Associations of reproductive breast cancer risk factors with expression of stem cell markers in benign breast tissue.

Background: We investigated the associations of reproductive factors known to influence breast cancer risk with the expression of breast stem cell markers CD44, CD24, and ALDH1A1 in benign breast biopsy samples. Methods: We included 439 cancer-free women with biopsy-confirmed benign breast disease within the Nurses' Health Study (NHS) and NHSII. The data on reproductive and other breast cancer risk factors were obtained from biennial questionnaires. Immunohistochemistry (IHC) was performed on tissue microarrays. For each core, the IHC expression was assessed using a semi-automated platform and expressed as % of cells that stained positive for a specific marker out of the total cell count. Generalized linear regression was used to examine the associations of reproductive factors with a log-transformed expression of each marker (in epithelium and stroma), adjusted for other breast cancer risk factors. Results: In multivariate analysis, the time between menarche and age at first birth was inversely associated with CD44 in epithelium (ß per 5 years = -0.38, 95% CI -0.69; -0.06). Age at first birth and the time between menarche and age at first birth were inversely associated with ALDH1A1 (stroma: ß per 5 years = -0.43, 95% CI -0.76; -0.10 and ß = -0.47, 95% CI -0.79; -0.15, respectively; epithelium: ß = -0.15, 95% CI -0.30; -0.01 and ß = -0.17, 95% CI -0.30; -0.03, respectively). Time since last pregnancy was inversely associated with stromal ALDH1A1 (ß per 5 years = -0.55, 95% CI -0.98; -0.11). No associations were found for CD24. The observed associations were similar in premenopausal women. In postmenopausal women, lifetime duration of breastfeeding was inversely associated with stromal ALDH1A1 expression (ß for ≥24 vs. 0 to <1 months = -2.24, 95% CI 3.96; -0.51, p-trend = 0.01). Conclusion: Early-life reproductive factors may influence CD44 and ALDH1A1 expression in benign breast tissue.

Associations of early-life and adult anthropometric measures with the expression of stem cell markers CD44, CD24, and ALDH1A1 in women with benign breast biopsies.

BACKGROUND: According to the stem cell hypothesis, breast carcinogenesis may be related to the breast stem cell pool size. However, little is known about associations of breast cancer risk factors, such as anthropometric measures, with the expression of stem cell markers in non-cancerous breast tissue. METHODS: The analysis included 414 women with biopsy-confirmed benign breast disease (BBD) in the Nurses' Health Study (NHS) and NHSII. Birthweight, weight at age 18, current weight, and current height were reported via self-administered questionnaire. Immunohistochemical staining of stem cell markers (CD44, CD24, ALDH1A1) in histopathologically normal epithelial and stromal breast tissue was quantified with an automated computational image analysis system. Linear regression was used to examine the associations of early-life and adult anthropometric measures with log-transformed stem cell marker expression, adjusting for potential confounders. RESULTS: Birthweight (≥10.0 vs. <5.5 lbs: ß [95% CI]=4.29 [1.02, 7.56]; p-trend=0.001 in stroma) and adult height (≥67.0 vs. <63.0 inch: 0.86 [0.14, 1.58]; p-trend=0.02 in epithelium and stroma combined) were positively associated with CD44 expression. Childhood body fatness was inversely (p-trend=0.03) and adult height was positively associated with CD24 expression in combined stroma and epithelium (p-trend=0.03). CONCLUSION: Our data suggest that anthropometric measures, such as birthweight, adult height, and childhood body fatness, may be associated with the stem cell expression among women with BBD. IMPACT: Anthropometric measures, such as birthweight, height, and childhood body fatness, may have long-term impacts on stem cell population in the breast.

Reproductive Factors Related to Childbearing and a Novel Automated Mammographic Measure, V.

