Nipple Aspirate Fluid Research Articles

Abstract P5-06-02: Proliferative breast disease identified by nipple aspirate fluid cytopathology has the laterality and asymmetry characteristics of breast cancer, supporting the thesis it is a cancer precursor

Abstract Introduction. Within the basket of conditions known as Benign Breast Disease, Proliferative Breast Disease (PBD) is a finding at biopsy or aspiration of either benign hyperplasia or atypical hyperplasia (ADH). PBD is known to confer increased risk of future breast cancer [Hartmann, NEJM 2015;372:78-89]. However, there is some controversy as to whether these lesions are precursors to breast cancer (BCa), in which their synchronicity and asymmetry should match BCa, or alternatively, if they are biomarkers of risk, in which future cancers would not necessarily be linked in time and space to earlier PBD lesions. The Left-dominant asymmetry was first described over half a century ago, but its molecular origins in embryonic development have only recently been characterized.[Wilting. J.Current Medic Chem 2011; 18:5519-27]. This study was conducted to explore if a similar pattern of asymmetry and unilateral v. bilateral incidence might exist in PBD. This could help to resolve the question of whether PBD is a precursor to BCa or merely a biomarker. Methods. We examined Nipple Aspirate Fluid (NAF) for evidence of proliferative cytopathology, defined as a finding of either hyperplasia or ADH. NAF was collected using either one of two models of aspirator devices (ForeCYTE™ or HALO™). NAF is aspirated, dispersed on a flower filter, sprayed with fixative and stained with the Pap stain prior to cytologic characterization. Filters mounted whole on glass are placed directly under the microscope. This method allows for a diagnostically interpretable specimen in NAF-droplets as little as 0.007 µL, 1000-fold smaller than the unassisted visual detection limit. A central lab evaluated all specimens. Results. Between 1/2012 and 6/2013, 1154 women without prior BCa (Age range 18-85; median 48, mean 47.9) consented to submit bilateral NAF specimens; 99.7% of breasts yielded NAF adequate for analysis, evidenced by presence of a duct-selective protein assay on the filter. PBD was found in 149 women, of whom 24 bilateral. The distribution is shown in the table below. Distribution of benign and atypical hyperplasia among 149 women with PBD Left-onlyRight onlyBilateralCCR 4594218CCR 51684CCR4 (L) and CCR5 (R)--2Total Women755024King Classification: CCR4 = Benign hyperplasia; CCR5 - Atypical Hyperplasia (ADH) Benign hyperplasia was found in 121 women: 10.5% of those tested. It was bilateral in 13% of cases and left-sided only in 57%. ADH was found in 30 women or 2.6% of those tested. When present, ADH was bilateral in 13% (4/30), L-sided only 53% (16/30) and R-sided only 33% (10/30) of the time. Total PBD identified by NAF cytopathology occurred in 13% (149/1154) of this population, was bilateral in 16.1% (24/149), L-only in 50.3% (75/149)and R-only in 33.5% 50/149). The excess left-sided occurrence of 17% was significant (p=0.048 by chi-square). Conclusion. This is the first report describing laterality and symmetry of PBD as made by non-invasive NAF collection and demonstrates that the pattern observed is similar to what is seen with invasive breast cancer and in situ lesions, supporting the hypothesis that PBD are precursor lesions to breast cancer. Citation Format: Kylstra JW, Kalnoski MH, Vo T, Lee ML, Chen S-C, Quay SC. Proliferative breast disease identified by nipple aspirate fluid cytopathology has the laterality and asymmetry characteristics of breast cancer, supporting the thesis it is a cancer precursor. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-06-02.

Abstract B50: Differential influence of vitamin D on the tumor promoting eicosanoid PGE2 in women at increased breast cancer risk

