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ObjectiveThe inflammatory signature of human milk is an indicator of mammary gland health and has implications for infant nutrition, growth, and immune system development. We hypothesized that measurement of mammary epithelium permeability would provide important context for the interpretation of the inflammatory signature of human milk.MethodsParticipants were drawn from a previous study of human milk cytokines and growth factors. Analysis included 162 participants (98 Black women, 64 White women) with infants ranging from 1-18 months of age. Levels of twelve inflammatory markers (IL-6, IL8, TNFα, IL-1β, FASL, VEGFD, FLT1, BFGF, PLGF, EGF, leptin, adiponectin) were evaluated previously. Milk sodium (Na) and potassium (K) were measured with ion selective probes. Increased mammary epithelium permeability (IMEP) was indicated by either Na levels or Na/K ratios greater than the 75thpercentile. Multivariate logistic regression models were used to examine associations between maternal-infant characteristics, inflammatory markers, and IMEP.ResultsThresholds of elevated Na and Na/K ratios at the 75thpercentile were found to be 8.0 mmol/L and 0.53 respectively. IMEP was most frequent (42.6%) during early lactation (1-3 months). The strongest predictors of IMEP as defined by Na > 75thpercentile were infant age, Black race, IL6, and BFGF, while maternal age and PLGF were important predictors of IMEP as defined by the Na/K ratio >75thpercentile. IMEP also moderated associations between maternal-infant characteristics and inflammatory markers.DiscussionThis study demonstrates the importance of mammary epithelium permeability measurement for the interpretation of human milk inflammatory profiles during established lactation.

Black women in the United States are disproportionately affected by early-onset, triple-negative breast cancer. DNA methylation has shown differences by race in healthy and tumor breast tissues. We examined associations between genome-wide DNA methylation levels in breast milk and breast cancer risk factors, including race, to explain how this reproductive stage influences a woman's risk for, and potentially contributes to racial disparities in, breast cancer. Breast milk samples and demographic, behavioral, and reproductive data, were obtained from cancer-free, uniparous, and lactating U.S. black (n = 57) and white (n = 82) women, ages 19–44. Genome-wide DNA methylation analysis was performed on extracted breast milk DNA using the Infinium HumanMethylation450 BeadChip. Statistically significant associations between breast cancer risk factors and DNA methylation beta values, adjusting for potential confounders, were determined using linear regression followed by Bonferroni Correction (P < 1.63 × 10−7). Epigenetic analysis in breast milk revealed statistically significant associations with race and lactation duration. Of the 284 CpG sites associated with race, 242 were hypermethylated in black women. All 227 CpG sites associated with lactation duration were hypomethylated in women who lactated longer. Ingenuity Pathway Analysis of differentially methylated promoter region CpGs by race and lactation duration revealed enrichment for networks implicated in carcinogenesis. Associations between DNA methylation and lactation duration may offer insight on its role in lowering breast cancer risk. Epigenetic associations with race may mediate social, behavioral, or other factors related to breast cancer and may provide insight into potential mechanisms underlying racial disparities in breast cancer incidence.

Introduction: Identifying biomarkers of breast cancer risk among young women would have value in developing effective screening and prevention strategies at early ages. We have proposed that DNA methylation analysis of breast milk may provide breast cancer risk information among young women, and could possibly provide etiologic clues related to the higher rates of early onset cancers among African American as compared with White women in the US. Objective: The purpose of this project was to identify associations between genome-wide DNA methylation levels in breast milk and race adjusted for other breast cancer risk factors. Study Population: Cancer-free, lactating U.S. Black (n=57) and White (n=82) women, ages 19 to 44, provided frozen breast milk samples, as well as demographic, behavioral, and reproductive data, to the Breastmilk Laboratory at University of Massachusetts Amherst. Women were uniparous and did not have a personal history of breast cancer at the time of milk donation. Methods: DNA was extracted from breast milk samples using the phenol-chloroform method. Genome-wide methylation analysis was performed on breast milk samples using the Infinium HumanMethylation450 BeadChip. Probes with 50% or more missing data, cross-reactive probes, as well as probes with minor allelic frequency greater than 0.05 in European- or African-Americans were removed, leaving 379,042 CpG sites for analysis. Multivariate generalized linear regression models were used to examine associations between race and other breast cancer risk factors and methylation beta values, adjusting for potential confounding factors. P-values less than 1E-7 were considered statistically significant. Results: Black women in this study were more likely to be never smokers, to not have used over-the-counter pain medication in the past week, and to breastfeed longer. After adjustment by age, BMI, smoking status, and batch number, race was significantly associated with differential methylation at 1143 CpG sites, including 1024 at which Black women demonstrated increased methylation levels. Additionally, breastfeeding duration was associated with 269 CpG sites, with 268 showing a significant inverse relationship with methylation. Methylation sites significantly associated with Black race and lactation duration were located within tumor suppressor and promoter genes as well as in genes implicated in obesity and diabetes. Conclusion: This preliminarily analysis of DNA methylation in breast milk suggests that Black women have increased methylation and longer breastfeeding is associated with reduced methylation. Further research to understand how etiologic factors related to breast cancer may alter DNA methylation patterns in normal breast may lead to improved understanding of breast cancer risk at a young age and potentially causes of racial disparities in breast cancer incidence between White and Black women. Citation Format: Davis Lynn BC, Bodelon C, Pfeiffer RM, Yang HP, Yang H, Lee M, Laird PW, Campan M, Weisenberger DJ, Murphy J, Sherman ME, Browne EP, Anderton DL, Arcaro KF, Gierach GL. Differences in genome-wide DNA methylation levels in breast milk by race and lactation duration [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P6-10-07.

