Nipple Aspirate Fluid Levels Research Articles

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

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

Abstract A96: Trans-resveratrol decreases breast proliferation and alters mammary promoter hypermethylation in women at increased risk for breast cancer

Abstract Trans-resveratrol, present in high concentration in the skin of red grapes and red wine, has a dose-dependent antiproliferative effect in vitro, prevents the formation of mammary tumors, and has been touted as a chemopreventive agent. We conducted in vitro experiments in hormone sensitive breast cancer cells and observed that trans-resveratrol downregulated DNA methyltransferase gene expression in a dose dependent fashion. Based upon this preclinical observation, we determined if transresveratrol had dose related anti-proliferative and methylation effects in humans. 16 adult women at increased breast cancer risk were randomized in double-blind fashion to placebo, 5 or 50 mg trans-resveratrol twice daily for 12 weeks. We collected serum, breast nipple aspirate fluid (NAF) and mammary duct (MD) specimens from all participants at all time points. Breast specific and systemic antiproliferative effects were assessed measuring the cancer promoting prostaglandin (PG)E2 in NAF and serum as well as changes in MD cytology, while methylation assessment of three cancer related genes (p16, RASSF-1α, APC) was performed on MD specimens. Serum resveratrol increased after consuming both trans-resveratrol doses (p&amp;lt;.001 for both). Cytology was not significantly changed by resveratrol treatment. RASSF-1α methylation significantly decreased after high (but not low) dose resveratrol, and the decrease correlated with increasing levels of serum resveratrol. RASSF-1α methylation also correlated with change in NAF levels of PGE2 (p=.0087). This work provides novel insights into the anticancer effects of trans-resveratrol in women at increased breast cancer risk, including prostaglandin inhibition and a decrease in methylation of the tumor suppressor gene RASSF-1α. Citation Information: Cancer Prev Res 2010;3(12 Suppl):A96.

Abstract A79: Human breast tissue bioavailability of topically applied limonene

Abstract Purpose: Limonene is a monocyclic monoterpene found at high concentration in citrus peel oil and has demonstrated anti-cancer effects in preclinical studies of mammary carcinogenesis. However, the effective oral dose determined in rodents extrapolates to a human dose that may not be feasible for long-term consumption. Limonene is highly lipophilic and has been long explored as an enhancer for percutaneous absorption of pharmaceutical drugs, including tamoxifen. Preliminary work from our group demonstrated that topically applied limonene is absorbed through the mouse skin and accumulates in the mammary glands. Methods: We conducted a pilot clinical study to determine whether limonene would be bioavailable in the breast following topical application. In the first study phase, we recruited 19 healthy women to apply limonene containing massage oil to the breast daily or 3x/wk. Nipple aspirate fluid (NAF) and plasma samples were collected before and after 4 weeks of limonene massage application. Sample limonene concentrations were determined using gas chromatography-mass spectrometry. In the second study phase, we recruited 25 healthy women to undergo 4 weeks of daily limonene massage oil application to the breast. Potential agent effect biomarkers were determined in NAF and plasma collected before and after the limonene massage application. Results: In samples collected from the first study phase, no significant increase in NAF and plasma limonene levels was observed following the limonene massage application, possibly because limonene deposits in the fatty breast tissue with minimal secretion to NAF or systemic circulation. Changes in agent effect biomarkers in NAF, including TGF-beta, EGF, and adiponectin, were evaluated in the second study phase for determination of the breast tissue limonene bioavailability. There were no consistent changes in the agent effect biomarkers overall or in pre-menopausal women following the limonene massage application. However, among the post-menopausal women (n =8), there were significant increases in NAF levels of TGF-beta (p = 0.005) and EGF (p = 0.032), and there was an increasing trend in adiponectin (p = 0.164, n=5 due to limited NAF volume). Conclusions: We conclude that breast massage application of limonene containing massage oil did not result in consistent changes in limonene secretion to the NAF and the potential drug effect biomarkers in NAF. However, limonene massage application increased the NAF expression of TGF-beta and EGF in post-menopausal women, indicative of the intervention effects in the breast tissue. Further studies are needed to explore the potential effects of topical limonene massage intervention on carcinogenesis biomarkers in a larger cohort of post-menopausal women. Supported by Department of Defense, BCRP Idea Award, BC061529 Citation Information: Cancer Prev Res 2010;3(1 Suppl):A79.

