Significance In Human Breast Cancer Research Articles

The immune checkpoint adenosine 2A receptor is associated with aggressive clinical outcomes and reflects an immunosuppressive tumor microenvironment in human breast cancer.

The crosstalk between the immune system and cancer cells has aroused considerable interest over the past decades. To escape immune surveillance cancer cells evolve various strategies orchestrating tumor microenvironment. The discovery of the inhibitory immune checkpoints was a major breakthrough due to their crucial contribution to immune evasion. The A2AR receptor represents one of the most essential pathways within the TME. It is involved in several processes such as hypoxia, tumor progression, and chemoresistance. However, its clinical and immunological significance in human breast cancer remains elusive. The mRNA expression and protein analysis were performed by RT-qPCR and immunohistochemistry. The log-rank (Mantel-Cox) test was used to estimate Kaplan-Meier analysis for overall survival. Using large-scale microarray data (METABRIC), digital cytometry was conducted to estimate cell abundance. Analysis was performed using RStudio software (7.8 + 2023.03.0) with EPIC, CIBERSORT, and ImmuneCellAI algorithms. Tumor purity, stromal and immune scores were calculated using the ESTIMATE computational method. Finally, analysis of gene set enrichment (GSEA) and the TISCH2 scRNA-seq database were carried out. Gene and protein analysis showed that A2AR was overexpressed in breast tumors and was significantly associated with high grade, elevated Ki-67, aggressive molecular and histological subtypes, as well as poor survival. On tumor infiltrating immune cells, A2AR was found to correlate positively with PD-1 and negatively with CTLA-4. On the other hand, our findings disclosed more profuse infiltration of protumoral cells such as M0 and M2 macrophages, Tregs, endothelial and exhausted CD8+ T cells within A2ARhigh tumors. According to the Single-Cell database, A2AR is expressed in malignant, stromal and immune cells. Moreover, it is related to tumor purity, stromal and immune scores. Our results also revealed that CD8+T cells from A2ARhigh patients exhibited an exhausted functional profile. Finally, GSEA analysis highlighted the association of A2AR with biological mechanisms involved in tumor escape and progression. The present study is the first to elucidate the clinical and immunological relevance of A2AR in breast cancer patients. In light of these findings, A2AR could be deemed a promising therapeutic target to overcome immune evasion prevailing within the TME of breast cancer patients.

Increased long noncoding RNA LINP1 expression and its prognostic significance in human breast cancer.

Mounting evidence suggests that the long noncoding RNAs (lncRNAs) function in multiple cancers. Dysregulation of lncRNA in non-homologous end joining (NHEJ) pathway 1 (LINP1) has been reported in breast cancer. However, its clinical significance in breast cancer remains unclear. The aim of this study was to investigate the prognostic relevance of LINP1 in breast cancer. Expression of LINP1 in tumor and their matched non-tumor tissues was determined by quantitative Real Time-PCR (qRT-PCR) in breast cancer patients. The relevance of LINP1 expression to the clinicopathological factors was assessed. Kaplan-Meier analysis was performed to evaluate the overall survival and disease-free survival of breast cancer patients with the expression level of LINP1. Cox proportional hazard regression analysis was performed at both univariate and multivariate levels. The results showed that LINP1 appeared to have higher expression in breast cancer tissues than in adjacent non-tumor tissues (p < 0.01). Increased expression of LINP1 was correlated with advanced TNM stage (p = 0.002), more lymph node metastasis (p = 0.000), and poorer pathological differentiation (p = 0.004). Furthermore, clinical assay indicated that patients with high LINP1 expression had a shorter overall survival and disease-free survival compared with the low LINP1 expression group. Finally, Cox regression analysis showed that LINP1 expression was an independent prognosis-predicting factor for breast cancer patients. LncRNA-LINP1 may be involved in the progression of the breast cancer and be a novel indicator of poor prognosis in patients with breast cancer.

