Nipple Aspirate Research Articles

C-Reactive Protein in Nipple Aspirate Fluid

Proteins expressed in nipple aspirate fluid (NAF) have been associated with breast cancer risk and may serve to detect inflammatory or premalignant states. Obesity, hyperlipidemia, and metabolic syndrome are associated with increased rates of breast cancer and are systemic markers of chronic inflammation. The aim of this study was to determine if one NAF protein, C-reactive protein (CRP), associated elsewhere in the human with cancer, relates to reproductive, nutrition, and body composition and activity factors. Women (n = 59) aged 30 to 64 years from the midwestern United States and Kenya were evaluated regarding the relation between reproductive (age, pregnancy, and breast-feeding indices), nutrition (serum lipids, serum, or NAF carotenoid), body composition and activity (activity, waist-hip ratio, fat percentage (% fat), and body mass index [BMI]) factors and CRP in NAF. Age at first pregnancy (p < .05), gravidity (p < .05), wean time from breast-feeding last baby (p < .05), serum triglycerides (p = .01), % fat (p < .05), and BMI (p < .05) were related significantly to CRP levels in NAF. A model derived from selective women's reproductive, nutritional, and body composition and activity factors significantly (p < .05) accounted for the variance in breast microenvironment inflammation as measured by CRP in NAFs. Detecting CRP in NAF may indicate local mammary inflammation, which has been associated with carcinogenesis. Specific NAF risk modeling that addresses reproductive, nutritional, and body composition and activity factors may be used for further advances in the prevention of breast cancer and the early detection and treatment of the disease.

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.

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

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

A descriptive study of variables associated with obtaining nipple aspirate fluid in a cohort of non-lactating women

BackgroundThe search for biologic endpoints and biomarkers in the study of breast cancer risk assessment and risk reduction strategies has led to an interest in obtaining cytologic information and other biomarkers from nipple aspirate fluid (NAF).MethodsThis descriptive study examined factors associated with an increased ability to obtain NAF in a cohort of 3043 women between the ages of 15 and 89 years of age. The majority of women were between the ages of 30–49 (N = 1529/50.2%). Variables examined in relation to obtaining fluid include: age, marital status, age at menarche, menopausal status, a history of pregnancy, a history of breast-feeding, estrogen use, oral contraceptive use, endocrine disorders and tranquilizer use.ResultsOn average, women from whom breast fluid was obtained were younger than women from whom breast fluid was attempted but not obtained (mean = 41.9 years versus 46.5 years, p < 0.0001). In unadjusted and age-adjusted comparisons, being married, a history of pregnancy, younger age at menarche (12 years of age or younger), tranquilizer use, oral contraceptive pill (OCP) use and endocrine problems were associated with an increased ability to obtain breast fluid. Post-menopausal status and exogenous estrogen use were associated with a decreased ability to obtain breast fluid. After age-adjustment, oral contraceptive use was no longer significantly associated with an increased ability to obtain fluid and post-menopausal status was no longer associated with a decreased ability to obtain breast fluid. After multivariate adjustment, age, being married, a history of pregnancy, tranquilizer use and a history of endocrine problems remained positively associated with the ability to obtain breast fluid. In addition, menopausal women who took estrogen were less likely to yield fluid than premenopausal women.ConclusionFour variables (being married, history of pregnancy, tranquilizer use and endocrine disorders) remained positively associated with the ability to obtain NAF in all analyses. A younger age was consistently associated with a greater ability to obtain NAF in this and other studies.

