Normal Breast Ducts Research Articles

Identification of Extracellular and Intracellular Signaling Components of the Mammary Adipose Tissue and Its Interstitial Fluid in High Risk Breast Cancer Patients

It has become clear that growth and progression of breast tumor cells not only depend on their malignant potential but also on factors present in the tumor microenvironment. Of the cell types that constitute the mammary stroma, the adipocytes are perhaps the least well studied despite the fact that they represent one of the most prominent cell types surrounding the breast tumor cells. There is compelling evidence demonstrating a role for the mammary fat pad in mammary gland development, and some studies have revealed the ability of fat tissue to augment the growth and ability to metastasize of mammary carcinoma cells. Very little is known, however, about which factors adipocytes produce that may orchestrate these actions and how this may come about. In an effort to shed some light on these questions, we present here a detailed proteomic analysis, using two-dimensional gel-based technology, mass spectrometry, immunoblotting, and antibody arrays, of adipose cells and interstitial fluid of fresh fat tissue samples collected from sites topologically distant from the tumors of high risk breast cancer patients that underwent mastectomy and that were not treated prior to surgery. A total of 359 unique proteins were identified, including numerous signaling molecules, hormones, cytokines, and growth factors, involved in a variety of biological processes such as signal transduction and cell communication; energy metabolism; protein metabolism; cell growth and/or maintenance; immune response; transport; regulation of nucleobase, nucleoside, and nucleic acid metabolism; and apoptosis. Apart from providing a comprehensive overview of the mammary fat proteome and its interstitial fluid, the results offer some insight as to the role of adipocytes in the breast tumor microenvironment and provide a first glance of their molecular cellular circuitry. In addition, the results open new possibilities to the study of obesity, which has a strong association with type 2 diabetes, hypertension, and coronary heart disease.

P63 expression in breast cancer: a highly sensitive and specific marker of metaplastic carcinoma.

p63, a member of the p53 gene family, is involved in cellular differentiation and is expressed in the nuclei of myoepithelial cells of normal breast ducts and lobules. Although p63 has been reported in metaplastic carcinomas of the breast, its expression pattern in breast carcinomas and sarcomas has not been fully characterized, and its potential diagnostic utility has not been defined. In this study, we determined p63 expression in a large number of breast carcinomas, including metaplastic carcinomas, and in Phyllodes tumors and sarcomas. We examined 189 invasive breast carcinomas, including 15 metaplastic carcinomas, as well as 10 Phyllodes tumors, and 5 pure sarcomas of the breast for pattern and intensity of p63 staining using an anti-p63 antibody (clone 4A4, Neomarkers). p63 was strongly expressed in 13 of 15 metaplastic carcinomas (86.7%). p63 was positive in all the metaplastic carcinomas with spindle cell and/or squamous differentiation (12 of 12), and in 1 of 3 metaplastic carcinomas with cartilage foci. In stark contrast, only 1 of 174 (0.6%) nonmetaplastic invasive carcinomas was positive for p63. All Phyllodes tumors and sarcomas were consistently negative for p63 expression. The sensitivity and specificity of p63 as a diagnostic marker for metaplastic carcinoma was 86.7% and 99.4%, respectively. We propose the inclusion of p63 as part of the diagnostic workup of challenging spindle cell tumors of the breast as a highly specific marker for metaplastic carcinomas.

Immunostaining patterns of myoepithelial cells in breast lesions: a comparison of CD10 and smooth muscle myosin heavy chain

Background: Recent studies have reported CD10 expression in myoepithelial cells (MEC) of the breast, supporting its use as a marker to help distinguish invasive breast carcinoma (IC) from ductal carcinoma...

WISP3 (CCN6) Is a Secreted Tumor-Suppressor Protein that Modulates IGF Signaling in Inflammatory Breast Cancer

Inflammatory breast cancer (IBC) is the most lethal form of locally advanced breast cancer. We have found that WISP3 is lost in 80% of human IBC tumors and that it has growth- and angiogenesis-inhibitory functions in breast cancer in vitro and in vivo. WISP3 is a cysteine-rich, putatively secreted protein that belongs to the CCN family. It contains a signal peptide at the N-terminus and four highly conserved motifs. Here, for the first time, we investigate the function of WISP3 protein in relationship to its structural features. We found that WISP3 is secreted into the conditioned media and into the lumens of normal breast ducts. Once secreted, WISP3 was able to decrease, directly or through induction of other molecule(s), the IGF-1-induced activation of the IGF-IR, and two of its main downstream signaling molecules, IRS1 and ERK-1/2, in SUM149 IBC cells. Furthermore, WISP3 containing conditioned media decreased the growth rate of SUM149 cells. This work sheds light into the mechanism of WISP3 function by demonstrating that it is secreted and that, once in the extracellular media, it induces a series of molecular events that leads to modulation of IGF-IR signaling pathways and cellular growth in IBC cells.