The effect of sulindac on growth differentiation factor 15 and 13,14-dihydro-15-keto prostaglandin A2 in nipple aspirate fluid

Use of non-steroidal anti-inflammatory drugs (NSAIDs) is associated in several large epidemiologic studies with a lower risk of epithelial cancers including breast though no randomized, placebo controlled phase III studies have been conducted to date. NSAID use was associated with a significantly lower risk of breast cancer among participants in a multiethnic cohort, though the nearly 50% reduction in risk was limited to women with estrogen receptor positive tumors; an observation previously reported by Terry et al., and supported by Gierach et al., and findings of Harris et al for cyclooxygenase-2 (COX-2) inhibitors. These findings in epidemiologic studies are supported by work in human mammary tissues and cell culture where overexpression of COX-2, one of two cyclooxygenase targets of NSAIDs, acts as an early event in the transition of normal breast cells to malignancy. Further, COX-2-associated prostaglandin E2 (PGE2) production has been shown to increase aromatase activity in mammary epithelial cells possibly explaining the observed benefit of NSAID use in hormone receptor positive disease and the recent observation of lower circulating estradiol levels among regular users of NSAIDs. Thus, there exists sufficient rationale for the use of NSAIDs for the prevention of breast cancer. While the best studied target of NSAIDs is the COX isoenzymes, the anti-tumor activities of each of the NSAID agents via 'non COX' pathways remains unknown. Sulindac, a non selective NSAID with well recognized COX-independent induction of apoptosis has remained a strong candidate for chemoprevention largely because of its broader anti-tumor activity. Growth differentiation factor 15 (GDF-15), a potent proapoptotic molecule in the TGF-β superfamily and potential tumor suppressor is upregulated by several NSAIDS independent of its activity as a COX inhibitor. Sulindac sulfone, a metabolite of sulindac and a COX-independent mediator of apoptosis, has been shown to be a potent inducer of GDF-15 at pharmacologically relevant doses whereas Celecoxib, a COX-2 specific inhibitor, has low GDF-15 induction potency even at toxic doses. Other NSAIDs such as aspirin and indomethacin as well as a number of candidate dietary chemopreventives including resveratrol, indole-3-carbinol, 3,3' Diindolylmethane (DIM), and lycopene as well as the PPAR agonists troglitazone, and 15-deoxy-Δ12,14-prostaglandin J2 all show potent induction of GDF-15 leading to the suggestion that GDF-15 might serve as a complementary molecular target to COX-2 for cancer chemoprevention and serve as a surrogate biomarker of apoptotic response. To better understand the effect of NSAIDs in the breast, we conducted a phase 1b dose study using sulindac as a representative drug of the non selective NSAIDs with known activity to induce GDF-15 and inhibit COX-2. To assess sulindac and its metabolites and their effects on a stable COX-2 derived prostaglandins 13,14-dihydro-15-keto prostaglandin A2 [PGEM], and the NSAID inducible growth differentiation factor (GDF-15) in nipple aspirate fluid (NAF), we randomized 30 women at increased risk for breast cancer to 150 mg once daily or twice daily sulindac for 6 weeks. Sulindac and sulfide were detectable in 57.7% of NAF samples with sulfone detectable in 11.6%. Sulindac was associated in a dose independent manner with a nonsignificant decrease in NAF PGEM levels (p = 0.1). Serum levels of sulindac, but not NAF sulindac, were correlated with a decrease in NAF PGEM levels (p = 0.03). GDF-15, showed a borderline significant trend towards higher levels in NAF with 300 mg daily sulindac (p = 0.07). This study suggests that COX inhibition in the breast may be achieved at 150 mg sulindac once daily while a higher daily dose is needed to exhibit activities via COX independent pathways. The study also suggests that NAF levels of PGEM and GDF-15 could be considered as drug effect biomarkers for future early phase chemoprevention trials.

Abstract CN04-03: Phase 1b sulindac study for women at high risk for breast cancer

Abstract CN04-03 Background Regular use of non steroidal anti-inflammatory agents (NSAIDs) has been associated with reduced risk of breast cancer. The breast specific activity of each of the numerous NSAID agents and their relative potency to prevent breast cancer remains unknown. Sulindac, a non selective NSAID with pro apoptotic activity is a strong candidate for chemoprevention. Objective and Methods To assess concentrations of sulindac and its metabolites as well as their effects on known molecular markers in nipple aspirate fluid (NAF) in 15 subjects receiving 150 mg once daily and 15 subjects receiving 150 mg twice daily sulindac. NAF and serum samples were collected before and after 6 weeks of sulindac dosing. Results Sulindac and sulfide were detectable in 57.7% of NAF samples with sulfone detectable in 11.6%. There were no statistically significant differences between sulindac or sulfide levels in NAF by dose. There was a significant positive correlation between NAF sulindac and NAF sulfide levels. NAF 13,14-dihydro-15-keto prostaglandin A2 (PGEM), a stable derivative of prostaglandins, exhibited a non significant trend towards decreased levels (p =0.1). Serum levels of sulindac, but not NAF sulindac, was correlated with a decrease in NAF PGEM levels (p=0.03). The NSAID inducible protein, growth differentiation factor GDF-15, showed a borderline significant trend towards higher levels in NAF in the 300 mg daily group (p = 0.07). Serum CRP levels were decreased with sulindac exposure with the greatest decrease observed among those women with elevated CRP levels at baseline. Conclusion Sulindac and its sulfide metabolites partition to the breast and are detectable in NAF. Sulindac exposure was associated with a trend towards reduction in PGEM that was correlated with serum levels of sulindac. GDF-15 levels increased in the high dose group. NAF levels of PGEM and GDF-15 may prove useful as NSAID response biomarkers if for larger efficacy based intervention studies in breast cancer prevention. Citation Information: Cancer Prev Res 2008;1(7 Suppl):CN04-03.

