Nipple Aspirate Fluid Research Articles

Non-Invasive Biomarkers for Early Detection of Breast Cancer.

Simple SummaryEarly diagnosis of breast cancer greatly increases the chance of cure and survival from the disease. The mammogram is widely used for early detection of breast cancer, but its effectiveness and accuracy have been a concern for a long time as well as its inability in detecting small cancers, especially in women with dense breast tissues. Therefore, it is an unmet clinical need to develop a simple, convenient test to overcome the shortcomings of mammography. Liquid biopsy, which is based on the analysis of body fluids, has attracted much attention in the search for cancer biomarkers. Recent advances in analytical techniques have gradually made it possible to detect breast cancer early through a biomarker analysis of blood, nipple aspirate fluid, sweat, urine, tears, or the breath. We envision that a simple blood or breath test holds great promise as a biomarker for early detection of breast cancer in the near future.Breast cancer is the most common cancer in women worldwide. Accurate early diagnosis of breast cancer is critical in the management of the disease. Although mammogram screening has been widely used for breast cancer screening, high false-positive and false-negative rates and radiation from mammography have always been a concern. Over the last 20 years, the emergence of “omics” strategies has resulted in significant advances in the search for non-invasive biomarkers for breast cancer diagnosis at an early stage. Circulating carcinoma antigens, circulating tumor cells, circulating cell-free tumor nucleic acids (DNA or RNA), circulating microRNAs, and circulating extracellular vesicles in the peripheral blood, nipple aspirate fluid, sweat, urine, and tears, as well as volatile organic compounds in the breath, have emerged as potential non-invasive diagnostic biomarkers to supplement current clinical approaches to earlier detection of breast cancer. In this review, we summarize the current progress of research in these areas.

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

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

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

Background: MicroRNAs (miRNAs) target 60% of human messenger RNAs and can be detected in tissues and biofluids without loss of stability during sample processing, making them highly appraised upcoming biomarkers for evaluation of disease. However, reporting of the abundantly expressed miRNAs in healthy samples is often surpassed. Here, we characterized for the first time the physiological miRNA landscape in a biofluid of the healthy breast: nipple aspirate fluid (NAF), and compared NAF miRNA expression patterns with publically available miRNA expression profiles of healthy breast tissue, breast milk, plasma and serum. Methods: MiRNA RT-qPCR profiling of NAF (n=41) and serum (n=23) samples from two healthy female cohorts was performed using the TaqMan OpenArray Human Advanced MicroRNA 754-Panel. MiRNA quantification data based on non-targeted or multi-targeted profiling techniques for breast tissue, breast milk, plasma and serum was retrieved from literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1-50, combined into an overall ranking per sample type and compared. Findings: NAF expressed 11 unique miRNAs and shared 21/50 miRNAs with breast tissue. Seven miRNAs were shared between the five sample types. Overlap between sample types varied between 42-62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis. Interpretation: This is the first study to characterize and compare the unique physiological NAF-derived miRNA landscape with the physiological expression pattern in breast tissue, breast milk, plasma and serum. Breast-specific sources did not mutually overlap more than with systemic sources. Given their established role in carcinogenesis, NAF miRNA assessment could be a valuable tool in breast tumor diagnostics. Funding: This work was supported by the Dutch Cancer Society (KWF) under grant number UU2016-8191. This was previously A Sister’s Hope grant with grant number 2012.WO38.C142. Declaration of Interests: The authors declare no potential conflicts of interest. Ethics Approval Statement: These studies were approved by the Institutional Review Boards of the UMC Utrecht and other participating hospitals and the UMC Utrecht Biobank Research Ethics Committee (TCBio). Informed consent was obtained from all participating women.

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

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

Disease Detection with Molecular Biomarkers: From Chemistry of Body Fluids to Nature-Inspired Chemical Sensors.

