Menstrual Phase and Menopausal Status Classification of Benign Breast Tissue Using Hormone-Regulated Gene Expression and Histomorphology: A Validation Study.

BACKGROUND: The validation of breast cancer risk biomarkers in benign breast samples (BBS) is a long-sought goal, hampered by the fluctuation of gene and protein expression with menstrual phase (MP) and menopausal status (MS). Previously, we identified hormone-related gene expression and histomorphology parameters to classify BBS by MS/MP. We now evaluate both together, to validate our prior results. PATIENTS AND METHODS: BBS were obtained from consenting women (86 premenopausal, 55 postmenopausal) undergoing reduction mammoplasty (RM) or contralateral unaffected breast (CUB) mastectomy. MP/MS was defined using classical criteria for menstrual dates and hormone levels on the day of surgery. BBS gene expression was measured with reverse transcription quantitative polymerase chain reaction (RT-qPCR) for three luteal phase (LP) genes (TNFSF11, DIO2, MYBPC1) and four menopausal genes (PGR, GREB1, TIFF1, CCND1). Premenopausal samples were classified into LP or non-LP, using published histomorphology parameters. Logistic regression and receiver-operator curve analysis was performed to assess area under the curve (AUC) for prediction of MP/MS. RESULTS: In all 131 women, menopausal genes plus age > 50 years predicted true MS [AUC 0.93, 95% confidence interval (CI) 0.89, 0.97]. Among premenopausal women, high TNFSF11 expression distinguished non-LP from LP samples (AUC 0.80, 95% CI 0.70, 0.91); the addition of histomorphology improved the prediction nonsignificantly (AUC 0.87, 95% CI 0.78, 0.96). In premenopausal subsets, addition of histomorphology improved LP prediction in RM (AUC 0.95, 95% CI 0.87, 1.0), but not in CUB (0.84, 95% CI 0.72, 0.96). CONCLUSIONS: Expression of five-gene set accurately predicts menopausal status and menstrual phase in BBS, facilitating the development of breast cancer risk biomarkers using large, archived sample repositories.

Relationship of Estradiol and Progesterone with Partnership and Parity Among Bangladeshi and British Women of European Origin.

Recent studies in social endocrinology have explored the effects of social relationships on female reproductive steroid hormones-estradiol and progesterone-investigating whether they are suppressed in partnered and parous women. Results have been mixed for these hormones although evidence is more consistent that partnered women and women with young children have lower levels of testosterone. These studies were sequential to earlier research on men, based on Wingfield's Challenge Hypothesis, which showed that men in committed relationships, or with young children, have lower levels of testosterone than unpartnered men or men with older or no children. The study described here explored associations between estradiol and progesterone with partnership and parity among women from two different ethnicities: South Asian and white British. We hypothesized that both steroid hormones would be lower among partnered and/or parous women with children ≤3 years old, regardless of ethnicity. In this study we analyzed data from 320 Bangladeshi and British women of European origin aged 18 to 50 who participated in two previous studies of reproductive ecology and health. Levels of estradiol and progesterone were assayed using saliva and/or serum samples and the body mass index calculated from anthropometric data. Questionnaires provided other covariates. Multiple linear regressions were used to analyze the data. The hypotheses were not supported. We argue here that, unlike links between testosterone and male social relationships, theoretical foundations for such relationships with female reproductive steroid hormones are lacking, especially given the primary role of these steroids in regulating female reproductive function. Further longitudinal studies are needed to explore the bases of independent relationships between social factors and female reproductive steroid hormones.

HPLC fractionation with immunoassay of steroids from nipple aspirate fluid.

