Barbara J. Fuhrman

Estrogen Metabolism and Risk of Breast Cancer in Postmenopausal Women

BACKGROUND Estrogens are recognized causal factors in breast cancer. Interindividual variation in estrogen metabolism may also influence the risk of breast cancer and could provide clues to mechanisms of breast carcinogenesis. Long-standing hypotheses about how estrogen metabolism might influence breast cancer have not been adequately evaluated in epidemiological studies because of the lack of accurate, reproducible, and high-throughput assays for estrogen metabolites. METHODS We conducted a prospective case-control study nested within the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO). Participants included 277 women who developed invasive breast cancer (case subjects) and 423 matched control subjects; at PLCO baseline, all subjects were aged 55-74 years, postmenopausal and not using hormone therapy, and provided a blood sample. Liquid chromatography-tandem mass spectrometry was used to measure serum concentrations of 15 estrogens and estrogen metabolites, in unconjugated and conjugated forms, including the parent estrogens, estrone and estradiol, and estrogen metabolites in pathways defined by irreversible hydroxylation at the C-2, C-4, or C-16 positions of the steroid ring. We calculated hazard ratios (HRs) approximating risk in highest vs lowest deciles of individual estrogens and estrogen metabolites, estrogens and estrogen metabolites grouped by metabolic pathways, and metabolic pathway ratios using multivariable Cox proportional hazards models. All statistical tests were two-sided. RESULTS Nearly all estrogens, estrogen metabolites, and metabolic pathway groups were associated with an increased risk of breast cancer; the serum concentration of unconjugated estradiol was strongly associated with the risk of breast cancer (HR = 2.07, 95% confidence interval [CI] = 1.19 to 3.62). No estrogen, estrogen metabolite, or metabolic pathway group remained statistically significantly associated with the risk of breast cancer after adjusting for unconjugated estradiol. The ratio of the 2-hydroxylation pathway to parent estrogens (HR = 0.66, 95% CI = 0.51 to 0.87) and the ratio of 4-hydroxylation pathway catechols to 4-hydroxylation pathway methylated catechols (HR = 1.34, 95% CI = 1.04 to 1.72) were statistically significantly associated with the risk of breast cancer and remained so after adjustment for unconjugated estradiol. CONCLUSIONS More extensive 2-hydroxylation of parent estrogens is associated with lower risk, and less extensive methylation of potentially genotoxic 4-hydroxylation pathway catechols is associated with higher risk of postmenopausal breast cancer.

Fecal microbial determinants of fecal and systemic estrogens and estrogen metabolites: a cross-sectional study

BackgroundHigh systemic estrogen levels contribute to breast cancer risk for postmenopausal women, whereas low levels contribute to osteoporosis risk. Except for obesity, determinants of non-ovarian systemic estrogen levels are undefined. We sought to identify members and functions of the intestinal microbial community associated with estrogen levels via enterohepatic recirculation.MethodsFifty-one epidemiologists at the National Institutes of Health, including 25 men, 7 postmenopausal women, and 19 premenopausal women, provided urine and aliquots of feces, using methods proven to yield accurate and reproducible results. Estradiol, estrone, 13 estrogen metabolites (EM), and their sum (total estrogens) were quantified in urine and feces by liquid chromatography/tandem mass spectrometry. In feces, β-glucuronidase and β-glucosidase activities were determined by realtime kinetics, and microbiome diversity and taxonomy were estimated by pyrosequencing 16S rRNA amplicons. Pearson correlations were computed for each loge estrogen level, loge enzymatic activity level, and microbiome alpha diversity estimate. For the 55 taxa with mean relative abundance of at least 0.1%, ordinal levels were created [zero, low (below median of detected sequences), high] and compared to loge estrogens, β-glucuronidase and β-glucosidase enzymatic activity levels by linear regression. Significance was based on two-sided tests with α=0.05.ResultsIn men and postmenopausal women, levels of total urinary estrogens (as well as most individual EM) were very strongly and directly associated with all measures of fecal microbiome richness and alpha diversity (R≥0.50, P≤0.003). These non-ovarian systemic estrogens also were strongly and significantly associated with fecal Clostridia taxa, including non-Clostridiales and three genera in the Ruminococcaceae family (R=0.57−0.70, P=0.03−0.002). Estrone, but not other EM, in urine correlated significantly with functional activity of fecal β-glucuronidase (R=0.36, P=0.04). In contrast, fecal β-glucuronidase correlated inversely with fecal total estrogens, both conjugated and deconjugated (R≤-0.47, P≤0.01). Premenopausal female estrogen levels, which were collected across menstrual cycles and thus highly variable, were completely unrelated to fecal microbiome and enzyme parameters (P≥0.6).ConclusionsIntestinal microbial richness and functions, including but not limited to β-glucuronidase, influence levels of non-ovarian estrogens via enterohepatic circulation. Thus, the gut microbial community likely affects the risk for estrogen-related conditions in older adults. Understanding how Clostridia taxa relate to systemic estrogens may identify targets for interventions.Trial registrationNot applicable.