Characterization of breast duct epithelia: a Raman spectroscopic study

Spectroscopic characterization of normal breast duct epithelia is the basis for understanding the spectral information obtained from epithelium-derived breast tumors. In this study, healthy breast tissue samples were examined by means of Raman spectroscopy with an excitation wavelength of 840 nm. Raman spectra of breast ducts were acquired using a microscopic mapping approach, and mapping measurements of breast duct epithelium were performed at a lateral resolution that enabled investigation of single epithelial cells. Different cellular compounds within the epithelial layer such as nuclei and cytoplasm were identified by principal components analysis (PCA) and K-means cluster analysis applied to their Raman spectra. Furthermore, spectra of breast secretion fluid that was found to cover the epithelium in most samples were analyzed. They contained mainly signal contributions from lipids. Two different classes of lipid secretion spectra were identified and both spectral classes could be found in the same sample. Spatially resolved analysis of breast duct fluid could provide new insights into breast secretion biochemistry in normal and pathological conditions.

Microinvasive lobular carcinoma associated with intraductal spread arising in a mammary hamartoma

A 53 year old woman presented with a lump in the inner lower quadrant of the left breast. Histological examination of the breast tumour confirmed that the lesion was a mammary hamartoma. Carcinoma with foci of microinvasion was observed in the lobules of the hamartoma concomitant with the intraductal spread of lobular carcinoma. Immunohistochemically, the cancer cells were negative for beta-catenin, which generally stained normal breast ducts and ductal carcinomas. This is only the sixth case of breast carcinoma arising in a mammary hamartoma to be reported and, moreover, the fourth case of lobular carcinoma occurring within a hamartoma. Despite the apparent rarity of this case, pathologists should be aware of the possibility of carcinomas arising within mammary hamartomas.

EMA: a differentiation antigen related to node metastatic capacity of breast carcinomas.

Epithelial membrane antigen (EMA), also known as MUC1, is a mucinous glycoprotein fixed to the luminal domain of the epithelial cell membrane of normal breast ducts. However, in breast cancer cells, it is usually dispersed in the cytoplasm. EMA staining patterns of 330 breast carcinomas were examined, and three groups formed: lineal (16%), cytoplasmic (75%), and negative (9%). Although these patterns were somewhat related to histological cancer types, this was not statistically significant. However, EMA showed statistically significant univariate relationships to tumor grade, tumor size, estrogen and progesterone receptors, and nodal stage. Logistic regression analysis showed that among these variables, all of which were univariately related to node metastasis, only tumor size and EMA were independent nodal stage predictors. A combined analysis of these two factors revealed that the statistical probability of a tumor metastasizing to four or more nodes increased in each tumor size group from 0.9% to 12% for pT1, from 2% to 29% for pT2 and from 10% to 63% for pT3, depending on the EMA staining. The tumors showing a lineal pattern were the least metastasizing, while the EMA-negative tumors were the most. After recognizing these relationships between EMA staining patterns and other well-known differentiation markers and the lymph node metastatic capacity of carcinomas, and considering the results obtained by others on survival, one might conclude that EMA is both a differentiation marker and a histological prognostic factor.

P53 Mutations and Expression in Breast Carcinoma in Situ

The p53 tumor suppressor gene is altered in approximately half of human cancers. Although p53 mutations are common in invasive breast carcinoma, few have been identified in breast carcinoma in situ (intraductal breast carcinomas). Most studies of p53 in breast carcinoma in situ are immunohistochemical studies of p53 staining in paraffin-embedded tissue sections. Few studies have isolated the tumor cells and subjected them to DNA sequence analysis. The current study was undertaken to characterize p53 in a cohort of breast carcinoma in situ cases, both with and without invasive disease. Fifty-eight frozen breast biopsy samples were used for these investigations. Twenty-seven cases had only ductal carcinoma in situ (CIS) and 31 cases had evidence of both invasive and in situ carcinoma. DNA sequence alterations in exons 2 through 11 of p53 were screened by the single-strand conformational polymorphism technique. Exons with altered mobility were sequenced. Among breast CIS cases without invasive disease, 22% had p53 mutations and 7% had DNA sequence alterations of unknown significance. Analysis of breast CIS with concurrent invasive disease demonstrated p53 mutations in 19% of cases and one (3%) DNA alteration of unknown significance. Each carcinoma having a p53 mutation in the breast CIS component had the identical mutation in the invasive component of the same tumor indicating a clonal relationship between the two tumor components. p53 protein overexpression was identified in 22% of pure intraductal breast carcinomas and in 35% of breast CIS with invasive disease. Comparison of immunostaining and DNA sequence alterations showed a significant association between overexpression and mutations (P = 0. 0037) in cases of CIS without invasion, and similarly between overexpression and mutations in cases of CIS with invasion (P = 0. 007). p53 mutations and p53 overexpression were relatively common in intraductal breast carcinomas but were not observed in adjacent normal breast lobules or ducts in 9 cases available for DNA analysis. The frequency of p53 alterations when comparing breast CIS with and without an invasive component indicated that p53 mutations usually occur before invasion during the progression of breast cancer, as is observed for a number of other adult solid tumors.