Fractionation of steroids allows for multiple assays to be run on a single low volume liquid biopsy, whereas performing the same number of assays without fractionation would require increasing the sample volume by dilution, rendering the concentration of steroids below the level of detection for most, if not all, downstream assays. Briefly, steroids are extracted from a biofluid sample using solvent phase extraction to separate the aqueous (conjugated) steroids from the non-aqueous (non-conjugated) steroids in the organic phase. The latter is further separated by high-performance liquid chromatography (HPLC) and collected in an automated fraction collector based on the UV detection of internal standards. Commercially available immunoassays are then used to quantify the < ng/ml concentrations of steroids in each fraction. This protocol was designed for small samples of nipple aspirate fluid (minimum 2 µL), but it can be modified to fractionate steroids from homogenized solid tissue samples or other liquid biopsies. Included in this protocol are precautions to help ensure reproducibility and minimize matrix effects and other errors of measurement, given that samples requiring fractionation are fundamentally precious and, like other quantitative procedures of small samples, can be prone to contamination by solvent residues and other factors.•The method permits quantitative analysis of multiple steroids from very small volumes of biofluid.•Fractionation by HPLC provides a highly purified sample for quantification.•The immunoassay end point provides specificity without expensive equipment.

Functions of dehydroepiandrosterone in relation to breast cancer.

Although DHEA sulfate (DS) is the most abundant steroid in the circulation, breast fluid contains an approximately 80-fold greater concentration than serum. Transport of DS into cells requires organic anion transporting polypeptides (OATPs), which are specific for cell type, cell location, and substrate, but may have a broader specificity for housekeeping functions. Specific classes, which may be modified by soluble factors including neutral steroids, have been identified in the breast. After transport, DS may be cleaved to DHEA by ubiquitous sulfatases, which may be modified by the cell milieu, or DHEA may enter by diffusion. Synthesis from cholesterol does not occur because CYP17B12 and cytochrome b5 are lacking in breast tissues. Case-control studies reveal a positive association of serum DS with risk of breast cancer. The association is even greater with DHEA, particularly in postmenopausal women with HR + invasive tumors. Metabolites of DHEA, androstenedione and testosterone, are associated with breast cancer but DHEA is likely to have an independent role as well. Mechanisms by which DHEA may promote breast cancer relate to its effect in increasing circulating IGF-I, by inhibiting the suppressive effect of glucocorticoids, and by promoting retention of pre-adipocytes with aromatase activity. In addition, DHEA may interact with the G-protein coupled receptor GPER for stimulation of miR-21 and subsequent activation of the MAPK pathway. DHEA also has antitumor properties that relate to stimulation of immunity, suppression of inflammation, and elevation of adipose tissue adiponectin synthesis. The net effect may depend on the which factors predominate.

Association of genetic polymorphisms with local steroid metabolism in human benign breasts.

PURPOSE: Although alterations of concentrations in circulating steroids have been linked to single nucleotide polymorphisms (SNPs) of steroidogenic enzymes, we hypothesized that SNPs of such enzymes located within the breast affect local steroid concentrations more than products of such SNPs absorbed from the circulation. METHODS: Steroids (estradiol, estrone, testosterone, androstenedione, DHEA, DHEA sulfate, progesterone) in nipple aspirate fluid (NAF) were purified by HPLC and they along with serum steroids were quantified by immunoassays. Polymorphisms of the transporter SLCO2B1 and enzymes HSD3B1, CYP19A1, HSD17B12, AKR1C3, CYP1B1, and SRD5A1 were measured in white blood cell DNA. RESULTS: Steroid concentrations in NAF of subjects with homozygous minor genotypes differed from those with heterozygotes, i.e., SLCO2B1 (rs2851069) decreased DHEAS (p = 0.04), HSD17B12 (rs11555762) increased estradiol (p < 0.004), and CYP1B1 (rs1056836) decreased estradiol (p = 0.017) and increased progesterone (p = 0.05). Also, in serum, CYP19A1 (rs10046 and rs700518) both decreased testosterone (p = 0.02) and SRD5A1 increased androstenedione (p = 0.006). Steroids in subjects with major homozygotes did not differ from those with heterozygotes indicating recessive characteristics. CONCLUSIONS: In the breast, SNPs were associated with decreased uptake of DHEAS (SLCO2B1), increased estradiol concentrations through increased oxidoreductase activity (HSD17B12), or decreased estradiol concentrations by presumed formation of 4-hydroxyestradiol (CYP1B1). CYP19A1 was associated with decreased testosterone concentrations in serum but had no significant effect on estrogen or androgen concentrations within the breast. The hormone differences observed in NAF were not usually evident in serum, indicating the importance of assessing the effect of these SNPs within the breast.