BACKGROUND: We investigated the associations between several reproductive factors related to childbearing and the variation (V) measure (a novel, objective, single summary measure of breast image intensity) by menopausal status. METHODS: Our study included 3,814 cancer-free women within the Nurses' Health Study (NHS) and NHSII cohorts. The data on reproductive variables and covariates were obtained from biennial questionnaires closest to the mammogram date. V-measures were obtained from mammographic images using a previously developed algorithm capturing the standard deviation of pixel values. We used multivariate linear regression to examine the associations of parity, age at first birth, time between menarche and first birth, time since last pregnancy, and lifetime breastfeeding duration with V-measure, adjusting for breast cancer risk factors, including the percentage of mammographic density (PMD). We further examined whether these associations were statistically accounted for (mediated) by PMD. RESULTS: Among premenopausal women, none of the reproductive factors were associated with V. Among postmenopausal women, inverse associations of parity and positive associations of age at first birth with V were mediated by PMD (percent mediated: nulliparity: 66.7%, P < 0.0001; parity: 50.5%, P < 0.01; age at first birth 76.1%, P < 0.001) and were no longer significant in PMD-adjusted models. Lifetime duration of breastfeeding was positively associated with V [>36 vs. 0 ≤1 months ß = 0.29; 95% confidence interval (CI) 0.07; 0.52, Ptrend < 0.01], independent of PMD. CONCLUSIONS: Parity, age at first birth, and breastfeeding were associated with postmenopausal V. IMPACT: This study highlights associations of reproductive factors with mammographic image intensity.

Effect of testosterone therapy on breast tissue composition and mammographic breast density in trans masculine individuals.

Objective: Determine the association between TT and breast tissue composition and breast tissue density in trans masculine individuals (TMIs). Design: This is a cross-sectional study. Setting: TMIs (n=444) underwent chest-contouring surgeries to treat their gender dysphoria between 2013 and 2019 at an urban medical center. Participants: Of the 444 TMIs, 425 had pathology images analyzed by our deep-learning algorithm to extract breast tissue composition. A subset of 42/444 TMIs had mammography prior to surgery; mammography files were available for 25/42 TMIs and analyzed using a breast density software, LIBRA. Main Outcomes and Measures: The first outcome was the association of duration of TT and breast tissue composition assessed by pathologists (categories of lobular atrophy and stromal composition) or by our algorithm (% epithelium, % fibrous stroma, and % fat). The second outcome is the association of TT and breast density as assessed by a radiologist (categorical variable) or by LIBRA (percent density, absolute dense area, and absolute non-dense area). Results: Length of TT was associated with increasing degrees of lobular atrophy ( p <0.001) but not fibrous content ( p =0.821) when assessed by the pathologists. Every six months of TT was associated with decreased amounts of both epithelium (exp(ß)=0.97, 95% CI 0.95-0.98, adj p =0.005) and stroma (exp(ß)=0.99, 95% CI 0.98-1.00, adj p =0.051), but not fat (exp(ß)=1.01, 95%CI 0.98-1.05, p =0.394) in fully adjusted models. There was no association between TT and radiologist's breast density assessment ( p =0.575) or LIBRA measurements ( p >0.05). Conclusions: TT decreases breast epithelium and fibrous stroma, thus potentially reducing the breast cancer risk of TMIs. Further studies are warranted to elucidate the effect of TT on breast density and breast cancer risk. Summary Box: Very little is known about the effect of gender-affirming testosterone therapy on cancer risks, such as breast cancer.Epidemiological studies had different conclusions about the association between testosterone and breast cancer in cisgender women (positive association) and trans masculine individuals (inverse association).More laboratory-based research are needed to understand the effect of testosterone on breast cancer risk in the understudied trans masculine population.Our study provides quantitative histological evidence to support prior epidemiological reports that testosterone may reduce breast cancer risk in trans masculine individuals.

Association of air pollution with postmenopausal breast cancer risk in UK Biobank.

BACKGROUND: We investigated the association of several air pollution measures with postmenopausal breast cancer (BCa) risk. METHODS: This study included 155,235 postmenopausal women (of which 6146 with BCa) from UK Biobank. Cancer diagnoses were ascertained through the linkage to the UK National Health Service Central Registers. Annual exposure averages were available from 2005, 2006, 2007, and 2010 for NO2, from 2007 and 2010 for PM10, and from 2010 for PM2.5, NOX, PM2.5-10 and PM2.5 absorbance. Information on BCa risk factors was collected at baseline. Cox proportional hazards regression was used to evaluate the associations of year-specific and cumulative average exposures with BCa risk, overall and with 2-year exposure lag, while adjusting for BCa risk factors. RESULTS: PM10 in 2007 and cumulative average PM10 were positively associated with BCa risk (2007 PM10: Hazard ratio [HR] per 10 µg/m3 = 1.18, 95% CI 1.08, 1.29; cumulative average PM10: HR per 10 µg/m3 = 1.99, 95% CI 1.75, 2.27). Compared to women with low exposure, women with higher 2007 PM10 and cumulative average PM10 had greater BCa risk (4th vs. 1st quartile HR = 1.15, 95% CI 1.07, 1.24, p-trend = 0.001 and HR = 1.35, 95% CI 1.25, 1.44, p-trend < 0.0001, respectively). No significant associations were found for any other exposure measures. In the analysis with 2-year exposure lag, both 2007 PM 10 and cumulative average PM10 were positively associated with BCa risk (4th vs. 1st quartile HR = 1.19, 95% CI 1.10, 1.28 and HR = 1.29, 95% CI 1.19, 1.39, respectively). CONCLUSION: Our findings suggest a positive association of 2007 PM10 and cumulative average PM10 with postmenopausal BCa risk.

