Differentiation of tumours of ductal and lobular origin: I. Proteomics of invasive ductal and lobular breast carcinomas

The vast majority of invasive breast tumors are ductal and lobular breast carcinomas. Despite the many similarities, some clinical follow-up data and the patterns of metastases suggest that these histological subtypes of breast cancer are biologically distinct. Few papers, however, describe immunohistochemical markers useful for differentiation of these carcinomas. Many investigations suggest that E cadherin protein expression is lost in lobular but not in ductal carcinoma. The absence of E-CD, as a partial loss of epithelial differentiation, may account for the extended spread of lobular carcinoma in situ and the peculiar diffuse invasion mode of invasive lobular carcinoma. Some investigations report the significance of E-CD associated proteins alpha-, beta-, gamma-catenin expression, as well as the usefulness of cytokeratins 5, 6, 8, 7 and thrombospondin in differentiating histological types of breast invasive carcinomas. Several reports have suggested the possibility that invasive ductal and lobular cancers differ with respect to expression of antigens involved in proliferation and cell cycle regulation. It has been shown that vascular endothelial growth factor expression, also the expression of maspin, a tumour suppressor gene product, is higher in ductal, than in lobular carcinoma. Expression of NKX3.1, a member of the NK-class of homeodomain, is highly restricted and is found primarily in lobular carcinoma. Some histological and immunohistochemical characteristics of pleomorphic lobular carcinoma are also discussed.     

Differentiation of tumours of ductal and lobular origin: II. Genomics of invasive ductal and lobular breast carcinomas

Breast cancer is considered to be a multifactorial disorder caused by both genetic and non-genetic factors. Different histological types of breast cancer differ in response to treatment and may have a divergent clinical course. Breast tissue is heterogeneous, with components of epithelial, mesenchymal, endothelial and lymphopoietic derivation. The genetic heterogeneity of invasive breast cancer is reflected by the wide spectrum of histological types and differentiation grades. Nevertheless, the influences of these cell types on the tumour's total pattern of gene expression can be estimated analytically. Microarrays permit total tissue analysis and provide a stable molecular portrait of tumours. Some investigations suggest differences in the gene expression profiling for ductal and lobular carcinomas. It has been reported that inactivating mutations of the E-cadherin gene are very frequent in infiltrating lobular breast carcinomas. Other than altered expression of E-cadherin, little is known about the underlying biology that distinguishes ductal and lobular tumour subtypes. However, about 8 genes have been identified differentially which are expressed in lobular and ductal cancers: E-CD, survivin, cathepsin B, TPI1, SPRY1, SCYA14, TFAP2B, and thrombospondin 4, osteopontin, HLA-G, and CHC1. Expression profiling of breast cancers can be used diagnostically to distinguish individual histologic subclassifications and may guide the selection of target therapeutics. However, future approaches will need to include methods for high throughput clinical validation and the ability to analyze microscopic samples.     

Characterization of spontaneous mammary gland carcinomas in female baboons

Spontaneous mammary gland carcinomas occurred in five baboons during a 13-year period at Southwest Foundation for Biomedical Research. The affected baboons ranged in age from 21 to 33 years. Menopause in the baboon occurs at approximately 26 years of age. All five animals had typical invasive ductal carcinoma. Morphologically, the tumors were characterized by neoplastic cells arranged from pseudopapillary and cribiform to more poorly differentiated solid cellular growth patterns. Additional features included lack of tubule formation (4/5), marked nuclear pleomorphism (5/5), a high mitotic rate (4/5) and tumor necrosis (4/5). Applying a grading system used for breast cancer in women, four tumors were graded as poorly differentiated carcinomas and one was graded as moderately differentiated. Co-existent ductal carcinoma in situ (DCIS) was observed in three of the mammary tumors. Metastases to the regional lymph nodes were confirmed in two animals, both with histological evidence of lymphovascular invasion in the primary tumor. Distant metastases were observed in only one animal. Immunohistochemical staining for human therapeutic markers revealed 2/5 tumors strongly positive for estrogen receptor, 1/5 strongly positive for progesterone receptor and 4/4 negative for HER2 expression. Although the incidence appears to be low, these five cases of mammary carcinoma in female baboons suggest that when present baboon mammary carcinoma is usually of ductal origin and behaves similar to a human breast carcinoma.     

