Indicator Of Breast Cancer Risk Research Articles

Abstract 3249: Differential impact of body mass index (BMI) on absolute and percent breast density; implications regarding their use as breast cancer risk (BCR) biomarkers

Abstract Percent breast density (PBD) is a commonly used biomarker of BCR. However, its use is greatly confounded by the strong influence of non-dense breast tissue on its measurement and factors, such as BMI, which have a direct impact on this non-dense tissue. Consequently, BMI, a potent BCR factor, is actually negatively correlated with PBD. We propose that absolute breast density (ABD), a more direct determinant of BCR which can be easily and accurately measured on a routine basis, is a more accurate and valid biomarker of BCR. To address this issue, we compared the correlation between PBD and ABD with baseline demographics (e.g., age, BMI, waist:hip ratio, reproductive parameters, family history of BC and personal history of breast biopsies) and dietary and physical activity variables (measured using a modified version of the NCI Diet History Questionnaire and the International Physical Activity Questionnaire, respectively) in a group of 169 healthy postmenopausal women enrolled in a chemoprevention trial testing the individual and combined effects of the antiestrogen Raloxifene and the omega-3 preparation Lovaza on BD used as a biomarker of BCR (NCT00723398). A volumetric breast density software (Volpara, Matakina, Wellington, New Zealand) was used for determination of ABD and PBD since it may be superior to the two-dimensional method in predicting BCR. As observed previously, a strong negative correlation between PBD and BMI was observed (Rho = -0.5, P<5e-12) due to the strong positive correlation of BMI with non-dense breast. However, we also observed a strong positive correlation of BMI with ABD (Rho = 0.41, P<2.5e-8), a relationship which is not well established in the literature and supports the use of ABD as a more accurate indicator of BCR. We also observed that correction of PBD by BMI did not frequently provide the same information as ABD, thus further indicating that the latter should be the gold standard for BD measurement. The strong positive association between BMI and ABD implies that even a modest change in BMI (as can be expected with dietary and physical activity manipulations) is likely to obscure the independent effect of an intervention on ABD. We indeed observed that many correlations between dietary variables and ABD did not emerge until adjustment was made for BMI. Our data support the following conclusions: 1) ABD instead of PBD (even after adjustment for BMI) should be used for assessment of BD; 2) based on our novel observation of the strong positive correlation between BMI and ABD, ABD should be adjusted for BMI when testing the influence of an intervention on BD as a biomarker of BCR. (Supported by grant KG081632 from Susan G. Komen for the Cure.) Citation Format: Susann Schetter, Terryl Hartman, Jason Liao, John P. Richie, Bogdan Prokopczyk, Cynthia Dubrock, Carina Signori, Christopher Hamilton, Laurence M. Demers, Karam El-Bayoumy, Andrea Manni. Differential impact of body mass index (BMI) on absolute and percent breast density; implications regarding their use as breast cancer risk (BCR) biomarkers. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3249. doi:10.1158/1538-7445.AM2014-3249

Postmortem validation of breast density using dual-energy mammography.

Mammographic density has been shown to be an indicator of breast cancer risk and also reduces the sensitivity of screening mammography. Currently, there is no accepted standard for measuring breast density. Dual energy mammography has been proposed as a technique for accurate measurement of breast density. The purpose of this study is to validate its accuracy in postmortem breasts and compare it with other existing techniques. Forty postmortem breasts were imaged using a dual energy mammography system. Glandular and adipose equivalent phantoms of uniform thickness were used to calibrate a dual energy basis decomposition algorithm. Dual energy decomposition was applied after scatter correction to calculate breast density. Breast density was also estimated using radiologist reader assessment, standard histogram thresholding and a fuzzy C-mean algorithm. Chemical analysis was used as the reference standard to assess the accuracy of different techniques to measure breast composition. Breast density measurements using radiologist reader assessment, standard histogram thresholding, fuzzy C-mean algorithm, and dual energy were in good agreement with the measured fibroglandular volume fraction using chemical analysis. The standard error estimates using radiologist reader assessment, standard histogram thresholding, fuzzy C-mean, and dual energy were 9.9%, 8.6%, 7.2%, and 4.7%, respectively. The results indicate that dual energy mammography can be used to accurately measure breast density. The variability in breast density estimation using dual energy mammography was lower than reader assessment rankings, standard histogram thresholding, and fuzzy C-mean algorithm. Improved quantification of breast density is expected to further enhance its utility as a risk factor for breast cancer.