Associations of gut microbiome with endogenous estrogen levels in healthy postmenopausal women.

PURPOSE: The gut microbiome is a potentially important contributor to endogenous estrogen levels after menopause. In healthy postmenopausal women, we examined associations of fecal microbiome composition with levels of urinary estrogens, their metabolites, and relevant metabolic pathway ratios implicated in breast cancer risk. METHODS: Eligible postmenopausal women (n = 164) had a body mass index (BMI) ≤ 35 kg/m2 and no history of hormone use (previous 6 months) or cancer/metabolic disorders. Estrogens were quantified in spot urine samples with liquid chromatography-high resolution mass spectrometry (corrected for creatinine). Bacterial DNA was isolated from fecal samples and the V1-V2 hypervariable regions of 16S rRNA were sequenced on the Illumina MiSeq platform. We examined associations of gut microbiome's indices of within-sample (alpha) diversity (i.e., Shannon, Chao1, and Inverse Simpson), phylogenetic diversity, and the ratio of the two main phyla (Firmicutes and Bacteroidetes; F/B ratio) with individual estrogens and metabolic ratios, adjusted for age and BMI. RESULTS: In this sample of 164 healthy postmenopausal women, the mean age was 62.9 years (range 47.0-86.0). We found significant inverse associations of observed species with 4-pathway:total estrogens (p = 0.04) and 4-pathway:2-pathway (p = 0.01). Shannon index was positively associated with 2-catechols: methylated 2-catechols (p = 0.04). Chao1 was inversely associated with E1:total estrogens (p = 0.04), and 4-pathway:2-pathway (p = 0.02) and positively associated with 2-pathway:parent estrogens (p = 0.01). Phylogenetic diversity was inversely associated with 4-pathway:total estrogens (p = 0.02), 4-pathway:parent estrogens (p = 0.03), 4-pathway:2-pathway (p = 0.01), and 4-pathway:16-pathway (p = 0.03) and positively associated with 2-pathway:parent estrogens (p = 0.01). F/B ratio was not associated with any of the estrogen measures. CONCLUSION: Microbial diversity was associated with several estrogen metabolism ratios implicated in breast cancer risk. Further studies are warranted to confirm these findings in a larger and more representative sample of postmenopausal women, particularly with enrichment of minority participants.

mTOR pathway candidate genes and energy intake interaction on breast cancer risk in Black women from the Women's Circle of Health Study.

BACKGROUND: Excessive energy intake has been shown to affect the mammalian target of the rapamycin (mTOR) signaling pathway and breast cancer risk. It is not well understood whether there are gene-environment interactions between mTOR pathway genes and energy intake in relation to breast cancer risk. METHODS: The study included 1642 Black women (809 incident breast cancer cases and 833 controls) from the Women's Circle of Health Study (WCHS). We examined interactions between 43 candidate single-nucleotide polymorphisms (SNPs) in 20 mTOR pathway genes and quartiles of energy intake in relation to breast cancer risk overall and by ER- defined subtypes using Wald test with a 2-way interaction term. RESULTS: AKT1 rs10138227 (C > T) was only associated with a decreased overall breast cancer risk among women in quartile (Q)2 of energy intake, odds ratio (OR) = 0.60, 95% confidence interval (CI) 0.40, 0.91 (p-interaction = 0.042). Similar results were found in ER- tumors. AKT rs1130214 (C > A) was associated with decreased overall breast cancer risk in Q2 (OR = 0.63, 95% CI 0.44, 0.91) and Q3 (OR = 0.65, 95% CI 0.48, 0.89) (p-interaction = 0.026). HIF-1α C1772T rs11549465 (C > T) was associated with decreased overall breast cancer risk in Q4 (OR = 0.29, 95% CI 0.14, 0.59, p-interaction = 0.007); the results were similar in ER+ tumors. These interactions became non-significant after correction for multiple comparisons. CONCLUSION: Our findings suggest that mTOR genetic variants may interact with energy intake in relation to breast cancer risk, including the ER- subtype, in Black women. Future studies should confirm these findings.

mTOR pathway candidate genes and physical activity interaction on breast cancer risk in black women from the women's circle of health study.