Effects of web-based cognitive behavioral stress management and health promotion interventions on neuroendocrine and inflammatory markers in men with advanced prostate cancer: A randomized controlled trial.

Cognitive behavioral stress management (CBSM) improves quality of life and mitigates stress biology in patients with early-stage cancer, including men with localized prostate cancer. However, treatments for advanced prostate cancer like androgen deprivation therapy (ADT) can lead to significant symptom burden that may be further exacerbated by stress-induced inflammation and cortisol dysregulation. The aim of this study was to examine the effects of CBSM (versus an active health promotion control) on circulating inflammatory markers and cortisol in men with advanced prostate cancer. METHODS: Men with stage III or IV prostate cancer (N = 192) who had undergone ADT within the last year were randomized to CBSM or health promotion. Both interventions were 10 weeks, group-based, and delivered online. Venous blood was drawn at baseline, 6 months, and 12 months to measure circulating levels of CRP, IL-6, IL-8, IL-10, and TNF-α. Saliva samples were collected at awakening, 30 min after awakening, evening, and night for two consecutive days at baseline, 6-months, and 12-months to measure diurnal cortisol slopes. RESULTS: Mixed modeling analyses demonstrated that changes in inflammatory markers and cortisol did not differ by intervention. Men in both CBSM and health promotion showed decreases in IL-10, IL-8, and TNF-α from baseline to 6 months (ß = -3.85--5.04, p's = 0.004-<0.001). However, these markers generally demonstrated a rebound increase from 6 to 12 months (ß = 1.91-4.06, p's = 0.06-<0.001). Men in health promotion also demonstrated a flatter diurnal cortisol slope versus men in CBSM at 6 months (ß = -2.27, p = .023), but not at 12 months. There were no intervention effects on CRP, IL-6, or overall cortisol output. CONCLUSIONS: Contrary to hypotheses, CBSM did not lead to changes in the circulating inflammatory markers and cortisol relative to health promotion. CBSM may be associated with healthy diurnal cortisol rhythm because of its focus on cognitive behavioral approaches to stress management. More research is needed to understand the impact of CBSM and health promotion on biomarkers among men with advanced prostate cancer.

Shift from androgen to estrogen action causes abdominal muscle fibrosis, atrophy, and inguinal hernia in a transgenic male mouse model.

Inguinal hernia develops primarily in elderly men, and more than one in four men will undergo inguinal hernia repair during their lifetime. However, the underlying mechanisms behind hernia formation remain unknown. It is known that testosterone and estradiol can regulate skeletal muscle mass. We herein demonstrate that the conversion of testosterone to estradiol by the aromatase enzyme in lower abdominal muscle (LAM) tissue causes intense fibrosis, leading to muscle atrophy and inguinal hernia; an aromatase inhibitor entirely prevents this phenotype. LAM tissue is uniquely sensitive to estradiol because it expresses very high levels of estrogen receptor-α. Estradiol acts via estrogen receptor-α in LAM fibroblasts to activate pathways for proliferation and fibrosis that replaces atrophied myocytes, resulting in hernia formation. This is accompanied by decreased serum testosterone and decreased expression of the androgen receptor target genes in LAM tissue. These findings provide a mechanism for LAM tissue fibrosis and atrophy and suggest potential roles of future nonsurgical and preventive approaches in a subset of elderly men with a predisposition for hernia development.