This article aims to review nature-inspired chemical sensors for enabling fast, relatively inexpensive, and minimally (or non-) invasive diagnostics and follow-up of the health conditions. It can be achieved via monitoring of biomarkers and volatile biomarkers, that are excreted from one or combination of body fluids (breath, sweat, saliva, urine, seminal fluid, nipple aspirate fluid, tears, stool, blood, interstitial fluid, and cerebrospinal fluid). The first part of the review gives an updated compilation of the biomarkers linked with specific sickness and/or sampling origin. The other part of the review provides a didactic examination of the concepts and approaches related to the emerging chemistries, sensing materials, and transduction techniques used for biomarker-based medical evaluations. The strengths and pitfalls of each approach are discussed and criticized. Future perspective with relation to the information and communication era is presented and discussed.

38P - Defining the physiological miRNA landscape in nipple aspirate fluid

38P - Defining the physiological miRNA landscape in nipple aspirate fluid

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

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

Abstract P6-05-03: Exploring the microRNA landscape of nipple aspirate fluid in the context of mammographic breast density

Abstract Background High mammographic density is an established risk factor for breast cancer but studies are needed to elucidate the etiological pathways underlying this relationship. The identification of factors (indirectly) regulating the accumulation of fat and connective tissue may help explain why high mammographic density is linked to breast cancer risk. Those factors may provide markers for predicting and even clues for modifying this risk. MicroRNAs could regulate specific cellular processes involved in regulating mammographic density, like fat accumulation, connective tissue density and immune response. Nipple aspirate fluid represents an excellent opportunity for biomarker detection within the breast microenvironment. Materials and Methods MicroRNA expression profiling was performed using TaqMan OpenArray Human MicroRNA Plates (ThermoFisher Scientific) in 40 pooled (paired left and right breast) nipple aspirate fluid samples obtained from age-matched women participating in a population-based breast cancer screening program with 'extremely dense' (n=20) and 'almost entirely fatty' breasts (n=20), without further abnormalities on mammography. Breast density was assessed using Volpara software in categories comparable to American College of Radiology (ACR) breast density categories. Results 25 significantly differentially expressed microRNAs were identified, including hsa-miR-19a-3p (p=0.001; upregulated in 'extremely dense' versus 'almost entirely fatty'), hsa-miR-324-5p (p=0.003; upregulated), hsa-miR-425-5p (p=0.001; upregulated), hsa-miR-660-5p (p=0.004; upregulated), hsa-miR-29b-3p (p=0.011; upregulated) and hsa-miR-187-3p (p=0.024; downregulated). Based on expression profiles of the 8 most significantly dysregulated microRNAs alone, nipple aspirate fluid samples could reliably be classified into 'extremely dense' and 'almost entirely fatty' mammographic density groups (31/40 (78%) samples correctly classified). Several of the dysregulated microRNAs have been associated with regulation of collagen deposition or crosslinking (e.g. miR-29, miR-222 and let-7) and adipocyte differentiation and proliferation (e.g. miR-29, miR-222, miR-155 and let-7), suggesting that these aberrant miRNAs may play a role in the development of breast tissue with high mammographic density. In addition, pathway enrichment analysis demonstrated an enrichment of the 'proteoglycans in cancer' pathway suggesting that proteoglycan dysregulation is intimately linked to microRNA dysregulation. Conclusions In summary, microRNA profiling has resulted in a panel of microRNAs that might help explain why high mammographic density is linked to breast cancer risk and may ultimately result in a biomarker panel for predicting this risk, or provide attractive targets for future breast cancer preventive strategies. Further validation in an independent set of nipple aspirate fluid samples from 'extremely dense' and 'almost entirely fatty' categories is currently ongoing. Citation Format: Moelans CB, Patuleia SI, Bakker MF, van der Wall E, van Diest PJ, van Gils CH. Exploring the microRNA landscape of nipple aspirate fluid in the context of mammographic breast density [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P6-05-03.

Contribution of the plasma and lymph Degradome and Peptidome to the MHC Ligandome.

