The changing microRNA landscape by color and cloudiness: a cautionary tale for nipple aspirate fluid biomarker analysis.

PURPOSE: Investigation of nipple aspirate fluid (NAF)-based microRNAs (miRNAs) as a potential screening tool for women at increased risk of developing breast cancer is the scope of our research. While aiming to identify discriminating NAF-miRNAs between women with different mammographic densities, we were confronted with an unexpected confounder: NAF sample appearance. Here we report and alert for the impact of NAF color and cloudiness on miRNA assessment. METHODS: Seven classes of NAF colors coupled with cloudiness appearance were established. Using 173 NAF samples from 154 healthy women (19 samples were bilaterally collected), the expression of 14 target and 2 candidate endogenous control (EC) miRNAs was investigated using Taqman Advanced miRNA assays to identify significant differential expression patterns between color-cloudiness classes. Inter- and intra-individual variation of miRNA expression was analyzed using the coefficient of variation (CV). RESULTS: We found that between the seven NAF classes, fold change miRNA expression differences ranged between 2.4 and 19.6 depending on the interrogated miRNA. Clear NAF samples exhibited higher miRNA expression levels compared to cloudy NAF samples with fold change differences ranging between 1.1 and 6.2. Inter-individual and intra-individual miRNA expression was fairly stable (CV < 15 %), but nevertheless impacted by NAF sample appearance. Within NAF classes, inter-individual variation was largest for green samples (CV 6-15 %) and smallest for bloody samples (CV 2-6 %). CONCLUSIONS: Our data indicate that NAF color and cloudiness influence miRNA expression and should, therefore, be systematically registered using an objective color classification system. Given that sample appearance is an inherent feature of NAF, these variables should be statistically controlled for in multivariate data analyses. This cautionary note and recommendations could be of value beyond the field of NAF-miRNAs, given that variability in sample color and cloudiness is likewise observed in liquid biopsies such as urine, cerebrospinal fluid and sputum, and could thereby influence the levels of miRNAs and other biomarkers.

The Physiological MicroRNA Landscape in Nipple Aspirate Fluid: Differences and Similarities with Breast Tissue, Breast Milk, Plasma and Serum.

BACKGROUND: MicroRNAs (miRNAs) target 60% of human messenger RNAs and can be detected in tissues and biofluids without loss of stability during sample processing, making them highly appraised upcoming biomarkers for evaluation of disease. However, reporting of the abundantly expressed miRNAs in healthy samples is often surpassed. Here, we characterized for the first time the physiological miRNA landscape in a biofluid of the healthy breast: nipple aspirate fluid (NAF), and compared NAF miRNA expression patterns with publically available miRNA expression profiles of healthy breast tissue, breast milk, plasma and serum. METHODS: MiRNA RT-qPCR profiling of NAF (n = 41) and serum (n = 23) samples from two healthy female cohorts was performed using the TaqMan OpenArray Human Advanced MicroRNA 754-Panel. MiRNA quantification data based on non-targeted or multi-targeted profiling techniques for breast tissue, breast milk, plasma and serum were retrieved from the literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1 and 50, combined into an overall ranking per sample type and compared. RESULTS: NAF expressed 11 unique miRNAs and shared 21/50 miRNAs with breast tissue. Seven miRNAs were shared between the five sample types. Overlap between sample types varied between 42% and 62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis. CONCLUSION: This is the first study to characterize and compare the unique physiological NAF-derived miRNA landscape with the physiological expression pattern in breast tissue, breast milk, plasma and serum. Breast-specific sources did not mutually overlap more than with systemic sources. Given their established role in carcinogenesis, NAF miRNA assessment could be a valuable tool in breast tumor diagnostics.

Application of Nipple Aspirate Fluid miRNA Profiles for Early Breast Cancer Detection and Management.

The authors of the recently published review, "Why the Gold Standard Approach by Mammography Demands Extension by Multiomics [...].

Differential proteomic comparison of breast cancer secretome using a quantitative paired analysis workflow.