BACKGROUND: Physical activity has been shown to affect the mammalian target of rapamycin (mTOR) signaling pathway and consequently breast carcinogenesis. Given that Black women in the USA are less physically active, it is not well understood whether there are gene-environment interactions between mTOR pathway genes and physical activity in relation to breast cancer risk in Black women. METHODS: The study included 1398 Black women (567 incident breast cancer cases and 831 controls) from the Women's Circle of Health Study (WCHS). We examined interactions between 43 candidate single-nucleotide polymorphisms (SNPs) in 20 mTOR pathway genes with levels of vigorous physical activity in relation to breast cancer risk overall and by ER-defined subtypes using Wald test with 2-way interaction term and multivariable logistic regression. RESULTS: AKT1 rs10138227 (C > T) and AKT1 rs1130214 (C > A) were only associated with a decreased risk of ER + breast cancer among women with vigorous physical activity (odds ratio [OR] = 0.15, 95% confidence interval (CI) 0.04, 0.56, for each copy of the T allele, p-interaction = 0.007 and OR = 0.51, 95% CI 0.27, 0.96, for each copy of the A allele, p-interaction = 0.045, respectively). MTOR rs2295080 (G > T) was only associated with an increased risk of ER + breast cancer among women with vigorous physical activity (OR = 2.24, 95% CI 1.16, 4.34, for each copy of the G allele; p-interaction = 0.043). EIF4E rs141689493 (G > A) was only associated with an increased risk of ER- breast cancer among women with vigorous physical activity (OR = 20.54, 95% CI 2.29, 184.17, for each copy of the A allele; p-interaction = 0.003). These interactions became non-significant after correction for multiple testing (FDR-adjusted p-value > 0.05). CONCLUSION: Our findings suggest that mTOR genetic variants may interact with physical activity in relation to breast cancer risk in Black women. Future studies should confirm these findings.

Associations of alcohol consumption with breast tissue composition.

BACKGROUND: We investigated the associations of alcohol with percentage of epithelium, stroma, fibroglandular tissue (epithelium + stroma), and fat in benign breast biopsy samples. METHODS: We included 857 cancer-free women with biopsy-confirmed benign breast disease within the Nurses' Health Study (NHS) and NHSII cohorts. Percentage of each tissue was measured on whole slide images using a deep-learning algorithm and then log-transformed. Alcohol consumption (recent and cumulative average) was assessed with semi-quantitative food frequency questionnaires. Regression estimates were adjusted for known breast cancer risk factors. All tests were 2-sided. RESULTS: Alcohol was inversely associated with % of stroma and fibroglandular tissue (recent ≥ 22 g/day vs. none: stroma: ß = - 0.08, 95% Confidence Interval [CI] - 0.13; - 0.03; fibroglandular: ß = - 0.08, 95% CI - 0.13; - 0.04; cumulative ≥ 22 g/day vs. none: stroma: ß = - 0.08, 95% CI - 0.13; - 0.02; fibroglandular: ß = - 0.09, 95% CI - 0.14; - 0.04) and positively associated with fat % (recent ≥ 22 g/day vs. none: ß = 0.30, 95% CI 0.03; 0.57; cumulative ≥ 22 g/day vs. none: ß = 0.32, 95% CI 0.04; 0.61). In stratified analysis, alcohol consumption was not associated with tissue measures in premenopausal women. In postmenopausal women, cumulative alcohol use was inversely associated with % of stroma and fibroglandular tissue and positively associated with fat % (≥ 22 g/day vs. none: stroma: ß = - 0.16, 95% CI - 0.28; - 0.07; fibroglandular: ß = - 0.18, 95% CI - 0.28; - 0.07; fat: ß = 0.61, 95% CI 0.01; 1.22), with similar results for recent alcohol use. CONCLUSION: Our findings suggest that alcohol consumption is associated with smaller % of stroma and fibroglandular tissue and a greater % of fat in postmenopausal women. Future studies are warranted to confirm our findings and to elucidate the underlying biological mechanisms.

mTOR pathway candidate genes and obesity interaction on breast cancer risk in black women from the Women's Circle of Health Study.