Author Correction: Childhood ecology influences salivary testosterone, pubertal age and stature of Bangladeshi UK migrant men.

In the version of this Article originally published, the units for the 'Weight' column in Table 1 were incorrect; they should have been kg. This has now been corrected.

Breast Hormone Concentrations in Random Fine-Needle Aspirates of Healthy Women Associate with Cytological Atypia and Gene Methylation.

Sex steroid hormones contribute to breast cancer development, but data on concentrations of these within breast tissue are limited. We performed simultaneous multiparameter measurement of breast sex steroids, breast epithelial cytology, and DNA methylation in 119 healthy women (54 pre- and 65 postmenopausal) without a history of breast cancer. Random fine-needle aspiration (rFNA) of the breast was performed simultaneously with blood collection. Breast samples were analyzed by LC/MS-MS for estrone, estradiol, progesterone, androstenedione, and testosterone. Blood samples were assayed for estradiol and progesterone by immunoassay. Cytomorphology was classified using the Masood Score, and DNA methylation of eight genes was analyzed using quantitative multiplexed methylation-specific PCR, and expressed as the cumulative methylation index (CMI). Serum and breast concentrations of estradiol and progesterone showed significant correlation (Spearman r = 0.34, Padj = 0.001 and r = 0.69, Padj < 0.0006, respectively). Progesterone concentration was significantly higher in the premenopausal breast (Padj < 0.0008), and showed a luteal surge. Breast estrone and estradiol concentrations did not differ significantly by menopause, but androstenedione concentration was higher in the breasts of postmenopausal women (P = 0.026 and Padj = 0.208). Breast androgens were significantly correlated with breast density (Spearman r = 0.27, Padj = 0.02 for testosterone) and CMI (Spearman r = 0.3, Padj = 0.038 for androstenedione). Our data indicate that future larger studies of breast steroid hormones along with other parameters are feasible. Significant associations of breast androgen concentrations with breast density and gene methylation warrant future study. Cancer Prev Res; 11(9); 557-68. ©2018 AACR.

Childhood ecology influences salivary testosterone, pubertal age and stature of Bangladeshi UK migrant men.

Male reproductive investment is energetically costly, and measures of human reproductive steroid hormones (testosterone), developmental tempo (pubertal timing) and growth (stature) correlate with local ecologies at the population level. It is unclear whether male reproductive investment in later life is 'set' during childhood development, mediated through adulthood, or varies by ethnicity. Applying a life-course model to Bangladeshi migrants to the United Kingdom, here we investigate plasticity in human male reproductive function resulting from childhood developmental conditions. We hypothesized that childhood ecology shapes adult trade-offs between reproductive investment and/or other fitness-related traits. We predicted correspondence between these traits and developmental timing of exposure to ecological constraints (Bangladesh) or conditions of surplus (United Kingdom). We compared: Bangladesh sedentees (n = 107); Bangladeshi men who migrated in childhood to the United Kingdom (n = 59); migrants who arrived in adulthood (n = 75); second-generation UK-born and raised children of Bangladeshi migrants (n = 56); and UK-born ethnic Europeans (n = 62). Migration before puberty predicted higher testosterone and an earlier recalled pubertal age compared with Bangladeshi sedentees or adult migrants, with more pronounced differences in men who arrived before the age of eight. Second-generation Bangladeshis were taller, with higher testosterone than sedentees and adult migrants, and higher waking testosterone than Europeans. Age-related testosterone profiles varied by group, declining in UK migrants, increasing in sedentees, and having no significant relationship within UK-born groups. We conclude that male reproductive function apparently remains plastic late into childhood, is independent of Bengali or European ethnicity, and shapes physiological trade-offs later in life.

The relationship of single-strand breaks in DNA to breast cancer risk and to tissue concentrations of oestrogens.