BACKGROUND: Worldwide, breast cancer is the main cause of cancer mortality in women. Most cases originate in mammary ductal cells that produce the nipple aspirate fluid (NAF). In cancer patients, this secretome contains proteins associated with the tumor microenvironment. NAF studies are challenging because of inter-individual variability. We introduced a paired-proteomic shotgun strategy that relies on NAF analysis from both breasts of patients with unilateral breast cancer and extended PatternLab for Proteomics software to take advantage of this setup. METHODS: The software is based on a peptide-centric approach and uses the binomial distribution to attribute a probability for each peptide as being linked to the disease; these probabilities are propagated to a final protein p-value according to the Stouffer's Z-score method. RESULTS: A total of 1227 proteins were identified and quantified, of which 87 were differentially abundant, being mainly involved in glycolysis (Warburg effect) and immune system activation (activated stroma). Additionally, in the estrogen receptor-positive subgroup, proteins related to the regulation of insulin-like growth factor transport and platelet degranulation displayed higher abundance, confirming the presence of a proliferative microenvironment. CONCLUSIONS: We debuted a differential bioinformatics workflow for the proteomic analysis of NAF, validating this secretome as a treasure-trove for studying a paired-organ cancer type.

New horizon for breast cancer biomarker discoveries: What might the liquid biopsy of nipple aspirate fluid hold?

The existence of cellular, molecular and biochemical heterogeneity of human breast cancers reveals the intricacy of biomarkers complexity, stimulating studies on new approaches (like "liquid biopsies") for the improvements in precision medicine. Breast cancer is recognized as a leading cause of morbidity and mortality worldwide with tumors significantly diverse and containing many types of cells showing different genetic and epigenetic profiles. In this field, the technology of liquid biopsy (applied to a fluid produced by breast gland, named nipple aspirate fluids, NAF) highlights the power of combining basic and clinical research. NAF is the mirror of the entire ductal/alveolar breast tree providing almost complete proteomic profile and a valuable source for biomarker discovery, in non-invasive manner than tissue biopsies. The liquid biopsy technology using NAF may represent the outstanding breakthrough of proteomic cancer research revealing novel diagnostic and prognostic applications. In conjunction to metabolomic and degradome profiling, the use of NAF as liquid biopsy approach will improve the detection of changes in the cellular microenvironment of the breast tumors, understanding molecular and biochemical mechanisms which drive breast tumor initiation, maintenance and progression, and finally enhancing the development of novel drug targets and new treatment strategies.

Nipple aspirate fluid-A liquid biopsy for diagnosing breast health.

PURPOSE: Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopathological features. Our aim, in this pilot study, was to investigate the intra- and interpatient protein composition of nipple secretions and the implications for their use as liquid biopsies. EXPERIMENTAL DESIGN: Matched pairs of nipple discharge/nipple aspirate fluid (NAF, n = 15) were characterized for physicochemical properties and SDS-PAGE. Four pairs were selected for semiquantitative proteomic profiling and trypsin-digested peptides analyzed using 2D-LC Orbitrap Fusion MS. The resulting data were subject to bioinformatics analysis and statistical evaluation for functional significance. RESULTS: A total of 1990 unique proteins were identified many of which are established cancer-associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals were observed. CONCLUSIONS AND CLINICAL RELEVANCE: This was the most complete proteomic study of nipple discharge/nipple aspirate fluid to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort.

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.

Proteomic profiling of nipple aspirate fluid (NAF): Exploring the complementarity of different peptide fractionation strategies.

NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To perform an in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30cm, SCX/RP-10cm, and OFFGEL/RP-10cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30cm approach, while SCX/RP-10cm and OFFGEL/RP-10cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies. BIOLOGICAL SIGNIFICANCE: High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic analysis of NAF samples from breast cancer patients, a precious and volume-limited biological sample. It reinforces the importance of optimizing sample-specific fractionation protocols for information retrieval from mass spectrometric analysis.

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.

Potential correlation between tumor aggressiveness and protein expression patterns of nipple aspirate fluid (NAF) revealed by gel-based proteomic analysis.

Breast cancer is the leading cause of cancer related deaths in women. Most breast cancers stem from mammary ductal cells that secrete nipple aspirate fluid (NAF), a biological sample that contains proteins associated with the tumor microenvironment. In this study, NAF samples from both breasts of 7 Brazilian patients with unilateral breast cancer were analyzed. These samples were systematically compared using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional fluorescence difference gel electrophoresis (2D-DIGE); substantial qualitative individual differences were observed. In general, when NAF samples were compared from both breasts within the same patient their electrophoretic patterns were very similar, regardless of their cancer status. A comparison of all patients identified 2 main NAF protein profiles. The HomEP, homogeneous expression profile, was characterized by typical SDS-PAGE and 2D-DIGE protein patterns that were observed in patients with a good breast cancer prognosis and were similar to previous Type I NAF classifications that used one-dimensional electrophoresis. The HetEP, heterogeneous expression profile, was characterized by distinct protein patterns that have not been reported in previous studies and have been primarily observed in breast cancer patients with a poor prognosis. The NAF samples were rich in metal-dependent proteolytic enzymes, as visualized by SDS-PAGE zymography. They varied qualitatively with respect to their gelatinolytic band distribution. However, there were no correlations between these characteristics and the pathologic features of these tumors. A comparative analysis of NAF samples taken from each breast in a single patient showed conserved zymographic patterns. In conclusion, the present study highlights important distinctions in the protein content of individual NAF samples and provides insight into the composition of the tumor microenvironment. These data reinforce breast cancer as a heterogeneous disease with a diverse natural history, which is becoming increasingly evident through other recent studies.