BACKGROUND: Obesity is known to stimulate the mammalian target of rapamycin (mTOR) signaling pathway and both obesity and the mTOR signaling pathway are implicated in breast carcinogenesis. We investigated potential gene-environment interactions between mTOR pathway genes and obesity in relation to breast cancer risk among Black women. METHODS: The study included 1,655 Black women (821 incident breast cancer cases and 834 controls) from the Women's Circle of Health Study (WCHS). Obesity measures including body mass index (BMI); central obesity i.e., waist circumference (WC) and waist/hip ratio (WHR); and body fat distribution (fat mass, fat mass index and percent body fat) were obtained by trained research staff. We examined the associations of 43 candidate single-nucleotide polymorphisms (SNPs) in 20 mTOR pathway genes with breast cancer risk using multivariable logistic regression. We next examined interactions between these SNPs and measures of obesity using Wald test with 2-way interaction term. RESULTS: The variant allele of BRAF (rs114729114 C > T) was associated with an increase in overall breast cancer risk [odds ratio (OR) = 1.81, 95% confidence interval (CI) 1.10-2.99, for each copy of the T allele] and the risk of estrogen receptor (ER)-defined subtypes (ER+ tumors: OR = 1.83, 95% CI 1.04,3.29, for each copy of the T allele; ER- tumors OR = 2.14, 95% CI 1.03,4.45, for each copy of the T allele). Genetic variants in AKT, AKT1, PGF, PRKAG2, RAPTOR, TSC2 showed suggestive associations with overall breast cancer risk and the risk of, ER+ and ER- tumors (range of p-values = 0.040-0.097). We also found interactions of several of the SNPs with BMI, WHR, WC, fat mass, fat mass index and percent body fat in relation to breast cancer risk. These associations and interactions, however, became nonsignificant after correction for multiple testing (FDR-adjusted p-value > 0.05). CONCLUSION: We found associations between mTOR genetic variants and breast cancer risk as well as gene and body fatness interactions in relation to breast cancer risk. However, these associations and interactions became nonsignificant after correction for multiple testing. Future studies with larger sample sizes are required to confirm and validate these findings.

Associations of stem cell markers in benign breast tissue with subsequent breast cancer risk.

We examined associations of stem cell markers CD44, CD24, and ALDH1A1 in benign breast biopsy samples with subsequent breast cancer (BCa) risk and explored if these associations were mediated by mammographic breast density (MBD). We included 101 BCa cases/375 controls, all with previous biopsy-confirmed benign breast disease (BBD) within the Nurses' Health Study (NHS) and NHSII. The data on BCa risk factors were obtained from biennial questionnaires. MBD was assessed with computer-assisted techniques. Immunohistochemistry (IHC) was done on BBD tissue microarrays. For each core, the IHC expression was assessed using a semi-automated method, and expressed as % of cells that stained positive for a specific marker out of the total cell count. Logistic regression was used to examine the associations of each marker's expression of each (in epithelium and stroma) with BCa risk, adjusted for risk factors. Stromal CD44 expression was inversely associated with BCa risk (OR for ≥10% vs. <10%=0.58, 95% CI 0.34, 1.00). Combined stromal + epithelial CD24 expression was inversely associated with BCa risk (>50% vs. 0-10% OR=0.17, 95% CI 0.04-0.81, p-trend =0.03). Stromal CD24 and ALDH1A1 as well as epithelial expression of any of the three markers were not associated with BCa risk. In a smaller subset of women with available MBD, these observed associations did not appear to be mediated by MBD. Our findings suggest inverse associations of CD44 in stroma and combined stromal + epithelial CD24 with BCa risk. Future studies are warranted to confirm our findings and to examine these associations by BBD subtype.

Associations of aspirin and other anti-inflammatory medications with breast cancer risk by the status of COX-2 expression.

BACKGROUND: We investigated the associations of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs) with breast cancer risk by the status of COX-2 protein expression. METHODS: This study included 421 cases and 3,166 controls from a nested case-control study within the Nurses' Health Study (NHS) and Nurses' Health Study II (NHSII) cohorts. Information on medication use was first collected in 1980 (NHS) and 1989 (NHSII) and was updated biennially. Medication use was defined as none, past or current; average cumulative dose and frequency were calculated for all past or current users using data collected from all biannual questionnaires preceding the reference date. Immunochemistry for COX-2 expression was performed using commercial antibody (Cayman Chemical and Thermo Fisher Scientific). We used polychotomous logistic regression to quantify associations of aspirin and NSAIDs with the risk of COX2+ and COX2- breast cancer tumors, while adjusting for known breast cancer risk factors. All tests of statistical significance were two-sided. RESULTS: In multivariate analysis, we found no differences in associations of the aspirin exposures and NSAIDs with breast cancer risk by COX2 expression status. In stratified analyses by COX2 status, significant associations of these medications with breast cancer risk were observed for dosage of aspirin among current users in COX2- tumors (OR for > 5 tablets per week vs. none 1.71, 95% CI 1.01-2.88, p-trend 0.04). Regular aspirin use was marginally associated with the risk of COX2- tumors (p-trend = 0.06). CONCLUSIONS: Our findings suggested no differences in associations of aspirin and other NSAIDs with COX2+ and COX2- tumors.