The potential role for prolactin-inducible protein (PIP) as a marker of human breast cancer micrometastasis.

The prolactin-inducible protein (PIP/GCPD15) is believed to originate from a limited set of tissues, including breast and salivary glands, and has been applied as a clinical marker for the diagnosis of metastatic tumours of unknown origin. We have investigated the potential role of PIP mRNA as a marker of human breast cancer metastasis. Using reverse transcription polymerase chain reaction and Southern or dot blot analysis, PIP mRNA was detected in 4/6 breast cell lines, independent of oestrogen receptor (ER) status. In breast primary tumours (n = 97), analysed from histologically characterized sections, PIP mRNA was detected in most cases. Higher PIP mRNA levels correlated with ER+ (P = 0.0004), progesterone receptor positive (PR+) (P = 0.0167), low-grade (P = 0.0195) tumours, and also PIP protein levels assessed by immunohistochemistry (n = 19, P = 0.0319). PIP mRNA expression was also detectable in 11/16 (69%) of axillary node metastases. PIP mRNA expression, however, was also detected in normal breast duct epithelium, skin, salivary gland and peripheral blood leucocyte samples from normal individuals. We conclude that PIP mRNA is frequently expressed in both primary human breast tumours and nodal metastases. However, the presence of PIP expression in skin creates a potential source of contamination in venepuncture samples that should be considered in its application as a marker for breast tumour micrometastases. © 1999 Cancer Research Campaign

The Fas/Fas-ligand system: a mechanism for immune evasion in human breast carcinomas.

Breast tumors are frequently associated with a predominantly lymphocytic infiltrate, which constitutes an immune response against the tumor. In spite of this massive infiltrate, the immune response appears to be inefficient and the tumor is able to evade it. We propose that in breast cancer, tumor escape from immunological surveillance results from the induction of apoptosis of Fas-bearing activated lymphocytes by FasL-bearing breast cancer cells. To test this proposal we studied the expression of FasL by human breast carcinomas and the MCF-7 breast cancer cell line by RT-PCR, immunohistochemistry, and Western Blot. Moreover, we describe the presence of apoptosis and Fas expression in the lymphocytic population surrounding the tumor. Strong membranous and cytoplasmic staining was detected in ductal carcinomas and hyperplastic breast tissue, but it was absent from normal breast tissue. No staining was found in normal glands in the non-tumor quadrants; however, the normal appearing ducts surrounding the carcinoma (tumor quadrant) showed intense immunoreactivity. Apoptosis was found predominantly among the lymphocytic population, as well as in the blood vessels and fibro-fatty tissue close to the tumor. Further characterization of apoptotic cells demonstrated that they were CD3+ cells. Our results suggest the breast tumors may elude immunological surveillance by inducing, via the Fas/FasL system, the apoptosis of activated lymphocytes. Recent data have demonstrated FasL RNA in other tumor types. Upregulation of FasL expression in hyperplastic and normal breast ducts close to the tumor also suggests a possible role in early neoplastic transformation and proliferation.

