Eva P. Browne

Determination of free Bisphenol A (BPA) concentrations in breast milk of U.S. women using a sensitive LC/MS/MS method

Bisphenol A (BPA) is a synthetic, endocrine-disrupting compound. Free BPA has been detected in human samples indicating that humans are internally exposed to estrogenically active BPA. The purpose of this study was to develop a sensitive method to detect free BPA in human breast milk. BPA was isolated from the milk of 21 nursing mothers in the U.S. by solid-phase extraction. It was then derivatized to improve sensitivity and subsequently analyzed by ultra high performance liquid chromatography-tandem mass spectrometry. Free BPA was detected in 62% of the milk samples (≤ 0.22-10.8 ng mL(-1), median 0.68 ng mL(-1), mean 3.13 ng mL(-1)). No statistical difference in BPA concentrations was observed between women with a low or high Body Mass Index (BMI) (<30 (n=11) and>30 (n=10), respectively). However, there was a significant association between BPA concentration and race. Caucasian women had significantly higher levels of free BPA in their breast milk than non-Caucasian women (mean=4.44 (n=14) and 0.52 (n=7), respectively; p<0.05). The difference between races could be attributed to variations in exposure, lifestyle or metabolism and suggests that certain populations should take extra precautions to limit BPA exposure, particularly during pregnancy and lactation.

Increased promoter methylation in exfoliated breast epithelial cells in women with a previous breast biopsy

Accurately identifying women at increased risk of developing breast cancer will provide greater opportunity for early detection and prevention. DNA promoter methylation is a promising biomarker for assessing breast cancer risk. Breast milk contains large numbers of exfoliated epithelial cells that are ideal for methylation analyses. Exfoliated epithelial cells were isolated from the milk obtained from each breast of 134 women with a history of a non-proliferative benign breast biopsy (Biopsy Group). Promoter methylation of three tumor suppressor genes, RASSF1, SFRP1 and GSTP1, was assessed by pyrosequencing of bisulfite-modified DNA. Methylation scores from the milk of the 134 women in the Biopsy Group were compared to scores from 102 women for whom a breast biopsy was not a recruitment requirement (Reference Group). Mean methylation scores for RASSF1 and GSTP1 were significantly higher in the Biopsy than in the Reference Group. For all three genes the percentage of outlier scores was greater in the Biopsy than in the Reference Group but reached statistical significance only for GSTP1. A comparison between the biopsied and non-biopsied breasts of the Biopsy Group revealed higher mean methylation and a greater number of outlier scores in the biopsied breast for both SFRP1 and RASSF1, but not for GSTP1. This is the first evidence of CpG island methylation in tumor suppressor genes of women who may be at increased risk of developing breast cancer based on having had a prior breast biopsy.

Differential expression of cancer associated proteins in breast milk based on age at first full term pregnancy.

BackgroundFirst full term pregnancy (FFTP) completed at a young age has been linked to low long term breast cancer risk, whereas late FFTP pregnancy age confers high long term risk, compared to nulliparity. Our hypothesis was that proteins linked to breast cancer would be differentially expressed in human milk collected at three time points during lactation based on age at FFTP.MethodsWe analyzed breast milk from 72 lactating women. Samples were collected within 10 days of the onset of lactation (baseline-BL), two months after lactation started and during breast weaning (W). We measured 16 proteins (11 kallikreins (KLKs), basic fibroblast growth factor, YKL-40, neutrophil gelatinase-associated lipocalin and transforming growth factor (TGF) β-1 and -2) associated with breast cancer, most known to be secreted into milk.ResultsDuring lactation there was a significant change in the expression of 14 proteins in women < 26 years old and 9 proteins in women > = 26 at FFTP. The most significant (p < .001) changes from BL to W in women divided by FFTP age (< 26 vs. > = 26) were in KLK3,6, 8, and TGFβ2 in women < 26; and KLK6, 8, and TGFβ2 in women > = 26. There was a significant increase (p = .022) in KLK8 expression from BL to W depending on FFTP age. Examination of DNA methylation in the promoter region of KLK6 revealed high levels of methylation that did not explain the observed changes in protein levels. On the other hand, KLK6 and TGFβ1 expression were significantly associated (r2 = .43, p = .0050).ConclusionsThe expression profile of milk proteins linked to breast cancer is influenced by age at FFTP. These proteins may play a role in future cancer risk.