Novel immunohistochemical markers for the differentiation of lobular and ductal invasive breast carcinomas

AIMS. Invasive ductal and lobular carcinomas are the most common histological types of breast cancer. The loss of E-cadherin expression has been suggested to be the most reliable marker for invasive lobular carcinoma. The aim of our study was to identify the diagnostic usefulness of novel markers in the differentiation of these tumor types. METHODS. We examined tissue microarrays (TMA) which were constructed from surgical specimens of 119 breast cancer patients. TMA consisted of 80 ductal carcinomas, 29 lobular carcinomas and special type cancers. TMA sections were stained using standard immunohistochemical methods. Monoclonal mouse antibodies against E-cadherin, cytokeratin 5/6 and 17, and polyclonal mouse antibodies against EMP1, DDR1, PRKCI and DVL1 were used. RESULTS. E-cadherin was absent in 93.3% of lobular tumors compared with only 15 % of ductal tumors (p<0.0001). EMP1 and DVL1 were overexpressed in lobular tumors (93.1% and 96.5%, respectively), whereas PRKCI and DDR1 were positive in ductal cancers (90% and 96.2%, respectively). Reduced expression or absence of both cytokeratins 5/6 and 17 was found in both tumor tissues in comparison to normal terminal duct lobular units (p<0.0001). CONCLUSIONS. Apart from the well-established marker, E-cadherin, proteins examined on TMA slides by immunohistochemistry (EMP1, DVL1, DDR1, PRKCI) may represent novel tissue markers helpful in the differentiation of ductal and lobular breast cancers. Further studies with larger sets of patients are desirable, to verify the complete immunohistochemical profiles of various histological types of breast cancer and determine the prognostic and predictive significance of novel markers.     

Identification of BCOX1, a novel gene overexpressed in breast cancer

The identification of tumor-associated antigens, which are specifically expressed in cancer tissues, is of utmost important for immunotherapy of breast cancer. We have combined in silico screening and experimental expression analysis to identify genes that are differentially expressed in breast carcinomas compared with their corresponding normal tissues. Using these approaches, we identified a novel gene, BCOX1, with overexpression in breast carcinoma. BCOX1 was highly homologous to KIAA0100, a hypothetical gene located on chromosome 17q11.2. RNA in situ hybridization shows that BCOX1 mRNA signal is mainly located in the cytoplasm of breast carcinoma epithelial cells, but not in those of normal epithelial cells, stroma cells and lymphocytes. Furthermore, mRNA expression of BCOX1 was moderately elevated in ductal in situ carcinoma (DCIS), peaked in invasive breast carcinoma (IBC) and metastatic breast carcinoma cells (MET) whereas absent in benign ductal epithelial cells. The predicted BCOX1 open reading frame of 666 bp encodes a putative protein of 222 amino acid residues with a calculated molecular weight of 2,4920 Da and a PI of 5.86. Computational analyses predict that the putative BCOX1 protein is a cytoplasmic protein. The functional relevance of this novel gene is yet to be determined. This study warrants further investigations to explore the molecular functions of BCOX1, and to determine its potential diagnostic and therapeutic applications for breast cancer.     

Differential expression of human tissue factor in normal mammary epithelial cells and in carcinomas.