Associations of coffee/caffeine consumption with postmenopausal breast cancer risk and their interactions with postmenopausal hormone use.

PURPOSE: We investigated the association of coffee and caffeine with breast cancer (BCa) risk, overall and by ER/PR status. We also examined potential interactions of coffee and caffeine with postmenopausal hormone use. METHODS: Our study included 77,688 postmenopausal participants from the Women's Health Initiative observational study cohort without a history of any cancer at baseline (except non-melanoma skin) and with valid Food Frequency Questionnaire data and complete data on dietary caffeine. Regular coffee (none, 1, 2-3, 4-5, and ≥ 6 cups/day) and caffeine (tertiles) were assessed at baseline. Information on BCa risk factors was collected at baseline. The associations were examined using survival analysis, accounting for death as a competing risk. RESULTS: The median follow-up time for our cohort was 18.3 years. During the follow-up, 5005 women developed invasive breast cancer. In multivariable analysis, coffee was not associated with the overall invasive BCa risk. Higher caffeine intake was mildly associated with increased BCa risk (2nd vs. 1st tertile SHR = 1.10, 95% CI 1.03-1.18, 3rd vs. 1st tertile SHR-1.05, 95% CI 0.98-1.13, overall p = 0.03). We found no interaction of coffee/caffeine with postmenopausal hormone use (p interaction = 0.44 and 0.42, respectively). In the exploratory analysis by ER/PR status, we found a positive association of caffeine with ER+ /PR+ BCa (2nd vs. 1st tertile SHR = 1.17, 95% CI 1.07-1.28, 3rd vs. 1st tertile SHR = 1.13, 95% CI 1.03-1.24, overall p = 0.002); no associations were observed for ER-/PR- tumors. Coffee was not associated with the risk of ER+ /PR+ or ER-/PR- tumors. CONCLUSION: We found no associations of coffee with BCa risk, overall and for ER/PR-defined tumor subtypes. The higher caffeine consumption was mildly and positively associated with the overall BCa risk and with ER+ /PR+ tumors.

Associations of established breast cancer risk factors with urinary estrogens in postmenopausal women.

PURPOSE: Circulating estrogens are an established risk factor for postmenopausal breast cancer (BCa). We describe the distribution of urinary estrogens, their metabolites, and relevant metabolic pathway ratios among healthy postmenopausal women and examine associations of several known BCa factors with these estrogen measures. METHODS: Eligible postmenopausal women (n = 167) had no history of hormone use (previous 6 months) and cancer/metabolic disorders and had a body mass index (BMI) ≤ 35 kg/m2. Estrogens were quantified in spot urine samples with liquid chromatography-high-resolution mass spectrometry and corrected for creatinine. We assessed overall distributions of estrogens and associations of age, BMI, race/ethnicity, parity/age at first birth, age at menarche, alcohol, and smoking with log-transformed estrogen measures using multivariate regression. RESULTS: BMI was positively associated with estrone (ß per unit = 0.04, 95% Confidence Interval [CI] 0.00; 0.07), combined parent estrogens (ß = 0.04, 95% CI 0.01; 0.07), and E2:total estrogens (ß = 0.04, 95% CI 0.02; 0.06), and inversely associated with 4-MeOE1 (ß = - 0.17, 95% CI - 0.33; - 0.02), E3:parent estrogens (ß = - 0.04, 95% CI - 0.07; - 0.00), and 16-pathway:parent (ß = - 0.04, 95% CI - 0.07; - 0.01). Being African American vs. white was associated with higher levels of 4-MeOE1 (ß = 3.41, 95% CI 0.74; 6.08), 17-epiE3 (ß = 1.19, 95% CI 0.07; 2.31), 2-pathway:parent (ß = 0.54, 95% CI 0.04; 1.04), and lower levels of E2:total estrogens (ß = - 0.48, 95% CI - 0.83; - 0.13). Having < 7 alcohol drinks/week vs. none was associated with higher levels of 16-ketoE2 (ß = 1.32, 95% CI 0.36; 2.27), 16-epiE3 (ß = 1.02, 95% CI 0.24; 1.79), and 17-epiE3 (ß = 0.55, 95% CI 0.02; 1.08). Smoking was positively associated with E3:parent (ß = 0.29, 95% CI 0.01; 0.57), 16-pathway:parent (ß = 0.25, 95% CI 0.01; 0.49), and inversely associated with estradiol (ß = - 0.52, 95% CI - 0.93; - 0.10). As compared to nulliparous, parous women with age at first birth ≥ 25 years had lower levels of estrone, combined parent estrogens, 2-OHE1, and 2-OHE2. CONCLUSION: Our findings suggest that BMI, race/ethnicity, and some reproductive and lifestyle factors may contribute to postmenopausal BCa through their effects on circulating estrogens.

