The prognostic significance of transforming growth factors in human breast cancer.

Transforming growth factor alpha (TGF alpha) and Transforming growth factor beta-1 (TGF-beta 1) are growth regulatory for breast cancer cell lines in vitro and several studies have suggested that levels of the receptor for TGF alpha, the epidermal growth factor (EGFR) in tumour biopsies predict relapse and survival. We have examined the prognostic significance of TGF alpha, TGF-beta 1 and EGFR mRNA expression in a series of patients with primary breast cancer with a median follow up period of 60 months. In 167 patients the expression of TGF-beta 1 was inversely correlated with node status (P = 0.065) but not ER status, tumour size or menopausal status. Patients with high levels of TGF-beta 1 had a longer disease free interval with a significantly longer probability of survival at 80 months although the overall relapse free survival was not increased. EGFR mRNA expression was measured in 106 patients and was inversely correlated with ER status (P = 0.018). EGFR levels did not predict for early relapse or survival. TGF alpha mRNA levels were measured in 104 patients, no correlation was seen tumour size, node status, Er status, or clinical outcome.     

Upregulation of basic fibroblast growth factor in breast carcinoma and its relationship to vascular density, oestrogen receptor, epidermal growth factor receptor and survival

Background: Angiogenesis, the process whereby endothelial cells divide and migrate to form new blood capillaries, has been assessed in tumours by measuring microvessel density. High microvessel density is a significant adverse prognostic factor in breast cancer. The angiogenic factor, basic fibroblast growth factor (bFGF), has been associated with tumourigenesis and metastasis in several human cancers. There are few quantitative studies of bFGF expression in normal tissues compared to cancer.Patients and methods: We have measured bFGF levels in 149 human primary breast carcinomas and assessed the findings in relation to microvessel density, oestrogen receptor (ER) and epidermal growth factor receptor (EGFR).Basic FGF levels were measured by ELISA. Western blotting and immunohistochemistry were carreid out to confirm the presence of bFGF.Results: Levels of bFGF were more than 10-fold higher in tumour cytosols compared to reduction mammoplasty tissue and 3-fold compared to non neoplastic cytosols from the same breast as the tumour (P < 0.0001). Immunohistochemistry showed bFGF protein was localised exclusively in the stroma whereas no bFGF staining was observed in the epithelial cells. High bFGF levels were significantly related to high ER (P = 0.01). Similarly, high bFGF levels were significantly related to low grade (P = 0.046) and to small tumour size (P = 0.04). No significant relationship was observed between bFGF and microvessel count, EGFR or age. In univariate analysis and in a Cox proportional hazard model bFGF did not reach significance for overall or relapse free survival.Conclusions: Our results show that although bFGF is elevated in breast carcinomas compared to normal breast tissue it is not related to microvessel density and it is not an independent predictor of survival in breast cancer patients. Basic FGF may be one of multiple factors that synergise with other growth factors such as VEGF to enhance angiogenesis.     

Estrogen Receptor Alpha Gene Expression in Breast Cancer Tissues from the Iranian Population - a Pilot Study

Estrogen receptor alpha (ERα) is one of the major sub-types of estrogen receptors. ERα plays an important rolein cellular proliferation and differentiation, chiefly in mammary tissues. In the present study we aimed to quantifyof ERα mRNA and protein expression in breast tissues from the Iranian population using a real-time PCR assay.Twenty nine breast tissues including 19 adenocarcinomas and 10 normal controls were recruited from the Iranianpopulation. mRNA extraction and cDNA synthesis were performed from these tissues using commercial kits.ERα mRNA and protein expression was quantified using real-time PCR and immunohistochemistry respectively.The results showed high expression of ERα mRNA (68%) and protein (53%) in the majority of breast cancertissues compared to normal breast tissues (p= 0.035). Also, high ERα mRNA was associated with tumour size ofbreast carcinomas. In this study, we first reported the expression of ERα in Iranian patients with breast cancersand demonstrated prevalence of the expression to be similar to breast cancers noted in other populations.     