A strong association between body fat mass and protein profiles in nipple aspirate fluid of healthy premenopausal non-lactating women

Fluid can be aspirated from the nipples of most non-lactating women. This nipple aspirate fluid (NAF) is a potential source for the discovery of new breast cancer biomarkers. NAF has two distinct protein profiles. Type I NAF is similar to the fluid associated with cystic disease of the breast, whereas type II NAF is enriched in milk-associated proteins. The prevalence of these two profiles differs in healthy women and in breast cancer patients. This study investigated the relationship of these two NAF profiles to reproductive history, body composition, diet, and levels of lipids, steroids and thyroid hormones in healthy premenopausal women (age 30-40 years) who had regular menstrual cycles and normal mammograms and were not taking contraceptive medications. On average, women with the type I NAF profile were older, had more years since last childbirth, were less likely to have breastfed their babies and had higher dietary saturated fat intake, body mass index, body fat mass, and levels of plasma low density lipoproteins than women with the type II profile (P <0.05). Using multiple logistic regression, type I NAF was predicted independently (P <0.05) by higher body fat mass [Odds Ratio (OR) = 3.0; 95% Confidence Interval (CI): 1.5-6.1], more years since last childbirth (OR = 2.6; 95% CI: 1.3-5.2) and a higher percentage of calories from saturated fat (OR = 4.1; 95% CI: 1.1-14.6). These results suggest that protein profiles of NAF might be influenced by amounts or types of dietary and body fat, but further study of the relationship of the two profiles to breast cancer risk is needed.

Identification of Biomarkers for Breast Cancer in Nipple Aspiration and Ductal Lavage Fluid

Identification of Biomarkers for Breast Cancer in Nipple Aspiration and Ductal Lavage Fluid

Cytologic and biochemical constituents of nipple aspirate fluid

Nipple aspirate fluid is secreted from breast ductal and acinous epithelial cells.Re- searches find that nipple aspirate fluid contains a variety of cellular and distinctive biochemical com- pounds,even including several important tumor markers which are correlated with early diagnosis and prognosis of breast cancer.

Ductal lavage (DL) of affected and unaffected breasts in women with newly diagnosed breast cancer: Can this technique be used for risk assessment?

1024 Background: Ductal lavage (DL) is designed to obtain cellular material from breast ducts. The use of DL in risk assessment is under investigation. The aim of this study was to evaluate cytologic findings in DL of affected and unaffected breasts in women undergoing definitive surgery for breast cancer. Methods: Women with newly diagnosed breast cancer participated prior to surgery. Women with prior breast cancer or receiving neoadjuvant therapy were excluded. The study was IRB approved; all women gave written informed consent. Women underwent nipple aspiration followed by ductal lavage of fluid-producing ducts for both the affected and unaffected breast in the operating room prior to surgery. Cytology was interpreted as insufficient cells to make a diagnosis (ICMD), benign, mildly or markedly atypical, or malignant. Results: Twenty-three women aged 32–74 years underwent nipple aspiration of both breasts prior to definitive surgery; 1 had bilateral breast cancer. One woman had DCIS, 1 T1a, 3 T1b, 9 T1c, 6 T2, and 4 T3 lesions. Node status was N0 in 13, N1mic in 3, N1 in 5 and N2 in 3 (15 underwent sentinel node evaluation). Five women produced no NAF and therefore did not undergo DL; 18 underwent DL of at least one breast. Cytology samples were available for 30 breasts, 16 affected and 14 unaffected. Two samples contained malignant cells (one from a patient with pre-existing malignant nipple discharge), 3 moderate atypia, all from affected breasts. Benign cytology was found in 20 samples (8 affected breasts) and ICMD was classified in 5 samples (3 affected breasts). Age (&lt;/&gt; 50), tumor size, nodal status, or sentinel node procedure were not associated with NAF production, success or cytology of DL. Conclusion: These findings suggest that DL is not effective in identifying known breast cancer. It brings into question its ability to identify women at increased risk. Future research should focus on molecular markers of risk or other means of tissue retrieval. No significant financial relationships to disclose.