Physiological COX-2 Expression in Breast Epithelium Associates with COX-2 Levels in Ductal Carcinoma in Situ and Invasive Breast Cancer in Young Women

Cyclooxygenase-2 (COX-2) overexpression is implicated in increased risk and poorer outcomes in breast cancer in young women. We investigated COX-2 regulation in normal premenopausal breast tissue and its relationship to malignancy in young women. Quantitative COX-2 immunohistochemistry was performed on adjacent normal and breast cancer tissues from 96 premenopausal women with known clinical reproductive histories, and on rat mammary glands with distinct ovarian hormone exposures. COX-2 expression in the normal breast epithelium varied more than 40-fold between women and was associated with COX-2 expression levels in ductal carcinoma in situ and invasive cancer. Normal breast COX-2 expression was independent of known breast cancer prognostic indicators, including tumor stage and clinical subtype, indicating that factors regulating physiological COX-2 expression may be the primary drivers of COX-2 expression in breast cancer. Ovarian hormones, particularly at pregnancy levels, were identified as modulators of COX-2 in normal mammary epithelium. However, serial breast biopsy analysis in nonpregnant premenopausal women suggested relatively stable baseline levels of COX-2 expression, which persisted independent of menstrual cycling. These data provide impetus to investigate how baseline COX-2 expression is regulated in premenopausal breast tissue because COX-2 levels in normal breast epithelium may prove to be an indicator of breast cancer risk in young women, and predict the chemopreventive and therapeutic efficacy of COX-2 inhibitors in this population.

Hemoglobin adducts as biomarkers of estrogen homeostasis: Elevation of estrogenquinones as a risk factor for developing breast cancer in Taiwanese Women

The aim of this study was to establish a methodology to analyze estrogen quinone-derived adducts, including 17β-estradiol-2,3-quinone (E2-2,3-Q) and 17β-estradiol-3,4-quinone (E2-3,4-Q), in human hemoglobin (Hb). The methodology was then used to measure the levels of these adducts in Hb derived from female breast cancer patients (n=143) as well as controls (n=147) in Taiwan. Our result confirmed that both E2-2,3-Q- and E2-3,4-Q-derived adducts, including E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb, were detected in all breast cancer patients with median levels at 434 (215–1472) and 913 (559–2384) (pmol/g), respectively. Levels of E2-2,3-Q-4-S-Hb correlated significantly with those of E2-3,4-Q-2-S-Hb (r=0.622–0.628, p<0.001). By contrast, median levels of these same estrogen quinone-derived adducts in healthy controls were 71.8 (35.7–292) and 139 (69.1–453) (pmol/g). This translated to ∼6-fold increase in mean values of E2-2,3-Q-4-S-Hb and E2-3,4-Q-2-S-Hb in breast cancer patients compared to those in the controls (p<0.001). Our findings add further support to the theme that cumulative body burden of estrogen quinones is an important indicator of breast cancer risk. We hypothesize that combination of genetic events and environmental factors may modulate estrogen homeostasis and enhance the production of estrogen quinones which lead to subsequent generation of pro-mutagenic DNA lesions in breast cancer patients.

