Analysis Of Nipple Aspirate Fluid Research Articles

Mass spectral analysis of nipple aspirate fluid for the early detection of breast cancer

Introduction: Methods for early breast cancer detection enable lesions to be treated at the earliest possible time-point, increasing survival and improving patient outcomes. Nipple aspirate fluid (NAF) produced by the lining of the ductal epithelial cells in asymptomatic women has been shown to contain substances which are potential targets for biomarkers of early cancer detection.

P125: Mass spectral analysis of nipple aspirate fluid - A potential for early breast cancer detection?

Introduction: Nipple aspirate fluid (NAF), produced by the lining of ductal epithelial cells has been observed to contain substances such as lipids and proteins, which may provide biological targets for early cancer detection. Such targets can be evaluated using liquid chromatography mass spectrometry (LCMS), a novel analytical chemistry technique that separates components of fluids according to their mass. Aim to assess the feasibility of testing NAF using LCMS metabolic analysis.

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

BackgroundWorldwide, 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.MethodsThe 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.ResultsA 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.ConclusionsWe 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.

Detection of Superoxide Dismutase-1 in Nipple Aspirate Fluids: A Reactive Oxygen Species—Regulating Enzyme in the Breast Cancer Microenvironment

Introduction Analysis of nipple aspirate fluid (NAF) allows the noninvasive identification of both cellular and biomolecular markers of human breast cancer, the most common female malignancy in developed countries. Cytosolic superoxide dismutase (SOD-1) represents a detoxifying enzyme able to regulate the balance between oncogenic and oncosuppressor reactive oxygen species. Patients and Methods We analyzed SOD-1 expression in 126 NAF samples collected from 67 women with and 59 without breast cancer, using enzyme-linked immunosorbent assay, immunoprecipitation, Western blotting, and immunocytochemistry. Results SOD-1 median values in plasma presented no difference between the no-cancer and cancer subgroups. No significant difference in the median level of SOD-1 between matched plasma and NAF from cancer patients was found, whereas SOD-1 median level in no-cancer NAF was significantly higher when compared with matched plasma. Finally, the SOD-1 median value in no-cancer NAF was significantly higher (about 2-fold) than in cancer NAF. Conclusion NAF measurement of SOD-1 is a useful tool to identify intracellular redox status of the breast microenvironment, mirroring the oxidative metabolic pathways occurring in breast tissue, both in physiologic and cancer conditions and in improving the identification of women at increased breast cancer risk. SOD-1 activity in the breast microenvironment may represent a functional "switch" between the detrimental/oncogenic properties and the oncosuppressor/proapoptotic role of this antioxidant enzyme.

Abstract 4574: Mining the proteome of breast cancer cell lines and nipple aspirate fluid in the quest for novel breast cancer biomarkers

Abstract Approximately 200,000 new cases of breast cancer are estimated in the United States for 2009, rendering breast cancer the most frequently diagnosed cancer in women. Patients diagnosed with early stage disease have significantly improved survival rates compared to late stage patients, underlining the need for identification of biomarkers for early detection. Breast cancer is highly heterogeneous and it can be categorized into five subtypes with distinct clinical outcome and different treatment modalities. In this study the secretome of breast cancer cell lines and nipple aspirate fluid (NAF) were analyzed by tandem mass spectrometry to identify novel breast cancer biomarkers. To reflect disease heterogeneity, three cell lines for each of three breast cancer types [estrogen (ER)/ progesterone (PR) receptor positive, triple negative, HER2/neu amplified] were selected (HCC-1428, BT483, MCF-7, MDA-MB-231, HCC-1143, HCC-38, SK-BR-3, HCC-202, UACC-812). NAF samples were obtained from 3 patients with ER positive breast cancer. Proteins of NAF samples and conditioned media of the cell lines were denatured, reduced and trypsin-digested. The peptides were separated by two-dimensional liquid chromatography and the fractions were analyzed in a linear ion-trap coupled to an orbitrap mass analyser. Spectra were searched with Mascot and X!Tandem engines using the IPI 3.46 human database. Scaffold software was used to cross-validate Mascot and X!Tandem results. Spectra were exported from Scaffold and uploaded into an in-house-program for further data analysis. Over 1,000 unique proteins were identified in the conditioned media of each cell line, resulting in more than 4,000 proteins from the 9 breast cancer cell lines. Additionally, 780 proteins were identified in the three NAF samples generating the most extensive NAF cancer proteome so far. Using an in-house program, we annotated the cellular localization and the biological function for each protein. Proteins identified in the three cell lines of each subtype were combined to generate non-redundant, subtype-specific proteomes. The proteomes of different subtypes were then compared, to distinguish proteins that may reveal subtype-specific signatures. The comparison between the ER-positive cancer cell line secretome and the NAF proteome revealed 400 common proteins which were selected for further investigation. A set of selection criteria were applied to generate a panel of the 30 most promising candidates for ER-positive breast cancer. Multiple reaction monitoring (MRM) assays for each of these proteins are being developed to verify their utility as potential biomarkers in serum. In conclusion, proteomic analysis of NAF and tissue culture supernatants of breast cancer cell lines holds promise for breast cancer-specific biomarker discovery. 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 4574.