Extending the benefits of adjuvant therapy in early HR+ breast cancer

INTRODUCTION: Oestrogen receptor alpha (ER alpha) is traditionally measured on all breast tumour specimens to identify those patients more likely to respond to anti-oestrogens. Progesterone receptor (PR) status has contributed useful information in defining more responsive subgroups. PR negativity may be a marker for increased signalling through growth factor receptor tyrosine kinase pathways. Progesterone acts through two PRs, PRA and PRB. PRB, the functionally active PR, can be silenced by promoter hypermethylation. METHODS: Following DNA and RNA extraction from 94 breast carcinomas, the methylation status of the PRB promoter was assessed by sodium bisulphite modification and methylation sensitive PCR (MSP). A quantitative realtime PCR analysis (QRTPCR) was used to determine the levels of PRB mRNA expression. Protein expression was evaluated immunohistochemically with a commercially available PRB antibody. RESULTS: 76% of the primary breast carcinoma samples demonstrated a methylated band for PRB. PRB methylation significantly compromised total PR immunohistochemistry (IHC) expression (P = 0.03). PRB mRNA correlated positively with total PR IHC (r = 0.58, P = 0.04), ER alpha IHC (P = 0.02), and tumour grade (P = 0.01). PRB protein expression was significantly associated with a number of favourable prognostic variables including smaller (P = 0.004) lower grade (P = 0.007), ER alpha IHC positive tumours (P < 0.001), and tumours with a low Nottingham Prognostic Index (NPI) (P = 0.0008). PRB mRNA levels were significantly associated with better overall survival (P = 0.04) in a univariate analysis. CONCLUSION: The majority of tumours were methylated for PRB. This did not directly compromise PRB expression suggesting that other factors may down regulate the PR gene. When PRB was expressed, it correlated with good prognostic markers and better overall survival.     

Stanniocalcin 2 is an estrogen-responsive gene coexpressed with the estrogen receptor in human breast cancer

Differences in gene expression are likely to explain the phenotypic variation between hormone-responsive and hormone-unresponsive breast cancers. In this study, DNA microarray analysis of approximately 10,000 known genes and 25,000 expressed sequence tag clusters was performed to identify genes induced by estrogen and repressed by the pure antiestrogen ICI 182 780 in vitro that correlated with estrogen receptor (ER) expression in primary breast carcinomas in vivo. Stanniocalcin (STC) 2 was identified as one of the genes that fulfilled these criteria. DNA microarray hybridization showed a 3-fold induction of STC2 mRNA expression in MCF-7 cells in < or = 3 h of estrogen exposure and a 3-fold repression in the presence of antiestrogen (one-way ANOVA, P < 0.0005). In 13 ER-positive and 12 ER-negative breast carcinomas, the microarray-derived mRNA levels observed for STC2 correlated with tumor ER mRNA (Pearson's correlation, r = 0.85; P < 0.0001) and ER protein status (Spearman's rank correlation, r = 0.73; P < 0.0001). The expression profile of STC2 was further confirmed by in situ hybridization and immunohistochemistry on a larger cohort of 236 unselected breast carcinomas using tissue microarrays. STC2 mRNA and protein expression were found to be associated with tumor ER status (Fisher's exact test, P < 0.005). The related gene, STC1, was also examined and shown to be associated with ER status in breast carcinomas (Fisher's exact test, P < 0.05). This study demonstrates the feasibility of using global gene expression data derived from an in vitro model to pinpoint novel estrogen-responsive genes of potential clinical relevance.     

Aromatase activity and estradiol in human breast cancer: its relationship to estradiol and epidermal growth factor receptors and to tumor-node-metastasis staging.

PURPOSE: The present report attempts to clarify whether there is a relationship between aromatase activity (ARAC) and estradiol (E2), hormonal receptors, E2 receptor (ER), and epidermal growth factor receptor (EGFR), as well as with tumor stage and histopathology in human breast cancers. MATERIALS AND METHODS: We studied 225 breast carcinomas, 67 of which were premenopausal and 158 postmenopausal. In each sample, ARAC, EGFR, ER, and E2 were quantified. ARAC was quantified by Thompson and Siiterii's method, EGFR was quantified with a two-point assay method using radioactive iodine (125I)-EGF as ligand, and ER was measured by the Scatchard method using 3H-E2. E2 was quantified by radioimmunoassay in the diethylether tumor extract. RESULTS: ARAC was found in 64% of the cancers studied. There is a strong direct association between ARAC and tumor size in postmenopausal patients (P = .001). In the postmenopausal group, the proportion of ARAC-positive (ARAC+) tumors is significantly higher among ER-positive (ER+) than ER-negative (ER-) ones (P less than .001). ER+ tumors also have significantly higher levels of E2 than do ER- ones (P less than .0001); similarly, ARAC+ tumors have significantly higher levels of E2 than do ARAC- ones (P less than .0001). There is a significant multiple linear correlation between the log of the levels of ARAC, ER, and EGFR and the log of tumor E2 (P less than .0001). The correlation coefficients obtained show that ARAC and ER have a positive effect on tumor E2. CONCLUSION: The results obtained suggest the importance of tumor ARAC in the tumoral levels of E2 and reinforce the possible biologic significance of tumor ARAC, especially in postmenopausal breast carcinoma patients.     

