Proteomic analysis to identify breast cancer biomarkers in nipple aspirate fluid.

PURPOSE: Proteomic analysis of breast nipple aspirate fluid (NAF) holds promise as a noninvasive method to identify markers of breast cancer. The objectives of the study were to: (a) describe the NAF proteome, (b) identify candidate markers of breast cancer in NAF by using proteomic analysis, and (c) validate the markers identified by using a quantitative, high-throughput ELISA analysis. EXPERIMENTAL DESIGN: For proteome analysis, NAF proteins from a single subject without breast cancer were separated by two-dimensional PAGE and were subjected to matrix-assisted laser desorption ionization time-of-flight mass spectometry identification. A total of 41 different proteins were identified, 25 of which were known to be secreted. To identify breast cancer markers, we separated 20 NAF samples (10 normal, 10 cancer) by two-dimensional PAGE. Three protein spots were detected that were up-regulated in three or more cancer samples. These spots were identified to be gross cystic disease fluid protein (GCDFP)-15, apolipoprotein D (apoD), and alpha1-acid glycoprotein (AAG). To validate these three potential biomarkers, 105 samples (53 from benign breasts and 52 from breasts with cancer) were analyzed using ELISA. RESULTS: Among all of the subjects, GCDFP-15 levels were lower (P <0.001) and AAG levels were higher (P=0.001) in breasts with cancer. This was also true in premenopausal (GCDFP-15, P=0.011; AAG, P=0.002) but not in postmenopausal women. GCDFP-15 levels were lowest (P=0.003) and AAG levels highest (P <0.001) in women with ductal carcinoma in situ (DCIS). Menopausal status influenced GCDFP-15 and AAG more in women without breast cancer than in women with breast cancer. apoD levels did not correlate significantly with breast cancer. CONCLUSIONS: Our study revealed that the NAF proteome, as defined by two-dimensional PAGE, consists of a limited number of proteins, and that the expression of AAG and GCDFP-15 correlates with disease presence and stage.     

Significant differences in nipple aspirate fluid protein expression between healthy women and those with breast cancer demonstrated by time-of-flight mass spectrometry

New approaches are needed for the early detection of breast cancer. Proteomic profiling technologies, such as surface-enhanced laser desorption ionization mass spectrometry (SELDI-MS), may be able to identify tumor markers in biological fluids. The objective of this study was to determine whether there are differences in protein expression patterns in nipple aspirate fluid (NAF) from the cancerous and noncancerous breasts of patients with unilateral breast cancer and the breasts of healthy volunteers. Paired NAF samples were obtained from 23 women with stage I or II unilateral invasive breast carcinoma and five healthy female volunteers. Aliquots of the samples were applied to SELDI Protein-chip arrays (WCX2 and IMAC3-Cu⁺⁺), and protein expression was analyzed using time-of-flight MS. A total of 463 distinct peaks were detected and analyzed. In breast cancer patients, no differences in protein expression were identified between the breast with the intact primary carcinoma and the contralateral noncancerous breast. Seventeen peaks were overexpressed in cancer-bearing breasts compared to breasts of healthy volunteers (p < 0.0005). When spectra from the nontumor-bearing breasts of breast cancer patients were compared with spectra from breasts of healthy volunteers, two peaks that were overexpressed in breast cancer patients and one peak that was underexpressed in breast cancer patients were detected (p < 0.0027). SELDI-MS was able to identify differences in the phenotypic proteomic profile of NAF samples obtained from patients with early-stage breast cancer and healthy women. Proteomic screening techniques such as SELDI-MS analysis of NAF may be useful for breast cancer screening and diagnosis.     

Screening for serological biomarkers of pancreatic cancer by two-dimensional electrophoresis and liquid chromatography-tandem mass spectrometry

Pancreatic cancer (PC) is one of the most lethal malignant tumors because of late diagnosis and the lack of response to various therapies. To identify potential biomarkers in cancerous serum from early stage PC patients, we carried out two-dimensional gel electrophoresis (2-DE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to compare the serum proteomic profiles from 45 patients with PC and 20 healthy volunteers. Seven spots showed differential expression on 2-DE gels and two up-regulated protein spots were identified by LC-MS/MS as α-1-antitrypsin (AAT). These protein spots were also confirmed by Western blotting. This is the first time that AAT isoforms have been identified as potential serum biomarkers for PC. The serum isoforms of AAT may be clinically useful for PC diagnosis and monitoring.     