Cytology in nipple aspirate fluid during a randomized soy food intervention among premenopausal women.

Because soy food consumption may influence breast tissue activity, we examined its effect on the presence of epithelial cells in nipple aspirate fluid (NAF). In a randomized, crossover design, 82 premenopausal women completed a high-soy and a low-soy diet for 6 mo each, separated by a 1-mo washout period. They provided NAF samples at baseline, 6 mo, and 13 mo during the midluteal phase of the menstrual cycle. Papanicolaou-stained cytology slides (for 33 women at baseline, 24 at low-soy, and 36 at high-soy) were evaluated in women with sufficient NAF. Mixed models evaluated the effect of the high-soy diet on epithelial cytology as compared to baseline and the low-soy diet. At the end of the high-soy diet, cytological subclass had decreased in 8 (24%) and increased in 3 (9%) women as compared to baseline, whereas the respective values were 3 (14%) and 6 (29%) for the low-soy diet samples (P = 0.32). Only the change in subclass indicated a trend in lower cytological class (P = 0.06). Contrary to an earlier report, the number of NAF samples with hyperplastic epithelial cells did not increase after a soy intervention in amounts consumed by Asians.

Influence of diet on nipple aspirate fluid production and estrogen levels.

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

The physiology of the normal human breast: an exploratory study.

The physiology of the nonlactating human breast likely plays a key role in factors that contribute to the etiology of breast cancer and other breast conditions. Although there has been extensive research into the physiology of lactation, few reports explore the physiology of the resting mammary gland, including mechanisms by which compounds such as hormones, drugs, and potential carcinogens enter the breast ducts. The purpose of this study was to explore transport of exogenous drugs into ductal fluid in nonlactating women and determine if their concentrations in the fluid are similar to those observed in the breast milk of lactating women. We selected two compounds that have been well characterized during lactation, caffeine and cimetidine. Caffeine passively diffuses into breast milk, but cimetidine is actively transported and concentrated in breast milk. After ingestion of caffeine and cimetidine, 14 nonlactating subjects had blood drawn and underwent ductal lavage at five time points over 12 h to measure drug levels in the fluid and blood. The concentrations of both caffeine and cimetidine in lavage fluid were substantially less than those observed in breast milk. Our results support recent evidence that the cimetidine transporter is not expressed in the nonlactating mammary gland, and highlight intriguing differences in the physiology and molecular transport of the lactating and nonlactating breast. The findings of this exploratory study warrant further exploration into the physiology of the nonlactating mammary gland to elucidate factors involved in disease initiation and progression.

Estrogen levels in nipple aspirate fluid and serum during a randomized soy trial.

BACKGROUND: 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. METHODS: 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. RESULTS: 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). CONCLUSION: Soy foods in amounts consumed by Asians did not significantly modify estrogen levels in NAF and serum. IMPACT: The trend toward lower estrogen levels in NAF during the high-soy diet counters concerns about adverse effects of soy foods on breast cancer risk.

The volume of nipple aspirate fluid is not affected by 6 months of treatment with soy foods in premenopausal women.

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

Carbohydrate antigens in nipple aspirate fluid predict the presence of atypia and cancer in women requiring diagnostic breast biopsy.

BACKGROUND: The goal of this prospective study was to determine (a) concentrations of the carbohydrate biomarkers Thomsen Friedenreich (TF) antigen and its precursor, Tn antigen, in nipple discharge (ND) collected from women requiring biopsy because of a suspicious breast lesion; and (b) if concentration levels predicted pathologic diagnosis. METHODS: Adult women requiring biopsy to exclude breast cancer were enrolled and ND obtained. The samples from 124 women were analyzed using an anti-TF and anti-Tn monoclonal antibodies in direct immunoassay. RESULTS: The highest median concentration in ND for TF and Tn was in women with ductal carcinoma in situ (DCIS). TF was higher in women with 1) cancer (DCIS or invasive) vs. either no cancer (atypia or benign pathology, p = .048), or benign pathology (p = .018); and 2) abnormal (atypia or cancer) versus benign pathology (p = .016); and was more predictive of atypia or cancer in post- compared to premenopausal women. Tn was not predictive of disease. High TF concentration and age were independent predictors of disease, correctly classifying either cancer or abnormal vs. benign pathology 83% of the time in postmenopausal women. CONCLUSIONS: TF concentrations in ND were higher in women with precancer and cancer compared to women with benign disease, and TF was an independent predictor of breast atypia and cancer. TF may prove useful in early breast cancer detection.

