450 (HM450K) Beadchip, after excluding CpGs that did not pass quality controls in all studies. Each of the four cohorts estimated odds ratios (ORs) and 95% confidence intervals (CI) for the association between each individual CpG and breast cancer risk. In addition, each study assessed the association between average methylation measures and breast cancer risk, adjusted and unadjusted for cell-type composition. Study-specific ORs were combined using fixed-effect meta-analysis with inverse variance weights. Stratified analyses were conducted by age at diagnosis (< 50, ≥ 50), estrogen receptor (ER) status (+/−), and time since blood collection (< 5, 5–10, > 10 years). The false discovery rate (q value) was used to account for multiple testing. Results The average age at blood draw ranged from 52.2 to 62.2 years across the four cohorts. Median follow-up time ranged from 6.6 to 8.4 years. The methylation measured at individual CpGs was not associated with breast cancer risk (q value > 0.59). In addition, higher average methylation level was not associated with risk of breast cancer (OR = 0.94, 95% CI = 0.85, 1.05; P = 0.26; P for study heterogeneity = 0.86). We found no evidence of modification of this association by age at diagnosis (P = 0.17), ER status (P = 0.88), time since blood collection (P = 0.98), or CpG location (P = 0.98). Conclusions Our data indicate that DNA methylation measured in the blood prior to breast cancer diagnosis in predominantly postmenopausal women is unlikely to be associated with substantial breast cancer risk on the HM450K array. Larger studies or with greater methylation coverage are needed to determine if associations exist between blood DNA methylation and breast cancer risk.

IMPORTANCE Clinically used breast cancer markers, such as tumor size, tumor grade, progesterone receptor (PR) status, and Ki-67 status, are known to be associated with short-term survival, but the association of these markers with long-term (25-year) survival is unclear. OBJECTIVETo assess the association of clinically used breast cancer markers with long-term survival and treatment benefit among postmenopausal women with lymph node-negative, estrogen receptor [ER]-positive and ERBB2-negative breast cancer who received tamoxifen therapy. DESIGN, SETTING, AND PARTICIPANTS This study was a secondary analysis of data from a subset of 565 women with ER-positive/ERBB2-negative breast cancer who participated in the Stockholm tamoxifen (STO-3) randomized clinical trial. The STO-3 clinical trial was conducted from 1976 to 1990 and comprised 1780 postmenopausal women with lymph node-negative breast cancer who were randomized to receive adjuvant tamoxifen therapy or no endocrine therapy. Complete 25-year follow-up data through December 31, 2016, were obtained from Swedish national registers. Immunohistochemical markers were reannotated in 2014. Data were analyzed from April to December 2020. INTERVENTIONS Patients in the original STO-3 clinical trial were randomized to receive 2 years of tamoxifen therapy vs no endocrine therapy. In 1983, patients who received tamoxifen therapy without cancer recurrence during the 2-year treatment and who consented to continued participation in the STO-3 study were further randomized to receive 3 additional years of tamoxifen therapy or no endocrine therapy. MAIN OUTCOMES AND MEASURES Distant recurrence-free interval (DRFI) by clinically used breast cancer markers was assessed using Kaplan-Meier and multivariable Cox proportional hazards analyses adjusted for age, period of primary diagnosis, tumor size (T1a and T1b [T1a/b], T1c, and T2),tumor grade (1-3), PR status (positive vs negative), Ki-67 status (low vs medium to high), and STO-3 clinical trial arm (tamoxifen treatment vs no adjuvant treatment). A recursive partitioning analysis was performed to evaluate which markers were able to best estimate long-term DRFI. RESULTSThe study population comprised 565 postmenopausal women (mean [SD] age, 62.0 [5.3] years) with lymph node-negative, ER-positive/ERBB2-negative breast cancer. A statistically significant difference in long-term DRFI was observed by tumor size (88% for T1a/b vs 76% for T1c vs 63% for T2 tumors; log-rank P < .001) and tumor grade (81% for grade 1 vs 77% for grade 2 vs 65% (continued)

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. Electronic supplementary material The online version of this article (10.1186/s13148-019-0664-7) contains supplementary material, which is available to authorized users.