Associations of Oral Contraceptives with Mammographic Breast Density in Premenopausal Women.

BACKGROUND: We investigated the associations of oral contraceptives (OC) with percent breast density (PD), absolute dense area (DA), nondense area (NDA), and a novel image intensity variation (V) measure in premenopausal women. METHODS: This study included 1,233 controls from a nested case-control study within Nurses' Health Study II cohort. Information on OCs was collected in 1989 and updated biennially. OC use was defined from the questionnaire closest to the mammogram date. PD, DA, and NDA were measured from digitized film mammograms using a computer-assisted thresholding technique; the V measure was obtained with a previously developed algorithm measuring the SD of pixel values in the eroded breast region. Generalized linear regression was used to assess associations between OCs and density measures (square root-transformed PD, DA, and NDA, and -untransformed V). RESULTS: OC use was not associated with PD [current vs. never: ß = -0.06; 95% confidence interval (CI), -0.37-0.24; past vs. never: ß = 0.10; 95% CI, -0.09-0.29], DA (current vs. never: ß = -0.20; 95% CI -0.59-0.18; past vs. never: ß = 0.13; 95% CI, -0.12-0.39), and NDA (current vs. never: ß = -0.19; 95% CI, -0.56-0.18; past vs. never: ß = -0.01; 95% CI, -0.28-0.25). Women with younger age at initiation had significantly greater V-measure (<20 years vs. never: ß = 26.88; 95% CI, 3.18-50.58; 20-24 years vs. never: ß = 20.23; 95% CI, -4.24-44.71; 25-29 years vs. never: ß = 2.61; 95% CI -29.00-34.23; ≥30 years vs. never: ß = 0.28; 95% CI, -34.16-34.72, P trend = 0.03). CONCLUSIONS: Our findings suggest that an earlier age at first OC use was associated with significantly greater V. IMPACT: These findings could guide decisions about the age for OC initiation.

Reliability of CD44, CD24, and ALDH1A1 immunohistochemical staining: Pathologist assessment compared to quantitative image analysis.

Background: The data on the expression of stem cell markers CD44, CD24, and ALDH1A1 in the breast tissue of cancer-free women is very limited and no previous studies have explored the agreement between pathologist and computational assessments of these markers. We compared the immunohistochemical (IHC) expression assessment for CD44, CD24, and ALDH1A1 by an expert pathologist with the automated image analysis results and assessed the homogeneity of the markers across multiple cores pertaining to each woman. Methods: We included 81 cancer-free women (399 cores) with biopsy-confirmed benign breast disease in the Nurses' Health Study (NHS) and NHSII cohorts. IHC was conducted with commercial antibodies [CD44 (Dako, Santa Clara, CA, USA) 1:25 dilution; CD24 (Invitrogen, Waltham, MA, USA) 1:200 dilution and ALDH1A1 (Abcam, Cambridge, United Kingdom) 1:300 dilution]. For each core, the percent positivity was quantified by the pathologist and Definiens Tissue Studio. Correlations between pathologist and computational scores were evaluated with Spearman correlation (for categorical positivity: 0, >0-<1, 1-10, >10-50, and >50%) and sensitivity/specificity (for binary positivity defined with 1 and 10% cut-offs), using the pathologist scores as the gold standard. Expression homogeneity was examined with intra-class correlation (ICC). Analyses were stratified by core [normal terminal duct-lobular units (TDLUs), benign lesions] and tissue type (epithelium, stroma). Results: Spearman correlation between pathologist and Definiens ranged between 0.40-0.64 for stroma and 0.66-0.68 for epithelium in normal TDLUs cores and between 0.24-0.60 for stroma and 0.61-0.64 for epithelium in benign lesions. For stroma, sensitivity and specificity ranged between 0.92-0.95 and 0.24-0.60, respectively, with 1% cut-off and between 0.43-0.88 and 0.73-0.85, respectively, with 10% cut-off. For epithelium, 10% cut-off resulted in better estimates for both sensitivity and specificity. ICC between the cores was strongest for CD44 for both stroma and epithelium in normal TDLUs cores and benign lesions (range 0.74-0.80). ICC for CD24 and ALDH1A ranged between 0.42-0.63 and 0.44-0.55, respectively. Conclusion: Our findings show that computational assessments for CD44, CD24, and ALDH1A1 exhibit variable correlations with manual assessment. These findings support the use of computational platforms for IHC evaluation of stem cell markers in large-scale epidemiologic studies. Pilot studies maybe also needed to determine appropriate cut-offs for defining staining positivity.