Every biological fluid, blood, interstitial fluid and lymph, urine, saliva, lacrimal fluid, nipple aspirate, and spinal fluid, contains a peptidome-degradome derived from the cellular secretome along with byproducts of the metabolic/catabolic activities of each parenchymal organ. Clement et al. (J Proteomics 78:172-187, 2013), Clement et al. (J Biol Chem 291:5576-5595, 2016), Clement et al. (PLoS One 5:e9863, 2010), Clement et al. (Trends Immunol 32:6-11, 2011), Clement et al. (Front Immunol 4:424, 2013), Geho et al. (Curr Opin Chem Biol 10, 50-55, 2006), Interewicz et al. (Lymphology 37:65‑72, 2004), Leak et al. (Proteomics 4:753‑765, 2004), Popova et al. (PLoS One 9:e110873, 2014), Zhou et al. (Electrophoresis 25:1289‑1298, 2004), D'Alessandro et al. (Shock 42:509‑517, 2014), Dzieciatkowska et al. (Shock 42:485‑498, 2014), Dzieciatkowska et al. (Shock 35:331‑338, 2011), Jordan et al. (J Surg Res 143:130‑135, 2007), Peltz et al. (Surgery 146:347‑357, 2009), Zurawel et al. (Clin Proteomics 8:1, 2011), Ling et al. (Clin Proteomics 6:175‑193, 2010), Sturm et al. (Nat Commun 4:1616, 2013). Over the last decade, qualitative and quantitative analysis of the biological fluids peptidome and degradome have provided a dynamic measurement of tissue homeostasis as well as the tissue response to pathological damage. Proteomic profiling has mapped several of the proteases and resulting degradation by-products derived from cell cycle progression, organ/tissue remodeling and cellular growth, physiological apoptosis, hemostasis, and angiogenesis. Currently, a growing interest lies in the degradome observed during pathological conditions such as cancer, autoimmune diseases, and immune responses to pathogens as a way to exploit biological fluids as liquid biopsies for biomarker discovery Dzieciatkowska et al. (Shock 42:485-498, 2014), Dzieciatkowska et al. (Shock 35:331-338, 2011), Ling et al. (Clin Proteomics 6:175-193, 2010), Ugalde et al. (Methods Mol Biol 622:3-29, 2010), Quesada et al. (Nucleic Acids Res 37:D239‑243, 2009), Cal et al. (Front Biosci 12, 4661-4669, 2007), Shen et al. (PLoS One 5:e13133, 2010a), Antwi et al. (Mol Immunol 46:2931-2937, 2009a), Antwi et al. (J Proteome Res 8:4722‑4731, 2009b), Bedin et al. (J Cell Physiol 231, 915‑925, 2016), Bery et al. (Clin Proteomics 11:13, 2014), Bhalla et al. (Sci Rep 7:1511, 2017), Fan et al. (Diagn Pathol 7:45, 2012a), Fang et al. (Shock 34:291‑298, 2010), Fiedler et al. (Clin Cancer Res 15:3812‑3819, 2009), Fredolini et al. (AAPS J 12:504‑518, 2010), Greening et al. (Enzymes 42:27‑64, 2017), He et al. (PLoS One 8:e63724, 2013), Huang et al. (Int J Gynecol Cancer 28:355‑362, 2018), Hashiguchi et al. (Med Hypotheses 73:760‑763, 2009), Liotta and Petricoin (J Clin Invest 116:26‑30, 2006), Petricoin et al. (Nat Rev Cancer 6:961‑967, 2006), Shen et al. (J Proteome Res 9:2339‑2346, 2010a), Shen et al. (J Proteome Res 5:3154‑3160, 2006), Smith (Clin Proteomics 11:23, 2014), Wang et al. (Oncotarget 8:59376‑59386, 2017), Yang et al. (Clin Exp Med 12:79‑87, 2012a), Yang et al. (J Clin Lab Anal 26:148‑154, 2012b), Yang et al. (Anat Rec (Hoboken) 293:2027‑2033, 2010), Zapico-Muniz et al. (Pancreas 39:1293‑1298, 2010), Villanueva et al. (Mol Cell Proteomics 5:1840‑1852, 2006), Robbins et al. (J Clin Oncol 23:4835‑4837, 2005), Klupczynska et al. (Int J Mol Sci 17:410, 2016). In this review, we focus on the current knowledge of the degradome/peptidome observed in two main biological fluids (plasma and lymph) during physiological and pathological conditions and its importance for immune surveillance.