Variation in Inflammatory Cytokine/Growth-Factor Genes and Mammographic Density in Premenopausal Women Aged 50–55

BackgroundMammographic density (MD) has been found to be an independent risk factor for breast cancer. Although data from twin studies suggest that MD has a strong genetic component, the exact genes involved remain to be identified. Alterations in stromal composition and the number of epithelial cells are the most predominant histopathological determinants of mammographic density. Interactions between the breast stroma and epithelium are critically important in the maturation and development of the mammary gland and the cross-talk between these cells are mediated by paracrine growth factors and cytokines. The potential impact of genetic variation in growth factors and cytokines on MD is largely unknown.MethodsWe investigated the association between 89 single nucleotide polymorphisms (SNPs) in 7 cytokine/growth-factor genes (FGFR2, IGFBP1, IGFBP3, TGFB1, TNF, VEGF, IL6) and percent MD in 301 premenopausal women (aged 50 to 55 years) participating in the Norwegian Breast Cancer Screening Program. We evaluated the suggestive associations in 216 premenopausal Singapore Chinese Women of the same age.ResultsWe found statistically significant associations between 9 tagging SNPs in the IL6 gene and MD in Norwegian women; the effect ranged from 3–5% in MD per variant allele (p-values = 0.02 to 0.0002). One SNP in the IL6 (rs10242595) significantly influenced MD in Singapore Chinese women.ConclusionGenetic variations in IL6 may be associated with MD and therefore may be an indicator of breast cancer risk in premenopausal women.

SU-C-116-07: Breast Density Quantification Using Liquid Phantoms with Dual-Energy Mammography

Purpose: Breast density has shown to be an important indicator for breast cancer risk. A computational calibration study using breast-equivalent liquid phantoms in dual-energy mammography is investigated to determine its feasibility and improvement in accuracy for breast density quantification. Methods: A computational model simulates a dual energy mammography system which is equipped with a tungsten anode x-ray tube and acquires dual energy images at 28 kVp with a 50 μm rhodium filter and 49 kVp with a 300 μm copper filter. Liquid phantoms, consisting of mixtures of water (H2O), glycerol (C3H6O3) and isopropyl alcohol (C3H6O) were used to simulate the X-ray attenuation properties of breast tissues. Calibration was accomplished with the simulated dual energy signals from the liquid phantoms using a rational polynomial function. The accuracy of liquid phantom calibration was tested with different phantom configurations of various thicknesses (2–8 cm) and densities (0%–100%), along with simulated glandular and adipose tissues. The performance of the liquid phantom calibration was compared to standard CIRS plastic phantom calibration by evaluating the errors in breast density quantification in simulated breast tissues. Results: Simulation results indicate that a mixture of liquid materials based on hydrogen, carbon and oxygen can be used to create glandular and adipose equivalent phantoms. For glandular and adipose tissues H2O, C3H6O3 and C3H6O percentages were estimated to be 42.68%, 22.62%, 34.69% and 0.11%, 16.37%, 83.51%, respectively. RMS errors for thicknesses were smaller (0.063 mm for glandular and 0.072 mm for adipose) using liquid phantoms than the results reported with CIRS phantoms. RMS errors for variable glandular densities were 1.93%. Conclusion: The results of this work indicate that a proper selection of liquid mixtures can be used as breast equivalent calibration phantoms in dual-energy mammography, which may potentially improve the accuracy in dual energy breast density quantification.

LBPV descriptors-based automatic ACR/BIRADS classification approach

Mammogram tissue density has been found to be a strong indicator for breast cancer risk. Efforts in computer vision of breast parenchymal pattern have been made in order to improve the diagnostic accuracy by radiologists. Motivated by recent results in mammogram tissue density classification, a novel methodology for automatic American College of Radiology Breast Imaging Reporting and Data System classification using local binary pattern variance descriptor is presented in this article. The proposed approach characterizes the local density in different types of breast tissue patterns information into the LBP histogram. The performance of macro-calcification detection methods is developed using FARABI database. Performance results are given in terms of receiver operating characteristic. The area under curve of the corresponding approach has been found to be 79%.