BackgroundPrior candidate gene studies have shown tumor suppressor DNA methylation in breast milk related with history of breast biopsy, an established risk factor for breast cancer. To further establish the utility of breast milk as a tissue-specific biospecimen for investigations of breast carcinogenesis we measured genome-wide DNA methylation in breast milk from women with and without a diagnosis of breast cancer in two independent cohorts.MethodsDNA methylation was assessed using Illumina HumanMethylation450k in 87 breast milk samples. After quality control, 368,171 autosomal CpG loci were analyzed. Cell type proportion estimates from RefFreeCellMix were calculated and adjusted for in this Epigenome Wide Association Study using linear mixed effects models adjusted for history of breast biopsy, age, time of delivery, cell type proportion estimates, array chip, and subject as random effect.ResultsEpigenome-wide analyses identified 58 differentially methylated CpG sites associated with a breast cancer diagnosis in the prospectively collected milk samples from the breast that would develop cancer compared with women without a diagnosis of breast cancer (q-value < 0.05). Nearly all CpG sites associated with a breast cancer diagnosis were hypomethylated in cases compared with controls, and were enriched for CpG islands. In addition, inferred repeat element methylation was lower in breast milk DNA from cases compared to controls, and cases exhibited increased estimated epigenetic mitotic tick rate as well as DNA methylation age compared with controls.ConclusionBreast milk has utility as a biospecimen for prospective assessment of disease risk, for understanding the underlying molecular basis of breast cancer risk factors, and improving primary and secondary prevention of breast cancer.

Epigenetics is a field of study that invites an interdisciplinary interaction of the social and biological sciences. This collaboration has, in fact, led to a blossoming research community over the past two decades, which is using new data, methods, and conceptual frameworks to address a host of old and emergent research questions. A recent (2014) search of PubMed found over a thousand articles on social, behavioral, and cognitive epigenetics. If one includes epidemiological epigenetic studies that incorporate either social causes or consequences in their research, the number expands nearly threefold. Yet, social epigenetics is a still nascent field, marginalized and misunderstood in social science. In this essay, we attempt to review basic epigenetic concepts and the way in which epigenetics has, and can be, of use to social and behavioral scientists in addressing some of the most fundamental sorts of questions their disciplines raise. Keywords: epigenetics; sociobiology; behavioral genetics; inheritance; environmental health; health and social behavior; cognitive neuroscience; methylation; histone modification

This article explores the social interactions of immigration, occupation, and wealth in two urban industrial cities of nineteenth-century New England that were largely built upon, and shaped by, immigration: the very rapidly growing factory town of Holyoke, Massachusetts, and a more mixed-market and steadily growing nearby community of Northampton, Massachusetts. Both communities were emergent, rapidly industrializing, inland cities, providing a quite distinct immigration context than large established cities of the East Coast. Both were destinations for the same general ethnic immigration waves over the late nineteenth century, but with very different, and differently impacted, social spaces into which immigrants arrived. Contrasting and considering both these emergent cities allows us to ascertain the extent to which the occupational distribution and accumulation of wealth by immigrant groups supports the broad pattern of nineteenth-century assimilation, and reveals ways in which other migration processes may have been at odds, or intertwined, with the long-term historical assimilation of immigrants in such communities. Our findings support a traditional assimilationist perspective in emergent urban-industrial centers. However, they also reveal the role of universal immiseration in an industrial city dual-labor market in facilitating or forcing assimilation, the temporal advantages for ethnic groups of arriving early in growing settlements, and the more individualistic nature of economic enclaves in gaining advantages over time that did not manifest across broad immigrant or occupational groups.

TGF-β plays a dual role in breast carcinogenesis, acting at early stages as tumor-suppressors and later as tumor-promoters. TGF-β isoforms are expressed in breast tissues and secreted in milk, suggesting that analysis of levels in milk might be informative for breast cancer risk. Accordingly, we assessed TGF-β2 levels in milk from women who had undergone a breast biopsy and related the concentrations to diagnosis. Milk donated by women who had undergone or were scheduled for a breast biopsy was shipped on ice for processing and testing. Breast cancer risk factors were obtained through a self-administered questionnaire, and biopsy diagnoses were extracted from pathology reports. TGF-β2 levels in milk, assessed as absolute levels and in relation to total protein, were analyzed in bilateral samples donated by 182 women. Linear regression was used to estimate relationships of log-transformed TGF-β2 levels and TGF-β2/ total protein ratios to biopsy category. Milk TGF-β2 levels from biopsied and non-biopsied breasts within women were highly correlated (r 2 = 0.77). Higher mean TGF-β2 milk levels (based on average of bilateral samples) were marginally associated with more severe breast pathological diagnosis, after adjusting for duration of nursing current child (adjusted p trend = 0.07). Our exploratory analysis suggests a borderline significant association between higher mean TGF-β2 levels in breast milk and more severe pathologic diagnoses. Further analysis of TGF-β signaling in milk may increase understanding of postpartum remodeling and advance efforts to analyze milk as a means of assessing risk of breast pathology.

