Nipple Aspirate Research Articles

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.

Aluminium, carbonyls and cytokines in human nipple aspirate fluids: Possible relationship between inflammation, oxidative stress and breast cancer microenvironment

The human breast is likely exposed to Al (aluminium) from many sources including diet and personal care products. Underarm applications of aluminium salt-based antiperspirant provide a possible long-term source of exposure, especially after underarm applications to shaved and abraded skin. Al research in breast fluids likely reflects the intraductal microenvironment. We found increased levels of aluminium in noninvasively collected nipple aspirate fluids (NAF) from 19 breast cancer patients compared with 16 healthy control subjects (268 vs 131μg/l, respectively; p<0.0001). In the same NAF samples we found significantly increased levels of protein oxidative carbonyls in cancer patients compared to healthy women (2.35 vs 0.41nmol/mg prot, respectively; p<0.0001). Aluminium content and carbonyl levels showed a significant positive linear correlation (r2 0.6628, p<0.0001). In cancer NAF samples (containing higher amounts of aluminium salts) we also found a significantly increased levels of pro-inflammatory cytokines (IL-1β, IL-6, IL-12 p70, and TNF-α) and chemoattractant CC and CXC chemokines (IL-8, MIP-1α and MCP-1). In 12 invasive cancer NAF samples we found a significant positive linear correlation among aluminium, carbonyls and pro-inflammatory IL-6 cytokine (Y=64.79x−39.63, r2 0.8192, p<0.0005), as well as pro-inflammatory monocyte chemoattractant MCP-1 cytokine (Y=2026x−866, r2 0.9495, p<0.0001). In addition to emerging evidence, our results support the possible involvement of aluminium ions in oxidative and inflammatory status perturbations of breast cancer microenvironment, suggesting aluminium accumulation in breast microenvironment as a possible risk factor for oxidative/inflammatory phenotype of breast cells.

Can Breast Nipple Fluid Collected with Automated Aspiration and Preserved in Based-Liquid Solution Improve the Cytological Samples?

Objective: Samples from breast nipples collected with the automated HALO™ Mamo Cito Test were studied in order to evaluate the cellularity for the diagnosis of breast cancer. Study Design: One hundred and fifty-nine asymptomatic women were prospectively examined. Women younger than 18 years, pregnant or lactating women, and women with a history of breast cancer, with previous radio- or chemotherapy and with nipple piercing were excluded from the study. Nipple samples from both breasts were collected. Results: In 107 (34.96%) of the 306 samples of nipple discharge analyzed by optical microscopy, adequate cellularity was observed after the HALO procedure. Cytological findings, previously categorized according to the National Health Service Breast Screening Program (NHSBSP), were grouped as unsatisfactory, benign, suspected for malignancy, and malignant. Of the cellular breast samples, 97.19% (104/107) were classified as benign, and 2.81% (3/107) were classified as suspicious for malignancy. In 199 cases there were samples without cells (65.03%). Most of the cells observed were easily recognized as well-preserved and adequately stained macrophages. None of the women reported discomfort with the nipple aspiration procedure. Conclusions: The results discussed here encouraged us to use samples collected automatically in routine procedures.

Abstract 3459: Metabolomic profiling reveals significant differences between the ductal fluid from cancerous compared to unaffected breasts.