Comparison of Liquid Chromatography-Tandem Mass Spectrometry, RIA, and ELISA Methods for Measurement of Urinary Estrogens

Absolute and relative concentrations of estrogens and estrogen metabolites are important for clinical decisions as well as for epidemiologic, experimental, and clinical research on hormonal carcinogenesis. RIA and ELISA are routinely used for measuring estrogen metabolites in blood and urine due to efficiency and low cost. Here, we compare absolute and ranked concentrations of estrone, estradiol, and estriol measured by indirect RIA and of 2-hydroxyestrone and 16α-hydroxyestrone measured by ELISA to the concentrations obtained using a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) method, which measures 15 estrogen metabolites concurrently. We used overnight urine samples collected from control women (362 premenopausal and 168 postmenopausal) participating in a population-based case-control study of breast cancer among Asian American women ages 20 to 55 years. When comparing RIA or ELISA levels to LC-MS/MS, absolute concentrations for the five estrogen metabolites ranged from 1.6 to 2.9 and 1.4 to 11.8 times higher in premenopausal and postmenopausal women, respectively (all P < 0.0001). However, LC-MS/MS measurements were highly correlated [Spearman r (rs) = 0.8-0.9] with RIA and ELISA measurements in premenopausal women and moderately correlated (rs = 0.4-0.8) in postmenopausal women. Measurements of the 2-hydroxyestrone:16α-hydroxyestrone ratio, a putative biomarker of breast cancer risk, were moderately correlated in premenopausal women (rs = 0.6-0.7) but only weakly correlated in postmenopausal women (rs = 0.2). LC-MS/MS had higher intraclass correlation coefficients (≥99.6%) and lower coefficients of variation (≤9.4%) than ELISA (≥97.2% and ≤14.2%) and RIA (≥95.2% and ≤17.8%). Comparison with the LC-MS/MS method suggests that the widely used RIA and ELISA estrogen metabolite measures may be problematic, especially at low estrogen metabolite levels characteristic of postmenopausal women. Cancer Epidemiol Biomarkers Prev; 19(1); 292–300

Erythrocyte Membrane Phospholipid Composition as a Biomarker of Dietary Fat

Background/Aims: In a cross-sectional study, we investigated the relationship between erythrocyte membrane phospholipid fatty acid composition and dietary fat; we also investigated roles of menopausal status, age, body mass index (BMI) and waist-to-hip ratio (WHR) in interindividual variation of the biomarker. Methods: Study participants were 204 women, aged 39–65 years, drawn from the ORDET cohort and selected as controls in a study of breast cancer. Membrane composition was assessed using capillary gas chromatography. Dietary fat composition was evaluated using a food frequency questionnaire. Results: In pre- and postmenopausal women, erythrocyte membrane phospholipid levels of linoleic acid, oleic acid, and mono-unsaturated fatty acids were significantly associated with corresponding dietary measures (partial correlation coefficients: 0.23 and 0.39; 0.45 and 0.47; 0.40 and 0.48; respectively, in pre- and postmenopausal women). Among postmenopausal women, membrane poly-unsaturated fatty acids were correlated with the corresponding dietary measure (r = 0.39, p < 0.001). Membrane eicosapentanoic and docosahexanoic acid levels were significantly correlated with intake of fish/shell fish : r = 0.21 and r = 0.43 (premenopausal), and r = 0.41 and r = 0.44 (postmenopausal). Age, BMI and WHR had independent effects on membrane lipid composition. Age was associated with delta-6 desaturase activity in postmenopausal women (r = 0.25, p < 0.05). BMI was negatively associated with delta-9 desaturase activity in both pre- and postmenopausal women (r = –0.29, p = 0.01 and r = –0.22, p < 0.01, respectively). WHR was negatively associated with delta-5 desaturase activity in pre-menopausal women (r = –024, p < 0.05). Conclusions: Erythrocyte membrane levels of some specific fatty acids can be used as biomarkers of these fatty acids as proportions of dietary fat.