Presence of Epithelial Cells in Nipple Aspirate Fluid is Associated with Subsequent Breast Cancer: a 25-Year Prospective Study

Fluid and epithelial cells obtained from the breasts of non-pregnant, non-lactating women by nipple aspiration, can be used for early diagnosis of breast neoplasms. However, since nipple aspirate fluid (NAF) with cells is obtainable from less than half of women sampled, the question arises: Is this method capable of targeting the women most likely to develop breast cancer? We approached this question with a 25-year prospective study to determine if subjects yielding NAF with or without epithelial cells were more likely to develop breast cancer during the follow-up period than subjects from whom no NAF or epithelial cells were obtained. Logistic regression analysis was used to determine relative risk (RR) with 95% confidence intervals (CI). The follow-up cohort of 972 was representative of the eligible cohort of 1605 for factors related to breast cancer risk and nipple aspiration outcome, and representative of the general population for breast cancer risk. After a mean follow-up period of 25 years, women with epithelial cells in NAF were significantly more likely to develop breast cancer (RR=1.92; CI=1.22-3.01; p<or=0.005), especially invasive breast cancer (RR=2.27; CI=1.27-4.03; p<or=0.005), than women with no NAF, or NAF without epithelial cells. These risks were higher for women<55 years of age at the time of sampling (RR=2.1 for any breast cancer, 2.5 for invasive breast cancer). We conclude that presence of NAF with epithelial cells is associated with subsequent breast cancer risk and may be a useful marker for women at higher risk.

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.

The Intraductal Approach to Breast Cancer Biomarker Discovery

Established methods of breast cancer detection have well-described limitations, and new diagnostic techniques are evolving continually to improve diagnostic accuracy. The intraductal approach encompasses the modalities of nipple aspiration, ductal lavage, and duct endoscopy, and is a means of directly accessing the microenvironment of the breast and either sampling or visualizing this intraductal milieu. The aim of sampling this mammary microenvironment is to obtain samples from the physical surroundings of cells that are undergoing malignant transformation, thereby providing a new method of detection before the development of a clinically or radiologically discernible mass. A literature review was conducted to investigate the evolution of the intraductal approach and its particular application in the field of biomarker discovery, primarily using the intraductal technique of nipple aspiration, in combination with emerging protein profiling techniques.

The clinical applications of mammary ductoscopy

Background Mammary ductoscopy (MD) is a newly developed endoscopic technique that allows direct visualization and biopsy examination of the mammary ductal epithelium where most cancers originate. The procedure can be performed under local anesthesia in the office setting. This article reviews the rationale, current clinical applications, and limitations of MD. Methods A literature search was performed using Pubmed for indexed articles published over the past 30 years using the key words “mammary ductoscopy,” “breast ductoscopy,” “ductal lavage,” and “nipple aspiration.” The most important articles were analyzed and discussed. Results MD is a useful diagnostic adjunct in patients with pathologic nipple discharge. Furthermore, it can reduce the number and extent of duct excision surgeries for pathologic nipple discharge. There is a clear need to design prospective clinical trials that evaluate the potential role of MD in breast cancer screening, guiding risk-reducing strategies, and as an adjunct to breast-conservation surgery. Conclusions MD is useful in the management of PND, but its potential role in the early detection or management of breast cancer requires further investigation.

Correlation of nipple aspiration and ductal lavage cytology with histopathologic findings for patients before scheduled breast biopsy examination

Background The use of ductal lavage to obtain cells from within the breast ducts has been described for patients at high risk for breast cancer. The purpose of this study was to correlate ductal lavage cytologic findings with the corresponding histology. Methods Twenty-two patients were evaluated and 20 patients underwent ductal lavage followed by breast biopsy examination as a result of positive nipple aspiration fluid. Ductal lavage samples were classified by a cytopathologist as negative, mild atypia, marked atypia, or malignant. A different pathologist interpreted the histologic findings of the biopsy examination. Results Adequate specimens for cellularity were obtained in 12 of 22 (53%) patients: 6 of the 12 (50%) had both benign cytology and histology, 2 (16.7%) had benign cytology with atypical ductal hyperplasia or atypical lobular hyperplasia on histology, 2 (16.7%) had marked atypia on cytology and benign histology, and 2 (16.7%) had malignant cytology and benign histology. The specificity of the procedure was 83.4%. Conclusions Ductal lavage yielding an adequate sample for analysis was successful in only 52% of patients. Of those, the cytologic–histologic correlation was discordant in 50%. The role of ductal lavage in accurately predicting lesions present on subsequent breast histologic evaluation of planned biopsy examinations requires further investigation.