Exosomal miRNAs in nipple aspirate fluid and breast cancer

Background: Exosomal vesicles transport genetic information between cells and thereby influence the cells they interact with. Exosomes have been detected in multiple body fluids, including blood, milk, urine, pleural fluid, cerebrospinal fluid and the aqueous humor of the eye. We performed a proof of principle study to determine: (I) if exosomes are detectable in breast nipple aspirate fluid (NAF); (II) if exosomes are present, do they contain micro (mi)RNAs; (III) if present and if they contain miRNAs, the ability of the miRNAs to predict: (i) breast cancer; and (ii) response to treatment with the anti-inflammatory/anti-cancer agents vitamin (vit) D and celecoxib. Methods: NAF was collected from 12 healthy women and 20 women with newly diagnosed breast cancer. NAF and matched blood were collected from 5 women before and after daily treatment for one menstrual cycle/one month with low (400 IU) or high dose (2,000 IU) vitD, or high dose vitD plus 400 mg celecoxib. Ten miRs (16, 21, 100, 129, 145, 155, 181, 199, 205, and 212) were measured in NAF and serum samples. Results: We were routinely able to detect exosomes in NAF and serum samples, and in the exosomes to measure multiple miRNAs. Although there was no significant difference in expression based on the presence or absence of breast cancer, miR16 and -155 expressions were higher (P=0.030 for each) in women with node positive compared to those with node negative breast cancer. ∆Ct expression was at higher (defined at ∆Ct difference ≥1) in serum than matched NAF in healthy women. Conclusions: Exosomes are detectable in NAF. The exosomes contain intracellular information that may be useful for evaluation of the breast.

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

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

Proteomics Analysis of Human Breast Milk to Assess Breast Cancer Risk

ObjectiveDetection of breast cancer (BC) in young women in the early stages is challenging for several reasons. First, mammography and imaging techniques are less effective because young women have dense breast tissue. Second, there is a possible relationship between pregnancy and BC risk. One of the potential options for detection of BC is biochemical monitoring of protein markers in different types of bodily fluids such as serum, nipple aspirate fluid and ductal lavage fluid, tear, urine, saliva and breast milk (BM). BM provides access to breast tissue in the form of exfoliated epithelial cells that are the origins of the most types of BCs. Analysis of BM is a noninvasive method that could be used for cancer diagnosis. Here we analyzed human BM by mass spectrometry (MS) based proteomics, to build biomarker signatures for early detection of BC, and for the prediction of the onset of BC.MethodsMilk samples were from donors with BC and matched controls, some of which donated before being diagnosed by BC which used for risk assessment studies. In addition, samples with the same genotype were investigated: the donor with BC donated milk from the breast affected by BC and from the healthy breast, used as control. We analyze human BM as an appropriate cancer microenvironment for BC biomarkers discovery using different analytical approaches such as in‐gel (1D or 2D SDS‐PAGE) and in‐solution digestion of human BM samples coupled with nanoliquid chromatography tandem mass spectrometry (nanoLC‐MS/MS) followed by data processing, data base search and statistical analysis.ResultsWe identified several alterations in protein expression; some of these proteins such as plasminogen, myeloperoxidase isoform CRA_b, tenascin C (hexabrachion) isoform CRA_a, alpha1‐antichymotrypsin, complement C4B precursor, apolipoprotein (J and A–I) and several other proteins were overexpressed (upregulated) in the BC samples and some of them such as different proteins from the casein family, lactoferrin, human bile salt, mannose receptor and several other proteins had a lower expression (downregulation) in the BC samples, as compared to healthy controls. These dysregulated proteins might be associated with BC risk. The upregulated and downregulated proteins that we found in human BM samples from BC suffering mothers, compared to controls, matched well with the current literature.ConclusionWe completed a set of preliminary studies for identification of potential BC protein biomarkers and found some dysregulated (upregulated and/or downregulated) proteins and the outcome of our experiments was well matched with literature in terms of dysregulated proteins (same proteins, or similar proteins in the same family of proteins that we identified in our experiments) found by others, who worked on different bodily fluids or tumor tissues and cell lines either in BC or in different types of cancers. Working on BM as a local cancer microenvironment and a noninvasive method, we found the possible BC associated proteins to be dysregulated in human BM from women with BC compared to controls. Most of our dysregulated proteins found in BM were related to BC development. This could strongly introduce those protein as a potential biomarker for BC diagnosis and in some cases risk assessment of BC.