Relationship of serum estrogens and estrogen metabolites to postmenopausal breast cancer risk: a nested case-control study

IntroductionElevated levels of circulating estrogens are linked to breast cancer risk among postmenopausal women but little is known about the importance of estrogen metabolism. A recently developed liquid chromatography tandem mass spectrometry-based method (LC-MS/MS) measuring a panel of 15 estrogen metabolites (EM) has been evaluated in one study, linking high levels of 2-pathway metabolites relative to the parent estrogens to reduced breast cancer risk. We analyzed this panel of EM in a nested case-control study of postmenopausal breast cancer.MethodsBetween 1977 and 1987, 6,915 women provided blood samples to the Columbia Missouri Serum Bank and were followed for incident breast cancer through December 2002. We studied 215 postmenopausal breast cancer cases and 215 matched controls who were postmenopausal and not using exogenous hormones at the time of blood draw. EM were examined individually, grouped by pathway (hydroxylation at the C-2, C-4 or C-16 positions of the steroid ring) and by ratios of the groupings. Logistic regression models controlling for matching and breast cancer risk factors were used to calculate quartile-specific odds ratios (ORs) and 95% CIs.ResultsSignificant elevated risks were not observed for individual EM, except for quartiles of 16-epiestriol (P trend = 0.07). The OR for total EM, the parent estrogens estrone and estradiol, and 2-pathway catechol EM (2-hydroxyestrone and 2-hydroxyestradiol) were elevated but the trends were not statistically significant. Among 2-pathway metabolites, risks for the highest levels of 2-hydroxyestrone-3-methyl ether and 2-methoxyestradiol were reduced; ORs for women in the highest versus lowest quartiles were 0.57 (95% CI = 0.33 to 0.99) and 0.53 (95% CI = 0.30 to 0.96), respectively. Overall, women with higher levels of 2-pathway EM had a reduced risk of breast cancer, which remained after accounting for levels of parent EM, 4-pathway EM and 16-pathway EM (all trends, P <0.11).ConclusionsWomen with more extensive hydroxylation along the 2-pathway may have a reduced risk of postmenopausal breast cancer. Further studies are needed to clarify the risks for specific EM and complex patterns of estrogen metabolism. This will require aggregation of EM results from several studies.

Associations of the Fecal Microbiome With Urinary Estrogens and Estrogen Metabolites in Postmenopausal Women