Heterogeneous expression of the lipocalin NGAL in primary breast cancers

We have previously shown that neu oncogene-initiated rat mammary carcinomas uniquely over-express neu-related lipocalin (NRL), a member of the calycin protein superfamily. Here, we characterize the putative human homolog of NRL, neutrophil gelatinase-associated lipocalin (NGAL). ngal gene expression was found at moderate levels in only 2 of 17 human tissues examined, breast and lung. When breast cancers were examined for NGAL mRNA and protein levels, they were found to exhibit heterogeneous expression. NGAL levels varied in these tumors from undetectable to exceeding those in normal breast parenchyma. Immuno-histochemical analysis confirmed the presence of NGAL within breast carcinoma cells but detected only low levels of this protein in normal ductal epithelium. In contrast, large amounts of the protein were localized to the lumen of normal breast ducts in the vicinity of NGAL-expressing tumors. Interestingly, unlike NRL in rat mammary carcinomas, no significant association between NGAL expression and HER-2/neu activation was found in human breast tumors. In contrast, a significant correlation between NGAL expression in breast cancer was found with several other markers of poor prognosis, including estrogen and progesterone receptor-negative status and high proliferation (S-phase fraction). NGAL levels were stratified as high or low in breast cancers from a cohort of node-positive patients with known outcome. No significant association between NGAL expression and disease-free or overall survival was observed. Int. J. Cancer (Pred. Oncol.) 79:565–572, 1998. © 1998 Wiley-Liss, Inc.

Heterogeneous expression of the lipocalin NGAL in primary breast cancers.

We have previously shown that neu oncogene-initiated rat mammary carcinomas uniquely over-express neu-related lipocalin (NRL), a member of the calycin protein superfamily. Here, we characterize the putative human homolog of NRL, neutrophil gelatinase-associated lipocalin (NGAL). ngal gene expression was found at moderate levels in only 2 of 17 human tissues examined, breast and lung. When breast cancers were examined for NGAL mRNA and protein levels, they were found to exhibit heterogeneous expression. NGAL levels varied in these tumors from undetectable to exceeding those in normal breast parenchyma. Immuno-histochemical analysis confirmed the presence of NGAL within breast carcinoma cells but detected only low levels of this protein in normal ductal epithelium. In contrast, large amounts of the protein were localized to the lumen of normal breast ducts in the vicinity of NGAL-expressing tumors. Interestingly, unlike NRL in rat mammary carcinomas, no significant association between NGAL expression and HER-2/neu activation was found in human breast tumors. In contrast, a significant correlation between NGAL expression in breast cancer was found with several other markers of poor prognosis, including estrogen and progesterone receptor-negative status and high proliferation (S-phase fraction). NGAL levels were stratified as high or low in breast cancers from a cohort of node-positive patients with known outcome. No significant association between NGAL expression and disease-free or overall survival was observed.

Upregulation of ATM in sclerosing adenosis of the breast.

The gene mutated in ataxia telangiectasia (ATM) has an established tumour suppressor role in breast cancer. ATM appears to be expressed in most normal cells, including breast epithelium, where it...

Immunohistochemical localization of prostate-specific antigen in benign and malignant breast tissues.

Prostate-specific antigen (PSA), a glycoprotein initially thought to be produced only by the epithelial cells of the prostate, has recently been found in 30% of female breast tumours using immunofluorometry. Our aim was to localize PSA immunohistochemically in a selected group of 27 paraffin-embedded breast tissues. A scoring system was developed for the histological assessment of PSA positivity within the breast tissue. One pathologist (DH) scored, classified and graded all tumours. Site-specific PSA staining was noted in the histology slides. Intense staining was identified in apocrine metaplasia and within the lining ductal epithelium of cystically dilated ducts. The epithelium in lesions of sclerosing adenosis was also frequently positive for PSA staining. Hyperplastic ductal epithelium (especially of mild degree) occasionally stained positive, as did normal breast ducts. Better differentiated tumours showed PSA staining [e.g. mucinous carcinoma (colloid)]. If an infiltrating duct carcinoma showed staining for PSA, adjacent intraductal carcinoma was also noted to stain positively, if present.

Clinical significance of bcl-2 gene expression in human breast cancer tissues.

The expression of estrogen receptor (ER) and bcl-2 (Bcl-2), an apoptosis protective oncogene, in normal and cancerous breast duct epithelia was immunohistochemically examined in fresh frozen tumor tissues from 142 Japanese breast cancer patients. The clinico-pathological characteristics and the disease free survival of the patients were analyzed. The expression of both the proteins was also observed in intraductal components of breast cancer. Although less than 1% of normal duct epithelia expressed ER, Bcl-2 was diffusely expressed. The expression of both these proteins in breast cancer significantly correlated with each other. Their expression significantly correlated negatively with tumor size but not with lymph node status. The papillo-tubular sub-type of invasive ductal carcinoma expressed Bcl-2 significantly more frequently than the solid-tubular sub-type. Patients with Bcl-2 expressing tumors survived without recurrence significantly more than those with tumors exhibiting reduced expression. Papillary-cribriform type intraductal components expressed both those proteins more often than the solid-comedo type.