Soy Food Intake and Biomarkers of Breast Cancer Risk: Possible Difference in Asian Women?

ABSTRACTSoy foods may protect against breast cancer in Asian but not in Western populations. We examined if the levels of various markers of breast cancer risk and inflammation, as well as the effects of soy food consumption on these markers, differ between Asian and non-Asian premenopausal women in two soy intervention trials. One study randomized 220 women to a 2-yr intervention and the other one randomized 96 women in a crossover design to examine the effects of consumption of 2 daily soy servings on nipple aspirate fluid (NAF) volume; estrogens in serum, NAF, and urine; insulin-like growth factor-1 (IGF-1), IGF-binding protein 3, and inflammatory markers in serum; and mammographic densities. Mixed linear models were applied to assess ethnic differences in biomarkers and response to the soy diet. Serum C-reactive protein, serum leptin, NAF volume, and NAF estrone sulfate were lower, while urinary isoflavones were higher in Asian than in non-Asian women. A significant interaction (pinteraction = 0.05) between ethnicity and soy diet was observed for IGF-1 but not for other biomarkers. The current findings suggest possible ethnic differences in levels of biomarkers for breast cancer risk but little evidence that Asian women respond differently to soy foods than non-Asian women.

A Quantitative Proteomics Approach to Clinical Research with Non-Traditional Samples.

The proper handling of samples to be analyzed by mass spectrometry (MS) can guarantee excellent results and a greater depth of analysis when working in quantitative proteomics. This is critical when trying to assess non-traditional sources such as ear wax, saliva, vitreous humor, aqueous humor, tears, nipple aspirate fluid, breast milk/colostrum, cervical-vaginal fluid, nasal secretions, bronco-alveolar lavage fluid, and stools. We intend to provide the investigator with relevant aspects of quantitative proteomics and to recognize the most recent clinical research work conducted with atypical samples and analyzed by quantitative proteomics. Having as reference the most recent and different approaches used with non-traditional sources allows us to compare new strategies in the development of novel experimental models. On the other hand, these references help us to contribute significantly to the understanding of the proportions of proteins in different proteomes of clinical interest and may lead to potential advances in the emerging field of precision medicine.

Vitamin D3 Treatment Influences PGE2 and TGFβ in Normal and Increased Breast Cancer Risk Women.

We reported that vitamin D3 increased transforming growth factor (TGF)β2 and decreased prostaglandin (PG)E2 in the breast of normal-risk women, suggesting a protective effect. We determined if the findings held for higher risk women. Seventy-eight women received daily for one month/menstrual cycle: placebo, 400 international units (IU) vitamin D3, 2,000 IU vitamin D3 or 2,000 IU vitamin D3/400 mg celecoxib. Nipple aspirate fluid (NAF) and/or serum were analyzed for PGE2, TGFβ1,-2, vitaminD binding protein (DBP) 25(OH)D; and plasma for celecoxib. 25(OH)D increased (p<0.001) in women receiving 2,000 IU vitamin D3. Two thousand IU vitamin D3 lowered NAF PGE2 in normal-risk women (p=0.029), whereas 2,000 IU vitamin D3/celecoxib lowered NAF PGE2 in high-risk women (p=0.063). Serum TGFβ1 was influenced by treatment (p=0.011). NAF TGFβ2 increase correlated with increase in 25(OH)D. DBP serum levels were higher than matched NAF, regardless of race, and did not appreciably change with treatment. Vitamin D3 influenced TGFβ1 and -β2 expression. PGE2 response to vitamin D3 treatment was influenced by a participant's breast cancer risk. The implications of these observations regarding breast cancer risk should be further evaluated.