Background Diets rich in fruits and vegetables (F/V) can reduce the inflammatory profile of circulating cytokines and potentially decrease the risk of breast cancer. However, the extent to which a diet rich in F/V alters cytokine levels in breast tissue remains largely unknown. Breast milk provides a means of assessing concentrations of secreted cytokines in the breast microenvironment and is a potential tool for studying the effects of diet on inflammation in breast tissue and breast cancer risk. Objective The aim of this pilot randomized trial was to test the feasibility of increasing F/V intake in breastfeeding women and of measuring changes in markers of inflammation in breast milk. Design and intervention Participants randomized to the intervention (n=5) were provided weekly boxes of F/V, along with dietary counseling, to increase consumption of F/V to 8 to 10 daily servings for 12 consecutive weeks. Controls (n=5) were directed to the US Department of Agriculture’s “ChooseMyPlate” diet for pregnancy and breastfeeding. Participants/setting Ten breastfeeding women consuming fewer than five servings of F/V per day, as estimated by the National Institutes of Health “All-Day” Fruit and Vegetable Screener (F/V Screener), were recruited through flyers and a lactation consultant between February and May 2016 in the Western Massachusetts area. Main outcome measures Baseline demographic and F/V intake data were collected during enrollment. At week 1 and week 13 (final) home visits, participants provided milk samples and anthropometric measurements were recorded. Participants completed F/V screeners at baseline and at study end. Adiponectin, leptin, C-reactive protein, and 11 additional cytokines were measured in breast milk collected at weeks 1 and 13. Statistical analyses F/V consumption at baseline and after the final visit, and between controls and intervention groups, was compared with dependent and independent t tests, respectively. Differences between cytokine levels at weeks 1 and 13 were assessed with a mixed-effects repeated-measures model. Results All women in the intervention increased F/V intake and were consuming more servings than controls by week 13; daily serving of F/V at baseline and final visit: controls=1.6 and 2.0, diet=2.6 and 9.9. Most cytokines were detected in the majority of milk samples: 12 were detected in 90% to 100% of samples, one was detected in 75% of samples, and one was detected in 7.5% of samples; coefficients of variation were below 14% for 11 of the cytokines. Conclusions These preliminary findings indicate that it is feasible to significantly increase F/V intake in breastfeeding women and provide support for conducting a larger diet intervention study in breastfeeding women, in which longer-term benefits of the intervention are assessed.

S-phase kinase-associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin-dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1 /S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)-negative breast cancers and demonstrated that triple-negative tumors were more likely to have high expression of SKP2 than were non-triple negative, ERα-negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple-negative than in ERα-positive breast cancers. Use of the triple-negative TMX2-28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple-negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers.

Roughly two-thirds of all breast cancers are ERα-positive and can be treated with the antiestrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 y. Aberrant DNA methylation, an epigenetic mechanism that alters gene expression in cancer, is thought to play a role in this resistance. To develop an understanding of Tamoxifen-resistance and identify novel pathways and targets of aberrant methylation, DNA from MCF-7 breast cancer cells and Tamoxifen-resistant derivatives, TMX2-11 and TMX2-28, were analyzed using the Illumina HumanMethylation450 BeadChip. Normalizing against MCF-7 values, ERα-positive TMX2-11 had 4000 hypermethylated sites and ERα-negative TMX2-28 had over 33 000. Analysis of CpG sites altered in both TMX2-11 and TMX2-28 revealed that the Tamoxifen-resistant cell lines share 3000 hypermethylated and 200 hypomethylated CpGs. ZNF350 and MAGED1, two genes hypermethylated in both cell lines, were examined in greater detail. Treatment with 5-aza-2ꞌdeoxycitidine caused a significant reduction in promoter methylation of both ZNF350 and MAGED1 and a corresponding increase in expression in TMX2-28. A similar relationship between methylation and expression was not detected in TMX2-11. Our findings are indicative of the variable responses to methylation-targeted breast cancer therapy and highlight the need for biomarkers that accurately predict treatment outcome.

1. IntroductionSocial, economic, and environmental factors all impacted mortality in 19th century Europe and the United States. The type of place (rural or urban, industrial or non-industrial area, stable or growing population) was a powerful predictor of mortality, especially for children, even when socioeconomic status (SES) is taken into account (e.g., Preston and Haines 1991; Vinovskis 1981; Preston and Van de Walle 1978). The effects of structural and social environmental mechanisms (sanitation, overcrowding, occupational structure, population stability) also varied within geographic regions (e.g., Preston and Van de Walle 1978; Melosi 1999). Yet individual-level socioeconomic circumstances influenced mortality across the wider disparities and inequalities of geographic circumstance in 19th century society (e.g., Condran 1995; Garrett et al. 2001; Preston and Haines 1991), and may have even been most salient where other circumstances were most dire (Reid 1997). We sought to expand on our own previous work in 19th century Massachusetts towns, by asking whether individual-level socioeconomic factors influenced mortality from infectious diseases characterized by epidemics in emerging urban, often stressful and harsh, environments.Holyoke, Massachusetts is located on the Connecticut River in the western portion of the state. The township was a planned industrial community and experienced rapid growth from the 1850s through 1910s, fueled primarily by immigration from Ireland, Canada, and Eastern Europe. Structural issues related to sanitation and overcrowding were continual problems in Holyoke during the last half of the 19th century and led to numerous recorded epidemic outbreaks (Green 1939). The industrial structure of the township created a high level of economic and social inequality, with immigrants entering primarily to work in low-paying mill jobs. The constant influx of new workers further impacted the township's demography by maintaining a relatively young population (Leonard, Beemer, and Anderton 2012). Immigrants of Irish and Canadian origin then experienced an accumulation in both personal and real estate wealth at a fast rate, eventually bringing their level of wealth close to that of the native-born. We argue that this accumulation of wealth over time represents a maturation effect, whereby later immigrants from a specific country of origin benefit from established social connections and resources of prior co-nationals. Overall mortality was high during periods of rapid growth, and only recent immigration significantly lowered the likelihood of death.2. Data and methodsWe utilized two datasets for this analysis. The first dataset includes records of all deaths in Holyoke between 1850 and 1912. The second dataset is an urban-based geographic sample of Federal census records, linked to property tax information and death records in the year following the census, allowing us to analyze personal and family characteristics not available from the death records. Gutman (1956) demonstrates the extraordinary completeness of Massachusetts death reporting and previous research (e.g., Leonard, Beemer, and Anderton 2012) demonstrates representation of the impoverished within the tax-linked dataset. We looked at trends in mortality rates for the entire population using the first dataset, and modeled mortality rates for the sampled census population using Poisson rate regression. Cause-specific counts of deaths are the numerators of the rates, with interpolated census population counts as the denominators. The model presented here was developed in prior research and applied to overall adult and child mortality (Leonard, Beemer, and Anderton 2012). In this note we replicate the same model, limited to mortality from the important infectious diseases of the time in this place.We focused on infectious diseases that caused significant epidemics in the 19th century United States (Condran and Murphy 2008) and were continually present in Holyoke between 1850 and 1912. …

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.