BACKGROUND: Tissue factor (TF) is a glycoprotein which binds factor VIIa. The TF-VIIa complex serves as a potent initiator of the coagulation pathways. TF, an immediate early gene, may also play a role in cell growth. Expression of TF was correlated with some types of cancers. MATERIALS AND METHODS: Normal, immortalized, and tumor human mammary epithelial cells were used in the experiments. The differential display (DD) technique was used to identify genes differentially expressed in the cells. TF expression patterns were examined by Northern blot analysis, immunofluorescence staining of cultured cells, and immunohistochemical staining in human cryostat sections. RESULTS: In a 5-way display, an amplified polymerase chain reaction (PCR) product was found in normal and immortalized human mammary epithelial cells but not in the breast cancer cells. The PCR fragment was cloned and sequenced. The result showed that the fragment was identical to human tissue factor. Northern blot analysis showed that expression level of tissue factor mRNA remained high in growing, quiescent, and senescent normal mammary epithelial cells. Immunofluorescence staining also confirmed tissue factor expression pattern in the cell lines tested. Immunohistochemical staining showed that tissue factor was expressed in the normal luminal and myoepithelial cells of some ducts but not others. No staining was observed in invasive carcinoma cells. However, myoepithelial cell staining was seen in some residual ductal structures in invasive tumors. CONCLUSIONS: This study shows the use of DD to reveal the loss of TF expression pattern in human breast cancer cell lines. Immunohistochemical staining results showed breast carcinoma cells expressed little TF, if any, suggesting that TF is not required for breast tumor cell invasion. The results also indicated that TF expression was independent of the proliferation status of the expressing cells. The expression pattern of TF may be a meaningful marker in the development of breast cancer.     

Arylamine N-acetyltransferase-1 is highly expressed in breast cancers and conveys enhanced growth and resistance to etoposide in vitro

Comparative two-dimensional proteome analysis was used to identify proteins differentially expressed in multiple clinical normal and breast cancer tissues. One protein, the expression of which was elevated in invasive ductal and lobular breast carcinomas when compared with normal breast tissue, was arylamine N-acetyltransferase-1 (NAT-1), a Phase II drug-metabolizing enzyme. NAT-1 overexpression in clinical breast cancers was confirmed at the mRNA level and immunohistochemical analysis of NAT-1 in 108 breast cancer donors demonstrated a strong association of NAT-1 staining with estrogen receptor-positive tumors. Analysis of the effect of active NAT-1 overexpression in a normal luminal epithelial-derived cell line demonstrated enhanced growth properties and etoposide resistance relative to control cells. Thus, NAT-1 may not only play a role in the development of cancers through enhanced mutagenesis but may also contribute to the resistance of some cancers to cytotoxic drugs.     

Promoter Hypermethylation of ARID1A Gene Is Responsible for Its Low mRNA Expression in Many Invasive Breast Cancers

ARID1A (AT-rich interactive domain 1A) has recently been identified as a tumor suppressor gene. Its mRNA expression is significantly low in many breast cancers; this is often associated with more aggressive phenotypes. However, the underlying molecular mechanism for its low expression has not been fully understood. This study was undertaken to evaluate the contribution of gene copy number variation, mutations, promoter methylation and histone modification to ARID1A’s low expression. 38 pairs of breast invasive ductal carcinomas and their normal breast tissue counterparts from the same patients were randomly selected for gene expression and copy number variation detection. Promoter methylation and histone modification levels were evaluated by MeDIP-qPCR and ChIP-qPCR, respectively. PCR product Sanger sequencing was carried out to detect the exon mutation rate. Twenty-two out of 38 invasive ductal carcinomas in the study (57.9%) revealed ARID1A mRNA low expression by realtime RT-PCR. The relative promoter methylation level was, significantly higher in ARID1A mRNA low expression group compared with its high expression group (p<0.001). In the low expression group, nineteen out of 22 invasive ductal carcinomas (86.4%) exhibited ARID1A promoter hypermthylation. In addition, the promoter hypermethylation was accompanied with repressive histone modification (H3K27Me3). Although five out of 38 invasive ductal carcinomas (13.2%) exhibited loss of ARID1A gene copy number by realtime PCR and nine exon novel mutations are seen from eight out of 33 invasive ductal carcinomas (24.2%), there was no statistically significant difference in both ARID1A mRNA low and high expression groups (p = 0.25,and p = 0.68, respectively). We demonstrate that promoter hypermethylation was the main culprit for ARID1A mRNA low expression in invasive ductal carcinomas. The influence of mutation and copy number variation on the expression were statistically insignificant at mRNA level, and were, therefore, not considered the main causes for ARID1A mRNA low expression in invasive breast cancer.     