CONTEXT: Clinical study of breast cancer patients in Chicago, IL, USA. OBJECTIVE: Ascertain the utility of measurements of single-strand breaks (SSB) in DNA for assessment of breast cancer risk. METHODS: Fine-needle aspirates of the breast, SSB by nick translation, percent breast density (PBD), Gail model risk, cumulative methylation index (CMI), enzymes of DNA repair and tissue antioxidants. RESULTS: DNA repair enzymes and 4-hydroxyestradiol were negatively associated with SSB; CMI and PBD were positively associated. CONCLUSIONS: Quantitative measurement of SSBs by this procedure indicates the relative number of SSBs and is related to promoter methylation, antioxidant availability and percent breast density.

AMP-activated protein kinase and energy balance in breast cancer.

Cancer growth and metastasis depends on the availability of energy. Energy-sensing systems are critical in maintaining a balance between the energy supply and utilization of energy for tumor growth. A central regulator in this process is AMP-activated protein kinase (AMPK). In times of energy deficit, AMPK is allosterically modified by the binding of increased levels of AMP and ADP, making it a target of specific AMPK kinases (AMPKKs). AMPK signaling prompts cells to produce energy at the expense of growth and motility, opposing the actions of insulin and growth factors. Increasing AMPK activity may thus prevent the proliferation and metastasis of tumor cells. Activated AMPK also suppresses aromatase, which lowers estrogen formation and prevents breast cancer growth. Biguanides can be used to activate AMPK, but AMPK activity is modified by many different interacting factors; understanding these factors is important in order to control the abnormal growth processes that lead to breast cancer neoplasia. Fatty acids, estrogens, androgens, adipokines, and another energy sensor, sirtuin-1, alter the phosphorylation and activation of AMPK. Isoforms of AMPK differ among tissues and may serve specific functions. Targeting AMPK regulatory processes at points other than the upstream AMPKKs may provide additional approaches for prevention of breast cancer neoplasia, growth, and metastasis.

Gene Methylation and Cytological Atypia in Random Fine-Needle Aspirates for Assessment of Breast Cancer Risk.

Methods to determine individualized breast cancer risk lack sufficient sensitivity to select women most likely to benefit from preventive strategies. Alterations in DNA methylation occur early in breast cancer. We hypothesized that cancer-specific methylation markers could enhance breast cancer risk assessment. We evaluated 380 women without a history of breast cancer. We determined their menopausal status or menstrual cycle phase, risk of developing breast cancer (Gail model), and breast density and obtained random fine-needle aspiration (rFNA) samples for assessment of cytopathology and cumulative methylation index (CMI). Eight methylated gene markers were identified through whole-genome methylation analysis and included novel and previously established breast cancer detection genes. We performed correlative and multivariate linear regression analyses to evaluate DNA methylation of a gene panel as a function of clinical factors associated with breast cancer risk. CMI and individual gene methylation were independent of age, menopausal status or menstrual phase, lifetime Gail risk score, and breast density. CMI and individual gene methylation for the eight genes increased significantly (P < 0.001) with increasing cytological atypia. The findings were verified with multivariate analyses correcting for age, log (Gail), log (percent density), rFNA cell number, and body mass index. Our results demonstrate a significant association between cytological atypia and high CMI, which does not vary with menstrual phase or menopause and is independent of Gail risk and mammographic density. Thus, CMI is an excellent candidate breast cancer risk biomarker, warranting larger prospective studies to establish its utility for cancer risk assessment. Cancer Prev Res; 9(8); 673-82. ©2016 AACR.

Protein Biomarkers for Breast Cancer Risk Are Specifically Correlated with Local Steroid Hormones in Nipple Aspirate Fluid.