CONTEXT The gut microbiota may influence the risk of breast cancer through effects on endogenous estrogens. OBJECTIVE The objective of the study was to investigate whether urinary estrogens and estrogen metabolites are associated with the diversity and composition of the fecal microbiome. DESIGN AND SETTING This was a cross-sectional study among women enrolled in Kaiser Permanente of Colorado. PARTICIPANTS A total of 60 women drawn from a random sample of healthy postmenopausal women (aged 55-69 y), without current or recent use of antibiotics or hormone therapy and no history of cancer or gastrointestinal disease participated in the study. OUTCOME MEASURES AND METHODS: Creatinine-standardized urinary estrogens (estrone and estradiol) and 13 hydroxylated estrogen metabolites were measured in spot urines by liquid chromatography-tandem mass spectrometry. The fecal microbiome was assessed using pyrosequencing of 16S rRNA amplicons. General linear models were used to test for associations of diversity and composition of the fecal microbiome with parent estrogen (estrone + estradiol), total estrogens, and estrogen metabolites and the ratio of estrogen metabolites to parent estrogen, which has been predictive of postmenopausal breast cancer risk in previous studies. RESULTS The ratio of metabolites to parents was directly associated with whole-tree phylogenetic diversity (R = 0.35, P = .01). Relative abundances of the order Clostridiales (R = 0.32, P = .02) and the genus Bacteroides (R = -0.30, P = .03) were also correlated with the ratio of metabolites to parents. Associations were independent of age, body mass index, and study design factors. CONCLUSIONS Our data suggest that women with a more diverse gut microbiome exhibit an elevated urinary ratio of hydroxylated estrogen metabolites to parent estrogen. Further research is warranted to confirm and relate these findings to clinical disease.

Estrogen metabolism and breast cancer risk among postmenopausal women: a case-cohort study within B~FIT

Although elevated circulating estrogens are associated with increased postmenopausal breast cancer risk, less is known regarding the role of estrogen metabolism in breast carcinogenesis. We conducted a case-cohort study within the Breast and Bone Follow-up to the Fracture Intervention Trial to assess serum estrogens and estrogen metabolites (EMs) in 407 incident breast cancer cases diagnosed during follow-up and a subcohort of 496 women. In 1992-93, women completed a baseline questionnaire and provided blood samples. Hazard ratios (HRs) and 95% confidence intervals (CIs), adjusted for geography and trial participation status, were estimated using Cox proportional hazard regression. Serum concentrations of EMs were measured by liquid chromatography-tandem mass spectrometry. EMs (quintiles, Q) were analyzed individually, as metabolic pathways (C-2, -4 or -16) and as ratios. Elevated circulating estradiol was associated with increased breast cancer risk (HRQ5vsQ1 = 1.86; 95% CI: 1.19-2.90; P trend = 0.04). An elevated ratio of the 2-hydroxylation pathway (HRQ5vsQ1 = 0.69; 95% CI: 0.46-1.05; P trend = 0.01) and 4-hydroxylation pathway (HRQ5vsQ1 = 0.61; 95% CI: 0.40-0.93; P trend = 0.004) to parent estrogens (estradiol and estrone) was inversely associated with risk. A higher ratio of the 2/16-hydroxylation pathways was associated with reduced risk (HRQ5vsQ1 = 0.60; 95% CI: 0.40-0.90; P trend = 0.002). Increased 2- or 4-hydroxylation of parent estrogens may lower risk of postmenopausal breast cancer. Analyses of metabolic pathways may help elucidate the role of estrogen metabolism in breast carcinogenesis.

A new approach to measuring estrogen exposure and metabolism in epidemiologic studies.

Endogenous estrogen plays an integral role in the etiology of breast and endometrial cancer, and conceivably ovarian cancer. However, the underlying mechanisms and the importance of patterns of estrogen metabolism and specific estrogen metabolites have not been adequately explored. Long-standing hypotheses, derived from laboratory experiments, have not been tested in epidemiologic research because of the lack of robust, rapid, accurate measurement techniques appropriate for large-scale studies. We have developed a stable isotope dilution liquid chromatography-tandem mass spectrometry (LC-MS(2)) method that can measure concurrently all 15 estrogens and estrogen metabolites (EM) in urine and serum with high sensitivity (level of detection=2.5-3.0fmol EM/mL serum), specificity, accuracy, and precision [laboratory coefficients of variation (CVs) < or =5% for nearly all EM]. The assay requires only extraction, a single chemical derivatization, and less than 0.5mL of serum or urine. By incorporating enzymatic hydrolysis, the assay measures total (glucuronidated+sulfated+unconjugated) EM. If the hydrolysis step is omitted, the assay measures unconjugated EM. Interindividual differences in urinary EM concentrations (pg/mL creatinine), which reflect total EM production, were consistently large, with a range of 10-100-fold for nearly all EM in premenopausal and postmenopausal women and men. Correlational analyses indicated that urinary estrone and estradiol, the most commonly measured EM, do not accurately represent levels of total urinary EM or of the other EM. In serum, all 15 EM were detected as conjugates, but only 5 were detected in unconjugated form. When we compared our assay methods with indirect radioimmunoassays for estrone, estradiol, and estriol and enzyme-linked immunosorbent assays for 2-hydroxyestrone and 16alpha-hydroxyestrone, ranking of individuals agreed well for premenopausal women [Spearman r (r(s))=0.8-0.9], but only moderately for postmenopausal women (r(s)=0.4-0.8). Our absolute readings were consistently lower, especially at the low concentrations characteristic of postmenopausal women, possibly because of improved specificity. We are currently applying our EM measurement techniques in several epidemiologic studies of premenopausal and postmenopausal breast cancer.