Associations of reproductive breast cancer risk factors with breast tissue composition.

BACKGROUND: We investigated the associations of reproductive factors with the percentage of epithelium, stroma, and fat tissue in benign breast biopsy samples. METHODS: This study included 983 cancer-free women with biopsy-confirmed benign breast disease (BBD) within the Nurses' Health Study and Nurses' Health Study II cohorts. The percentage of each tissue type (epithelium, stroma, and fat) was measured on whole-section images with a deep-learning technique. All tissue measures were log-transformed in all the analyses to improve normality. The data on reproductive variables and other breast cancer risk factors were obtained from biennial questionnaires. Generalized linear regression was used to examine the associations of reproductive factors with the percentage of tissue types, while adjusting for known breast cancer risk factors. RESULTS: As compared to parous women, nulliparous women had a smaller percentage of epithelium (ß = - 0.26, 95% confidence interval [CI] - 0.41, - 0.11) and fat (ß = - 0.34, 95% CI - 0.54, - 0.13) and a greater percentage of stroma (ß = 0.04, 95% CI 0.01, 0.08). Among parous women, the number of children was inversely associated with the percentage of stroma (ß per child = - 0.01, 95% CI - 0.02, - 0.00). The duration of breastfeeding of ≥ 24 months was associated with a reduced proportion of fat (ß = - 0.30, 95% CI - 0.54, - 0.06; p-trend = 0.04). In a separate analysis restricted to premenopausal women, older age at first birth was associated with a greater proportion of epithelium and a smaller proportion of stroma. CONCLUSIONS: Our findings suggest that being nulliparous as well as having a fewer number of children (both positively associated with breast cancer risk) is associated with a smaller proportion of epithelium and a greater proportion of stroma, potentially suggesting the importance of epithelial-stromal interactions. Future studies are warranted to confirm our findings and to elucidate the underlying biological mechanisms.

Gut microbiome, body weight, and mammographic breast density in healthy postmenopausal women.

PURPOSE: We examined gut microbiome (GM) profiles in relation to mammographic breast density (BD) and body mass index (BMI) in healthy postmenopausal women. METHODS: Eligible women were postmenopausal, had a BMI ≤ 35 kg/m2, and had not recently taken oral/IV antibiotics. All women provided a fecal sample and information on breast cancer risk factors. Mammographic BD was classified with the American College of Radiology's BI-RADS BD classification system. Bacterial DNA was isolated from fecal samples and the V1-V2 hypervariable regions of 16S rRNA were sequenced on the Illumina MiSeq platform. We examined associations of GM with indices of within-sample (alpha) diversity and the ratio of the two main phyla (Firmicutes and Bacteroidetes; F/B ratio) with BD and BMI. RESULTS: Among 69 women with BD data, 39 had low BD (BI-RADS I/II) and 30 had high BD (BI-RADS III/IV). BMI was inversely associated with BD (mean BMI = 23.8 and 28.0 in women with high and low BD, respectively, p = 1.07 × 10-5). Similar levels of GM diversity were found across weight groups according to Shannon (p = 0.83); Inverse Simpson (p = 0.97); and Chao1 (p = 0.31) indices. F/B ratio and microbiota diversity were suggestively greater in women with high vs. low BD (p = 0.35, 0.14, 0.15, and 0.17 for F/B ratio, Shannon, Inverse Simpson and Chao1, respectively). CONCLUSION: Suggestive differences observed in women with high and low BD with respect to GM alpha diversity and prevalence of specific GM taxa need to be confirmed in larger studies.