Biomarkers associated with breast cancer are associated with obesity

Background: Obesity is linked to the development of postmenopausal breast cancer, and some studies indicate obesity predicts a worse prognosis for premenopausal women who develop the disease. It was our hypothesis that proteins associated with breast cancer would be associated with body mass index (BMI). Methods: We searched our database of women enrolled in breast health translational research trials for information on BMI and markers predictive of breast cancer (basic fibroblast growth factor (bFGF), prostate-specific antigen (PSA), human kallikrein (hK)2, and urinary plasminogen activator (uPA). Information on BMI and one or more nipple aspirate fluid (NAF) or serum biomarkers was available from 382 women. Results: In this data set, NAF and serum levels of PSA (nPSA and sPSA), and NAF levels hK2, bFGF and uPA were each associated with pre- and/or postmenopausal breast cancer. sPSA was inversely associated with BMI in both pre- ( r = −.56, p = .001) and postmenopausal women ( r = −.62, p = .0035) without breast cancer. This association was lost when controlling for plasma volume. In women without breast cancer, NAF bFGF ( p = .07, premenopausal subjects) and NAF hK2 ( p = .09, postmenopausal subjects) were borderline associated with BMI. In women with breast cancer, nPSA was inversely ( r = −.53, p = .049) associated with BMI in premenopausal women and directly associated with BMI in postmenopausal women ( r = .37, p = .017). nPSA trended higher in hormone sensitive cancers, especially those that expressed progesterone receptor ( p = .059). Conclusions: sPSA was inversely associated with BMI in all pre- and postmenopausal women and specifically in pre- and postmenopausal women without breast cancer. NAF PSA was associated with BMI in pre- and postmenopausal women with breast cancer. Evaluating the change in PSA with changes in weight may provide clues regarding a subject's breast cancer risk.

Increased expression of the inflammatory protein YKL‐40 in precancers of the breast

Serum levels of YKL-40 have been associated with inflammatory diseases and breast cancer. Our purpose was to determine if YKL-40 in breast tissue, nipple aspirate fluid (NAF) and serum is (i) concentrated in NAF compared to matched serum, (ii) increased in the NAF, serum or tissue of women with biopsy proven precancer or cancer compared to healthy women and (iii) influenced by menopausal status. 118 women (61 healthy subjects, 10 with precancer and 47 with breast cancer) aged 17-95 years provided NAF with or without serum samples for analysis. Matched tissue was analyzed from a subset of subjects who underwent breast biopsy. All NAF and serum samples had detectable levels of YKL-40. Median YKL-40 levels for the entire cohort were 683 fold higher in NAF than serum. Premenopausal subjects had higher NAF and lower serum levels of YKL-40 than postmenopausal subjects. YKL-40 levels in NAF but not serum were higher in women with precancer (atypical hyperplasia and lobular carcinoma in situ) than in either healthy subjects (p = 0.025) or subjects with breast cancer (p = 0.015). In women with precancer, YKL-40 distribution in tissue correlated with YKL serum level (p = 0.043). YKL-40 is concentrated in NAF, with the highest concentrations in premenopausal women. NAF levels of YKL-40 are significantly higher in women with precancers than healthy subjects, suggesting that measuring YKL-40 in NAF may improve the identification of women at increased breast cancer risk.

Celecoxib decreases prostaglandin E2 concentrations in nipple aspirate fluid from high risk postmenopausal women and women with breast cancer.