Abstract Numerous lines of evidence point to the tumor promoting properties of prostaglandin (PG)E2. Preclinical studies have demonstrated the ability of both vitamin D (vitD) and celecoxib to downregulate the eicosanoid. We previously reported that vitD, but not the combination, significantly downregulated PGE2 in normal risk women. We hypothesized that there would be a similar or greater benefit of vitD alone, or in combination with celecoxib, in women at increased breast cancer risk. 40 women at increased breast cancer risk were prospectively enrolled and randomized in double blind fashion for one month (postmenopausal) or one menstrual cycle (premenopausal) to one of four daily treatment regimens: placebo, 400 IU vitD, 2000 IU vitD, or 2000 IU vitD + 400 mg celecoxib. Breast nipple aspirate fluid (NAF) and serum were collected. Both breast specific and systemic levels of PGE2 were measured. There was a significant increase (p=.02) in circulating levels of vitD after treatment in women receiving 2000 IU vitD with or without celecoxib, but not in women receiving placebo or 400 IU vitD. Celecoxib levels increased in those treated with celecoxib but not in the other groups. In the breast, as reflected in NAF, there was a 20% increase (p=.07) in PGE2 after treatment with 2000 IU vitD, with little change after treatment in the other groups. No treatment significantly changed circulating levels of PGE2. These findings suggest that, unlike in normal risk women, 2000 IU vitD may increase levels of a key cancer promoting enzyme in women of increased breast cancer risk. Supported in part with funds from the Komen Foundation and the Department of Defense. Citation Format: Wenyi Qin, Charles Walker, Edward Sauter. Differential influence of vitamin D on the tumor promoting eicosanoid PGE2 in women at increased breast cancer risk. [abstract]. In: Proceedings of the Thirteenth Annual AACR International Conference on Frontiers in Cancer Prevention Research; 2014 Sep 27-Oct 1; New Orleans, LA. Philadelphia (PA): AACR; Can Prev Res 2015;8(10 Suppl): Abstract nr B50.

Nipple Aspirate Fluid (NAF) cytology supports prediction of breast cancer risk using the IBIS model

Nipple Aspirate Fluid (NAF) cytology supports prediction of breast cancer risk using the IBIS model

Is nipple aspirate fluid (NAF) a reliable source to identify biomarkers in breast cancer? A feasibility study.

e22101 Background: Elevated expression of MUC1 and Cyclin B1 has been reported in malignant breast tissue and is associated with more aggressive tumor characteristics. We hypothesized that in NAF, ...

Nipple Aspirate Fluid Cytology in the Office-Based Screening for Breast Cancer Risk [333

Nipple Aspirate Fluid Cytology in the Office-Based Screening for Breast Cancer Risk [333

Protein identification from dried nipple aspirate fluid on Guthrie cards using mass spectrometry.

Nipple aspirate fluid (NAF) requires investigation as a potential source of biomarkers for early diagnosis or risk assessment in breast cancer and other breast disorders. The present study demonstrated that proteins were easily extracted from dried NAF spots on Guthrie cards and were suitable for mass spectrometry analysis. NAF was obtained from 80 women, collected on Guthrie cards, between 2007 and 2010. The NAF-proteins were extracted from the card by incubating the card in water. These proteins were then quantified and separated using one-dimensional, 12% SDS-PAGE, gel electrophoresis and on high-resolution gradient gels at different concentrations (4–12, 8–16 and 4–20%). The bands with the most abundant proteins were excised from the gradient gels and the proteins were identified by liquid chromatography quadrupole time of flight. Immunoglobulins, Zn-α2-glicoprotein, apoliprotein D and prolactin inducible protein were among those identified. The NAF-Guthrie card collection method has not been applied previously, however, NAF proteins have been identified using other collecting techniques, confirming the feasibility of the NAF Guthrie card collection method for analyzing the proteins within NAF. The NAF-Guthrie card collecting method has multiple advantages, including being inexpensive, non-invasive, reliable and painless, and the cards can be stored at room temperature. Examining NAF may assist in identifying individuals at a higher risk of breast cancer and in improving patient prognosis.

Anti-MUC1 Antibody in Nipple Aspirate Fluids Correlates with Tumor Aggressiveness in Breast Cancer: A Feasibility Study

Antibodies against MUC1 are found in circulation of breast cancer (BC) patients. We hypothesized that anti-MUC1 antibodies might be present in even a higher concentration in nipple aspirate fluid (NAF) and could be used to predict aggressiveness of BC. Serum and NAF samples were collected from high risk lesions, BC, and healthy contralateral breasts. ELISA was used to measure the amount of IgG, IgM, and IgA against a tumor-specific MUC1 peptide derived from the extracellular tandem repeat domain of MUC1. Tumor characteristics were recorded prospectively; 120 NAF samples were obtained from a total of 77 women in the study. There was no significant difference of anti-MUC1 antibody levels compared to BC with other lesions. Anti-MUC1 IgG level in NAF was higher in triple negative tumors (P = 0.02); serum anti-MUC1 IgG levels were significantly higher in patients with ER (−) tumor and recurrent disease (P = 0.01); NAF anti-MUC1 IgA levels were significantly higher in patients with LVI and Her2-neu (+) tumors (P < 0.05). These results show that NAF could be a reliable biomarker to predict tumor aggressiveness in BC. A larger study will be needed to confirm these data and to investigate the potential of anti-MUC1 antibodies in NAF and serum to predict disease outcome.