The role of background diet on the effects of eicosapentaenoic acid and docosahexaenoic acid supplementation in healthy pre-menopausal women: a randomized, cross-over, controlled study

BackgroundThe links between dietary fat intake, polyunsaturated fatty acid intake and breast cancer risk remain equivocal, with some studies pointing to improvements in risk upon omega-3 supplementation. However, the background diet is poorly controlled in most studies, potentially confounding this link. Therefore, this study examined the hypothesis that in order to see the benefits of omega-3 fatty acid supplementation, the background diet must be low in fat.MethodsOf the 56 healthy, pre-menopausal women randomized to one of two experimental arms, consisting of a two-treatment, randomized, cross-over design, 41 completed the 10 month intervention. The two diet phases (habitual and low-fat) were separated by a washout phase, each lasting 3 menstrual cycles. During each diet phase, women were supplemented with 1.2 g eicosapentaenoic acid + docosahexaenoic acid per day.ResultsRed blood cell fatty acid composition indicated that more eicosapentaenoic acid and docosahexaenoic acid was incorporated in the low-fat diet than the habitual diet, though both diet phases resulted in significant increases in the omega-3 to omega-6 ratio. In the context of omega-3 supplementation in breast cancer risk reduction, we also measured fatty acid incorporation into nipple aspirate fluid. Similar changes to red blood cells were noted in nipple aspirate fluid, with higher incorporation of eicosapentaenoic acid in the low-fat diet phase.ConclusionsThese data suggest that the total level of dietary fat has some direct impact on fatty acid partitioning in addition to the recognized importance of fatty acid ratios, and supports the hypothesis that dietary fat intake must be considered a confounder in supplementation trials. Additionally, we demonstrate that n3 supplementation both reaches and imparts improvements in lipid content and n3:n6 at the target breast tissue.Trial registrationTrial was been retrospectively registered at clinicaltrials.gov (RegNCT02816125).

Characterization of the microbiome of nipple aspirate fluid of breast cancer survivors.

The microbiome impacts human health and disease. Until recently, human breast tissue and milk were presumed to be sterile. Here, we investigated the presence of microbes in the nipple aspirate fluid (NAF) and their potential association with breast cancer. We compared the NAF microbiome between women with a history of breast cancer (BC) and healthy control women (HC) using 16S rRNA gene amplicon sequencing. The NAF microbiome from BC and HC showed significant differences in community composition. Two Operational Taxonomic Units (OTUs) showed differences in relative abundances between NAF collected from BC and HC. In NAF collected from BC, there was relatively higher incidence of the genus Alistipes. By contrast, an unclassified genus from the Sphingomonadaceae family was relatively more abundant in NAF from HC. These findings reflect the ductal source DNA since there were no differences between areolar skin samples collected from BC and HC. Furthermore, the microbes associated with BC share an enzymatic activity, Beta-Glucuronidase, which may promote breast cancer. This is the first report of bacterial DNA in human breast ductal fluid and the differences between NAF from HC and BC. Further investigation of the ductal microbiome and its potential role in breast cancer are warranted.

Protein Biomarkers for Breast Cancer Risk Are Specifically Correlated with Local Steroid Hormones in Nipple Aspirate Fluid.

The local endocrine environment of the breast may have stronger relations to breast cancer risk than systemic hormones. Nipple aspiration fluid (NAF) provides a window into this milieu. We hypothesized that the correlations between proteins and steroid hormones in NAF are stronger, and specific relationships may reveal links to breast cancer risk. NAF and blood samples were obtained simultaneously from 54 healthy women and from the contralateral unaffected breast of 60 breast cancer patients. The abundance of five proteins, superoxide dismutase (SOD1), C-reactive protein (CRP), chitinase-3-like protein 1 (YKL40), cathepsin D (CatD), and basic fibroblast growth factor (bFGF) in NAF was measured using ELISA. The NAF and serum concentrations of estradiol, estrone, progesterone, androstenedione, testosterone, and dehydroepiandrostrerone (DHEA) were measured using ELISA or RIA. The correlations between proteins and hormones revealed that NAF proteins correlated with each other: SOD1 with CRP (R = 0.276, P = 0.033) and CatD (R = 0.340, P = 0.0036), and bFGF with CRP (R = 0.343, P = 0.0021). NAF proteins displayed significant correlations with NAF steroids, but not with serum steroids: SOD1 with DHEA (R = 0.333, P = 0.019), YKL40 with testosterone (R = 0.389, P = 0.0012), and bFGF negatively correlated with testosterone (R = -0.339, P = 0.015). The regulation of YKL40 and bFGF by testosterone was confirmed in breast cancer cell lines. In summary, NAF proteins were more strongly related to local hormone levels than to systematic hormone levels. Some proteins were specifically correlated with different NAF steroids, suggesting that these steroids may contribute to breast cancer risk through different mechanisms.