Computer Aided Detection of Breast Density and Mass, and Visualization of Other Breast Anatomical Regions on Mammograms Using Graph Cuts

Breast cancer mostly arises from the glandular (dense) region of the breast. Consequently, breast density has been found to be a strong indicator for breast cancer risk. Therefore, there is a need to develop a system which can segment or classify dense breast areas. In a dense breast, the sensitivity of mammography for the early detection of breast cancer is reduced. It is difficult to detect a mass in a breast that is dense. Therefore, a computerized method to separate the existence of a mass from the glandular tissues becomes an important task. Moreover, if the segmentation results provide more precise demarcation enabling the visualization of the breast anatomical regions, it could also assist in the detection of architectural distortion or asymmetry. This study attempts to segment the dense areas of the breast and the existence of a mass and to visualize other breast regions (skin-air interface, uncompressed fat, compressed fat, and glandular) in a system. The graph cuts (GC) segmentation technique is proposed. Multiselection of seed labels has been chosen to provide the hard constraint for segmentation of the different parts. The results are promising. A strong correlation (r = 0.93) was observed between the segmented dense breast areas detected and radiological ground truth.

Abstract P4-12-04: Association of single-strand breaks (SSBs) in normal breast DNA with estimates of breast cancer risk

Abstract Background: Formation of single strand breaks in DNA is a constant process, estimated to occur 10,000 times per day in each cell, either from endogenous biosynthetic errors or from interference by endogenous or exogenous agents. Base excision repair in some cases may be impaired or may be exceeded by the rate of DNA damage, leading to cancer. Nick translation of with labeled nucleotides can be used as a quantitative indicator of SSBs. Methods: Healthy women were recruited through the Love-Avon Army of Women and breast clinics of Northwestern and John Hopkins Universities. Digital or digitized mammograms were evaluated for percent density using Cumulus software. The medians (ranges) were age 51 (36 to 60); BMI 28.2 (18.7 to 51.3); life-time Gail estimate 12.5 (5.6 to 28.3); % breast density 16.5 (2.4 to 52.5); Masood score 13 (0 to 18). Breast tissue was obtained by random fine needle aspiration (rFNA). Specimens were rinsed into ice-cold phosphate buffered saline and were stored at −80°C. Thawed samples were centrifuged at 2200 g for 60 min. A kit from Norgen Inc., was used to separate DNA, RNA, and protein from the pellet. The lipids were extracted with ethyl acetate-hexane (3:2) from the supernatant fluid, and triglycerides were precipitated from cold 90% methanol, leaving a purified lipid fraction containing the steroids. Steroids were then fractionated by HPLC on a C18 column, and estradiol was analyzed by a radioimmunoassay. Blood was obtained at the same time as the rFNA samples and was frozen prior to analysis. An aliquot containing 200 ng of DNA (260/280 ratio 1.3 to 2.3) was taken for the nick translation assay. Incorporation of free nucleotides at SSBs with dCTP labeled with3H was catalyzed by Polymerase I from E. coli. A quality control preparation prepared from calf thymus DNA and a reagent blank without DNA was included with each set of 6 samples. Incorporation of3H-dCTP was linear with dose of DNA and reached a maximal at 30 min. Separation of free from incorporated3H-dCTP was accomplished by gel chromatography on an 80 × 15 mm column. The concordance of interassay values was 0.96. Results: Incorporation of nucleotides into DNA ranged from 0.03 to 8.59 pmol/μg, median 0.63 pmol/μg DNA. The association with other factors associated with breast cancer is shown in the Table. A significant correlation, was found with % breast density, life-time risk by the Gail model, but not serum estradiol concentrations. Conclusions: Assessment of SSBs by the nick translation procedure may be a useful indicator of breast cancer risk. Future studies will relate this method with actual risk as assessed by analysis of pre-diagnosis specimens with subsequent occurrence of breast cancer. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P4-12-04.

Gene methylation in random FNA samples as biomarkers for breast cancer.