Development of a novel approach for breast cancer prediction and early detection using minimally invasive procedures and molecular analysis: how cytomorphology became a breast cancer risk predictor.

With enhanced public awareness, advances in breast imaging, and emphasis on early breast cancer detection and prevention, more women are seeking consultation to assess the status of their breast health. Risk assessment has become an integral part of established multi-disciplinary breast care, and breast cancer risk reduction interventions have received a great deal of attention. Similarly, interest in identification of high-risk individuals has increased significantly. Atypical proliferative changes in breast epithelial cells are ranked high among various known breast cancer risk factors and, in recent years, have been the subject of several investigations. Breast tissue and fluid in the ductal system provide a rich source of cells and biomarkers that have the potential to aid in the assessment of short-term risk of breast cancer development, and assess responses to interventional prevention efforts. There are three minimally invasive procedures currently being utilized to sample breast tissue in asymptomatic high-risk individuals. These procedures are: fine-needle aspiration biopsy, nipple aspiration fluid, and ductal lavage. In this review article, the merits and limitations of each procedure are presented, and the contribution of cytomorphology and molecular analysis in breast cancer prediction is highlighted. In addition, the role of Masood Cytology Index as a surrogate endpoint biomarker in chemopreventative trials is discussed.

Biological sample collection for clinical proteomics: existing SOPs.

Proteomic study of clinical samples aims at the better understanding of physiological and pathological conditions, as well as the discovery of diagnostic and prognostic markers for the latter. Quantitative and/or qualitative variations of body fluid proteome may reflect health- or disease-associated events connected to the adjacent or distant body regions of the fluid production/secretion/excretion and/or systemic reactions to the presence of disease. Sample collection and preparation is a critical step in order to obtain useful and valid information in clinical proteomics analysis. In this chapter, we present the current protocols and guidelines for human body fluid collection and storage, prior to proteomic analysis. A variety of body fluids that are currently being used in proteomic analysis and have potential interest in clinical practice are presented including blood plasma and serum, urine, cerebrospinal fluid, cerumen, nasal secretions, saliva, tears, breast milk, bronchoalveolar fluid, nipple aspirate fluid, amniotic fluid, bile, cervico-vaginal fluid, and seminal plasma. With no doubt these body fluids differ in the extent of their application in clinical proteomics investigations, hence in some cases the presented SOPs are established following more extensive testing (e.g., plasma, serum, urine, CSF) than others (nasal secretions, saliva, tears, breast milk, bronchoalveolar fluid, nipple aspirate fluid, amniotic fluid, bile, cervico-vaginal fluid, and seminal plasma). However, even in these latter cases, the presented protocols were reported by at least two independent groups according to the literature. We hope they can thus serve as a reliable guide for sample collection based on our current knowledge in the field and excellent starting points for proteomics investigators. It should also be pointed that variations to these protocols exist and their further refinement in the future is foreseen following the evolution of the proteomics technologies.

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.

Circulating estrogens and estrogens within the breast among postmenopausal BRCA1/2 mutation carriers.