Genetic Determinants of Nipple Aspiration Fluid Yield.

Nipple aspiration fluid (NAF) is a non-invasively-acquired biosample that can provide a window into the breast environment, but NAF yield is highly variable. Its determinants must be better understood for studies of breast cancer risk. The wet earwax phenotype was identified as one determinant of NAF yield in the 1970s, and is linked to single nucleotide polymorphisms (SNPs) in the ATP-binding cassette transporter gene ABCC11. We have investigated this, as well as SNPs in the prolactin (PRL) and prolactin receptor (PRLR) genes, in relation to NAF yield. DNA was extracted from white blood cells of 557 NAF yielders and 359 non-yielders, and was used to genotype ABCC11 (rs17822931), PRL (rs849870, rs849872, rs849886, rs2244502, rs1341239), and PRLR (rs37364, rs34024951, rs1610218, rs9292575, rs7718468) using Taqman genotyping assay. The association between NAF yield and each single SNP was analyzed using logistic regression adjusting for age, race, and menopausal status. ABCC11 rs17822931 showed a negative association with NAF yield [odds ratio (OR) 0.66, 95% confidence interval (CI) 0.49-0.88; p=0.004]. The PRL rs849870 and the haplotype combination with other SNPs showed a marginal association with NAF yield. In addition, the years since last birth also showed negative association with NAF yielding (OR 0.98, 95% CI 0.96-0.99; p=0.001). The combination of the years since last birth with ABCC11 SNP revealed significant interaction between reproductive factor and genetic factor. Our results confirmed the association between NAF yield and earwax phenotype through ABCC11 genotype. Combined with the recency of last birth, ABCC11 genotype should be considered in the design of studies utilizing NAF as a biosample.

Nipple Aspirate Fluid Hormone Concentrations and Breast Cancer Risk.

Prior reports identify higher serum concentrations of estrogens and androgens as risk factors for breast cancer, but steroids in nipple aspirate fluid (NAF) may be more related to risk. Incident breast cancer cases and mammography controls were recruited. Sex steroids were measured in NAF from the unaffected breasts of cases and one breast of controls. Menopausal status and menstrual cycle phase were determined. NAF steroids were purified by HPLC and quantified by immunoassays. Conditional logistic regression models were used to examine associations between NAF hormones and case-control status. NAF samples from 160 cases and 157 controls were evaluable for hormones. Except for progesterone and dehydroepiandrosterone (DHEA), the NAF and serum concentrations were not significantly correlated. NAF estradiol and estrone were not different between cases and controls. Higher NAF (but not serum) DHEA concentrations were associated with cases, particularly among estrogen receptor (ER)-positive cases (NAF odds ratio (OR) = 1.18, 95 % confidence interval (CI) 1.02, 1.36). NAF DHEA was highly correlated with NAF estradiol and estrone but not with androstenedione or testosterone. Higher progesterone concentrations in both NAF and serum were associated with a lower risk of ER-negative cancer (NAF OR = 0.69, 95 % CI 0.51, 0.92). However, this finding may be explained by case-control imbalance in the number of luteal phase subjects (2 cases and 19 controls). The significantly higher concentration of DHEA in NAF of cases and its correlation with NAF estradiol indicates a potentially important role of this steroid in breast cancer risk; however, the negative association of progesterone with risk is tentative.