Patient-centered research: how do women tolerate nipple fluid aspiration as a potential screening tool for breast cancer?

BACKGROUND: Nipple fluid aspiration (NFA) is a technique to acquire nipple aspirate fluid (NAF), which is considered a rich source of breast-specific biomarkers. Originating directly from the mammary ducts, this liquid biopsy can offer insight into the process of carcinogenesis at its earliest stage and therefore could be of added value to the current imaging-based breast cancer screening tools. With that in mind, it is necessary to know how well NFA is tolerated. AIM: To evaluate the participants' tolerability of NFA compared to breast imaging screening methods and blood draws. MATERIALS AND METHODS: Three cohorts of women underwent NFA: healthy women (n = 190), women diagnosed with breast cancer (n = 137) and women at high risk of developing breast cancer (n = 48). A 0-10 discomfort score of NFA, mammography, breast MRI and blood draws, was filled in at the study visits, which took place once or annually. RESULTS: The median discomfort rate of NFA was 1, which was significantly lower than the median discomfort of mammography and breast MRI (5 and 3, respectively, p < 0.001), but significantly higher than median discomfort for blood draws (0, p < 0.001). The great majority of women would undergo the procedure again (98%) and recommend it to others (97%). CONCLUSION: This study shows that NFA was well tolerated by healthy women, women diagnosed with breast cancer and high-risk women. This makes NFA a feasible method to pursue as a potential future breast cancer early detection tool, based on resident biomarkers. TRIAL REGISTRATION: NL41845.041.12 , NL57343.041.16 and NL11690.041.06 in trialregister.nl.

The changing microRNA landscape by color and cloudiness: a cautionary tale for nipple aspirate fluid biomarker analysis.

PURPOSE: Investigation of nipple aspirate fluid (NAF)-based microRNAs (miRNAs) as a potential screening tool for women at increased risk of developing breast cancer is the scope of our research. While aiming to identify discriminating NAF-miRNAs between women with different mammographic densities, we were confronted with an unexpected confounder: NAF sample appearance. Here we report and alert for the impact of NAF color and cloudiness on miRNA assessment. METHODS: Seven classes of NAF colors coupled with cloudiness appearance were established. Using 173 NAF samples from 154 healthy women (19 samples were bilaterally collected), the expression of 14 target and 2 candidate endogenous control (EC) miRNAs was investigated using Taqman Advanced miRNA assays to identify significant differential expression patterns between color-cloudiness classes. Inter- and intra-individual variation of miRNA expression was analyzed using the coefficient of variation (CV). RESULTS: We found that between the seven NAF classes, fold change miRNA expression differences ranged between 2.4 and 19.6 depending on the interrogated miRNA. Clear NAF samples exhibited higher miRNA expression levels compared to cloudy NAF samples with fold change differences ranging between 1.1 and 6.2. Inter-individual and intra-individual miRNA expression was fairly stable (CV < 15 %), but nevertheless impacted by NAF sample appearance. Within NAF classes, inter-individual variation was largest for green samples (CV 6-15 %) and smallest for bloody samples (CV 2-6 %). CONCLUSIONS: Our data indicate that NAF color and cloudiness influence miRNA expression and should, therefore, be systematically registered using an objective color classification system. Given that sample appearance is an inherent feature of NAF, these variables should be statistically controlled for in multivariate data analyses. This cautionary note and recommendations could be of value beyond the field of NAF-miRNAs, given that variability in sample color and cloudiness is likewise observed in liquid biopsies such as urine, cerebrospinal fluid and sputum, and could thereby influence the levels of miRNAs and other biomarkers.

Diagnostic Accuracy of Nipple Aspirate Fluid Cytology in Asymptomatic Patients: A Meta-analysis and Systematic Review of the Literature.

PURPOSE: To calculate the diagnostic accuracy of nipple aspirate fluid (NAF) cytology. BACKGROUND: Evaluation of NAF cytology in asymptomatic patients conceptually offers a non-invasive method for either screening for breast cancer or else predicting or stratifying future cancer risk. METHODS: Studies were identified by performing electronic searches up to August 2019. A meta-analysis was conducted to attain an overall pooled sensitivity and specificity of NAF for breast cancer detection. RESULTS: A search through 938 studies yielded a total of 19 studies. Overall, 9308 patients were examined, with cytology results from 10,147 breasts [age (years), mean ± SD = 49.73 ± 4.09 years]. Diagnostic accuracy meta-analysis of NAF revealed a pooled specificity of 0.97 (95% CI 0.97-0.98), and sensitivity of 0.64 (95% CI 0.62-0.66). CONCLUSIONS: The diagnostic accuracy of nipple smear cytology is limited by poor sensitivity. If nipple fluid assessment is to be used for diagnosis, then emerging technologies for fluid biomarker analysis must supersede the current diagnostic accuracy of NAF cytology.