The local endocrine environment of the breast may have stronger relations to breast cancer risk than systemic hormones. Nipple aspiration fluid (NAF) provides a window into this milieu. We hypothesized that the correlations between proteins and steroid hormones in NAF are stronger, and specific relationships may reveal links to breast cancer risk. NAF and blood samples were obtained simultaneously from 54 healthy women and from the contralateral unaffected breast of 60 breast cancer patients. The abundance of five proteins, superoxide dismutase (SOD1), C-reactive protein (CRP), chitinase-3-like protein 1 (YKL40), cathepsin D (CatD), and basic fibroblast growth factor (bFGF) in NAF was measured using ELISA. The NAF and serum concentrations of estradiol, estrone, progesterone, androstenedione, testosterone, and dehydroepiandrostrerone (DHEA) were measured using ELISA or RIA. The correlations between proteins and hormones revealed that NAF proteins correlated with each other: SOD1 with CRP (R = 0.276, P = 0.033) and CatD (R = 0.340, P = 0.0036), and bFGF with CRP (R = 0.343, P = 0.0021). NAF proteins displayed significant correlations with NAF steroids, but not with serum steroids: SOD1 with DHEA (R = 0.333, P = 0.019), YKL40 with testosterone (R = 0.389, P = 0.0012), and bFGF negatively correlated with testosterone (R = -0.339, P = 0.015). The regulation of YKL40 and bFGF by testosterone was confirmed in breast cancer cell lines. In summary, NAF proteins were more strongly related to local hormone levels than to systematic hormone levels. Some proteins were specifically correlated with different NAF steroids, suggesting that these steroids may contribute to breast cancer risk through different mechanisms.

Genetic Determinants of Nipple Aspiration Fluid Yield.

PURPOSE: Nipple aspiration fluid (NAF) is a non-invasively-acquired biosample that can provide a window into the breast environment, but NAF yield is highly variable. Its determinants must be better understood for studies of breast cancer risk. The wet earwax phenotype was identified as one determinant of NAF yield in the 1970s, and is linked to single nucleotide polymorphisms (SNPs) in the ATP-binding cassette transporter gene ABCC11. We have investigated this, as well as SNPs in the prolactin (PRL) and prolactin receptor (PRLR) genes, in relation to NAF yield. METHODS: DNA was extracted from white blood cells of 557 NAF yielders and 359 non-yielders, and was used to genotype ABCC11 (rs17822931), PRL (rs849870, rs849872, rs849886, rs2244502, rs1341239), and PRLR (rs37364, rs34024951, rs1610218, rs9292575, rs7718468) using Taqman genotyping assay. The association between NAF yield and each single SNP was analyzed using logistic regression adjusting for age, race, and menopausal status. RESULTS: ABCC11 rs17822931 showed a negative association with NAF yield [odds ratio (OR) 0.66, 95 % confidence interval (CI) 0.49-0.88; p = 0.004]. The PRL rs849870 and the haplotype combination with other SNPs showed a marginal association with NAF yield. In addition, the years since last birth also showed negative association with NAF yielding (OR 0.98, 95 % CI 0.96-0.99; p = 0.001). The combination of the years since last birth with ABCC11 SNP revealed significant interaction between reproductive factor and genetic factor. CONCLUSIONS: Our results confirmed the association between NAF yield and earwax phenotype through ABCC11 genotype. Combined with the recency of last birth, ABCC11 genotype should be considered in the design of studies utilizing NAF as a biosample.

Nipple Aspirate Fluid Hormone Concentrations and Breast Cancer Risk.