Automated microscopic image analysis and the prognosis of preneoplasms and carcinomas of the mammary gland

Studies of preneoplasias and carcinomas of the mammary gland have been conducted by means of automated microscopic image analysis, on the basis of previous results as well as with references to the international literature, for 2 purposes: 1. Determination in the context of a clinical follow-up study of the individual carcinoma risk for patients with proliferative fibrocystic breast disease (mastopathy) and 2. Preparation of an objective automated grading of ductal breast carcinomas for better assessment of prognosis. Against the background of the assumption that the majority of carcinomas and precancerous lesions of the breast originate from the terminal ductal lobular unit, an effort is made to determine, independent severity of the mastopathy, the biological valence of solid, cribriform, and papillary ductal epithelial proliferations (no, possible or inevitable preneoplasia). Proliferation patterns without and with atypical features are checked for their similarity with intraductal and invasive carcinomas (similarity principle) and are additionally examined for differences, depending on localization and distance from tumour (topological principle). Automated histological tumour grading is to distinguish with greater subtlety within the large heterogeneous group of moderately differentiated carcinomas and is to objectify and thus facilitate the difficult task of delimitation of moderately and poorly differentiated carcinomas. Nuclear grading will be the major basis for classification along these lines. Objectified and reproducible tumour grading is believed to be extremely helpful in better prognostication of breast cancer.     

M-CSF (monocyte colony stimulating factor) and M-CSF receptor expression by breast tumour cells: M-CSF mediated recruitment of tumour infiltrating monocytes?

Infiltrating immune cells in 30 primary human epithelial breast tumours were studied using specific anti-CD3 (T cells), anti-CD68 (macrophages), anti-CD57 (NK cells), and an anti-pan-B cell antibody (L26). The majority of tumour infiltrating inflammatory cells are T cells (40-50%) and monocytes/macrophages (15-35%). The macrophage specific chemo-attractant and growth factor CSF-1 is detected by immunohistochemical techniques (IHC) at the level of invasive breast cancer cells in 46/50 tumours but not at the level of in-situ (pre-invasive) cancer. A mosaic staining pattern was usually observed, with a very high expression in areas of obvious stromal invasion (90% cells positive) and absent or trace staining in intraductal carcinoma. Macrophages and plasma cells are equally intensely positive. In-situ hybridisation experiments confirm the production of CSF-1 (mRNA) by tumour cells and show the same pattern of expression. Expression of the CSF-1 receptor protein (fms) was also observed by IHC in 41/48 invasive tumours, albeit at weaker intensities than in tumour infiltrating monocytes/macrophages. A concomitant expression of both CSF-1 and fms in in-situ carcinoma was never seen (n = 14). It is therefore proposed that the associated expression of CSF-1 and its receptor may be linked to the invasive potential of breast cancer, the monocytic infiltrate being an indication of the quantitative importance of CSF-1 production by the tumour.     

Increased angiotensin II type-2 receptor density in hyperplasia,DCIS and invasive carcinoma of the breast is paralleled with increased iNOS expression

Binding of angiotensin II on the angiotensin II type-1 receptor induces cell growth, while triggering via the angiotensin II type-2 (AT(2)) receptor causes an opposing effect of growth inhibition and apoptosis. AT(2) receptor stimulation has also been shown to enhance inducible nitric oxide synthase (iNOS) expression, an enzyme associated with cancer. To study the involvement of the angiotensin II receptors and iNOS in the carcinogenesis of human breast, we visualised both factors in tissues from patients with hyperplasia, ductal carcinoma in situ (DCIS) and invasive carcinoma using immunocytochemistry and in situ hybridisation. In normal ducts, levels for AT(2) protein and mRNA are low, but these are markedly increased in all pathological tissues. While in normal tissue both negative and positive ducts are found, the staining patterns in hyperplasia, DCIS and invasive carcinoma have a homogeneous positive appearance. Similarly, iNOS enzyme expression was very low in the ductal epithelium of normal tissues, but highly increased in all pathologies, with the highest expression found in hyperplastic ducts. Three human cell lines were assayed for the presence of AT(2) receptor. Normal HMec 1001-3 cells were weakly positive, but only one of the adenocarcinoma cell lines, designated SK-BR-3, was shown to express both AT(2) protein and its mRNA. We show that AT(2) receptor and iNOS overexpression are associated with breast disease, further confirming the involvement of the components of the renin-angiotensin system in the aetiology of breast cancer.