Association of kallikrein expression in nipple aspirate fluid with breast cancer risk

Human kallikreins (hK) 2, 3, 6 and 10 are expressed in breast and prostate tissue. hK2 and hK3 (prostate-specific antigen, PSA) are used to screen for prostate cancer. hK6 and hK10 are downregulated in breast cancer compared to normal breast tissue. We demonstrated that levels of PSA in nipple aspirate fluid (NAF) are lower in women with breast cancer than in normal women. We hypothesize that the expression of hK2, 3, 6 and 10 are related and important in detecting breast cancer. The goals of this study are to determine the level of expression of kallikreins in NAF and serum, the association of hK2, 3, 6 and 10 in NAF, and the association of each of the kallikreins with breast cancer. In NAF from 275 women, hK3, 6 and 10 were detectable in >/= 90% and hK2 in 74% of samples analyzed. NAF levels were highest for hK6 and lowest for hK2, regardless of cancer and menopausal status. hK3 was detectable in 15/29 (52%) and hK2 in 0/29 serum samples collected from 6 women. hK2 and hK3 were concentrated in NAF vs. matched serum. The 4 kallikreins were associated with the exception of hK2 with hK6 or hK10. PSA levels were higher in normal pre- than postmenopausal subjects (but not women with breast cancer), whereas levels of hK2, 6 and 10 did not differ by menopausal status. hK2 and PSA were associated with both pre- and postmenopausal breast cancer; hK6 and 10 were not. hK2 and PSA were more associated with pre- than postmenopausal breast cancer. Using logistic regression, PSA and menopausal status provided the best model of breast cancer prediction, with a sensitivity of 91% and specificity of 39%. In conclusion, 4 kallikreins are expressed in NAF. hK2 and PSA, and hK6 and hK10 are highly associated. Higher premenopausal PSA levels suggest the influence of ovarian steroids. PSA shows the most promise in aiding in the early detection of breast cancer.     

Mitochondrial DNA mutations in breast cancer tissue and in matched nipple aspirate fluid

Unlike nuclear (n)DNA, of which there is one paired copy per cell, there are many copies of mitochondrial (mt)DNA per cell, making PCR amplification of mtDNA easier in samples of limited cellularity. The aims of this study were to (i) determine the mutation patterns of breast cancers through a comprehensive screen of mtDNA mutations, and (ii) assess if mutations in the cancers are also detectable in breast nipple aspirate fluid (NAF), a physiologic fluid which contains shed ductal epithelial cells. Fifteen breast cancers, matched benign tissues and NAF were collected. Nine overlapping primer sets were used to sequence the entire mitochondrial genome from tissue samples. For NAF samples, we focused on the 19 nucleotide positions (np) where mutations were found in a 3701 bp region (np 15331 to 2463), which includes the displacement (D)-loop, a mtDNA mutation hot spot. Fourteen of the fifteen (93%) cancer samples had > or =1 somatic mtDNA mutation for a total of 45 at 35 np (9 np reported previously, 26 new). Nine of fifteen tumors had > or =2 mutations. The D-loop contained 17 of 45 (38%) and non-D-loop (coding) regions contained 28 (62%) mutations. Of the 28 mutations in the coding loci, 11 led to an amino acid change. The frequency of mtDNA mutations was higher in the D-loop region (1.5 versus 0.18% of loci). 155 polymorphisms were identified (98 reported previously, 57 new). Sixteen of forty-five (36%) mutations were located at polymorphism sites. Four of nineteen mtDNA mutations in 10 cancers located between np 15331 and 2463 were found in matched NAF (two of eleven mutations in the D-loop and two of eight in non-D-loop regions). No mutations were found in five matched NAF samples from women whose cancers lacked a mutation in the same region. In conclusion, mtDNA mutations in breast cancer occur both within and outside of the D-loop, though the mutation rate in the D-loop is over 7-fold higher than in coding areas. We identified 26 new mutation loci (25 in regions sequenced by others, one in an area not). The high frequency of mtDNA mutations at polymorphic loci requires further investigation. Specific mtDNA mutations can be detected in a subset of NAF samples from women with breast cancer.     