Prior reports identify higher serum concentrations of estrogens and androgens as risk factors for breast cancer, but steroids in nipple aspirate fluid (NAF) may be more related to risk. Incident breast cancer cases and mammography controls were recruited. Sex steroids were measured in NAF from the unaffected breasts of cases and one breast of controls. Menopausal status and menstrual cycle phase were determined. NAF steroids were purified by HPLC and quantified by immunoassays. Conditional logistic regression models were used to examine associations between NAF hormones and case-control status. NAF samples from 160 cases and 157 controls were evaluable for hormones. Except for progesterone and dehydroepiandrosterone (DHEA), the NAF and serum concentrations were not significantly correlated. NAF estradiol and estrone were not different between cases and controls. Higher NAF (but not serum) DHEA concentrations were associated with cases, particularly among estrogen receptor (ER)-positive cases (NAF odds ratio (OR) = 1.18, 95 % confidence interval (CI) 1.02, 1.36). NAF DHEA was highly correlated with NAF estradiol and estrone but not with androstenedione or testosterone. Higher progesterone concentrations in both NAF and serum were associated with a lower risk of ER-negative cancer (NAF OR = 0.69, 95 % CI 0.51, 0.92). However, this finding may be explained by case-control imbalance in the number of luteal phase subjects (2 cases and 19 controls). The significantly higher concentration of DHEA in NAF of cases and its correlation with NAF estradiol indicates a potentially important role of this steroid in breast cancer risk; however, the negative association of progesterone with risk is tentative.

Local transdermal therapy to the breast for breast cancer prevention and DCIS therapy: preclinical and clinical evaluation.

PURPOSE: Women at high risk of breast cancer and those with carcinoma in situ need non-toxic, well-tolerated preventive interventions. One promising approach is drug delivery through the breast skin (local transdermal therapy, LTT). Our goal was to test novel drugs for LTT, to establish that LTT is applicable to non-steroidal drugs. METHODS: Athymic nude rats were treated with oral tamoxifen, transdermal 4-hydroxytamoxifen (4-OHT) or endoxifen gel applied daily to the axillary mammary gland for 6 weeks (Study 1). Study 2 was identical to Study 1, testing transdermal telapristone acetate (telapristone) gel versus subcutaneous implant. At euthanasia, mammary glands and blood were collected. In Study 3, consenting women requiring mastectomy were randomized to diclofenac patch applied to the abdomen or the breast for 3 days preoperatively. At surgery, eight tissue samples per breast were collected from predetermined locations, along with venous blood. Drug concentrations were measured using liquid chromatography-tandem mass spectroscopy. RESULTS: Mammary tissue concentrations of 4-OHT, endoxifen, and telapristone were significantly higher in the axillary glands of the gel-treated animals, compared to inguinal glands or to systemically treated animals. Plasma concentrations were similar in gel and systemically treated animals. The clinical trial showed significantly higher mammary concentrations when diclofenac was applied to the breast skin versus the abdominal skin, but concentrations were variable. CONCLUSIONS: These results demonstrate that lipophilic drugs can be developed for LTT; although the nude rat is suitable for testing drug permeability, delivery is systemic. In human, however, transdermal application to the breast skin provides local delivery.

Methodological considerations in estrogen assays of breast fluid and breast tissue.

Estradiol (E2) in nipple aspirate fluid (NAF), ductal lavage fluid (DLF), and random fine needle aspirates (rFNA) are compared. Quantification was by immunoassay or tandem MS. The percent of women yielding NAF varied between 24% and 48% and for DLF was 86.3%. Variation between ducts within a breast was not less than variation between breasts within women but variation between breasts and within women over time was significantly less than variation between women. Serum E2 was highly significantly different among phases of the menstrual cycle but NAF E2 was not different. The correlation between serum and breast fluid E2 concentrations in premenopausal women had coefficients of determination of less than 15%. The correlation between serum and NAF in studies of postmenopausal women varied greatly and may depend on patient selection. The difference between NAF E2 between pre- and postmenopausal women was only 22%; for rFNA it was non-significantly 44% lower in a similar group of postmenopausal women. Progesterone was 96% and 98% lower in postmenopausal NAF and rFNA samples, respectively. Measurements of E2 in breast fluid or breast tissue appears to provide similar estimates of E2 exposure. E2 levels in breast fluid do not reflect the rapid changes that occur in serum and, thus, serum availability of E2 is only one factor determining its levels in the breast. The similarity of levels between breasts and between ducts suggests that estimates of estrogen exposure does not require multiple samples, however, unavailability of fluid may require rFNA in some cases.