Risk of Breast Cancer Associated with Estrogen DNA Adduct Biomarker

Prior research suggests elevated concentrations of estrogen DNA adducts (EDA) may confer an increased breast cancer risk (1). Three prior studies found that breast cancer cases had elevated concentrations of EDA relative to estrogen metabolites and conjugates (EDA:EMC), particularly for the 4-hydroxy pathway, compared with healthy controls in urine and blood samples (2–4). Moreover, these studies showed women at high versus low risk of breast cancer (defined by Gail score) had elevated EDA:EMC, suggesting that elevated EDA:EMC precedes cancer development (2–4). However, these studies had small sample sizes (<80 cases) and biospecimens in cases were not collected prior to breast cancer diagnosis. As these prior studies could not rule out the possibility that breast cancer itself led to elevated EDA:EMC, we conducted an analysis using prospectively collected samples from the Women's Health Initiative (WHI) study, in which EDA:EMC was examined in biospecimens collected prior to breast cancer.

This data analysis was conducted among 385 postmenopausal women from the WHI using a nested case–control design (n = 191 cases and n = 194 controls). Eligibility criteria included the following: ages 50 to 79 years at enrollment, enrollment in either the observational study or diet modification trial, and availability of biospecimen, which for cases must have been ≥1 year prior to diagnosis (5). Cases had a physician-adjudicated diagnosis of incident breast cancer. Controls were 1:1 matched on age at enrollment, race/ethnicity, biospecimen collection time point, hysterectomy status, clinical site, and WHI study (6). Subsequent to sample selection, some participants were removed from analysis (i.e., removal of ductal carcinoma in situ cases). A priori calculations indicated this study was powered to detect an OR = 2.2. All participants provided informed consent. This study was approved by the Fred Hutch Cancer Research Center Institutional Review Board (Seattle, WA).

Laboratory methods were based on previous reports for EDA:EMC and creatinine (4), with the exception of instrumentation and extraction methodology. Briefly, ascorbic acid was added to urine at collection, and then urine was stored at −80°C. After thawing, a solution was made of 200 μL of urine and 10 μL of a 10 ng/mL solution of 2-OH-3-OCH₃E₁ in water (as internal standard). The sample was vortexed for 15 seconds, 600 μL of methanol was added, the sample was vortexed again for 30 seconds, and the supernatant was removed and evaporated to dryness under a steady stream on nitrogen. The sample was then reconstituted with 50 μL of a solution of 80:20 water:acetonitrile both containing 0.1% formic acid. Injections (20 μL) were made using a Waters I-class UPLC Platform coupled with a Waters Xevo TQ-S Mass Spectrometer.

Statistical analysis included unconditional logistic regression to estimate ORs of breast cancer associated with EDA:EMC. As described previously (1–3), the numerator sums six EDA compounds (i.e., catechol estrogens in 4-hydroxy and 2-hydroxy pathways for both estrones and estradiols bound to a DNA base pair; Supplementary Table S1). The denominator sums EMC (i.e., those inactivated) in the same pathways but not bound to a DNA base pair (e.g., 2-OHE₂). The intraassay coefficient of variation (CV) for all batches in this analysis was <30%. Secondary analyses included investigation of the 2- and 4-hydroxy pathways separately, and investigation of whether estrogen receptor (ER) status or a greater time between sample collection and cancer impacted results.

In this dataset, the mean age was 63 years, and women were predominantly White (Table 1). With the exception of Gail score, smoking history, and estrogen + progestin hormone therapy, cases and controls were similar. Overall, EDA:EMC did not differ between cases and controls (Table 2), and no difference in either EDA or EMC concentrations between cases and controls was seen. Risk did not differ when restricted to ER⁺ breast cancer, nor to those with a biospecimen collected ≥2 years prior to breast cancer.

In this analysis, in which biospecimens were collected prior to cancer, we did not detect an association between EDA:EMC overall and breast cancer, nor an elevation in breast cancer risk associated with the 4-hydroxy or 2-hydroxy pathway when investigated separately.

The biologic plausibility for EDA's role in breast cancer has been described in numerous studies (1, 7, 8). Certain catechol estrogen metabolites, particularly 4-hydroxy EDAs, are genotoxic as they are capable of forming DNA adducts, which through depurination may result in DNA mutation similarly to benzene, a known carcinogen (1). On the basis of these preclinical observations, three human studies reported EDA:EMC was significantly elevated in women with breast cancer relative to control women (1–3).

Limitations of this analysis include the relatively high %CVs for some compounds (Supplementary Table S1). The mean %CV across all compounds was 15%. For the most part, EDA compounds had lower %CVs and still those were not associated with elevated breast cancer risk. Prior to this study, no study had evaluated this research question with prospectively collected biospecimens, a strength of this study. This study did not provide evidence that breast cancer risk was increased in relation to EDA:EMC, nor as hypothesized with an increased risk in relation to 4-hydroxy EDAs.

R.T. Chlebowski reports personal fees from Novartis, AstraZeneca, Genentech, Merck, Immunomedics, and Puma during the conduct of the study. L.F. Tinker reports other from the National Heart, Lung, and Blood Institute (contract) during the conduct of the study. No potential conflicts of interest were disclosed by the other authors.

K.W. Reding: Conceptualization, funding acquisition, methodology, writing–original draft. C.J. Han: Formal analysis, investigation, writing–review and editing. D. Whittington: Formal analysis, writing–review and editing. M. Zahid: Conceptualization, investigation, writing–review and editing. E.G. Rogan: Conceptualization, writing–review and editing. D. Langford: Methodology, writing–review and editing. T.E. Rohan: Writing–review and editing. R.T. Chlebowski: Writing–review and editing. T.-Y.D. Cheng: Writing–review and editing. W.E. Barrington: Writing–review and editing. L.F. Tinker: Resources, project administration, writing–review and editing.

This study was funded by NCI grant R21CA178373. The Women's Health Initiative program was funded by the National Heart, Lung, and Blood Institute, NIH, and U.S. Department of Health and Human Services through contracts, HHSN268201600018C, HHSN268201600001C, HSN268201600002C, HHSN268201600003C, and HHSN268201600004C. The authors wish to thank the study participants for their contribution to this research project.

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