Intracrinology of breast microenvironment: hormonal status in nipple aspirate fluid and its relationship to breast cancer

Breast cancer, a complex and multifactorial disease, is the most commonly diagnosed malignancy affecting women. Methods currently available for breast cancer detection have well-described limitations; in this respect, the intraductal approaches directly assess the microenvironment of the breast. Nipple aspirate fluid (NAF) can be noninvasively obtained from the breast in most women and represents a promising biological tool to assess metabolic, hormonal and molecular changes occurring in the cells lining the ducts, from which breast cancer arises. The aim of this review is to highlight the application of NAF studies in the field of biomarker discovery, which provide results useful for early detection and prevention of breast cancer risk; in fact, the analysis of NAF (mirroring the ductal–lobular microenvironment) is a reliable method for assessment of metabolic/hormonal pathways within the mammary gland, identifying biomolecular mechanisms of breast cancer initiation and progression. The intracrinology of breast microenvironment (i.e., hormonal status in NAF) may provide independent diagnostic/prognostic factors, highlighting the importance of early altered hormonal metabolism (e.g., aromatase, estrogen sulfotransferase and steroid sulfatase pathway) in relation to breast cancer initiation. The possible application of targeted therapies through the inhibition of intratumoral enzymes involved in steroid metabolism is also discussed. The intraductal approach to hormone analyses may provide a further panel of biomarkers providing clinical benefits and strengthening the armory against breast cancer.

Protein profile ana lysis of the breast microenvironment to differentiate healthy women from breast cancer patients

Protein and proteomic high-throughput technologies provide the polypeptide signatures of nipple aspirate fluid (NAF), a breast secretion collected noninvasively from healthy individuals and cancer patients. As breast cancer develops from ductal–lobular epithelium, the analysis of NAF (mirroring the ductal–lobular microenvironment) is a useful tool for the analysis of metabolic pathways within the mammary gland, deepening our knowledge of the biomolecular mechanisms of breast cancer initiation and progression. The different protein expression of major NAF proteins, separated using 1D polyacrylamide gels, has proven valuable for the early detection of women with increased risk of cancer. The failure to recognize a single marker with sufficient clinical sensitivity and/or specificity has driven the identification of breast cancer multiple proteins by 2D electrophoresis. Mass spectrometry-based proteomic approaches (SELDI- and MALDI-TOF technologies) have allowed the characterization of differential NAF proteomic fingerprints between healthy individuals and breast cancer patients. The intraductal approach of protein and proteomic analyses may provide a panel of biomarkers to strengthen the armory against breast cancer.

Proteomic Analysis of Breast Tissue and Nipple Aspirate Fluid for Breast Cancer Detection

Clinical proteomics has great promise, but many hurdles. Proteomic approaches may prove to be the answer to decades of frustrating searches for markers that are specific and selective for breast cancer. In order to achieve this goal, the challenge arises that breast cancer is a heterogeneous disease and individual variations are enormous. To identify sensitive and specific breast cancer markers will require standardization of every method, from tissue collection through statistical analysis and diligence. Small pilot studies of tumors with varied characteristics will not suffice; large, homogeneous sample sets will be the most efficient route to identify breast cancer markers. Proteomic analysis of nipple aspirate fluid may identify markers that can be used for screening with this noninvasive technique. Proteomic analysis of breast biopsies will allow more detailed characterization of the individual's cancer, with therapy tailored to the disease based on this information.