Accurately quantifying parent estrogens (PE) estrone (E1) and estradiol (E2) and their metabolites (EM) within breast tissue and serum may permit detailed investigations of their contributions to breast carcinogenesis among BRCA1/2 mutation carriers. We conducted a study of PE/EM in serum, nipple aspirate fluid (NAF), and ductal lavage supernatant (DLS) among postmenopausal BRCA1/2 mutation carriers. PE/EM (conjugated and unconjugated) were measured in paired serum/NAF (n = 22 women) and paired serum/DLS samples (n = 24 women) using quantitative liquid chromatography-tandem mass spectrometry (LC/MS/MS). The relationships between serum and tissue-specific PE/EM were measured using Pearson's correlation coefficients. Conjugated forms of PE/EM constituted the majority of estrogen in serum (88 %), NAF (59 %) and DLS (69 %). PE/EM in NAF and serum were highly correlated [E1 (r = 0.97, p < 0.0001), E2 (r = 0.90, p < 0.0001) and estriol (E3) (r = 0.74, p < 0.0001)] as they were in DLS and serum [E1 (r = 0.92, p < 0.0001; E2 (r = 0.70, p = 0.0001; E3 (r = 0.67, p = 0.0004)]. Analyses of paired total estrogen values for NAF and serum, and DLS and serum yielded ratios of 0.22 (95 % CI 0.19-0.25) and 0.28 (95 % CI 0.24-0.32), respectively. This report is the first to employ LC/MS/MS to quantify PE/EM in novel breast tissue-derived biospecimens (i.e., NAF and DLS). We demonstrate that circulating PE and EM are strongly and positively correlated with tissue-specific PE and EM measured in NAF and DLS among postmenopausal BRCA1/2 mutation carriers. If confirmed, future etiologic studies could utilize the more readily obtainable serum hormone levels as a reliable surrogate measure of exposure at the tissue level.

Metabolomic characterization of nipple aspirate fluid by (1)H NMR spectroscopy and GC-MS.

Nipple aspirate fluid (NAF) is a noninvasively obtained biofluid from the duct openings of the breast. NAF components are constantly secreted, metabolized, and reabsorbed by the epithelial lining of the lactiferous ducts of the breast. NAF has been studied as a potential breast tissue surrogate for the discovery of novel breast cancer risk, early detection, and treatment response biomarkers. We report the first unsupervised metabolite characterization of nipple aspirate fluid using NMR and GC-MS using convenience samples previously collected from four premenopausal and four postmenopausal women. A total of 38 metabolites were identified using the two analytical techniques, including amino acids, organic acids, fatty acids, and carbohydrates. Analytical reproducibility of metabolites in NAF by GC-MS was high across different extraction and analysis days. Overall, 31 metabolites had a coefficient of variation below 20%. By GC-MS, there were eight metabolites unique to NAF, 19 unique to plasma, and 24 shared metabolites. Correlative analysis of shared metabolites between matched NAF and plasma samples from pre- and postmenopausal women shows almost no correlations, with the exception being lactic acid, which was significantly negatively correlated (R(2) = 0.57; P = 0.03). These results suggest that NAF is metabolically distinct from plasma and that the application of metabolomic strategies may be useful for future studies investigating breast cancer risk and intervention response biomarkers.

Early Diagnosis of Canine Mammary Tumours Using Nipple Aspirate Fluid Cytology and Fine Needle Aspiration Cytological Techniques

In the present study clinical evaluation of canine mammary tumour cases to the Small Animal Surgical Out Patient Ward of the Madras Veterinary College Teaching Hospital, Department of Clinics, Chennai – 600 007 between September 2005 and June 2006 was done. During the study period of 10 months, out of 100 dogs screened for mammary gland involvement, 88 showed mammary neoplasms and 12 were nonneoplastic. . NAF (Nipple aspirate fluid) was attempted in 651 glands of 83 dogs, of which, 346 glands of 72 dogs yielded NAF. Out of 346 glands, 224 were asymptomatic NAF yielders were mostly 6 10 years (44.44 per cent). Pure breeds (73.61 per cent) yielded NAF mostly and 94.44 per cent were intact. There was 30.63 per cent yield from the inguinal glands. NAF was mostly creamy white, dirty, watery and sticky. Cellularity was moderate (52.89 per cent). Immunostained NAF smears revealed high epithelial:macrophage ratio in malignant type. Fine needle aspiration biopsy (FNAB) was taken from 144 glands of 101 dogs including one necropsy case, 93 were malignant and 14 were benign tumours. NAF findings correlated well with FNAB and histopathology in the diagnosis of canine mammary tumours (88.89 per cent).