Nipple aspirate fluid proteome of healthy females and patients with breast cancer.

BACKGROUND: The ductal/alveolar system of the female breast constantly secretes and reabsorbs fluid in nonpregnant/nonlactating women. This fluid, referred to as nipple aspirate fluid (NAF), can be obtained by a noninvasive procedure and it is part of the microenvironment where more than 95% of breast cancers arise. METHODS: Using an Orbitrap mass analyzer coupled to a linear ion trap, we performed an in-depth proteomic analysis of NAF samples obtained from 3 healthy individuals and 3 patients with breast cancer. Multiple fractionation methods such as size-exclusion and anion-exchange chromatography were applied for protein separation before mass spectrometric analysis. RESULTS: We identified more than 800 unique proteins in total, generating the most extensive NAF proteome thus far. Using gene ontology, we classified the identified proteins by their subcellular localization and found that more than 50% were extracellular or plasma membrane proteins. By searching against the Plasma Proteome Database, we confirmed that 40% of the proteins were also found in the plasma. Unigene database searching for transcripts of the proteins not found in the plasma revealed that the vast majority were expressed in the mammary gland. CONCLUSIONS: Our extensive proteome database for NAF may be helpful in the identification of novel cancer biomarkers.

Sulindac and sulindac metabolites in nipple aspirate fluid and effect on drug targets in a phase I trial.

Regular use of nonsteroidal anti-inflammatory drugs (NSAID) has been associated with reduced risk of breast cancer. Sulindac, a nonselective NSAID with both cyclooxygenase-2-dependent and -independent activities, is a candidate for breast chemoprevention. We conducted a phase Ib trial in 30 women at increased risk for breast cancer to evaluate the breast tissue distribution of sulindac at two dose levels (150 mg daily and 150 mg twice daily for 6 weeks), using nipple aspirate fluid (NAF) as a surrogate of breast tissue drug exposure. We also explored the effect of sulindac on drug-induced biomarkers in NAF. We show that sulindac and its metabolites partition to human breast as measured by NAF levels. Sulindac intervention did not decrease 13,14-dihydro-15-keto prostaglandin A(2), a stable derivative of prostaglandin E(2), in NAF, but exposure was associated with a significant trend towards higher levels of growth differentiation factor 15 in NAF in women receiving 150 mg twice daily (P = 0.038). These results are the first to show partitioning of sulindac and metabolites to human breast tissue and the first evidence for a potential dose-dependent effect of sulindac on growth differentiation factor 15 levels in NAF.

Patterns of sex steroid hormones in nipple aspirate fluid during the menstrual cycle and after menopause in relation to serum concentrations.

Previous studies have shown that progesterone concentrations in serum and nipple aspirate fluid (NAF) are significantly correlated in premenopausal women, but estradiol concentrations are not. We therefore sought to ascertain the patterns of both steroids in NAF throughout the menstrual cycle and in postmenopausal women. Simultaneous samples of blood and NAF were obtained from 40 premenopausal and 16 postmenopausal women. Premenopausal samples were backdated from the following menstrual period. Steroids were purified by high-performance liquid chromatography before quantification by immunoassays. Serum steroids and NAF progesterone followed the expected pattern across the menstrual cycle, with a midcycle peak of estradiol and a midluteal peak of progesterone. However, the estradiol peak in NAF occurred about a week after the serum peak in the midluteal phase, when serum estradiol had declined to less than half the value at midcycle. NAF estrone was also elevated at the midluteal phase. Potential estrogen precursors androstenedione, estrone sulfate, and dehydroepiandrosterone sulfate declined in NAF from midcycle to the midluteal phase as NAF estradiol was increasing. Progesterone concentrations were significantly lower in NAF in postmenopausal women than in premenopausal women, but estrogen concentrations were not. This is the first description of the temporal relationships of sex steroids in NAF and serum relative to the menstrual cycle. These results provide insights into the lack of correlation of NAF and breast tissue estrogens with serum estrogens, and generate new hypotheses.