Estrogen and Progesterone Levels in Nipple Aspirate Fluid of Healthy Premenopausal Women: Relationship to Steroid Precursors and Response Proteins

Concentrations of estrogen and progesterone within the breast could provide a better reflection of breast cancer risk than levels in the circulation. We developed highly sensitive immunoassays for multiple steroid hormones and proteins in the nipple aspirate fluid (NAF), which can be obtained noninvasively with a simple suction device. Previous studies showed that NAF hormone levels are strongly correlated between breasts and within a single breast over time and are predictably related to hormone replacement therapy or use of oral contraceptives. This study evaluates the relationship of NAF estrogen and progesterone levels to those in serum and saliva, the relationship of NAF estradiol to androgenic and estrogenic precursors in NAF, and the relationship of NAF hormone levels to those of response proteins such as cathepsin D and epidermal growth factor (EGF). Normal premenopausal women collected saliva daily and donated blood and NAF in the midluteal phases of menstrual cycles at intervals of 0, 4, 12, and 15 months. Analytes were measured by immunoassays after solvent fractionation. Log-transformed values were fit to repeated measures analysis of covariance models to ascertain associations between analytes. Small nonsignificant associations were found between NAF and serum or salivary estradiol. However, progesterone in NAF was significantly associated with progesterone in serum and saliva (R=0.18 and 0.32, respectively). Within NAF, the estradiol precursors estrone sulfate, androstenedione, and dehydroepiandrosterone were significantly associated with estradiol concentration (P<0.06), and a multiprecursor model explained the majority of variance in NAF estradiol (model R(2)=0.83). Cathepsin D and EGF in NAF could not be predicted from serum or salivary steroid measurements; however, both could be predicted from estradiol and its precursors in NAF (model R(2)=0.70 and 0.93, respectively). By showing consistent associations between estradiol and its precursors and response proteins, these data provide support for the biological validity of NAF hormone measurements and for the importance of steroid interconversion by aromatase and sulfatase within the breast. The low correlation between estrogen levels in NAF and those in serum or saliva suggests that the degree of association between estrogen or its androgen precursor levels and risk of breast cancer observed in epidemiologic studies using serum estimates might be highly attenuated.

Nipple aspirate fluid color is associated with breast cancer

Background: Fluid can be non-invasively aspirated from the breast nipple (nipple aspirate fluid, NAF). NAF may have many colors, including clear, white, yellow, green, and red/brown. While bloody spontaneous nipple discharge has been linked with breast cancer, the association of NAF color with cancer is not established. Our hypothesis was that red/brown NAF color was associated with breast cancer. The purpose of this study was to assess (1) if red/brown NAF is associated with the presence and progression of breast cancer, (2) the influence of prior needle or surgical biopsy on NAF color, and (3) if an association between NAF color and breast cancer was found, to develop a cancer predictive model including NAF color and cytology, and clinical information. Methods: Specimens were obtained from 848 breasts between 1999 and 2004 after subjects enrolled in an IRB approved protocol to evaluate biologic markers of breast cancer. Cytologic evaluation was performed on Papanicolaou-stained cytospin preparations of NAF. Results: Red/brown NAF was associated with breast cancer when considering all samples ( p < 0.001) and samples from women who did not undergo recent surgery ( p = 0.005). Needle biopsy did not, but surgical biopsy did influence NAF color. For the 327 women with NAF collected from both breasts, there was a significant association between red/brown NAF color and the presence of breast cancer ( p = 0.005). Red/brown NAF was more common in breasts with ductal carcinoma in situ than atypical hyperplasia ( p = 0.008). The optimal model, included NAF color, cytology, and age, was 92% sensitive and 61% specific in predicting if a woman had breast cancer. Conclusions: NAF color was associated with the presence of breast cancer and the progression from precancer to cancer in a population of women who presented to a breast cancer evaluation clinic. NAF color contributed to a highly predictive breast cancer detection model. Additional studies are warranted to determine the usefulness of NAF color in the assessment of women who present for breast cancer evaluation.