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 were retrieved from the literature by means of a systematic search. MiRNAs from each individual study were orderly ranked between 1 and 50, combined into an overall ranking per sample type and compared. RESULTS: 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% and 62%. Highly ranked NAF miRNAs have established roles in breast carcinogenesis. CONCLUSION: 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.

Calidad higiénico-sanitaria de dos sistemas de ordeño en fincas bovinas ubicadas en el sector Vuelta Larga, municipio Maturín, estado Monagas (Venezuela) / Hygienic-sanitary quality of two milking systems in bovine farms located in sector Vuelta Larga, Maturín municipal state, Monagas state (Venezuela)

RESUMEN El objetivo de la presente investigación fue evaluar la calidad higiénico-sanitaria de dos sistemas de ordeño en fincas bovinas del municipio Maturín estado Monagas (Venezuela). El ensayo tuvo una duración de seis semanas, se calculó la eficiencia higiénica (%EH) de las instalaciones, el personal, la rutina de ordeño, la limpieza-desinfección y el almacenamiento-transporte de dos unidades de producción. Adicionalmente, se determinó la población de bacterias aerobias mesófilas (BAM) que se cuantificaron en un experimento en bloques al azar, con arreglo factorial de los tratamientos (2x2) donde el factor A, correspondió al sistema de ordeño manual y mecánico y el factor B, a la aplicación y omisión de secado de los pezones de cada animal previo al ordeño. La variable se examinó por análisis de varianza y sus valores promedios comparados por Tukey al 5% de probabilidad. El %EH de las fincas evaluadas fue de 66,66% para la finca 2 y de 54,84% para la finca 1, considerados no satisfactorios. Los resultados obtenidos para la variable BAM indican que el factor sistema de ordeño mostró diferencia altamente significativa (p ≤ 0,01); el sistema manual presentó el menor conteo con 5,24 Log10 UFC/ mL; mientras que para el factor condición de secado se constató diferencia significativa (p ≤ 0,05), ya que la aplicación de secado presentó la menor población de bacterias aerobias mesófilas con 5,25 Log10 UFC/mL. Las medidas higiénicas y sanitarias implementadas en la rutina de ordeño influyeron directamente en la calidad de la leche cruda.

ABSTRACT The objective of the present investigation, was to evaluate the 'Hygienic-Sanitary Quality of two Milking Systems in Bovine Farms' in Vuelta Larga Sector, Maturín City, Monagas State (Venezuela). The trial lasted six weeks, in which the Hygienic Efficiency (% EH) of the facilities, the workers, the milking routine, the cleaning and disinfection measures and the storage and transport of both milking systems, were calculated. Furthermore, the population of Mesophilic Aerobic Bacteria (BAM) was determined ; The data were processed following a distribution in random blocks with factorial arrangement (2x2) whose factor A represented the milking system: manual and mechanical, and the factor B was the application and omission of manual drying of teats for each animal prior to milking. The percentage of Hygienic Efficiency (% EH) of the farms evaluated was not satisfactory, corresponding to 66.66% in the farm 2 and 54.84% in the farm 1. The variable was examined by analysis of variance and its mean values compared by Tukey test at 5% of probability. Factor A achieved a highly significant difference (p ≤0.01), with the manual system obtaining the lowest count with 5.24 Log10 CFU / mL. In factor B significant difference was found (p≤0.05), the drying application decreased the BAM population with 5.25 Log10 CFU / mL. The Hygienic and Sanitary measures implemented in the milking routine directly influence the quality of raw milk.

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 [...].

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

BACKGROUND: Worldwide, 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. METHODS: The 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. RESULTS: A 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. CONCLUSIONS: We 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.

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.

Nipple aspirate fluid-A liquid biopsy for diagnosing breast health.

PURPOSE: Nipple secretions are protein-rich and a potential source of breast cancer biomarkers for breast cancer screening. Previous studies of specific proteins have shown limited correlation with clinicopathological features. Our aim, in this pilot study, was to investigate the intra- and interpatient protein composition of nipple secretions and the implications for their use as liquid biopsies. EXPERIMENTAL DESIGN: Matched pairs of nipple discharge/nipple aspirate fluid (NAF, n = 15) were characterized for physicochemical properties and SDS-PAGE. Four pairs were selected for semiquantitative proteomic profiling and trypsin-digested peptides analyzed using 2D-LC Orbitrap Fusion MS. The resulting data were subject to bioinformatics analysis and statistical evaluation for functional significance. RESULTS: A total of 1990 unique proteins were identified many of which are established cancer-associated markers. Matched pairs shared the greatest similarity (average Pearson correlation coefficient of 0.94), but significant variations between individuals were observed. CONCLUSIONS AND CLINICAL RELEVANCE: This was the most complete proteomic study of nipple discharge/nipple aspirate fluid to date providing a valuable source for biomarker discovery. The high level of milk proteins in healthy volunteer samples compared to the cancer patients was associated with galactorrhoea. Using matched pairs increased confidence in patient-specific protein levels but changes relating to cancer stage require investigation of a larger cohort.