Differential expression of cancer-related proteins in paired breast milk samples from women with breast cancer.

Background: Breast cancer risk increases during pregnancy and remains elevated for a number of years thereafter. Cancer-associated proteins that are secreted into breast milk may provide a means to detect cancer in the lactating breast or to assess future breast cancer risk. Objective: To determine whether proteins linked to breast cancer would be differentially expressed in matched (both breasts from each participant) human milk samples collected from women with unilateral breast cancer. Methods: Five cancer-associated proteins (basic fibroblast growth factor [bFGF], YKL-40, neutrophil gelatinase-associated lipocalin, and transforming growth factor β1 and β2) were analyzed in milk provided by 5 lactating women, 4 of whom were known to have cancer in 1 breast (and the opposite breast clinically disease free) at the time of milk collection and 1 who developed breast cancer 2 years after milk collection. Results: Expression was significantly higher for TGFβ2 (P = .03) and bFGF (P =.03) in the breasts with cancer. Conclusion: These proteins may play a role in assessing a woman’s risk of pregnancy-associated breast cancer. Because of variable protein concentration among patients and the limited sample size, the results are considered preliminary.

Potential of breastmilk analysis to inform early events in breast carcinogenesis: rationale and considerations

This review summarizes methods related to the study of human breastmilk in etiologic and biomarkers research. Despite the importance of reproductive factors in breast carcinogenesis, factors that act early in life are difficult to study because young women rarely require breast imaging or biopsy, and analysis of critical circulating factors (e.g., hormones) is often complicated by the requirement to accurately account for menstrual cycle date. Accordingly, novel approaches are needed to understand how events such as pregnancy, breastfeeding, weaning, and post-weaning breast remodeling influence breast cancer risk. Analysis of breastmilk offers opportunities to understand mechanisms related to carcinogenesis in the breast, and to identify risk markers that may inform efforts to identify high-risk women early in the carcinogenic process. In addition, analysis of breastmilk could have value in early detection or diagnosis of breast cancer. In this article, we describe the potential for using breastmilk to characterize the microenvironment of the lactating breast with the goal of advancing research on risk assessment, prevention, and detection of breast cancer.

Promoter Methylation in Epithelial-Enriched and Epithelial-Depleted Cell Populations Isolated from Breast Milk

Background: Breast cancer is the most frequently diagnosed cancer among Turkish women and both the incidence and associated mortality appear to be increasing. Of particular concern is the percentage of young women diagnosed with breast cancer; roughly 20% of all breast cancer diagnoses in Turkey are in women younger than 40 years. Increased DNA methylation in the promoter region of tumor suppressor genes is a promising molecular biomarker, and human milk provides exfoliated breast epithelial cells appropriate for DNA methylation analyses. Comparisons between DNA methylation patterns in epithelial (epithelial-enriched) and nonepithelial (epithelial-depleted) cell fractions from breast milk have not been reported previously. Objective: In the present study, we examined promoter methylation of 3 tumor suppressor genes in epithelial-enriched and epithelial-depleted cell fractions isolated from breast milk of 43 Turkish women. Methods: Percentage methylation in the promoter region of Rass association domain family 1 (RASSF1), secreted frizzle related protein 1 (SFRP1), and glutathione-S-transferase class pi 1 was determined by pyrosequencing of the epithelial-enriched and epithelial-depleted cell fractions. Results: Pyrosequencing identified a few subjects with significantly increased methylation in 1 or more genes. There was little correlation between the 2 cell fractions within individuals; only 1 woman had increased methylation for 1 gene (SFRP1) in both her enriched and depleted cell fractions. Methylation was positively associated with age for SFRP1 (epithelial-depleted fraction) and with body mass index for RASSF1 (epithelial-enriched cell fraction), respectively. Conclusion: Overall, results show that the methylation signals vary between different cell types in breast milk and suggest that breast milk can be used to assess DNA methylation patterns associated with increased breast cancer risk.