518 Background: Current methods to determine breast cancer risk are insufficiently sensitive to select women most likely to benefit from preventive strategies. We hypothesized that candidate gene promoter hypermethylation may provide an individualized risk profile. We performed a prospective study to determine whether DNA cumulative methylation index (CMI) varies by menstrual phase or menopausal status, and to correlate CMI with established risk factors. Methods: We obtained random fine needle aspiration (rFNA) samples from healthy women age 35-60 and determined their menopausal and menstrual status, lifetime Gail risk, mammographic breast density, and cytologic atypia assessed as the Masood score. We evaluated CMI of 11 candidate genes in rFNA cells using the Quantitative Multiplex Methylation-Specific PCR (QM-MSP) technique. We used Wilcoxon test and ANOVA model to compare CMI across menopausal and menstrual (follicular, mid-cycle, luteal) categories, respectively. We used linear regression model to adjust for age and BMI. Methylation scores were log-transformed in the analysis. Results: We enrolled 390 women at the Avon Breast Centers at Johns Hopkins and Northwestern, the majority through the Love/Avon Army of Women, and 380 completed study procedures. Median age 50 (36-60), mean BMI 28 (18.7-50.8), 52% were postmenopausal. Mean life-time Gail risk 14.6 (5.6-54.1), mean percent mammographic density 19.6 (2.5-72.8), and mean Masood score (N=354) 13.6 (7-18). QM-MSP analysis was completed on 229 samples. We did not observe differences in CMI among menopausal (P=0.4895) or menstrual categories (P=0.2333). There was no association between CMI and life-time Gail risk (P=0.706) or breast density (P=0.4116). We observed a significant correlation between CMI and Masood score (P=0.0167). Conclusions: CMI correlates with degree of cytologic atypia and is potentially a robust indicator of breast cancer risk since it does not vary with menstrual or menopausal status. Next, we will select genes that best reflect changes in the clinical parameters to create a gene methylation signature that will be validated in other studies and correlated with breast cancer risk.

Estimation of breast density: An adaptive moment preserving method for segmentation of fibroglandular tissue in breast magnetic resonance images

Breast density has been found to be a potential indicator for breast cancer risk. The estimation of breast density can be seen as a segmentation problem on fibroglandular tissues from a breast magnetic resonance image. The classic moment preserving is a thresholding method, which can be applied to determine an appropriate threshold value for fibroglandular tissue segmentation. This study proposed an adaptive moment preserving method, which combines the classic moment preserving and a thresholding adjustment method. The breast MR images are firstly performed to extract the fibroglandular tissue from the breast tissue. The next step is to obtain the areas of the fibroglandular tissue and the whole breast tissue. Finally, breast density can be estimated for the given breast. The Friedman test shows that the qualities of segmentation are insignificant with p<0.000 and Friedman chi-squared=1116.12. The Friedman test shows that there would be significant differences in the sum of the ranks of at least one segmentation method. Average ranks indicate that the performance of the four methods is ranked as adaptive moment preserving, fuzzy c-means, moment preserving, and Kapur's method in order. Among the four methods, adaptive moment preserving also achieves the minimum values of MAE and RMSE with 9.2 and 12. This study has verified that the proposed adaptive moment preserving can identify and segment the fibroglandular tissues from the 2D breast MR images and estimate the degrees of breast density.

OT2-04-02: Metformin for Breast Cancer Prevention: A Pilot Study.

Abstract Background There is a body of accumulating evidence connecting metformin, a biguanide derivative which reduces hepatic gluconeogensis and improves skeletal muscle insulin sensitivity, to reduced breast cancer risk. This, in addition to a well established drug safety record among diabetics and non-diabetics, has provoked an interest in further study of metformin's effects on breast cancer risk indicators. In our study, we examine the effects of metformin on breast density and proliferative cell marker Ki67, both indicators of breast cancer risk. This is a small observational pilot study to determine the feasibility of subject recruitment, data collection through fine-needle aspiration, and breast density measurement. Trial Design: Recruitment of 10 breast surgery patients with high risk histology to: 1. Undergo peri-areolar fine needle aspiration (RPFNA) used to collect breast cells for analysis of proliferative marker Ki67. 2. Breast density assessment by a) mammography (the standard method) and b) whole-breast ultrasound (an innovative method recently developed at UCSD). Each subject will undergo a single view mammogram, whole-breast ultrasound, and intra-operative RPFNA. No drug or therapeutic intervention will take place in this pilot study. Eligibility: Female undergoing excisional high risk breast surgery. Specific Aims: Aim 1. To determine the feasibility of recruiting high risk breast surgery patients to an observational study involving RPFNA, mammography, and whole-breast ultrasound. Aim 2. a) To compare mammography and whole-breast ultrasound for measurement of breast density. b) To examine potential for detecting biomarkers of cell proliferation through RPFNA. Statistical Method: Breast density will be determined by the ratio of fibroglandular tissue to fat measured on mammogram and WBU. Cell proliferation will be assessed by the percentage of positivity for biomarker Ki67 in breast ductal cells. Accrual and target accrual: Five of ten breast excisional high risk breast surgery subjects have been accrued at this time. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr OT2-04-02.