Growth modulation of mouse keratinocytes by transforming growth factors

The effects of exogenously added transforming growth factor (TGF alpha and TGF beta on the growth of BALB/MK cells were examined. TGF alpha supplanted the epidermal growth factor (EGF) requirement in these cells. In contrast, TGF beta reversibly inhibited the growth of BALB/MK cells by abrogating the stimulatory actions of EGF or TGF alpha. The inhibitory effects of TGF beta appeared to be mediated by events distal to EGF ligand-receptor interactions. Growth inhibition of BALB/MK cells by TGF beta did not result in the induction of differentiation. This finding is different from the growth inhibition of these cells induced by elevated calcium levels (1.5 mM) which was tightly coupled to terminal differentiation. The BALB/MK cells were found to express TGF alpha mRNA, as well as TGF beta mRNA and protein. In addition, TGF alpha, as well as EGF, enhanced TGF alpha gene expression. These studies suggest a role for endogenous TGFs in regulating BALB/MK proliferation. TGF alpha provides a positive growth signal, while TGF beta is a potent inhibitor of growth even in the presence of such positive modulators as TGF alpha and EGF.     

MeCP2 and MBD2 expression in human neoplastic and non-neoplastic breast tissue and its association with oestrogen receptor status

This study analysed mRNA expression of two members of the methyl-CpG-binding protein family - MeCP2 and MBD2 - in human non-neoplastic (n=11) and neoplastic (n=57) breast tissue specimens using a quantitative real-time PCR method. We observed higher expression levels of MeCP2 mRNA in neoplastic tissues than in non-neoplastic tissues (P=0.001), whereas no significant differences for MBD2 were detected. When studying the relations between the most important clinicopathologic features of breast cancer and the mRNA expression level of both MBDs, we found that oestrogen receptor (OR)-positive breast cancer specimens contained higher levels of MeCP2 mRNA than did OR-negative cancers (P=0.005). Furthermore, we observed statistically significantly higher levels of MeCP2 in non-neoplastic tissues expressing high levels of OR as compared to those expressing low levels (P=0.017). Finally, using a linear regression model, we identified a statistically significant association between OR expression and MeCP2 mRNA expression in neoplastic and non-neoplastic breast tissue specimens (P=0.003). In conclusion, we were able to demonstrate for the first time that there exists a strong association between OR status and MeCP2 mRNA expression. Furthermore, we speculate that MeCP2, regulated by OR, plays a key role in the differentiation processes in human breast tissues.     

Comparison of EGFR, c-erbB-2 product and ras p21 immunohistochemistry as prognostic markers in primary breast cancer.

In a prospective study of 75 breast carcinomas, monoclonal antibodies EGFR1, Anti-serum 21N (As21N) and RAP-5 were used to assess immunohistochemically expression of Epidermal Growth Factor Receptor (EGFR), c-erbB-2 oncoprotein and ras protein p21. A careful comparison was made of their relative prognostic significance. Positive staining was seen with EGFR1 in 12/71 cancers (17%) and with As21N in 16/75 cancers (21%). Positive staining with RAP-5 occurred in all cancers and benign breast tissue, but varied in intensity. EGFR expression correlated with the number of involved lymph nodes, histological grade, estrogen and progesterone receptor (ER, PR) levels and the Melbourne Prognostic Index. C-erbB-2 and ras expression both correlated with ER levels and EGFR, but not with the Prognostic Index. Based on an immunohistochemical technique, EGFR expression emerges as the parameter with strongest prognostic associations.     