Biologic markers of breast cancer in nipple aspirate fluid and nipple discharge are associated with clinical findings

BACKGROUND: The aim of this prospective study was to assess predictive markers in nipple aspirate fluid (NAF) and pathologic nipple discharge (PND) collected prior to excisional breast biopsy, as well as clinical factors available prior to biopsy, with histopathologic results in women with a radiographically suspicious and/or palpable breast lesion. METHODS: 208 NAF samples from 191 women were evaluated for the following candidate predictive proteins and cellular markers: prostate-specific antigen (PSA), human glandular kallikrein 2 (hK2), basic fibroblast growth factor (bFGF), S phase fraction (SPF), DNA index, and cytology. Clinical factors included whether or not the lesion was palpable, menopausal status, history of pregnancy, history of birth control or hormone replacement use, and PND. RESULTS: Considering all women, bFGF (p=0.005) and SPF (0.031) were associated, and abnormal cytology approached an association (p=0.056) with the presence of breast cancer. Women with PND were less likely to have breast cancer (4 vs. 37%, p<0.001) or palpable lesions (10 vs.43%, p<0.001), were younger, had lower PSA levels (p=0.046), and were more likely to have atypical NAF cytology (p=0.002). Excluding PND, increased age, postmenopause (both p<0.01), high bFGF (p=0.004) and low PSA (p=0.05) were associated with cancer. The best breast cancer predictive model included cytology, bFGF, and age (88% sensitive and 57% specific). When the data were divided by menopausal status, the optimal models to predict breast cancer, which included NAF hK2 or PSA and age, were 100% sensitive and 41% specific in pre- vs. 93% sensitive and 12% specific in postmenopausal women. CONCLUSION: NAF and clinical biomarkers are sensitive predictors of whether a breast contains cancer, and may ultimately help guide treatment. Future studies to determine the optimal combination of predictive markers are warranted.     

Prostate-specific antigen expression in nipple aspirate fluid is associated with advanced breast cancer

We previously demonstrated that prostate-specific antigen (PSA) is present in breast nipple aspirate fluid (NAF) and its expression is inversely associated with the presence of breast cancer. The purpose of this study was to determine if PSA levels in NAF decrease with disease progression from DCIS to metastatic breast cancer. One hundred and forty-nine women underwent nipple aspiration before or in conjunction with surgery to treat their breast cancer. PSA levels decreased with more advanced disease stage (P = 0.016), larger tumor size (P = 0.031), and nodal involvement (P = 0.041). PSA levels were lower in women with than without distant disease spread (P = 0.049). We also evaluated the association of PSA with these clinical parameters based on menopausal status. In general, PSA predicted disease involvement better in pre- than in post-menopausal women. There was no association between PSA and race. Spearman's rank analysis demonstrated that PSA was inversely related to tumor size (P = 0.009), nodal status (P = 0.005), disease stage (P = 0.004), and distant metastases (P = 0.04). NAF PSA provides useful prognostic information which may assist with breast cancer treatment.     

Proteomic analysis of nipple aspirate fluid to detect biologic markers of breast cancer

The early detection of breast cancer is the best means to minimise disease-related mortality. Current screening techniques have limited sensitivity and specificity. Breast nipple aspirate fluid can be obtained noninvasively and contains proteins secreted from ductal and lobular epithelia. Nipple aspirate fluid proteins are breast specific and generally more concentrated than corresponding blood levels. Proteomic analysis of 1 microl of diluted nipple aspirate fluid over a 5-40 kDa range from 20 subjects with breast cancer and 13 with nondiseased breasts identified five differentially expressed proteins. The most sensitive and specific proteins were 6500 and 15 940 Da, found in 75-84% of samples from women with cancer but in only 0-9% of samples from normal women. These findings suggest that (1) differential expression of nipple aspirate fluid proteins exists between women with normal and diseased breasts, and (2) analysis of these proteins may predict the presence of breast cancer.     