Estrogen Levels in Nipple Aspirate Fluid and Serum during a Randomized Soy Trial

On the basis of hypothesized protective effect, we examined the effect of soy foods on estrogens in nipple aspirate fluid (NAF) and serum, possible indicators of breast cancer risk. In a crossover design, we randomized 96 women who produced 10 μL or more NAF to a high- or low-soy diet for 6 months. During the high-soy diet, participants consumed 2 soy servings of soy milk, tofu, or soy nuts (∼50 mg of isoflavones per day); during the low-soy diet, they maintained their usual diet. Six NAF samples were obtained using a FirstCyte aspirator. Estradiol (E(2)) and estrone sulfate (E(1)S) were assessed in NAF and estrone (E(1)) in serum only, using highly sensitive radioimmunoassays. Mixed-effects regression models accounting for repeated measures and left-censoring limits were applied. Mean E(2) and E(1)S were lower during the high-soy than the low-soy diet (113 vs. 313 pg/mL and 46 vs. 68 ng/mL, respectively) without reaching significance (P = 0.07); the interaction between group and diet was not significant. There was no effect of the soy treatment on serum levels of E(2) (P = 0.76), E(1) (P = 0.86), or E(1)S (P = 0.56). Within individuals, NAF and serum levels of E(2) (r(s) = 0.37; P < 0.001) but not of E(1)S (r(s) = 0.004; P = 0.97) were correlated. E(2) and E(1)S in NAF and serum were strongly associated (r(s) = 0.78 and r(s) = 0.48; P < 0.001). Soy foods in amounts consumed by Asians did not significantly modify estrogen levels in NAF and serum. The trend toward lower estrogen levels in NAF during the high-soy diet counters concerns about adverse effects of soy foods on breast cancer risk.

The Volume of Nipple Aspirate Fluid Is Not Affected by 6 Months of Treatment with Soy Foods in Premenopausal Women1–4

Based on the hypothesis that soy food consumption may influence breast tissue activity, we examined its effect on the production of nipple aspirate fluid (NAF), a possible indicator of breast cancer risk. Of 310 premenopausal women screened, 112 (36%) produced at least 10μL of NAF, the minimum for study participation. In a crossover design, we randomized 96 women to 2 groups who, in reverse order, consumed a high-soy diet with 2 soy servings/d (1 serving = 177 mL soy milk, 126 g tofu, or 23 g soy nuts) and a low-soy diet with <3 servings/wk of soy for 6 mo each separated by a 1-mo washout period. During each diet period, 3 NAF samples were obtained (baseline and 3 and 6 mo) using a FirstCyte Aspirator and 4 urine samples (baseline and 1, 3, and 6 mo) were analyzed for isoflavonoids by liquid chromatography tandem MS. Adherence to the study protocol according to 24-h dietary recalls and urinary isoflavonoid excretion was high. The drop-out rate was 15% (n= 14); 82 women completed the intervention. The 2 groups produced similar mean NAF volumes at baseline (P= 0.95) but differed in age and previous soy intake and in their response to the intervention (P= 0.03). In both groups, NAF volume decreased during the first 3 mo of the high-soy diet period and returned to baseline at 6 mo, but there was no effect of the high-soy diet on NAF volume (P= 0.50 for diet;P-interaction = 0.21 for diet with time). Contrary to an earlier report, soy foods in amounts consumed by Asians did not increase breast tissue activity as assessed by NAF volume.