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.

Purpose Poor living conditions are posited as an underlying cause of American Indian (AI) infant mortality, which is unusually high in the postneonatal period. We explore whether the effects of neighborhood poverty on AI infant death are identifiable by using observational data. Methods Vital records for infants born between 1990 and 1999 to AI women in a metropolitan area (n = 4751) are linked with tract-level poverty data. A counterfactual framework, an explicit causal contrast study design, and propensity score matching methods were used. For each comparison, we created exchangeable groups by matching infants with the same probability of exposure to poverty when one was exposed and the other was not. Results Our results suggest that neighborhood poverty has little effect on AI infant death outcomes. Importantly, the study design makes transparent the challenge of identifying appropriate analytic comparison groups in studies of neighborhood poverty and health. Conclusions Collecting additional data will likely not overcome the fact that AIs with a high probability of living in poverty rarely reside in low-poverty neighborhoods. Yet, some of them must if a meaningful counterfactual comparison is to be made and the effects of neighborhood poverty on AI infant death are to be identified.

In this study, we investigate the extent to which women and men differ in patterns of medication use, based on quantitative and qualitative data from a household survey in Western Massachusetts. Using a broad definition of medications, 96% of the sample reported taking one or more medications in the month preceding the survey (86% if vitamins, supplements, and alternative medications are excluded). Twenty-one percent of respondents reported taking five or more medications, and women were significantly more likely to report taking five or more medications in the month preceding the survey. For both sexes, analgesics and vitamins were the most commonly used medications, but women were more likely to report having taken hormones, supplements, and antihistamines. The likelihood of medicating reported health conditions did not differ by sex, but the frequency of reporting health conditions was higher among women, and the difference was significant for body aches and psychosomatic conditions. Analyses of qualitative data indicate that female networks of relatives and friends are an important source of advice on medications for both men and women. Responses to open-ended questions suggest that women's discourse about the effect of medications differs from men's in terms of the range and detail of descriptions of symptoms and side effects.

Historical mortality analysis is often confounded by changing disease environments, diagnostic criteria, and terminology. Recorded causes of death are shaped by these local and historical contexts. We analyze changing literal causes of death during the shift from miasmatic to germ theories of disease using death records from two Massachusetts towns for selected years spanning 1850 to 1912. This analysis demonstrates that (1) International Classification of Diseases (ICD) classifications are more stable, yet potentially less informative, than the literal causes recorded in death accounts, (2) recorded causes of death often include additional qualifications and elaborations beyond basic literal causes of death, and the use of such qualifiers rose dramatically during the late nineteenth century, (3) social biases are clearly evident in the extent to which causes of death were further described or qualified, and (4) the additional descriptive qualification of deaths during this period of often ambiguous historical causes of death can potentially aid in efforts to classify causes of death and derive robust estimates of cause-specific mortality trends.

Prior candidate gene studies have shown that methylation-induced silencing of tumor suppressor genes in breast milk is related with history of breast biopsy, an established risk factor for breast cancer. To further establish the utility of breast milk as a tissue-specific biospecimen for investigations of breast carcinogenesis we measured genome-wide DNA methylation in breast milk from women with and without a diagnosis of breast cancer. Breast milk provides a source of cells without invasive procedure that is available during a window of increased breast cancer risk. Actively lactating women were enrolled and consented through the University of Massachusetts Amherst breast milk study targeting high risk subjects. Participants completed a questionnaire to provide demographic and breast cancer risk factor data. Genome-wide DNA methylation levels were quantified using the Illumina 450K HumanMethylation array. Data was processed in R using RnBeads. Beta-values were normalized (FUNnorm), low quality probes were filtered and transformed to M-values. Linear mixed effects models were fit including a random effect for subject and adjusted for history of breast biopsy, age, body mass index, age of the baby, and estimated cell type proportions, an interaction term for time of diagnosis was included. DNA methylation in women with breast cancer (n=9), was compared with women without a history of breast cancer (n=22). Among women with a diagnosis of breast cancer there were two groups: those diagnosed within one year of donating breast milk, and those diagnosed more than one year before sample collection. Four women in the control and five women in the cancer group provided bilateral breast milk samples at recruitment and both samples were included in the analyses. CpGs with significant differential methylation were tested for enrichment in gene pathways using the KEGG database. We identified 215 differentially methylated CpG sites in women diagnosed within one year of sample donation compared with women without a diagnosis of breast cancer (Q Citation Format: Lucas A. Salas, Sara N. Lundgren, Eva P. Browne, Elizabeth C. Punska, Douglas L. Anderton, Kathleen F. Arcaro, Brock C. Christensen. Altered DNA methylation in breast milk from women with breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2017-4355

Recent research has considerably increased our understanding of the factors associated with the American epidemiological transition in the late nineteenth century. However, uncertainty remains regarding the impact on mortality of specific changes ancillary to urbanization and industrialization in American cities and towns. The broad objective of the Connecticut Valley Historical Demography Project is to examine changing relationships between socioeconomic status, the rise of new urban-industrial communities, and cause-specific mortality trends during the rapid development of New England manufacturing. To address these issues, the present analysis examines two emergent urban centers in Massachusetts, adopting a micro-demographic approach to explore late-nineteenth-century and turn-of-the-century determinants of mortality.

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.