Abstract P3-07-11: Nipple aspiration fluid yielders and non-yielders: Genetic characteristics

Abstract Background: Nipple aspiration fluid (NAF) is a noninvasively acquired biosample that can provide a window of observation into the breast environment, but NAF yield is variable across studies, and is related to a variety of demographic and reproductive factors. We have recently observed a positive correlation between serum prolactin level and NAF yield status. The genetic traits determining NAF yield are unknown, but data from the 1970s linked wet type ear wax to NAF yield, and recent studies point to single nucleotide polymorphisms (SNPs) in the ABCC11 gene as a determinant of earwax type. Further, prolactin and prolactin receptor SNPs are related to serum prolactin level. We have investigated associations between NAF yield and SNPs in prolactin, prolactin receptor and ABCC11 genes in a recently completed case-control study. Method: NAF and/or blood were collected from 916 women. Subjects were defined as yielder if NAF volume was equal or more than 2μL and non-yielder if it was less than 2μL. According to this definition, our subjects categorized as 557 yielders and 359 non-yielders. DNA was extracted from blood using Qiagen kit and 20 ng of DNA was used to amplify and genotype for ABCC11 (rs17822931), PRLR (rs37364), PRL (rs2244502), PRL (rs849872) SNPs using Taqman genotyping assay on an Applied Biosystems 7900HT machine. Results: The median age of Yielders and Non-yielders was 51 and 54, respectively, with the same range of 31-70 for both groups (P&amp;lt;0.0001). There were 209(38%) premenopausal, 257(46%) postmenopausal and 81(15%) perimenopausal of yielders vs. 99(28%) premenopausal, 229(64%) postmenopausal and 26(7%) perimenopausal of non-yielders (P&amp;lt;0.0001). ABCC11 genotype distribution was significantly different between yielders and non-yielders (P = 0.00163). However, genotype distribution was not significantly different for PRLR (rs37364), PRL (rs2244502) and PRL (rs849872). YNYTotalP (N vs. NY)ABCC11 (rs17822931)5413578980.00163CC437 (81%)269 (75%)706 TT5 (1%)16 (4%)21 CT99 (18%)72 (20%)171 PRLR (rs37364)5483599070.176GG89 (16%)43 (12%)132 TT272 (50%)181 (50%)453 GT187 (34%)135 (38%)322 PRL (rs2244502)5393588970.087AA73 (14%)32 (9%)105 TT233 (43%)155 (43%)388 AT233 (43%)171 (48%)404 PRL (rs849872)5433599020.102CC17 (3%)11 (3%)28 TT332 (61%)244 (68%)576 CT194 (36%)104 (29%)298 Conclusion: Our analysis demonstrates a correlation between ABCC11 (rs17822931) SNP and NAF yield status, with the major homozygous CC allele associated with NAF yielder and minor homozygous TT allele associated with non-yielder status. If NAF-based risk biomarkers are to be developed, a better understanding of the genetics of NAF yield is required. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-07-11.

Hormonal Determinants of Nipple Aspirate Fluid Yield among Breast Cancer Cases and Screening Controls

Nipple aspiration fluid (NAF) use as a biosample is limited by the variable yield across studies. We investigated the endocrine determinants of yield in an ongoing breast cancer case-control study. One-hundred and eighteen women yielding ≥2 μL NAF and 120 non-yielders were included; serum hormones were measured; differences in median hormones were assessed using the Wilcoxon rank-sum test. ORs and 95% confidence intervals (95% CI) for yielder status relative to hormone levels were estimated using logistic regression, adjusting for parity and lactation, and, in premenopausal women, menstrual cycle phase (MCP). Prolactin concentrations were higher in yielders than non-yielders (premenopausal: 7.6 and 2.5 ng/mL, P < 0.01; postmenopausal 5.3 and 2.2 ng/mL; P < 0.01). Among premenopausal-yielders, estradiol was lower (64.3 vs. 90.5 pg/mL, MCP-adjusted P = 0.02). In separate menopausal status and parity-adjusted models, significant case-control differences persisted in prolactin: case OR 1.93 (95% CI, 1.35-2.77), control OR 1.64 (95% CI, 1.17-2.29). Premenopausal control yielders had higher progesterone (OR, 1.70; 95% CI, 1.18-2.46) and sex-hormone binding-globulin (OR, 2.09; 95% CI, 1.08-4.05) than non-yielders. Among parous women, further adjustment for lactation suggested a stronger positive association of serum prolactin with yield in cases than controls. NAF-yielders show higher prolactin than non-yielders, regardless of menopause and parity; implications of this and other endocrine differences on NAF biomarkers of breast cancer risk deserve further study. NAF yield is associated with a distinct endocrine environment that must be considered in studies of NAF-based breast cancer risk markers.