Intra- and inter-individual variability in measurements of biomarkers for oxidative damage in vivo: Nutrition and Breast Health Study

Oxidative stress has been implicated in the pathogenesis of various chronic diseases, such as cancer, cardiovascular disease and inflammatory conditions, as well as in ageing. Although a number of markers are now available, little is known about the reliability of single measurements of such markers in healthy individuals. The study examined the distribution of variance for three oxidative stress markers, 8-oxo-2′-deoxyguanosine (8-oxodG), 5-hydroxymethyl-2′-deoxyuridine (5-OHmdU) and total 8-isoprostane-F2α, which were measured every 3–6 months over 1 year in blood and breast nipple aspirate fluid (NAF) for 103 premenopausal women. For both plasma and NAF, the between-subject variances of 8-isoprostane-F2α were consistently greater than the within-subject variances. Consequently, their reliability coefficients were close to the level of those for cholesterol. On the other hand, the within-subject variances were much greater than the between-subjects variances for blood 5-OHmdU, resulting in low reliability coefficients, i.e. <0.3. Overall, the reliability coefficients for blood 8-oxodG were between those of 8-isoprostane-F2α and 5-OHmdU, but closer to those of 8-isoprostane-F2α. The results suggest that the reliability of oxidative stress markers may vary considerably depending on the type of marker. Caution should be exercised in selecting markers as well as in determining the number of study subjects or the number of samples per subject in a study. There also may be ample room to optimize laboratory techniques to quantify markers of oxidative DNA damage.

Comparison of cytomorphology in specimens obtained by random periareolar fine needle aspiration and ductal lavage from women at high risk for development of breast cancer

Ductal lavage (DL) and random periareolar fine needle aspiration (RPFNA) are both being used to harvest epithelial cells for risk assessment as well as response evaluation in chemoprevention trials. The magnitude of increase in relative risk has been defined in a prospective study for RPFNA but not for DL atypia. We attempted both procedures in 26 women at high risk for development of breast cancer. Median age was 43 (range 32-57); 15 women were premenopausal, with 6 of the postmenopausal women on HRT. Collection of nipple aspirate fluid (NAF) was attempted and, if successful, was followed by DL; RPFNA was then performed on all women. Both procedures were attempted the same day (follicular phase of menstrual cycle if premenopausal) in 24 subjects and within three months for two subjects. Twenty-three subjects produced NAF, 17 of the 23 (74%) had a successful duct cannulation as part of the DL procedure, with 16 yielding sufficient (10) ductal cells for morphologic assessment. Twenty-five of 26 (96%) subjects had a successful RPFNA procedure with adequate cellularity for morphology. There was concordance between DL and RPFNA specimens for traditional cytologic category assessment in 10/16 (63%), Masood index score in 13/16 (82%), and Consensus Panel assessment in 12/16 (75%) of specimens. We conclude that same day DL and RPFNA is feasible, with 62% and 96% of high-risk women having a successful procedure with evaluable cytomorphology. RPFNA was more likely to yield an evaluable specimen, but if a cellular DL specimen was obtained, morphology was generally similar.