TROP2 methylation and expression in tamoxifen-resistant breast cancer

BackgroundThe DNA methyltransferase 1 inhibitor, 5-Aza-2′-deoxycytidine (5-Aza-dC) is a potential treatment for breast cancer. However, not all breast tumors will respond similarly to treatment with 5-Aza-dC, and little is known regarding the response of hormone-resistant breast cancers to 5-Aza-dC.MethodsWe demonstrate that 5-Aza-dC-treatment has a stronger effect on an estrogen receptor-negative, Tamoxifen-selected cell line, TMX2-28, than on the estrogen receptor-positive, MCF7, parental cell line. Using data obtained from the HM450 Methylation Bead Chip, pyrosequencing, and RT-qPCR, we identified a panel of genes that are silenced by promoter methylation in TMX2-28 and re-expressed after treatment with 5-Aza-dC.ResultsOne of the genes identified, tumor associated calcium signal transducer 2 (TACSTD2), is altered by DNA methylation, and there is evidence that in some cancers decreased expression may result in greater proliferation. Analysis of DNA methylation of TACSTD2 and protein expression of its product, trophoblast antigen protein 2 (TROP2), was extended to a panel of primary (n = 34) and recurrent (n = 34) breast tumors. Stratifying tumors by both recurrence and ER status showed no significant relationship between TROP2 levels and TACSTD2 methylation. Knocking down TACSTD2 expression in MCF7 increased proliferation however; re-expressing TACSTD2 in TMX2-28 did not inhibit proliferation, indicating that TACSTD2 re-expression alone was insufficient to explain the decreased proliferation observed after treatment with 5-Aza-dC.ConclusionsThese results illustrate the complexity of the TROP2 signaling network. However, TROP2 may be a valid therapeutic target for some cancers. Further studies are needed to identify biomarkers that indicate how TROP2 signaling affects tumor growth and whether targeting TROP2 would be beneficial to the patient.

Pro-inflammatory cytokines and growth factors in human milk: an exploratory analysis of racial differences to inform breast cancer etiology

BackgroundAnalysis of cytokines and growth factors in human milk offers a noninvasive approach for studying the microenvironment of the postpartum breast, which may better reflect tissue levels than testing blood samples. Given that Black women have a higher incidence of early-onset breast cancers than White women, we hypothesized that milk of the former contains higher levels of pro-inflammatory cytokines, adipokines, and growth factors.MethodsParticipants included 130 Black and 162 White women without a history of a breast biopsy who completed a health assessment questionnaire and donated milk for research. Concentrations of 15 analytes in milk were examined using two multiplex and 4 single-analyte electrochemiluminescent sandwich assays to measure pro-inflammatory cytokines, angiogenesis factors, and adipokines. Mixed-effects ordinal logistic regression was used to identify determinants of analyte levels and to compare results by race, with adjustment for confounders. Factor analysis was used to examine covariation among analytes.ResultsThirteen of 15 analytes were detected in ≥ 25% of the human milk specimens. In multivariable models, elevated BMI was significantly associated with increased concentrations of 5 cytokines: IL-1β, bFGF, FASL, EGF, and leptin (all p-trend < 0.05). Black women had significantly higher levels of leptin and IL-1β, controlling for BMI. Factor analysis of analyte levels identified two factors related to inflammation and growth factor pathways.ConclusionThis exploratory study demonstrated the feasibility of measuring pro-inflammatory cytokines, adipokines, and angiogenesis factors in human milk, and revealed higher levels of some pro-inflammatory factors, as well as increased leptin levels, among Black as compared with White women.

Selective adhesive cell capture without molecular specificity: new surfaces exploiting nanoscopic polycationic features as discrete adhesive units