Epithelial cells in nipple aspirate fluid and subsequent breast cancer risk: A historic prospective study

Background

Breast and prostate cancer patients often develop lesions of locally high bone turnover, when the primary tumor metastasizes to the bone causing an abnormal high bone resorption at this site. The objective of the present study was to determine whether local increased bone turnover in breast and prostate cancer patients is associated with an increase in cartilage degradation and to test in vitro whether osteoclasts or cathepsin K alone generate CTXII from human bone.

Methods

The study included 132 breast and prostate cancer patient, where presence of bone metastases was graded according to the Soloway score. Total bone resorption (CTXI_total) and cartilage degradation (CTXII) were determined.

Results

Breast and prostate cancer patients with bone metastases revealed significant increased levels of CTXI_total at Soloway scores 1 and higher compared to patients without bone metastases (p < 0.001). CTXII was statistically elevated at score 3 and 4 (p < 0.01). CTXII/CTXI_total significantly decreased at score 3 and 4 (p < 0.001). Levels of CTXI_total, CTXII and CTXII/CTXI_total changed +900%, +130%, and -90%, respectively at Soloway score 4 compared to score 0. The in vitro experiments revealed that osteoclasts released CTXI fragments but not CTXII from bone specimens. The same was observed for cathepsin K.

Conclusion

Data suggest that an uncoupling between bone resorption and cartilage degradation occurs in breast and lung cancer patient.     

Identification of biomarkers for breast cancer in nipple aspiration and ductal lavage fluid.

PURPOSE: To establish a comprehensive proteomic approach for biomarker discovery and validation in breast fluid. EXPERIMENTAL DESIGN: 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. RESULTS: 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. CONCLUSIONS: 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.     

Discovery of oral fluid biomarkers for human oral cancer by mass spectrometry

Proteomic analysis of human oral fluid (whole saliva) holds promise as a non-invasive method to identify biomarkers for human oral cancer, a high impact local disease in the oral cavity affecting 38,000 Americans and with 350,000 cases worldwide annually. In this study, matrix-assisted laser desorption/ionization--mass spectrometry (MALDI-MS) was used to profile oral fluid samples from oral cancer and control subjects, and 46 peptides/proteins were found at significantly different levels between the two groups. To identify a candidate protein biomarker, oral fluid samples were separated by liquid chromatography (LC) using a C4 reversed-phase column. The collected LC fractions were monitored by MALDI-MS and the fraction containing the candidate biomarker was digested for LC-MS/MS analysis to identify it. The use of nanospray MS/MS for the identification of candidate peptide biomarkers was also demonstrated. This approach can be useful for the identification of protein or peptide biomarkers following MALDI-MS or surface-enhanced laser desorption/ionization MS profiling of clinical samples. This study clearly demonstrated that oral fluid contains proteomic signatures that may serve as biomarkers for human diseases such as oral cancer. Once discovered and validated on a large and independent clinical cohort, oral fluid proteomic biomarkers may be extensively used for future disease diagnosis.     

Pilot studies on the p53 gene in nipple aspirate fluid from patients with breast cancer

Nipple Aspirate Fluid (NAF) from patients with breast cancer is a potential source of exfoliated tumour material amenable to molecular biological study, but few such data have been reported. In this study we demonstrate that polymerase chain reaction (PCR) amplification of p53 gene DNA is achievable in a proportion of NAF samples from breast cancer patients. Subsequently four NAF samples from patients whose primary tumours were identified as having a defined p53 mutation were studied by single stranded conformational polymorphism analysis (SSCP). Two samples yielded PCR product indistinguishable from wild type and two yielded no product. Whilst no cancer-related genetic mutations were demonstrated in NAF samples, further study is warranted.     

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

Background  

Isotope-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.     