Abstract Introduction: There is significant need to develop new methods to improve breast cancer (BC) screening and early detection. Metabolomic analysis of breast ductal fluid is an exciting opportunity for improving the detection of BC at an early stage. We hypothesize that differences between metabolomic profiles of tumor cells compared to normal cells can be used to develop an assay for early detection of BC. Breast ductal fluid can be obtained in a non-invasive way from the breast via nipple aspiration or ductal lavage. Approach: Metabolomic profiles were evaluated in ductal lavage fluid samples obtained from 54 patients with breast tumors (DCIS and Invasive Breast Cancer). The samples were collected in the operating room from patients with unilateral disease and confirmed diagnosis, prior to their surgery. For each patient, two samples were obtained: one from the affected breast and the other from the contralateral normal breast (control). Each patient served as her own control. Metabolite extraction was performed using a sequential solvent extraction protocol. Metabolomic analysis was performed using UPLC-TOF MS analysis and the data were acquired in duplicate (two technical replicates) for each sample. We used the XCMS package (Scripps Center for Metabolomics, La Jolla, CA) for peak detection, peak matching, and correction of retention time shift. This is followed by ion annotation to identify derivative ions originating from the same compound in the forms of adduct, isotopes, and in-source fragments. We performed statistical analysis to identify ions with significant difference between the tumor and normal samples. To correct for multiple testing, we used q-values based on Storey's pFDR method. Significant ions with q-value &amp;lt; 0.1 were selected. In addition, we calculated fold change (FC) based on the median of the relative intensity of tumor/normal samples for each pair. A subset of the significant ions with |FC| &amp;gt; 1.5 was selected. Putative identifications of the selected ions were obtained through mass-based search against four databases: the Human Metabolite DataBase (HMDB), Metlin, Madison Metabolomics Consortium Database (MMCD), and LIPID MAPS. Results: We analyzed the differences between normal and tumor samples excluding the subjects who received preoperative treatment (n=8). Based on statistical significance and FC, we selected 39 ions. Verification of putative IDs for these metabolites is underway. Conclusion: Our metabolomics analysis of the breast ductal fluid reveals significant differences between cancerous and normal breasts. These differences may be used to develop an assay that may improve early detection of BC. Supported by a Grant from the Avon Foundation for Women Citation Format: Luisa Matos do Canto, Shawna C. Willey, Elizabeth D. Feldman, Amrita Cheema, Mohammad R. Nezami Ranjbar, Habtom W. Ressom, Bassem R. Haddad. Metabolomic profiling reveals significant differences between the ductal fluid from cancerous compared to unaffected breasts. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3459. doi:10.1158/1538-7445.AM2013-3459

Expression of epidermal growth factor, transforming growth factor-β1 and adiponectin in nipple aspirate fluid and plasma of pre and post-menopausal women.

BackgroundNipple aspirate fluid (NAF) contains large amounts of protein thought to reflect the microenvironment of the breast, and is of interest in breast cancer prevention research. The correlation between specific NAF proteins to plasma concentrations have not been well studied in healthy women. We collected matched NAF and plasma from 43 healthy pre and postmenopausal women participating in an early phase clinical study to compare the levels of putative cancer protein biomarkers. We compared baseline NAF and plasma levels of epidermal growth factor (EGF), transforming growth factor-beta 1 (TGF-β1), and adiponectin and evaluated menopausal status and body mass index (BMI) as potential modifying factors.FindingsNAF and plasma levels of EGF, TGF-β1 and adiponectin were not correlated. EGF and TGF-β1 levels in NAF of premenopausal women were significantly higher than postmenopausal women (P’s < 0.01). These differences by menopausal status were not observed in plasma. Both NAF and plasma adiponectin levels were non-significantly higher in postmenopausal women. NAF biomarker levels were not associated with BMI whereas plasma EGF, TGF-β1 and adiponectin levels in postmenopausal women were all inversely correlated with BMI (P’s < 0.05).ConclusionsProtein biomarkers differ significantly between NAF and plasma and are affected differently by both BMI and menopausal status. This study demonstrates important differences in biological information gained by characterizing biomarkers in NAF compared to plasma and suggests each sample source may independently inform on breast cancer risk.

Abstract 2524: Cytology in nipple aspirate fluid during a randomized soy food intervention among premenopausal women.