P152 Metabolic syndrome: Breast cancer risk indicator in postmenopausal women

P152 Metabolic syndrome: Breast cancer risk indicator in postmenopausal women

Influence of diet on nipple aspirate fluid production and estrogen levels

One possible mechanism how nutritional factors may affect breast cancer risk is through an influence on estrogen levels. Nipple aspirate fluid (NAF) is thought to provide a more direct insight into hormonal influences on breast tissue than serum. The ability to produce NAF may be an indicator of breast cancer risk. The current analysis was conducted as part of a soy trial in 92 premenopausal women and evaluated the relation of usual dietary intake with NAF volume and the most predominant steroidal estrogens in NAF and serum at baseline. Estradiol (E(2)) and estrone sulfate (E(1)S) were assessed in NAF and E(2), estrone (E(1)), and E(1)S, in serum using highly sensitive radioimmunoassays. The statistical analysis applied multivariate, log-linear regression models. Intake of saturated fat and cheese (p = 0.06 for both) indicated a positive trend with NAF volume whereas isoflavonoid and soy consumption suggested inverse associations (p = 0.01 and p = 0.08). For estrogens in NAF, total fat and monounsaturated fat intake was positively associated with E(2) (p = 0.05 and p = 0.02) and in serum, alcohol intake was associated with higher E(1)S levels (p = 0.02). These findings suggest a weak influence of dietary composition on NAF production and estrogen levels in serum and NAF.

Quantitative analysis of promoter methylation in exfoliated epithelial cells isolated from breast milk of healthy women

Promoter methylation analysis of genes frequently silenced in breast cancer is a promising indicator of breast cancer risk, as these methylation events are thought to occur long before presentation of disease. The numerous exfoliated epithelial cells present in breast milk may provide the breast epithelial DNA needed for detailed methylation analysis and assessment of breast cancer risk. Fresh breast milk samples and health, lifestyle, and reproductive history questionnaires were collected from 111 women. Pyrosequencing analysis was conducted on DNA isolated from the exfoliated epithelial cells immunomagnetically separated from the total cell population in the breast milk of 102 women. A total of 65 CpG sites were examined in six tumor suppressor genes: PYCARD (also known as ASC or TMS1), CDH1, GSTP1, RBP1 (also known as CRBP1), SFRP1, and RASSF1. A sufficient quantity of DNA was obtained for meaningful analysis of promoter methylation; women donated an average of 86 ml of milk with a mean yield of 32,700 epithelial cells per ml. Methylation scores were in general low as expected of benign tissue, but analysis of outlier methylation scores revealed a significant relationship between breast cancer risk, as indicated by previous biopsy, and methylation score for several CpG sites in CDH1, GSTP1, SFRP1, and RBP1. Methylation of RASSF1 was positively correlated with women’s age irrespective of her reproductive history. Promoter methylation patterns in DNA from breast milk epithelial cells can likely be used to assess breast cancer risk. Additional studies of women at high breast cancer risk are warranted.

Breast cancer precursors revisited: molecular features and progression pathways

Increasingly more coherent data on the molecular characteristics of benign breast lesions and breast cancer precursors have led to the delineation of new multistep pathways of breast cancer progression through genotypic-phenotypic correlations. It has become apparent that oestrogen receptor (ER)-positive and -negative breast lesions are fundamentally distinct diseases. Within the ER-positive group, histological grade is strongly associated with the number and complexity of genetic abnormalities in breast cancer cells. Genomic analyses of high-grade ER-positive breast cancers have revealed that a substantial proportion of these tumours harbour the characteristic genetic aberrations found in low-grade ER-positive disease, suggesting that at least a subgroup of high-grade ER-positive breast cancers may originate from low-grade lesions. The ER-negative group is more complex and heterogeneous, comprising distinct molecular entities, including basal-like, HER2 and molecular apocrine lesions. Importantly, the type and pattern of genetic aberrations found in ER-negative cancers differ from those of ER-positive disease. Here, we review the available molecular data on breast cancer risk indicator and precursor lesions, the putative mechanisms of progression from in situ to invasive disease, and propose a revised model of breast cancer evolution based on the molecular characteristics of distinct subtypes of in situ and invasive breast cancers.