Proteomic analysis of nipple aspirate fluid throughout the menstrual cycle in healthy pre-menopausal women

A proteomic approach to nipple aspiration fluid (NAF) has been used in a number of studies comparing women with breast cancer and healthy women. However, to make useful comparisons between women with breast cancer and healthy women it is necessary to establish whether there is physiological variation in the proteomic profiles of NAF. The purpose of this study was, for the first time, to examine how the proteomic profile of NAF using surface-enhanced laser desorption ionisation time-of-flight mass spectrometry varies across the menstrual cycle in healthy pre-menopausal women. Twelve women were recruited and nipple aspiration was carried out weekly from both breasts of each subject for two menstrual cycles. Matching serum samples for luteinising hormone, follicle stimulating hormone and oestradiol were obtained at each aspiration attempt. Statistically significant peaks were found for three healthy volunteers (p < 0.05). However, the peaks that varied across the menstrual cycle were different from one healthy volunteer to another and the differences were small compared with the large variation in proteomic profiles between healthy volunteers. This study provides proof of concept that the NAF proteomic profile does not vary substantially during the menstrual cycle and that therefore it is valid to compare NAF profiles from pre-menopausal women that have been taken at different stages in the menstrual cycle.

Proteomic analysis of nipple aspirate fluid from women with early-stage breast cancer using isotope-coded affinity tags and tandem mass spectrometry reveals differential expression of vitamin D binding protein

BackgroundIsotope-coded affinity tag (ICAT) tandem mass spectrometry (MS) allows for qualitative and quantitative analysis of paired protein samples. We sought to determine whether ICAT technology could quantify and identify differential expression of tumor-specific proteins in nipple aspirate fluid (NAF) from the tumor-bearing and contralateral disease-free breasts of patients with unilateral early-stage breast cancer.MethodsPaired NAF samples from 18 women with stage I or II unilateral invasive breast carcinoma and 4 healthy volunteers were analyzed using ICAT labeling, sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), liquid chromatography, and MS. Proteins were identified by sequence database analysis. Western blot analysis of NAF from an independent sample set from 12 women (8 with early-stage breast cancer and 4 healthy volunteers) was also performed.Results353 peptides were identified from tandem mass spectra and matched to peptide sequences in the National Center for Biotechnology Information database. Equal numbers of peptides were up- versus down-regulated. Alpha2HS-glycoprotein [Heavy:Light (H:L) ratio 0.63] was underexpressed in NAF from tumor-bearing breasts, while lipophilin B (H:L ratio 1.42), beta-globin (H:L ratio 1.98), hemopexin (H:L ratio 1.73), and vitamin D-binding protein precursor (H:L ratio 1.82) were overexpressed. Western blot analysis of pooled samples of NAF from healthy volunteers versus NAF from women with breast cancer confirmed the overexpression of vitamin D-binding protein in tumor-bearing breasts.ConclusionICAT tandem MS was able to identify and quantify differences in specific protein expression between NAF samples from tumor-bearing and disease-free breasts. Proteomic screening techniques using ICAT and NAF may be used to find markers for diagnosis of breast cancer.

Analysis of nipple aspirate fluid for diagnosis of breast cancer: an alternative to invasive biopsy

Over 40,000 women in the USA will die this year of breast cancer. Current generally accepted techniques to detect breast cancer are limited to breast examination and mammography. Abnormalities found by these techniques require an invasive needle or surgical biopsy to determine if cancer is present. The author’s ultimate goal is to determine if a woman has breast cancer without the need for invasive biopsies, and do this before the abnormality is detectable by standard screening techniques. Herein, the technology is reviewed as it was, as it is today, and its future potential is discussed.

Influence of Lactation History on Breast Nipple Aspirate Fluid Yields and Fluid Composition

Analysis of nipple aspirate fluid (NAF) can be useful for understanding the impact that various lifestyle factors have on the biology of the breast. In this study, breast NAF was obtained at baseline from premenopausal women who volunteered for a dietary intervention trial. The influence of lactation history on both fluid yields and fluid composition was explored. We examined the levels of fat-soluble micronutrients (tocopherols, carotenoids, retinol), one lipid oxidation product (8-isoprostane), cholesterol, and protein in NAF. Roughly half of the women in the trial had never lactated, but this did not affect fluid yields appreciably. Carotenoid and tocopherol levels were significantly higher in NAF from women who lactated 6 months or more versus women who had lactated for shorter periods of time or never, but 8-isoprostane, protein, and cholesterol levels were not affected appreciably by lifetime lactation history. Longer times after weaning were associated with higher cholesterol levels, and there also was a suggestion the fat-soluble micronutrients declined with time after weaning. This is of interest since high cholesterol levels in breast fluid have been associated with an increased breast cancer risk, while carotenoids and tocopherols are thought to be protective. The results of this study provide further evidence of the potential benefits of prolonged lactation via its influence on NAF composition.

Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer.

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 μl of diluted nipple aspirate fluid over a 5–40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75–84% of samples from women with cancer but in only 0–9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer.British Journal of Cancer (2002) 86, 1440–1443. DOI: 10.1038/sj/bjc/6600285 www.bjcancer.com© 2002 Cancer Research UK