BackgroundCelecoxib inhibits PGE2 production in cancerous tissue. We previously reported that PGE2 levels in nipple aspirate fluid (NAF) and plasma were not decreased in women at increased breast cancer risk who received celecoxib 200 mg twice daily (bid). The endpoints of the current study were to determine if a short course of celecoxib 400 mg bid would decrease PGE2 levels in women 1) at increased breast cancer risk, and 2) with established breast cancer.MethodsNAF and plasma samples were collected before, 2 weeks after taking celecoxib 400 mg bid, and two weeks after washout from 26 women who were at increased breast cancer risk. From 13 women with newly diagnosed breast cancer, NAF from the incident breast and plasma were collected before and on average 2 weeks after taking celecoxib. Additionally, in nine of the 13 women with breast cancer, NAF was collected from the contralateral breast.ResultsNo consistent change in NAF or plasma PGE2 levels was noted in high risk premenopausal women. NAF PGE2 levels decreased after celecoxib administration in postmenopausal high risk women (p = 0.02), and in both the NAF (p = 0.02) and plasma (p = 0.03) of women with breast cancer.ConclusionCelecoxib 400 mg bid taken on average for 2 weeks significantly decreased NAF, but not plasma, PGE2 levels in postmenopausal high risk women, and decreased both NAF and plasma PGE2 levels in women with newly diagnosed breast cancer. PGE2 levels may predict celecoxib breast cancer prevention and treatment efficacy. Our observations are preliminary, and larger studies to confirm and extend these findings are warranted.

Leptin expression in breast nipple aspirate fluid (NAF) and serum is influenced by body mass index (BMI) but not by the presence of breast cancer.

While obesity is a known risk factor for postmenopausal breast cancer, the molecular mechanisms involved are unclear. Systemic levels of leptin, the product of the ob (obesity) gene, are increased in obese individuals (body mass index, BMI, over 25) and are higher in women than men. Leptin has been found to stimulate the growth of breast cancer cells in vitro. Our goal was to determine whether leptin was 1) present in nipple aspirate fluid (NAF), and 2) whether NAF leptin levels were associated with a) levels in serum, b) obesity, and c) breast cancer. We collected and evaluated NAF specimens from 83 subjects and serum specimens from 49 subjects. NAF leptin was detectable in 16/41 (39 %) of premenopausal and 21/42 (50 %) postmenopausal subjects. NAF leptin was significantly lower (p = 0.042) in premenopausal than postmenopausal women with a BMI < 25, but not in those with a higher BMI. NAF leptin was significantly associated with BMI in premenopausal (p = 0.011) but not in postmenopausal women. Serum leptin was associated with BMI in both premenopausal and postmenopausal women (p = 0.0001 for both). NAF and serum leptin were associated in premenopausal (p = 0.02) but not postmenopausal women. Neither NAF nor serum leptin was associated with premenopausal or postmenopausal breast cancer. Our findings include that 1) leptin is present in the breast and detectable in a subset of NAF samples, 2) NAF leptin in premenopausal but not postmenopausal women parallels serum leptin levels, and 3) neither NAF nor serum levels of leptin were associated with premenopausal or postmenopausal breast cancer.

Circulating testosterone and prostate-specific antigen in nipple aspirate fluid and tissue are associated with breast cancer.

Preliminary evidence has associated testosterone and prostate-specific antigen (PSA) with breast cancer. Our objective was to determine whether a) testosterone levels in nipple aspirate fluid (NAF), serum, or breast tissue are associated with breast cancer; b) testosterone levels in serum are associated with levels in NAF; c) PSA in NAF, serum, or breast tissue is associated with breast cancer; and d) serum PSA is associated with NAF PSA levels. We obtained 342 NAF specimens from 171 women by means of a modified breast pump. Additionally, we collected 201 blood samples from 99 women and 51 tissue samples from 41 subjects who underwent surgical resection for suspected disease. Women currently using birth control pills or hormone replacement therapy were excluded from the study. Controlling for age and menopausal status, serum testosterone was significantly increased in women with breast cancer (p = 0.002). NAF and serum testosterone levels were not associated. Neither NAF nor tissue testosterone was associated with breast cancer. Controlling for menopausal status and age, NAF PSA was significantly decreased in women with breast cancer (p < 0.001). We did not find serum PSA to be associated with breast cancer, although we found an indication that, in postmenopausal women, its levels were lower in women with cancer. Serum PSA was associated with NAF PSA in postmenopausal women (p < 0.001). PSA levels in cancerous tissue were significantly lower than in benign breast specimens from subjects without cancer (p = 0.011), whereas levels of PSA in histologically benign specimens from subjects with cancer were intermediate. Our results suggest that serum testosterone is increased and NAF PSA is decreased in women with breast cancer, with PSA expression being higher in normal than in cancerous breast tissues. NAF and serum PSA levels in postmenopausal women are correlated, suggesting that as laboratory assessment of PSA becomes more sensitive, serum PSA may become useful in identifying women with breast cancer.