Non-steroidal anti-inflammatory drug (NSAID) use and levels of a lipid oxidation marker in plasma and nipple aspirate fluids

Non-steroidal anti-inflammatory drugs (NSAIDs) are thought to reduce cancer risk by inhibiting cyclo-oxygenases, resulting in deceased formation of inflammatory mediators and oxidative stress. We examined whether the level of one oxidative stress marker, 15-F(2t)-isoprostane, was affected by NSAID use in plasma and breast nipple aspirate fluids (NAF) of pre-menopausal women who were participating in a dietary intervention trial (n=121). Baseline levels of 15-F(2t)-isoprostane were lower in NSAID users than non-users in both NAF and plasma, although the differences did not persist after intervention. Over the duration of the study, information on NSAID use was collected five times, and average 15-F(2t)-isoprostane levels in both NAF and plasma exhibited a statistically significant trend for decreases with increased frequency of NSAID use. These results indicate that NSAID use can result in lower levels of 15-F(2t)-isoprostane, which may have implications for the effects of NSAID use on breast cancer risk.

Identification of Biomarkers for Breast Cancer in Nipple Aspiration and Ductal Lavage Fluid

To establish a comprehensive proteomic approach for biomarker discovery and validation in breast fluid. A total of 95 specimens from three institutions were used including 10 nipple aspiration fluid (5 stage I/II cancerous breasts and 5 age-matched healthy controls), 42 ductal lavage fluid from 14 patients with unilateral stage I/II cancer (25 from 9 cancerous breasts and 17 from 7 contralateral breasts), and 42 ductal lavage fluid from 14 high-risk women (multiple ducts repeated lavage). Differentially expressed protein/peptides were discovered by proteomic analysis of training sample, using ProteinChip arrays and surface-enhanced laser desorption ionization (SELDI) time-of-flight mass spectrometry, and validated on independently collected testing samples. After protein identification, ELISA was done to confirm the SELDI findings. We were able to obtain reproducible protein profiles using minimal amount of protein (1 mug) by applying an optimized chip protocol and SELDI. We were able to select cancer-associated biomarkers despite large individual variability by applying both unsupervised and supervised cluster analysis. Furthermore, we were able to train and test candidate biomarkers on independently collected samples and identified one component of a multimarker panel as human neutrophil peptides 1 to 3. Breast fluid is a rich source of breast cancer biomarkers. In combination with high-throughput novel proteomic profiling technology and multicenter study design, markers that are highly specific to breast cancer can be discovered and validated. Our observations also suggest that persistent elevation of human neutrophil peptide in high-risk women may imply early onset of cancer not yet detectable by current detection method. Proof of this hypothesis requires follow-up on a larger study population.

Nutritional predictors for cellular nipple aspirate fluid: Nutrition and Breast Health Study

The presence of epithelial cells in breast nipple aspirate fluid (NAF), irrespective of abnormality, has been associated with increased risk of breast cancer in previous studies. We sought to investigate whether the presence of epithelial cells in NAF is associated with nutritional parameters among 71 healthy premenopausal women who participated in the Nutrition and Breast Health Study and provided any samples of NAF during the study. Total of 142 samples which were obtained over a 1-year period of intervention with low-fat and/or high vegetable-fruit diets were available for cytological evaluation. The odds ratios (ORs) and 95% confidence intervals (CIs) for the detection of epithelial cells in NAF were estimated by fitting generalized estimating equations models by quartile level of nutritional parameters. The probability of yielding epithelial cell-positive NAF progressively increased with increasing total fat intake (p=0.001). The OR for the highest quartile level of fat intake, compared with lowest, was 7.22 (95% CI 1.14-45.82). On the other hand, there were a marginally significant inverse association with total fiber intake as well as an weak inverse association with the number of servings of fruit and vegetables. Furthermore, the probability of detecting epithelial cells in NAF decreased with increasing plasma levels of lutein and alpha-carotene (p-values for linear trend; 0.001 and 0.049, respectively). The ORs for the highest versus lowest quartile levels are 0.17 (95% CI 0.04-0.65) and 0.19 (95% CI 0.04-0.91), respectively. These results are generally in support of roles of nutritional factors in breast cancer and thus further studies are warranted.