Abstract 3764: Promoter methylation in exfoliated breast cells isolated from breast milk of women who have had a breast biopsy

Abstract Analysis of promoter methylation patterns in genes frequently downregulated in breast cancer may serve as an early indicator of breast cancer risk. To date, studies of methylation patterns in breast epithelial cells from asymptomatic women have been limited to cells obtained from fine nipple aspirate and ductal lavage. Analysis of promoter hypermethylation in exfoliated epithelial cells isolated from breast milk provides a non-invasive opportunity to assess breast cancer risk. Our preliminary data indicate that we can detect a cancer methylation phenotype in the milk of women at high risk. We propose that epithelial cells isolated from breast milk of lactating women scheduled to have a breast biopsy provide a resource for assessing early changes in DNA related to increased breast cancer risk. Two hundred and fifty breast milk samples from women who are scheduled to have, or have had a breast biopsy will be collected. Of these women, we expect that roughly ten percent will be diagnosed with breast cancer. Breast milk will be collected from both the biopsied and contralateral breast of each participant. To date, 153 participants have donated breast milk, yielding 300 breast milk samples with an average size of 50 ml (±30 ml) per breast. After collection, the epithelial enriched and epithelial depleted cells from each breast were isolated. Subsequent DNA isolation of both the epithelial enriched and depleted fraction of cells yielded an average of 372 ng (range 12-5000 ng) of high quality DNA. Using the DNA extracted from epithelial enriched and depleted fractions of cells isolated from breast milk, the promoter methylation level of 9 genes known to be methylated in breast cancer will be examined using pyrosequencing technology. We will assess the promoter methylation levels of: CDH1, DAP-K, ER-α, GSTP1, HIN1, P16, RAR-ß, RASSF1A, and SFRP1 in the epithelial enriched and depleted bisulfite treated DNA of each breast milk sample. Preliminary pyrosequencing assays conducted in our lab have shown that there is a difference in RASSF1A methylation levels between the epithelial enriched DNA from the cancerous breast compared to the epithelial depleted DNA from the non-cancerous breast of the same participant. Further methylation analysis may prove that breast milk can be used as non-invasive alternative to ductal lavage or fine needle aspirate, which may yield accurate breast cancer risk assessment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3764.

Quantification of breast density with dual energy mammography: An experimental feasibility study

Breast density, the percentage of glandular breast tissue, has been shown to be a strong indicator of breast cancer risk. A quantitative method to measure breast density with dual energy mammography was investigated using physical phantoms. The dual energy mammography system used a tungsten anode x-ray tube with a 50 microm rhodium beam filter for low energy images and a 300 microm copper beam filter for high energy images. Glandular and adipose equivalent phantoms of uniform thickness were used to calibrate a dual energy basis decomposition algorithm. Four different phantom studies were used to evaluate the technique. The first study consisted of phantoms with thicknesses of 2.5-8.5 cm in 0.5 cm steps with variable densities centered at a mean of 28%. The second study consisted of phantoms at a fixed thickness of 4.0 cm, which ranged in densities from 0% to 100% in increments of 12.5%. The third study consisted of 4.0 cm thick phantoms at densities of 25%, 50% and 75% each imaged at three areal sizes, approximately 62.5, 125, and 250 cm2, in order to assess the effect of breast size on density measurement. The fourth study consisted of step phantoms designed to more closely mimic the shape of a female breast with maximal thicknesses from 3.0 to 7.0 cm at a fixed density of 50%. All images were corrected for x-ray scatter. The RMS errors in breast density measurements were 0.44% for the variable thickness phantoms, 0.64% for the variable density phantoms, 2.87% for the phantoms of different areal sizes, and 4.63% for step phantoms designed to closely resemble the shape of a breast. The results of the phantom studies indicate that dual energy mammography can be used to measure breast density with an RMS error of approximately 5%.