S-phase kinase-associated protein 2 (SKP2) is an important cell cycle regulator, targeting the cyclin-dependent kinase (CDK) inhibitor p27 for degradation, and is frequently overexpressed in breast cancer. p27 regulates G1 /S transition by abrogating the activity of cyclin/CDK complexes. p27 can undergo phosphorylation at serine 10 (pSer10p27). This phosphorylation event is associated with increased cell proliferation and poor prognosis in patients with glioma. The relationship between SKP2 and pSer10p27 in breast cancer has not been previously investigated. Immunohistochemistry (IHC) of SKP2, p27, pSer10p27, and other genes involved in this pathway, was analyzed in 188 breast tumors and 50 benign reduction mammoplasty samples. IHC showed SKP2 to be more highly expressed in estrogen receptor α (ERα)-negative breast cancers and demonstrated that triple-negative tumors were more likely to have high expression of SKP2 than were non-triple negative, ERα-negative tumors. A significant positive relationship was discovered for SKP2 and pSer10p27. High levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple-negative than in ERα-positive breast cancers. Use of the triple-negative TMX2-28 breast cancer cell line to address the role of SKP2 in cell cycle progression confirmed that SKP2 contributes to a more rapid cell cycle progression and may regulates pSer10p27 levels. Together, the results indicate that presence of high SKP2 plus high pSer10p27 levels in triple-negative breast cancers is associated with aggressive growth, and highlight the validity of using SKP2 inhibitors as a therapeutic approach for treating this subset of breast cancers.

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) and30 (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; p0.05). 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.

The mortality transition in Western Europe and the United States encompassed a much more complex set of conditions and experiences than earlier thought. Our research addresses the complex set of relationships among growing urban communities, family wealth, immigration and mortality in New England by examining individual-level, sociodemographic mortality correlates during the nineteenth-century mortality plateau and its early twentieth-century decline. In contrast to earlier theories that proposed a more uniform mortality transition, we offer an alternative hypothesis that focuses on the impact of family wealth and immigration on individual-level mortality during the early stages of the mortality transition in Northampton and Holyoke, Massachusetts.

Early “environmental equity” studies claimed hazardous waste facilities (TSDFs) were disproportionately located in minority and disadvantaged communities. Recently, research has challenged such claims. Regardless of conclusions, no national study has evaluated inequity in the siting process or the impact of TSDFs on the demographic composition of communities after TSDFs began operations, or distinguished between TSDF-caused change and general population trends. This article presents findings from the first national tract-level longitudinal study of communities with TSDFs. We find no stark evidence of environmental inequity or disparate impact. Results suggest compositional change in host communities may best be explained by general population trends.

Research addressing “environmental equity” and “environmental racism” claims that facilities for treatment, storage, and disposal of hazardous wastes (TSDFs) are located disproportionately in minority areas. In the first comprehensive study of TSDFs to use census tract-level data, we find no nationally consistent and statistically significant differences between the racial or ethnic composition of tracts which contain commercial TSDFs and those which do not. TSDFs are more likely to be found in tracts with Hispanic groups, primarily in regions with the greatest percentage of Hispanics. Different geographic units of analysis elaborate on, but are consistent with, these results.

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.

Many local agencies that provide services to the homeless have only limited data and resources to estimate the extent of need or the size of the local population at risk of requiring the services they provide. Methods exist, however, that are capable of providing reasonable estimates of underlying service populations with minimal record keeping. In this paper one group of such models, first‐capture models, is discussed as a resource‐efficient technique for estimating the underlying population consistently at risk ("residents") and those occasionally at risk ("transients") of requiring agency services.

This paper evaluates age, period, and cohort effects on marital fertility during onset of the Utah fertility transition (1880–1909). Computerized genealogies are used to derive age–period–cohort fertility rates for 49,842 once-married couples. Age, period and cohort effects on marital fertility are then estimated using Johnson's (1985) relational model. Declining marital fertility in Utah is shown to be explained by both lower fertility levels across periods and increasing age-specific limitation across cohorts. Direct cohort effects on fertility are insignificant. These results are consistent with prior research, and the view that fertility levels were adaptive (in part through birth spacing across ages) to immediate contexts of childbearing while age-specific fertility truncation increased across cohorts (in part through the more general diffusion of contraceptive innovations).

Nearly all developed countries have experienced dramatic declines in the seasonality of demographic behavior over the last century. This article examines seasonal behavior in the population of Taiwan between 1906 and 1942, during Japanese colonial rule. First, we analyze seasonal demographic cycles and their ties to the cultural calendar and agriculture. Second, we compare seasonality in two regions (Xinzhu and Tainan) that experienced different agricultural development under the colonial regime. Findings demonstrate that demographic seasonality was less pronounced when urban development and colonial agricultural intensification produced more heterogeneous seasonal patterns of labor. We find that changes in agricultural organization, rather than purely a shift to nonagricultural production, may significantly reduce established seasonal timing of demographic behavior.

Emerging industrial communities of nineteenth-century New England experienced both rapid population growth and lagging development of public health infrastructures. In turn, high mortality in these newly urban cities contributed to a delay in the regional mortality transition of the late nineteenth century. Analyzing death records and a file of linked cause-specific death and manuscript census records for the industrializing community of Northampton, Massachusetts, we show that early in the city's development, mortality clustered near industrial activities and open sewers. When industrial areas were sewered, clustering of mortality abated, and differences between industrial and commercial areas of the town were no longer significant. These findings illustrate Szreter's emphasis on considering both the benefits and costs of development. (1) Initial development contributed to high mortality in newly emergent urban-industrial centers like Northampton and was abated only when lagging public health infrastructures caught up with rapid growth and development near the turn of the century.

Using a national survey of facilities governed by the Resource Conservation and Recovery Act (RCRA), we examine the sociospatial distribution of a much larger group of hazardous materials handlers than did previous environmental equity studies. Overall we find that RCRA-governed facilities are more likely to be sited in working-class neighborhoods with lower percentages of minority residents. We do not find evidence of stark environmental inequities. RCRA facilities, however, are close to neighborhoods with a higher percentage of minority residents. And in nonmetropolitan areas, they are slightly more likely to be located in neighborhoods with a higher percentage of black residents.

This article presents findtngs of the first national tract-level analysis of the distribution of residential characteristics, including the percentage of selected minorities and socioeconomically disadvan taged groups, in relation to uncontrolled toxic waste sites (i. e., CERCLIS and NPL sites). In contrast to prevailing notions, the authors find that uncontrolled toxic waste sites are not disproportionately located in minority neighborhoods or neighborhoods of soctoeconomtcally disadvantaged residents. However, multivariate analyses of site distribution and a hazard regression analysis of the site prioritization process suggest that communities with a higher percentage of Black residents are less likely to receive NPL (National Priorities List) designation, delaying potential remediation. Biases in the prioritization process are, however, substantively small.