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.

Analytical Validation of the ForeCYTE Breast Health Test

Introduction: The ForeCYTE Breast Health Test provides a 10-year and lifetime breast cancer risk stratification using family history of breast and ovarian cancer, personal reproductive history, and cytological evaluation of exfoliated cells present in nipple aspirate fluid (NAF) in the risk stratification algorithm developed by TyrerCuzick. Materials and Methods: Specimens of NAF are collected on membrane filters with one of two FDA-cleared biopsy instruments, the Halo or the Mammary Aspiration Specimen Cytology Test (MASCT) System devices, are fixed upon collection by spraying the membrane filters with a 0.1 mL mist of Saccomanno's fixative. The specimen filters are processed in situ for NAF protein using a colloidal gold assay with a limit of detection of 0.1 ng of protein (corresponding to 1 pL of NAF) and a linear range to 1,000 ng. The filters are then stained with a modified Papanicolaou stain and morphological criteria applied to establish a diagnosis. Results: Spiked MCF-7 breast cancer cells demonstrate quantitative recovery (94.7%) in the range of 1-10 cancer cells/membrane filter with a sensitivity for identifying a cellular specimen of 100% at 4-100 cells/membrane and 75% at 2 cells/membrane and 50% at 1 cell /membrane. If hyperplasia is noted and morphology is inadequate

Resolving breast cancer heterogeneity by searching reliable protein cancer biomarkers in the breast fluid secretome.

BackgroundOne of the major goals in cancer research is to find and evaluate the early presence of biomarkers in human fluids and tissues. To resolve the complex cell heterogeneity of a tumor mass, it will be useful to characterize the intricate biomolecular composition of tumor microenvironment (the so called cancer secretome), validating secreted proteins as early biomarkers of cancer initiation and progression. This approach is not broadly applicable because of the paucity of well validated and FDA-approved biomarkers and because most of the candidate biomarkers are mainly organ-specific rather than tumor-specific. For these reasons, there is an urgent need to identify and validate a panel of biomarker combinations for early detection of human tumors. This is especially important for breast cancer, the cancer spread most worldwide among women. It is well known that patients with early diagnosed breast cancer live longer, require less extensive treatment and fare better than patients with more aggressive and/or advanced disease.ResultsIn the frame of searching breast cancer biomarkers (especially using nipple aspirate fluid mirroring breast microenvironment), studies have highlighted an optimal combination of well-known biomarkers: uPA + PAI-1 + TF. When individually investigated they did not show perfect accuracy in predicting the presence of breast cancer, whereas the triple combination has been demonstrated to be highly predictive of pre-cancer and/or cancerous conditions, approaching 97-100% accuracy.ConclusionDespite the heterogeneous composition of breast cancer and the difficulties to find specific breast cancer biomolecules, the noninvasive analysis of the nipple aspirate fluid secretome may significantly improve the discovery of promising biomarkers, helping also the differentiation among benign and invasive breast diseases, opening new frontiers in early oncoproteomics.

Effect of aluminium on migratory and invasive properties of MCF-7 human breast cancer cells in culture

Aluminium (Al) has been measured in human breast tissue, nipple aspirate fluid and breast cyst fluid, and recent studies have shown that at tissue concentrations, aluminium can induce DNA damage and suspension growth in human breast epithelial cells. This paper demonstrates for the first time that exposure to aluminium can also increase migratory and invasive properties of MCF-7 human breast cancer cells. Long-term (32weeks) but not short-term (1week) exposure of MCF-7 cells to 10−4M aluminium chloride or 10−4M aluminium chlorohydrate increased motility of the cells as measured by live cell imaging (cumulative length moved by individual cells), by a wound healing assay and by migration in real time through 8μm pores of a membrane using xCELLigence technology. Long-term exposure (37weeks) to 10−4M aluminium chloride or 10−4M aluminium chlorohydrate also increased the ability of MCF-7 cells to invade through a matrigel layer as measured in real time using the xCELLigence system. Although molecular mechanisms remain to be characterized, the ability of aluminium salts to increase migratory and invasive properties of MCF-7 cells suggests that the presence of aluminium in the human breast could influence metastatic processes. This is important because mortality from breast cancer arises mainly from tumour spread rather than from the presence of a primary tumour in the breast.