This work explored how molecularly non-specific polycationic nanoscale features on a collecting surface control kinetic and selectivity aspects of mammalian cell capture. Key principles for selective collector design were demonstrated by comparing the capture of two closely related breast cancer cell lines: MCF-7 and TMX2-28. TMX2-28 is a tamoxifen-selected clone of MCF-7. The collector was a silica surface, negatively-charged at pH 7.4, containing isolated molecules (~ 8 nm diameter) of the cationic polymer, poly(dimethyl-aminoethylmethacrylate), pDMAEMA. Important in this work is the non-selective nature of the pDMAEMA interactions with cells: pDMAEMA generally adheres negatively charged particles and cells in solution. We show here that selectivity towards cells results from collector design: this includes competition between repulsive interactions involving the negative silica and attractions to the immobilized pDMAEMA molecules, the random pDMAEMA arrangement on the surface, and the concentration of positive charge in the vicinity of the adsorbed pDMAEMA chains. The latter act as nanoscopic cationic surface patches, each weakly attracted to negatively-charged cells. Collecting surfaces engineered with an appropriate amount pDMAEMA, exposed to mixtures of MCF-7 and TMX2-28 cells preferentially captured TMX2-28 with a selectivity of 2.5. (This means that the ratio of TMX2-28 to MCF cells on the surface was 2.5 times their compositional ratio in free solution.) The ionic strength-dependence of cell capture was shown to be similar to that of silica microparticles on the same surfaces. This suggests that the mechanism of selective cell capture involves nanoscopic differences in the contact areas of the cells with the collector, allowing discrimination of closely related cell line-based small scale features of the cell surface. This work demonstrated that even without molecular specificity, selectivity for physical cell attributes produces adhesive discrimination.

Abstract 4298: Cytokines and adipokines in breastmilk of black and white women

Introduction: Giving birth may be associated with a transient increase in breast cancer risk post-delivery and with elevated risk of basal breast cancers, especially in the absence of breastfeeding, whereas parity is related to lower risk of postmenopausal ER-positive tumors. Black women develop more early-onset and basal breast cancers than White women, but factors contributing to this disparity are poorly understood. Accordingly, we compared levels of 15 proteins with hypothesized roles in breast cancer risk in breastmilk from healthy Black and White women. Methods: We tested breastmilk donated by 130 Black and 162 White women, annotated with breast cancer risk factor questionnaire data. Following pilot testing using the MesoScale Discovery system (Rockville, MD), the following analytes were measured in breastmilk, adjusted for total protein concentration: interferon-γ, IL1-β, IL-6, IL-8, TNF-α, FLT-1, TIE-2, PlGF, VEGFC, VEGFD, adiponectin, leptin, and FAS-L. M30-apoptosis and Bradford protein were measured with Molecular Devices VersaMax reader (Sunnyvale, CA). Univariate associations of race with women9s characteristics were computed using chi square tests and t-tests. Multivariable logistic regression models with a random effect to account for plate effects were used to assess the associations (odds ratios (ORs) and 95% confidence intervals (CIs)) of analyte levels with: race, family history of breast cancer, smoking, baby9s age in days, body mass index at time of sample donation, age, menarche, age at first birth, return of menses, parity, and over-the-counter pain medication. Results: Of the 15 analytes measured, all but VEGFC and TIE2 were detectable and reliably measured. Black women compared to White women had higher BMI (p = 0.05) and higher parity (p = 0.02), and were younger age at menarche (p = 0.005) and younger age at first birth (p = 0.01). White women had higher levels of smoking (p = 0.01) and more frequently had a first degree relative with breast cancer (p = 0.03). Compared with White women, Black women had significantly higher levels of IL1-β (OR 1.77, 95% CI 1.13 - 2.77, p = 0.01) adjusting for the baby9s age in days. Black women had significantly higher levels of leptin (OR 2.01, 95% CI 1.28 - 3.15, p = 0.002) and leptin/adiponectin ratio (OR 2.36, 95% CI 1.49 - 3.72, p = 0.0004), even after adjusting for body mass index. Discussion: Preliminary data demonstrate differences in levels of putative markers of breast cancer risk between White and Black women, including IL1-β, leptin and the leptin/adiponectin ratio. These data suggest that further analyses of biomarkers in breastmilk may be useful for understanding breast cancer risk and to identify possible factors that may be associated with racial disparities in early onset and basal breast cancers. Citation Format: Jeanne Murphy, Mark E. Sherman, Ruth M. Pfeiffer, Hannah P. Yang, Ana I. Caballero, Eva P. Browne, Gretchen L. Gierach, Kathleen F. Arcaro. Cytokines and adipokines in breastmilk of black and white women. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4298.