Background: TGF-β isoforms play a dual role in breast carcinogenesis, acting at early stages as tumor-suppressors and later as tumor-promoters. TGF-β isoforms are expressed in breast tissues and secreted in milk, suggesting that analysis of levels in milk might have value for understanding the effect of pregnancy and lactation on breast carcinogenesis. Data suggest that TGF-β2 levels in milk are higher than TGF-β1, show heterogeneity among women, and may vary with psychosocial stress, maternal immunity, and length of nursing. We assessed whether TGF-β2 levels in milk from each breast were correlated with a history of prior breast biopsy (biopsied vs. not biopsied) and biopsy diagnosis. Methods: Fresh breast milk donated by lactating women who had either undergone or were scheduled for a breast biopsy was shipped on ice to a central laboratory. Breast cancer risk factors were obtained via questionnaire and biopsy diagnoses were extracted from pathology reports. Milk was processed to create cellular-enriched and cellular-depleted fractions and frozen at -20[≤]C prior to testing. TGF-β2 levels in milk were analyzed using the Quantikine Human TGF-β2 Immunoassay (R&D Systems DB250) and quantified using the Coomassie Plus (Bradford) Assay (Thermo Scientific) kits in bilateral samples donated by182 women, including duplicate samples from 1 woman (n= 366). We compared log transformed TGF-β2 (pg/ml) and TGF-β2/total protein ratio (pg/μg) levels for biopsied and non-biopsied breasts using t-tests and ordinary least squares regressions adjusted for confounders and clustering of observations by woman. Results: TGF-β2 levels ranged 0 - 68,070 pg/ml and total protein levels ranged 1,558- 36,198 μg/ml. The levels of TGF-β2 in the biopsied and non-biopsied breasts were correlated (r2 = 0.64), albeit not statistically significant. Controlling for mother's attained age, age at first birth, age of baby (surrogate for length of nursing), number of live births, time since breast biopsy, and race, did not alter the finding for TGF-β2 or the TGF-β2/total protein ratio. But, mean levels of TGF-β2 proteins were highest in milk among women with cancer (9971 pg/ml, n=16), followed by proliferative lesions with atypia (8842 pg/ml; n=4) biopsy diagnoses. TGF-β2 levels were lower among women with non-proliferative biopsy diagnoses (4131 pg/ml; n=292) as compared with those who had proliferative lesions with atypia or cancer (P Conclusion: More severe pathologic diagnoses were associated with higher mean TGF-β2 levels in breast milk, although levels of biopsied and non-biopsied breasts were correlated. These data suggest that higher TGF-β2 levels in milk are linked to higher breast cancer risk for a woman, but do not specifically indicate which breast is at risk. Citation Format: Hannah P. Yang, Mark E. Sherman, Sallie Smith Schneider, Christina M. Chisholm, Eva P. Browne, Sidra Mahmood, Sarah Lenington, Douglas L. Anderton, Kathleen A. Arcaro. Association of TGF-β2 levels in breast milk with breast biopsy diagnosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 117. doi:10.1158/1538-7445.AM2013-117

Estrogen receptor (ER) status remains one of the most important breast cancer diagnostic and prognostic biomarkers. Roughly two-thirds of all breast cancers are ER-positive and can be treated with the anti-estrogen, Tamoxifen, however resistance occurs in 33% of women who take the drug for more than 5 years. MCF-7 breast cancer cells were previously cultured with 1 uM Tamoxifen, and after 6 months the clonal lines TMX2-11 and TMX2-28 with acquired resistance were isolated. TMX2-28 lost ER-α gene expression while a second clonal line, TMX2-11, did not. TMX2-28 cells are highly invasive in vitro, in contrast to TMX2-11 and the parent cell line, MCF-7. Studies of endocrine resistance and global gene expression in Tamoxifen-resistant cell cultures and human tumors have detected alterations in numerous pathways including ER-signaling, growth factor receptor signaling, cell cycle, and apoptosis. Aberrant DNA promoter methylation is a major epigenetic mechanism affecting gene expression in cancer. Our focus is on its role in Tamoxifen resistance. Hypermethylation of normally unmethylated cytosines in CpG-rich promoter regions frequently results in gene silencing, while hypomethylation in CpGs outside promoter regions leads to genetic instability. To develop a broad understanding of Tamoxifen resistance and to identify novel pathways and targets of aberrant methylation, DNA from MCF-7, TMX2-11, and TMX2-28 was analyzed using the Illumina HumanMethylation450 BeadChip. The three cell lines have similar hypermethylation patterns in 306,000 CpG sites and similar hypomethylation patterns in 132,000 CpG sites across the genome. A subset of CpG sites was identified where the two Tamoxifen-selected lines share a methylation pattern distinct from the MCF-7 parent line. When normalized to MCF-7, TMX2-11 and TMX2-28 share 722 hypermethylated and 65 hypomethylated CpG sites in the promoter region encompassing 539 and 55 genes respectively. Pathway analysis indicates the hypermethylated genes are involved in processes relevant to acquired Tamoxifen resistance. These pathways include cell signaling, adhesion, transcriptional activation and repression, differentiation, proliferation, and apoptosis. Ongoing studies on a subset of the genes found differentially methylated between parental and Tamoxifen-selected cell lines are aimed at confirming methylation findings and examining gene expression. Additionally, the cells will be treated with the demethylating agent 5-aza-deoxycitidine to determine whether reversal of the hypermethylation restores Tamoxifen sensitivity. Greater knowledge of the molecular modifications accompanying Tamoxifen-resistance is needed, as it will lead to discovery of new therapeutic targets and improved treatment, possibly allowing prediction of resistance from biopsy DNA. Citation Format: Kristin E. Williams, Douglas L. Anderton, Maxwell P. Lee, Brian T. Pentecost, Kathleen F. Arcaro. Epigenetic changes in breast cancer cells associated with acquired Tamoxifen resistance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5368. doi:10.1158/1538-7445.AM2013-5368