Epidemiologic studies of estrogen metabolism and breast cancer.

Early epidemiologic studies of estrogen metabolism measured only 2-hydroxyestrone and 16α-hydroxyestrone and relied on direct enzyme immunoassays without purification steps. Eight breast cancer studies have used these assays with prospectively collected blood or urine samples. Results were inconsistent, and generally not statistically significant; but the assays had limited specificity, especially at the low concentrations characteristic of postmenopausal women. To facilitate continued testing in population-based studies of the multiple laboratory-based hypotheses about the roles of estrogen metabolites, a novel liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed to measure concurrently all 15 estrogens and estrogen metabolites in human serum and urine, as unconjugated and total (glucuronidated+sulfated+unconjugated) concentrations. The assay has high sensitivity (lower limit of quantitation ∼1-2 pmol/L), reproducibility (coefficients of variation generally ⩽5%), and accuracy. Three prospective studies utilizing this comprehensive assay have demonstrated that enhanced 2-hydroxylation of parent estrogens (estrone+estradiol) is associated with reduced risk of postmenopausal breast cancer. In the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) cohort, the serum ratio of 2-hydroxylation pathway metabolites to parent estrogens was associated with a 28% reduction in breast cancer risk across extreme deciles (p-trend=.05), after adjusting for unconjugated estradiol and breast cancer risk factors. Incorporating this ratio into a risk prediction model already including unconjugated estradiol improved absolute risk estimates substantially (by ⩾14%) in 36% of the women, an encouraging result that needs replication. Additional epidemiologic studies of the role of estrogen metabolism in the etiology of hormone-related diseases and continued improvement of estrogen metabolism assays are justified.

Equol Status Modifies the Association of Soy Intake and Mammographic Density in a Sample of Postmenopausal Women

Only 30% to 50% of people produce the daidzein-metabolite equol after eating soy. We conducted a cross-sectional study of the associations between equol status, intake of soy foods, and mammographic density in a sample of postmenopausal women recruited at a radiology clinic near Buffalo, New York. Participants were 48 to 82 years old, had no history of cancer or breast reduction/augmentation, and no recent use of antibiotics or hormones. Percent density was measured by computer-assisted analysis of digitized images of craniocaudal films. Equol status was assessed using a soy-challenge protocol and usual soy intake by questionnaire. General linear models were used to assess independent and joint effects of equol status and intake of soy on multivariate adjusted percent density (covariates included age, body mass index, parity, age at first birth, and ever use of combined hormone therapy). Of 325 enrolled, 232 (71%) participants completed study assessments and are included in the present analysis. Mean percent density was 34% (±18%). Seventy-five (30%) participants were producers of equol. Forty-three (19%) participants reported regularly eating >1 soy food or supplement/wk. There were no significant independent associations of equol status or soy intake with percent density, but the interaction between these factors was significant (P < 0.01). Among equol producers, those with weekly soy intake had lower percent density (30.7% in weekly consumers of soy versus 38.9% in others; P = 0.08); among nonproducers, weekly soy intake was associated with higher percent density (37.5% in weekly soy consumers versus 30.7% in others; P = 0.03). Results suggest that equol producers and nonproducers may experience different effects of dietary soy on breast tissue. (Cancer Epidemiol Biomarkers Prev 2008;17(1):33–42)