Abstract Background. Epidemiologic studies support the hypothesis that soy foods protect against breast cancer, in particular among Asian populations with high intake. A previous report that observed an increase in the number of women with hyperplasic epithelial cells after consuming soy raised concerns that isoflavones in soy may elevate breast cancer risk due to their estrogen-like structure. We examined the effect of a high-soy diet on the presence of epithelial cells in nipple aspirate fluid (NAF), a non-invasive method to obtain breast fluid and cells. Methods. In a crossover design, 82 premenopausal women completed a randomized intervention with a high-soy diet of 2 servings/day and a low-soy diet with &amp;lt;3 servings/week for 6 months each separated by a 1-month washout period. Adherence to the intervention strategy was excellent as assessed by urinary isoflavonoid excretion and by 7 unannounced 24-hour dietary recalls. NAF samples were obtained at baseline, after 6 months, and after 13 months. Papanicolaou-stained cytology slides were evaluated in women with sufficient NAF and classified as inadequate (&amp;lt;10) mammary epithelial cells (class I), benign mammary epithelial cells (class II), atypical mammary epithelial cells (class III), and malignant cells present (class IV). Mixed models were applied to examine the effect of the high-soy diet on epithelial cytology as compared to baseline and the low-soy diet.Results. The mean NAF volumes for women with and without cytology specimens differed significantly; they were 58±49 vs. 19±16 μL at baseline, 59±37 vs. 13±14 μL at the end of the high-soy diet period, and 51±40 vs. 10±9 μL at the end of the low-soy diet period. At baseline, 33 women produced sufficient NAF for cytological evaluation, 36 women at the end of the high-soy diet, and 24 women at the end of the low-soy diet. For 22 (67%) participants, no change in cytological class was seen at the end of the high-soy diet and for 12 (57%) at the end of the low-soy diet, but a decrease from baseline to the end of the high-soy diet occurred in 8 (24%) and an increase in 3 (9%) women. In comparison, a decrease in 3 (14%) and an increase in 6 (29%) women was seen at the end of the low-soy diet. Although not statistically significant (p=0.05), the high-soy diet was associated with a trend in lower cytological class. Conclusions. Contrary to an earlier report, the number of NAF samples with hyperplastic epithelial cells did not increase after an intervention with soy foods in amounts consumed by Asians. Citation Format: Gertraud Maskarinec, Shana Suzuki, Yukiko Morimoto, Ian Pagano, Adrian A. Franke, Hormoz Ehya. Cytology in nipple aspirate fluid during a randomized soy food intervention among premenopausal women. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2524. doi:10.1158/1538-7445.AM2013-2524

Aberrantly Methylated DNA as a Biomarker in Breast Cancer

Aberrant DNA hypermethylation at gene promoters is a frequent event in human breast cancer. Recent genome-wide studies have identified hundreds of genes that exhibit differential methylation between breast cancer cells and normal breast tissue. Due to the tumor-specific nature of DNA hypermethylation events, their use as tumor biomarkers is usually not hampered by analytical signals from normal cells, which is a general problem for existing protein tumor markers used for clinical assessment of breast cancer. There is accumulating evidence that DNA-methylation changes in breast cancer patients occur early during tumorigenesis. This may open up for effective screening, and analysis of blood or nipple aspirate may later help in diagnosing breast cancer. As a more detailed molecular characterization of different types of breast cancer becomes available, the ability to divide patients into subgroups based on DNA biomarkers may improve prognosis. Serial monitoring of DNA-methylation markers in blood during treatment may be useful, particularly when the cancer burden is below the detection level for standard imaging techniques. Overall, aberrant DNA methylation has a great potential as a versatile biomarker tool for screening, diagnosis, prognosis and monitoring of breast cancer. Standardization of methods and biomarker panels will be required to fully exploit this clinical potential.

A phase I safety and efficacy study of the mammary aspirate specimen cytology test device for collection of spe-cimens for exfoliative cytopathology of the breast ducts

Background: The ability to identify asymptomatic women at high risk for breast cancer using known pre-malignant changes in exfoliative cytopathology of nipple aspirate fluid is of clinical importance. Exfoliative cytopathology of Nipple Aspirate Fluid (NAF) has been shown to be an important adjunct to the currently accepted standard of medical care, i.e. mammography, coupled with physical examination, for the diagnosis of breast cancer. This is especially important for the subset of women aged 18-50 who are not identified as “high risk”, and therefore, for whom mammography is not routinely recommended. The objective of this study was to determine if a new, patented Class II medical device, the Mammary Aspirate Specimen Cytology Test (MASCT) System, designed to collect NAF for subsequent cytological examination is safe and effective. Methods: The MASCT medical device is a modified breast pump and was used to obtain bilateral specimens from 34 healthy, non-pregnant, female subjects for cytopathological examination. A conventional breast disease work-up was performed (medical history/risk factor collection, clinical breast examination and mammogram) and NAF specimens collected. Specimen weight was measured and a cytopathological examination was performed. Vital signs measurements, clinical laboratory analysis, and adverse event reporting were performed. Results: Based on cytopathological evaluation and/or measurable weight changes on the specimen collection membrane filter, all breasts evaluated (100%) yielded nipple aspirate fluid. Specimen weights ranged from in 60 breasts (representing 30 subjects) being evaluated cytologically. Fifty-eight of sixty breasts evaluated cytopathologically (97%) were reported as cytology Class I, and 2 of 60 (3%) were reported as cytology Class IIa. Cytopathological findings correlated well with mammogram and clinical breast exam results. No adverse events, including pain from the collection procedure, were reported. Conclusion: Based on this clinical study, we conclude that the Mammary Aspiration Specimen Cytology Test device is safe and effective for the collection of mammary aspirate specimens for laboratory cytopatho-logical testing.