Bisphenol A (BPA) is a synthetic compound with structural similarities to the hormone 17β-estradiol. BPA is a major component of polycarbonate plastics and epoxy resins, which are used in the production of plastic containers, metal can linings, dental sealants, thermal receipt paper and household paper products. Incomplete polymerization of BPA as well as exposure to high temperatures and acidic or basic conditions can cause BPA monomers to leach from these products. Therefore, most people are exposed to BPA and levels have been quantified in human urine, blood, saliva, amniotic fluid, placental tissue, colostrum and breast milk. BPA is a weak estrogen and is considered a potential endocrine disrupting compound in humans. Initially it was thought that BPA was rapidly conjugated and excreted from the body. However, free BPA has been detected in human samples indicating that humans are internally exposed to estrogenically active BPA. The length of time that free BPA remains in circulation in the body and the extent to which it accumulates in tissues, such as the breast, is unknown. Given the known sources of BPA exposure, it is likely that lifestyle habits lead to varying levels of BPA exposure. Repeated use of products that contain or are packaged in materials with a high BPA content can lead to higher exposure rates. The same lifestyle choices resulting in high BPA exposure may also lead to high body mass index (BMI); for example, frequent consumption of carbonated soft drinks is associated with a high BMI in women and these beverages are often packaged in plastic bottles or metal cans that may contain BPA. Two recent studies have demonstrated a relationship between BPA levels in urine and obesity in adults and children. Because of the difficulty in measuring BPA in a complex fatty matrix such as milk, few studies have reported on BPA levels in breast milk, and none have examined the relationship between BMI and BPA. In the present study we optimized a sensitive method for assessing free BPA in breast milk and determined whether the levels of BPA were related to characteristics including age, race, BMI, child's age, and number of children nursed. BPA was separated from breast milk samples from 21 nursing women in the U.S. by solid-phase extraction and subsequently analyzed by HPLC-MS/MS. Free BPA was detected in 71% (15/21) of the breast milk samples. The method detection limit in water was determined to be 0.11 ng/mL. Free BPA concentrations were detected ranging from below the method detection limit to 10.81 ng/mL (median 0.49, mean 3.06, SD 3.85 ng/mL). No statistical difference in BPA concentrations was observed between women with a low BMI (< 21.00, n = 10) and a high BMI (> 27.00, n = 11). However, there was a significant association between BPA concentration and race. Caucasian women (n = 14) had significantly higher levels of free BPA in their breast milk than non-Caucasian women (n = 7) (Two-tailed test: t = 2.54, p < 0.05). Citation Format: Stephanie M. Zimmers, Eva P. Browne, Patrick O'Keefe, Douglas L. Anderton, David A. Reckhow, Kathleen F. Arcaro. Determination of free bisphenol A (BPA) concentrations in breast milk of U.S. women using a sensitive LC/MS/MS method. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2520. doi:10.1158/1538-7445.AM2013-2520

Breast cancer is expected to be the most frequently diagnosed form of cancer and the second leading cause of cancer deaths among women in 2012. The primary objective of the present study was to provide insights into the molecular mechanisms underlying the aggressive growth of triple-negative and basal-like breast cancers. In this study, we use the Tamoxifen-selected, MCF-7 derivative, TMX2-28 breast cancer cell line. TMX2-28 cells are triple-negative, have acquired a mixed basal/luminal cytokeratin profile, suggestive of a more basal-like phenotype, and are highly proliferative. We also examined thirty frozen breast carcinoma samples for mRNA, and 188 breast tumors and 50 benign reduction mammoplasty samples (formalin-fixed paraffin embedded) for protein expression. S-phase kinase-associated protein 2 (SKP2) plays an important role in cell cycle regulation by targeting p27 for degradation. The cyclin-dependent kinase (CDK) inhibitor p27 regulates G1/S transition by binding cyclin/CDK complexes and abrogating its activity. By targeting p27 for degradation, SKP2 frees the complexes needed to progress into the S phase of the cell cycle. Evaluation of SKP2 expression in TMX2-28 revealed significantly higher levels than in other breast cancer cell lines. Despite the high levels of SKP2 expression, p27 protein was not reduced. However, levels of the serine-10 phosphorylated form of p27 (pSer10p27), which has been associated with increased proliferation rates, was found to be upregulated. Furthermore, suppression of SKP2 completely eliminated pSer10p27 expression and slowed cell cycle progression confirming the role of SKP2 in the aggressive growth of TMX2-28 cells. Assessment of mRNA from 30 frozen human breast cancers demonstrated that SKP2 is more highly expressed in ERα-negative and basal-like breast cancers. Immunohistochemical analysis confirmed that SKP2 is more highly expressed in ERα-negative breast cancers and for the first time demonstrated that triple-negative breast cancers are more likely to overexpress SKP2 than are non-triple negative, but still ERα-negative tumors. We did not observe an inverse relationship between SKP2 and p27 expression, however a significant positive relationship was discovered between SKP2 and pSer10p27. Furthermore, high levels of SKP2 and pSer10p27 were observed significantly more often in ERα-negative and triple-negative than in ERα-positive breast cancers. The relationship between SKP2 and pSer10p27 is a better indicator of increased tumor proliferation in triple-negative breast cancers based on these results and those of our cell culture model. Inhibiting SKP2 in tumors expressing high levels of both SKP2 and pSer10p27 may be a valid therapeutic approach. Citation Format: Katerina D. Fagan-Solis, Joseph M. Gogzit, Brook A. Bentley, Sharon M. Marconi, Christopher N. Otis, Douglas L. Anderton, Sallie Smith Schneider, Brian T. Pentecost, Kathleen F. Arcaro. SKP2 overexpression is associated with increased serine-10 phosphorylation of p27 (pSer10p27) in triple-negative breast cancers. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 563. doi:10.1158/1538-7445.AM2013-563