The myoepithelial defense: a host defense against cancer

The behavior of human tumors depends not only on the nature of the tumor cells themselves but also on the modifying effects of various normal host cells such as fibroblasts and endothelial cells. One cell type, however — the myoepithelial cell — has not been studied scientifically. Myoepithelial cells normally surround ducts and acini of glandular organs such as the breast and salivary glands and contribute to the synthesis of a surrounding basement membrane. This relationship suggests that myoepithelial cells may exert paracrine effects on glandular epithelium and also regulate the progression of ductal carcinoma in situ (DCIS) to invasive carcinoma. Myoepithelial tumors, in turn, tend to be benign or low-grade neoplasms that exhibit the rare property of accumulating rather than degrading extracellular matrix material. To better understand the nature of myoepithelial tumors, as well as the possible role of normal myoepithelial host cells in cancer, we have established immortal cell lines and a number of transplantable xenografts from various human myoepithelial tumors of the salivary gland and breast. The cell lines exhibit a normal myoepithelial phenotype and the xenografts continue to accumulate an abundant extracellular matrix. Further ultrastructural, immunocytochemical, molecular, and bochemical studies reveal that myoepithelial cells secrete relatively low levels of matrix-degrading proteinases but relatively high levels of maspin and various other anti-invasive proteinase inhibitors, that some of these inhibitors accumulate within the myoepithelial matrix, and that myoepithelial cells can induce epithelial morphogenesis (spheroid formation) and inhibit tumor-cell invasion in vitro. Myoepithelial cells, which surround normal breast ducts and DCIS, have also been found to selectively express maspin and certain proteinase inhibitors in situ. These inherent myoepithelial properties are likely to contribute to the low-grade nature of myoepithelial neoplasms and advance our hypothesis that host myoepithelial cells regulate the progression of in situ to invasive carcinoma by providing an important host defense against cancer invasion.

Immunohistochemical localisation of hydroxysteroid sulphotransferase in human breast carcinoma tissue: a preliminary study

Understanding the function and regulation of the metabolism of steroid hormones by breast tumours will be instrumental to the development of novel treatments for this widespread disease. We have examined the expression of hydroxysteroid sulphotransferase, an enzyme which inactivates many steroids, in particular androgens, in normal breast tissue and in six ductal-type mammary carcinoma using immunohistochemistry. The enzyme is not expressed in the epithelial cells which line the normal breast duct, but is present in significant amounts in neoplastic cells, suggesting that the gene encoding this protein is activated at some stage of the neoplastic transformation. The implications of this finding for the role of steroid metabolism in breast cancer are discussed.

Breast Carcinoma with Positive Results for Melanoma Marker (HMB-45): HMB-45 Immunoreactivity in Normal and Neoplastic Breast

HMB-45 (melanocytic cell-specific monoclonal antibody) immunoreactivity has been detected by the immunoperoxidase technique in the cytoplasm of 2 of 100 breast carcinomas. In addition, normal breast lobules and ducts were occasionally found to have positive results in 6 of 100 cases. These findings indicate the need to exercise caution in the interpretation of HMB-45 immunoreactivity for immunohistochemical characterization of neoplasms.

Cell proliferation in normal human breast ducts, fibroadenomas, and other ductal hyperplasias measured by nuclear labeling with tritiated thymidine: Effects of menstrual phase, age, and oral contraceptive hormones

The proliferative rates of terminal duct cells were studied by tritiated thymidine labeling in vitro in 104 specimens of benign breast tissue from 92 women. The tritiated thymidine labeling index showed a cyclic pattern consistent with regulation by female sex hormones. It was low during the segment of the menstrual cycle encompassing days 2 through 15 (early cycle) and often was elevated during the segment encompassing days 16 through 1 (late cycle). The early cycle geometric mean index for morphologically normal terminal ducts was 0.19, and for ducts of fibroadenomas it was 0.77. The geometric means for late cycle were 1.03 and 1.67 respectively. The difference for normal ducts between early and late cycles was highly significant (P<0.001). For both normal ducts and fibroadenomas the tritiated thymidine labeling index correlated negatively with the age of the patient, and the higher index value of fibroadenoma ducts relative to normal ducts was entirely accounted for by the younger mean age of the fibroadenoma patients. Oral use of contraceptive hormones could not be shown to change the index in either normal ducts or fibroadenoma ducts. Based on a postulated duration of DNA synthesis of 12 hours, the mean turnover time of the cells of normal terminal ducts would be approximately 22 days for 20 year old women with regular menstrual cycles, and would increase to 147 days at age 40. the mean potential doubling times of fibroadenomas would be approximately 33 days. The index values of other benign ductal proliferative lesions of the breast were similar to those of fibroadenomas and normal terminal ducts.