Expression of epidermal growth factor receptor mRNA and protein in primary breast carcinomas

The expression of epidermal growth factor receptor (EGFR) mRNA and protein has been determined in a group of breast carcinomas and compared to oestrogen and progesterone receptor (ER, PgR) status, as well as pathological features. In situ hybridization using a digoxigenin-labelled oligonucleotide probe was applied to formalin-fixed paraffin-embedded sections, and immunohistochemistry was used to determine EGFR protein.EGFR mRNA was detected in 66% of carcinomas with a third having labelling similar to normal breast tissue, 22% heterogeneous weak to strong labelling, and 11% strong labelling. EGFR protein was detected in 36% and these tumours had a strong correlation to lack of ER and high histological grade. The presence of EGFR protein was strongly correlated with more intense labelling for EGFR mRNA (p < 0.0001). This contrasted with normal breast in which both EGFR protein and mRNA were present with varying degrees in both tumours and a normal breast control. The ER-/PgR- carcinomas showed the full range of EGFR mRNA labelling. It is postulated that oestrogen or oestrogen regulated proteins are involved in regulation of EGFR mRNA and protein. In a proportion of tumours lacking steroid receptors regulation is lost, leading to EGFR overexpression.     

Prognostic value of maspin mRNA expression in ER alpha-positive postmenopausal breast carcinomas

Maspin, a member of the serpin family, has a role in cell migration, angiogenesis and apoptosis. Little is known of the clinical significance of maspin gene expression in human cancers. We developed a real-time quantitative RT-PCR assay to quantify the full range of maspin mRNA copy numbers in a series of 10 ER alpha-positive and 10 ER alpha-negative breast tumours. We observed a statistical link between low maspin mRNA levels and positive oestrogen status (P=0.0012). In consequence, to better assess the prognostic value of maspin gene expression in breast cancer, we then quantified maspin mRNA content in an additional independent well-defined cohort of 105 ER alpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. Maspin expression varied widely in tumour tissues (by nearly four orders of magnitude), being underexpressed in 33 out of 105 tumours (31.4%) and overexpressed in 24 out of 105 tumours (22.9%) relative to normal breast tissues. Immunohistochemical studies demonstrated that maspin protein was strictly expressed in myoepithelial cells of normal breast tissue and in tumour epithelial cells, exclusively in maspin-overexpressing tumours. Patients with tumours overexpressing the maspin gene had significantly shorter relapse-free survival after surgery than patients whose tumours normally expressed or underexpressed maspin (P=0.0011). The prognostic significance of maspin overexpression persisted in Cox multivariate regression analysis (P=0.0024). These findings show that the maspin mRNA level can have important prognostic significance in human breast cancer, and point to the maspin gene as a putative molecular predictor of hormone responsiveness in breast cancer.     

Expression of HER2 and estrogen receptor alpha depends upon nuclear localization of Y-box binding protein-1 in human breast cancers

In our present study, we examined whether nuclear localization of Y-box binding protein-1 (YB-1) is associated with the expression of epidermal growth factor receptors (EGFR), hormone receptors, and other molecules affecting breast cancer prognosis. The expression of nuclear YB-1, clinicopathologic findings, and molecular markers [EGFR, HER2, estrogen receptor (ER)alpha, ER beta, progesterone receptor, chemokine (C-X-C motif) receptor 4 (CXCR4), phosphorylated Akt, and major vault protein/lung resistance protein] were immunohistochemically analyzed. The association of the expression of nuclear YB-1 and the molecular markers was examined in breast cancer cell lines using microarrays, quantitative real-time PCR, and Western blot analyses. Knockdown of YB-1 with siRNA significantly reduced EGFR, HER2, and ER alpha expression in ER alpha-positive, but not ER alpha-negative, breast cancer cell lines. Nuclear YB-1 expression was positively correlated with HER2 (P = 0.0153) and negatively correlated with ER alpha (P = 0.0122) and CXCR4 (P = 0.0166) in human breast cancer clinical specimens but was not correlated with EGFR expression. Nuclear YB-1 expression was an independent prognostic factor for overall (P = 0.0139) and progression-free (P = 0.0280) survival. In conclusion, nuclear YB-1 expression might be essential for the acquisition of malignant characteristics via HER2-Akt-dependent pathways in breast cancer patients. The nuclear localization of YB-1 could be an important therapeutic target against not only multidrug resistance but also tumor growth dependent on HER2 and ER alpha.