Breast cancer is the second most commonly diagnosed cancer among women, and is difficult to diagnose during pregnancy and lactation. Frequent methylation of the promoter region of key tumor suppressor genes occurs in the early etiology of breast cancer. We aimed to evaluate the feasibility of using DNA extracted from the epithelial cells exfoliated in breast milk as a biomarker for 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. Breast milk samples from both the biopsied and non-biopsied breasts of women scheduled for or who had had a breast biopsy were collected and processed within 24 hours of expression. Epithelial enriched cell fractions were isolated via immunomagnetic separation and DNA subsequently isolated and bisulfite treated for RASSF1, GSTP1, and SFRP1 promoter methylation analysis via pyrosequencing. When available, pathology reports from each participant were also obtained to categorize the outcome of the biopsy. Over the past two years, we have collected breast milk samples from 230 women who were scheduled for or had a breast biopsy. Of those 230 participants, we obtained the biopsy reports from 85% of participants (196 biopsy reports). Using pyrosequencing, we analyzed 30 individual CpG sites on 3 genes (RASSF1, GSTP1 and SFRP1) in epithelial enriched cell fractions of the biopsied and non-biopsied breast. In women whose biopsy resulted in a non-proliferative lesion (n=104), there was no significant difference in the average epithelial DNA methylation of their biopsied breast compared to their non-biopsied breast for RASSF1 (p=0.15) and GSTP1 (p=0.92), but for SFRP1 the average methylation was significantly higher in the biopsied breast (p = 0.02). In women whose biopsy result revealed cancer (n=10), there was a significant increase in average RASSF1 methylation of their biopsied breast compared to their non-biopsied breast (p=0.05). This study is the first to collect breast milk and obtain epithelial cells and DNA from a population of women at increased risk of breast cancer. Utilizing this unique population, we have been able to assess promoter methylation in 3 genes known to be methylated in breast cancer. Long term follow-up is ongoing for women enrolled in this study to determine breast health outcomes. Knowledge of promoter methylation along with long term follow-up may help us to better assess individual breast cancer risk. Given the high percentage of women that give birth and therefore produce milk, if even for just a few days, a breast cancer risk screen based on epithelial cells in milk has the potential to affect a significant portion of all women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-443. doi:10.1158/1538-7445.AM2011-LB-443

Breast cancers discovered during pregnancy or lactation (gestational) are known to be very aggressive and of poor prognosis. We have found a Mouse Mammary Tumor Virus (MMTV) like virus in human breast cancer, but not in adjacent normal tissue, indicating it exogenous origin. This virus, Human Mammary Tumor Virus (HMTV) recognized by its unique env sequence is present in 62% of gestational breast cancer, compared with 38% of sporadic breast cancers (Wang et al, 2003. Medical Oncology 20:233-236). The aggressive phenotype of gestational breast cancer was suggested to be due to the effect of hormones that stimulate cellular growth. HMTV- LTR contained sequences which are responsive to several hormones (glucocorticoid, estrogen, prolactin, and progesterone) and a transcription factor (TEF-1 family) (Wang et al, 2003. Medical Oncology 20:233-236). To find if HMTV is present in other tissues that respond to hormones, we have investigated the presence of HMTV sequences in two of these tissues. Endometrium is a classical example of such tissue. Endometrial cancers and normal endometrium were analyzed for HMTV env sequences. The results indicated that HMTV env sequences were present in 3.4% of the normal tissues and 37% of the tumors analyzed. Recently, it was reported that MMTV-like sequence was found in 10% of endometrial cancers (Johal et al. 2010, J. Med. Virology; 82: 1044-50). During lactation, the mammary gland experiences major metabolic changes due to hormonal stimulation. We have examined 88 normal milks and 63 milks from women, who have undergone biopsies. Whereas in the cells of normal milks 8% showed env sequences cells in the milk of the biopsied group showed that 23.8% contain HMTV sequences. These results confirmed electron microscope observations that a betaretrovirus was present in milk from healthy women and those with breast cancer (Moore et al. 1971, Nature 229: 611-615). Taken together, the results presented here suggest that tissues and tumors stimulated by hormones showed high prevalence of HMTV sequences. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2302. doi:10.1158/1538-7445.AM2011-2302

Gene specific promoter methylation in breast epithelial cells is considered one of the most promising biomarkers for assessing breast cancer risk. Unfortunately, obtaining breast epithelial cells is difficult. We recently demonstrated that human breast milk provides a source of epithelial cells useful for assessing breast cancer risk through analysis of DNA promoter methylation patterns. However, it has not been determined whether separation of the epithelial cells from the total cell population is necessary to obtain reliable promoter methylation patterns useful in assessing this risk. The present study determines the extent to which separation of the cell populations in breast milk alters the observed methylation patterns. A fresh breast milk sample (average size of 118 mL ± 29 mL) was collected from six lactating women between 25 and 43 years of age. For each milk sample, three groups of cells were collected: an unseparated (or total) cell population, an epithelial-enriched cell fraction and an epithelial-depleted cell fraction. The depleted and enriched cell fractions were obtained by immunomagnetic separation. Cell fractions were frozen and shipped to EpigenDX for DNA isolation, bisulfite treatment and pyrosequencing. Promoter methylation of five tumor suppressor genes (APC, CyclinD2, p16, RARβ, and RASSF1A) was examined. Cell separation resulted in distinct methylation patterns. Overall, the highest methylation scores were detected in the epithelial-enriched cell fractions: mean methylation scores were significantly higher in the epithelial-enriched fraction as compared to the depleted fraction or total cell population for APC, CyclinD2, RARβ, and RASSF1A. Only p16 showed very low methylation: Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1995. doi:10.1158/1538-7445.AM2011-1995

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.