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IMPORTANCE Benign breast diseases (BBDs) are common and associated with breast cancer risk, yet the etiology and risk of BBDs have not been extensively studied. OBJECTIVE To investigate the risk of BBDs by age, hormonal factors, and family history of breast cancer. DESIGN, SETTING, AND PARTICIPANTS This retrospective cohort study assessed 70 877 women from the population-based Karolinska Mammography Project for Risk Prediction of Breast Cancer (KARMA) who attended mammographic screening or underwent clinical mammography from January 1, 2011, to March 31, 2013, at 4 Swedish hospitals. Participants took part in a comprehensive questionnaire on recruitment. All participants had complete follow-up through high-quality Swedish national registers until December 31, 2015. Pathology medical records on breast biopsies were obtained for the participants, and BBD subtypes were classified according to the latest European guidelines. Analyses were conducted from January 1 to July 31, 2020. EXPOSURES Hormonal risk factors and family history of breast cancer. MAIN OUTCOMES AND MEASURESFor each BBD subtype, incidence rates (events per 100 000 person-years) and multivariable Cox proportional hazards ratios (HRs) with time-varying covariates were estimated between the ages of 25 and 69 years.RESULTS A total of 61 617 women within the mammographic screening age of 40 to 69 years (median age, 53 years) at recruitment with available questionnaire data were included in the study.Incidence rates and risk estimates varied by age and BBD subtype. At premenopausal ages, nulliparity (compared with parity Ն3) was associated with reduced risk of epithelial proliferation without atypia (EP; HR, 0.62; 95% CI, 0.46-0.85) but increased risk of cysts (HR, 1.38; 95% CI, 1.03-1.85). Current and long (Ն8 years) oral contraceptive use was associated with reduced premenopausal risk of fibroadenoma (HR, 0.65; 95% CI, 0.47-0.90), whereas hormone replacement therapy was associated with increased postmenopausal risks of epithelial proliferation with atypia (EPA; HR, 1.81; 95% CI, 1.07-3.07), fibrocystic changes (HR, 1.60; 95% CI, 1.03-2.48), and cysts (HR, 1.98; 95% CI, 1.40-2.81). Furthermore, predominantly at premenopausal ages, obesity was associated with reduced risk of several BBDs (eg, EPA: HR, 0.31; 95% CI, 0.17-0.56), whereas family history of breast cancer was associated with increased risk (eg, EPA: HR, 2.11; 95% CI, 1.48-3.00). CONCLUSIONS AND RELEVANCEThese results suggest that the risk of BBDs varies by subtype, hormonal factors, and family history of breast cancer and is influenced by age. Better understanding of BBDs is important to improve the understanding of benign and malignant breast diseases.

Background Use of immunohistochemistry (IHC) based surrogates of molecular breast cancer subtypes is common in research and clinical practice, but information on their comparative validity and prognostic capacity is scarce. Methods Data from two PAM50-subtyped Swedish breast cancer cohorts were used; STO-3 with 561 patients diagnosed 1976-1990, and Clinseq with 237 patients diagnosed 2005-2012. We evaluated three surrogate classifications; the IHC3 surrogate classifier based on ER/PR/HER2, and the StGallen and Prolif surrogate classifiers also including Ki-67. Accuracy, kappa, sensitivity and specificity were computed as compared to PAM50. Alluvial diagrams of misclassification patterns were plotted. Distant recurrence-free survival was assessed using Kaplan-Meier plots, and tamoxifen treatment benefit for luminal subtypes was modeled using flexible parametric survival models. Results The concordance with PAM50 ranged from poor to moderate (kappa = 0.36–0.57, accuracy = 0.54-0.75), with best performance for the Prolif surrogate classification in both cohorts. Good concordance was only achieved when luminal subgroups were collapsed (kappa = 0.71- 0.69, accuracy = 0.90-0.91). The StGallen surrogate classification misclassified luminal A into luminal B, the reverse pattern was seen with the others. In distant recurrence-free survival, surrogates were more similar to each other than PAM50. The difference in tamoxifen treatment benefit between luminal A and B for PAM50 was not replicated with any surrogate classifier. Conclusions All surrogate classifiers had limited ability to distinguish between PAM50 luminal A and B, but patterns of misclassifications differed. PAM50 subtyping appeared to yield larger separation of survival between luminal subtypes than any of the surrogate classifications.

PURPOSE To assess the long-term (20-year) endocrine therapy benefit in premenopausal patients with breast cancer. METHODS Secondary analysis of the Stockholm trial (STO-5, 1990-1997) randomly assigning 924 premenopausal patients to 2 years of goserelin (3.6 mg subcutaneously once every 28 days), tamoxifen (40 mg orally once daily), combined goserelin and tamoxifen, or no adjuvant endocrine therapy (control) is performed. Random assignment was stratified by lymph node status; lymph node–positive patients (n = 459) were allocated to standard chemotherapy (cyclophosphamide, methotrexate, and fluorouracil). Primary tumor immunohistochemistry (n = 731) and gene expression profiling (n = 586) were conducted in 2020. The 70-gene signature identified genomic low-risk and high-risk patients. Kaplan-Meier analysis, multivariable Cox proportional hazard regression, and multivariable time-varying flexible parametric modeling assessed the long-term distant recurrence-free interval (DRFI). Swedish high-quality registries allowed a complete follow-up of 20 years. RESULTS In estrogen receptor–positive patients (n = 584, median age 47 years), goserelin, tamoxifen, and the combination significantly improved long-term distant recurrence-free interval compared with control (multivariable hazard ratio [HR], 0.49; 95% CI, 0.32 to 0.75, HR, 0.57; 95% CI, 0.38 to 0.87, and HR, 0.63; 95% CI, 0.42 to 0.94, respectively). Significant goserelin-tamoxifen interaction was observed ( P = .016). Genomic low-risk patients (n = 305) significantly benefitted from tamoxifen (HR, 0.24; 95% CI, 0.10 to 0.60), and genomic high-risk patients (n = 158) from goserelin (HR, 0.24; 95% CI, 0.10 to 0.54). Increased risk from the addition of tamoxifen to goserelin was seen in genomic high-risk patients (HR, 3.36; 95% CI, 1.39 to 8.07). Moreover, long-lasting 20-year tamoxifen benefit was seen in genomic low-risk patients, whereas genomic high-risk patients had early goserelin benefit. CONCLUSION This study shows 20-year benefit from 2 years of adjuvant endocrine therapy in estrogen receptor–positive premenopausal patients and suggests differential treatment benefit on the basis of tumor genomic characteristics. Combined goserelin and tamoxifen therapy showed no benefit over single treatment. Long-term follow-up to assess treatment benefit is critical.