Paired Ductal CarcinomaIn Situand Invasive Breast Cancer Lesions in the D-Loop of the Mitochondrial Genome Indicate a Cancerization Field Effect

Alterations in the mitochondrial genome have been chronicled in most solid tumors, including breast cancer. The intent of this paper is to compare and document somatic mitochondrial D-loop mutations in paired samples of ductal carcinoma in situ (DCIS) and invasive breast cancer (IBC) indicating a potential breast ductal epithelial cancerization field effect. Paired samples of these histopathologies were laser-captured microdissected (LCM) from biopsy, lumpectomy, and mastectomy tissues. Blood samples were collected as germplasm control references. For each patient, hypervariable region 1 (HV1) in the D-loop portion of the mitochondrial genome (mtGenome) was sequenced for all 3 clinical samples. Specific parallel somatic heteroplasmic alterations between these histopathologies, particularly at sites 16189, 16223, 16224, 16270, and 16291, suggest the presence of an epithelial, mitochondrial cancerization field effect. These results indicate that further characterization of the mutational pathway of DCIS and IBC may help establish the invasive potential of DCIS. Moreover, this paper indicates that biofluids with low cellularity, such as nipple aspirate fluid and/or ductal lavage, warrant further investigation as early and minimally invasive detection mediums of a cancerization field effect within breast tissue.

Breast cancer biomarkers: proteomic discovery and translation to clinically relevant assays

Although the molecular classification and prognostic assessment of breast tumors based on gene expression profiling is well established, a number of proteomic studies that propose potential breast cancer biomarkers has not yet led to any new diagnostic, prognostic or predictive test in wide clinical use. This review examines the current status of breast cancer biomarkers, discusses sample types (including plasma, tumor tissue, nipple aspirate and ductal lavage, as well as cell culture models) and different electrophoretic and mass spectrometry methods that have been widely used for the discovery of proteomic biomarkers in breast cancer, and also considers several approaches to biomarker validation. The pathway leading from the initial proteomic discovery and validation process to translation into a clinically useful test is also discussed.

The Human Mammary Gland as a Target for Isoflavones: How Does the Relation Vary in Individuals with Different Ethnicity?

Based on observational studies, it appears that soy food consumption provides protection against breast cancer primarily in Asian but not in Western populations. Given the problems in examining the effects of isoflavones directly in the human mammary gland, this review describes epidemiologic studies that investigated the association with biomarkers reflecting hormonal activity of isoflavones, in particular sex steroid levels, mammographic densities, nipple aspirate fluid, and tissue specimens from biopsies or surgeries. Three possible mechanisms that may be responsible for ethnic-specific health effects from these compounds are discussed: genetic variation in metabolic enzymes, timing of exposure, and intestinal metabolism by microbiota. Only a limited number of comparative studies and even fewer nutritional interventions have examined effects and addressed differences in biomarkers between Asian and Western populations. Investigations that looked at estrogens and mammographic densities as endpoints observed some associations in Asian women that were not seen in Caucasians. On the other hand, the low rate of nipple aspirate fluid production and a lack of breast tissue studies make it impossible to evaluate effects of isoflavones on these biomarkers in Asian women. Based on the current evidence, it appears likely that the timing of exposure is the most important determinant of beneficial health effects from soy foods. This may be the result of gut microbiota, which colonize the intestine during childhood and facilitates the hydrolysis of glycosides and the formation of equol from dadzein, a pathway that may result in beneficial health effects. The current evidence is insufficient to answer the question whether women of diverse ethnic groups experience distinct effects from soy isoflavones in breast tissue, but as knowledge about the role of early life nutrition and the development of gut microbiota increases, the potential for diverse metabolic pathways of isoflavones in individuals with different ethnic backgrounds and dietary exposures may be clarified.

The Use of Halo Breast Test (Halo Mamo Cito Test) to Improve Cytology Examination of Breast Nipple Aspirate Samples Reserved in Surepath Medium

The Use of Halo Breast Test (Halo Mamo Cito Test) to Improve Cytology Examination of Breast Nipple Aspirate Samples Reserved in Surepath Medium