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.

PURPOSE: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

DNA promoter hypermethylation in nipple fluid: a potential tool for early breast cancer detection.

INTRODUCTION: Nipple fluid aspiration provides direct non-invasive sampling of fluid from the mammary ductal system, where the majority of breast cancers originate. DNA promoter hypermethylation ("methylation") occurs early and at high frequency in breast carcinogenesis, bearing the potential as a biomarker for cancer detection at its earliest stages. We assessed methylation in nipple fluid from breasts of healthy women, of women with sporadic breast cancer and of their contralateral breasts. Our goal was to investigate whether nipple fluid can be used as a reliable methylation biomarker source. MATERIALS AND METHODS: Methylation levels of 13 genes were analysed by quantitative multiplex-methylation specific PCR (QM-MSP) in nipple fluid samples from breasts of healthy women, and from the affected and contralateral breasts of breast cancer patients. RESULTS: Methylation analysis of the low-volume nipple fluid samples was feasible. Despite the generally low methylation levels, cancerous and healthy breasts nipple fluid could be discriminated with an area under the receiver operating characteristic curve (AUC) of 0.64 (p<0.01) based on a multivariate model including AKR1B1, ALX1, RASSF1A and TM6SF1. Within-patient differences between cancerous and contralateral nipple fluid samples were less prominent. CONCLUSIONS: Cancerous nipple fluid contains increased levels of methylation of tumor suppressor genes that potentially could serve as a biomarker for early breast cancer detection.

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.

The In's and Out's of Ductography: A Comprehensive Review.

Nipple discharge is a common clinical problem, which is most frequently benign. However, nipple discharge may be the presenting symptom of an underlying malignancy. Ductography can provide valuable information in identifying the cause of the discharge. As ductography is a relatively low-volume procedure in most practices, many radiologists and trainees often lack familiarity and experience with the procedure. This article is intended to review the indications, technique, relevant ductal anatomy, and potential imaging findings of ductography.

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

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

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

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

Proteomic profiling of nipple aspirate fluid (NAF): Exploring the complementarity of different peptide fractionation strategies.

NAF is a breast fluid that is closely related to the tumor microenvironment and a valuable sample for studying breast cancer. To perform an in-depth proteomic analysis of this sample, aliquots of a single NAF digest were analyzed by the following peptide-centric fractionation strategies: a) 30-cm reversed-phase (RP) column on-line with an LTQ-Orbitrap XL; b) off-line strong cation-exchange (SCX) column; and c) pI-based OFFGEL fractionation. All fractions from approaches (b) and (c) were further analyzed on a 10-cm RP column hyphenated to the same mass spectrometer. The RP-30cm, SCX/RP-10cm, and OFFGEL/RP-10cm approaches identified 1676, 2930, and 3240 peptides, which corresponded to 193, 390 and 528 proteins, respectively. In our cumulative dataset, 4466 distinct NAF peptides corresponded to a total of 557 proteins, of which only 34% were identified by all three approaches. No exclusive protein identification was associated to the RP-30cm approach, while SCX/RP-10cm and OFFGEL/RP-10cm contributed to 28 and 166 exclusive identifications, respectively. Each approach provided additional information related to energy metabolism (fermentation process/carbohydrate biosynthesis). In conclusion, the pre-fractionation platforms used were complementary for the comprehensive characterization of NAF and our work provides methodological information for future quantitative cancer-related NAF sample studies. BIOLOGICAL SIGNIFICANCE: High-resolution peptide separation is a sine qua non condition for achieving extensive proteome coverage. Various techniques have been employed to improve peptide fractionation prior to LC-MS/MS, thus allowing a comprehensive characterization of complex biological samples. Although fractionation efficiency is very sample-dependent, this issue is commonly overlooked, and a "cookbook" approach is routinely used during this critical step. The present study provides a systematic comparison of analytical information needed for the successful large-scale differential proteomic analysis of NAF samples from breast cancer patients, a precious and volume-limited biological sample. It reinforces the importance of optimizing sample-specific fractionation protocols for information retrieval from mass spectrometric analysis.

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

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