乳头抽吸液中细胞及生物化学成分的研究

乳头抽吸液是由乳腺导管上皮和腺泡上皮细胞分泌的物质。研究表明乳头抽吸液中含有多种细胞和生物化学成分,其中不乏对乳腺癌诊断和预后具有指导意义的肿瘤标记物。     

乳头抽吸液中细胞及生物化学成分的研究

乳头抽吸液是由乳腺导管上皮和腺泡上皮细胞分泌的物质。研究表明乳头抽吸液中含有多种细胞和生物化学成分,其中不乏对乳腺癌诊断和预后具有指导意义的肿瘤标记物。     

Prostate-specific antigen and insulin-like growth factor binding protein-3 in nipple aspirate fluid are associated with breast cancer

Insulin-like growth factor-1 (IGF-1) is an important growth factor for breast cancer cells and insulin-like growth factor binding protein-3 (IGFBP-3) its most prevalent binding protein. Prostate-specific antigen (PSA) enzymatically cleaves IGFBP-3 into fragments (BP3-FR). Our purpose was to determine the association of these markers in nipple aspirate fluid (NAF) and serum with the presence of breast cancer. NAF from 175 and serum from 215 subjects were collected from women with or without breast cancer. In unadjusted analysis low NAFPSA (P < 0.001) and high NAFIGFBP-3 (P = 0.023) were associated with breast cancer. Low serum PSA was associated with postmenopausal breast cancer (P = 0.034). In separate multivariate analyses, controlling for age, menopausal status, and age at menarche, NAF PSA and IGFBP-3 were each associated with breast cancer. The association was significant for NAF IGFBP-3 in all women (P = 0.031), but for NAF PSA only in premenopausal women (P < 0.001). When considered jointly, only NAF PSA was significant. Therefore, NAF PSA, and to a lesser extent NAF IGFBP-3 and serum PSA, seem to be important predictors of breast cancer.     

Patterns of reduced nipple aspirate fluid production and ductal lavage cellularity in women at high risk for breast cancer

Introduction

Nipple aspiration is a noninvasive technique for obtaining breast fluids from the duct openings of the nipple for the evaluation of abnormalities associated with breast cancer. Nipple aspirate fluid (NAF) can be elicited from 48 to 94% of healthy women, and its production has been linked to an increased relative risk for breast cancer development. NAF production has been used in studies to guide the selection of ducts for ductal lavage, a procedure in which ducts are cannulated and flushed with saline to collect cells. In a previous multicenter trial to evaluate intraductal approaches in women at high-risk for breast cancer, NAF production was observed in 84% of the subjects. However, we observed a significantly lower proportion of fluid-yielding subjects in a similar series of high-risk women. The purpose of the present study was to identify variables associated with this reduction.

Method

Nipple aspiration was performed on 33 high-risk women (defined as having a 5-year Gail model index of more than 1.7, a personal or family history of breast cancer, and/or a BRCA1 or BRCA2 germline mutation) to identify ductal orifices for lavage procedures. Lavage was performed on all fluid-yielding ducts and on nine non-fluid-yielding ducts.

Results

Fluid-yielding ducts were identified in 12 of 33 (36%) of the subjects in the present series, compared with 16 of 19 (84%) of the subjects undergoing identical procedures at our facility during a multicenter trial (P = 0.001). Reduced NAF yields were associated with postmenopausal status (P = 0.02), BRCA germline mutations (P = 0.004), and risk reduction therapies, including bilateral salpingo-oophorectomy (BSO) and/or selective estrogen receptor modulators (SERMs; P = 0.009). All nine (100%) of the ductal lavage specimens collected from non-fluidyielding ducts were acellular, in comparison with 3 of 13 specimens from fluid-yielding ducts (P < .001).

Conclusion

Analysis of high-risk women in the present series revealed patterns of reduced NAF production and ductal lavage cellularity compared with a previous multicenter trial. The present series included more BRCA-positive women, many of whom had undergone BSO and/or were using SERMs. Our data suggest that endocrine mechanisms associated with these risk-reducing therapies may be related to patterns of diminished breast fluid production.