Expression of Axl and its prognostic significance in human breast cancer.

Breast cancer is the most common malignant cancer and second leading cause of cancer-related death among women, and its prevalence continues to increase. Axl overexpression has been identified in the many types of human cancer, and it has been demonstrated to participate in signaling pathways related to carcinogenesis and cancer development. In the present study, Axl expression was examined by performing immunohistochemical staining in 60 breast cancer tumors and 40 benign breast lesions (25 mammary dysplasia and 15 breast fibroadenoma). In total, 34 (56.67%) cancer tissues and 13 (32.5%) benign breast lesions were classified as exhibiting high levels of Axl expression, indicating a significant association between malignancy and high Axl expression. High Axl expression was also associated with estrogen receptor (ER) positivity (P=0.028), progesterone receptor (PR) positivity (P=0.007), and poor tumor differentiation (P=0.033). No significant associations were observed between Axl expression and age, tumor size, lymph node metastasis, tumor node metastasis staging, human epidermal growth factor receptor 2 and Ki67 antigen. The Kaplan-Meier survival analysis and Cox proportional hazard model both demonstrated that there was no statistical difference between Axl expression and breast cancer prognosis. However, it remains unclear whether the expression of Axl is correlated with the prognosis of luminal type breast cancer patients.

Retraction Note: Expression of microRNA-497 and its prognostic significance in human breast cancer

Dysregulation of microRNAs (miRNAs) plays critical roles in tumor progression. The aim of this study was to investigate the clinicopathologic and prognostic significance of miR-497 expression in human breast cancer (BC). Taqman qRT-PCR assay was performed to detect the expression of microRNA (miR)-497 in 30 pairs of BC tissues and corresponding noncancerous breast tissues. Additionally, the expression of this miRNA was detected in another 128 BC tissues and its correlations with clinicopathologic features of patients were analyzed. Kaplan-Meier analyses were used to assess survival of patients. Univariate and multivariate analyses were performed using the Cox proportional hazards model to analyze the prognostic significance of miR-497 expression. Our data indicated that the relative level of miR-497 expression in BC tissues was significantly lower than that in corresponding noncancerous breast tissues (P = 0.0046). Of 128 BC patients, 74 (57.8%) were placed in the high-miR-497 group and 54 (42.2%) were placed in the low-miR-497 group. By statistical analyses, low miR-497 expression was observed to be closely correlated with higher differentiation grade, positive HER-2 expression, higher incidence of lymph node metastasis and advanced clinical stage. Moreover, patients with high miR-497 expression had better 5-year disease-free and overall survival compared with the low miR-497 group (P = 0.0124 and 0.0018, respectively). Univariate and multivariate analyses indicated that low miR-497 expression was an independent poor prognostic factor for BC patients. Our data provided the first evidence that downregulation of miR-497 was correlated with BC progression, and miR-497 might be a potential molecular biomarker for predicting the prognosis of patients. The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2025828761093488

Abstract 3281: TGFβ pathway alteration, body mass index, physical activity and breast cancer outcomes: The Shanghai Breast Cancer Survival Study

Abstract Background: Cumulative evidence suggests that the transforming growth factor-beta (TGF-β) pathway may have prognostic significance in human breast cancer. It also has been shown that the TGF-β pathway may play a role in glucose regulation and energy homeostasis. However, no previous study has examined the potential modifying effects of obesity and physical activity on associations between TGF-β pathway biomarkers, such as TGFβRII and pSmad2, and breast cancer outcomes. Methods: Breast cancer patients aged 20 to 75 years were recruited 6 months after diagnosis between 2002 and 2005 and participated in in-person baseline and follow-up surveys that collected information on cancer diagnosis, treatment, lifestyle exposures, anthropometrics, and physical activity. The expression of TGFβRII and nuclear pSmad2 in breast cancer cells and breast stromal tissue cells was evaluated immunohistochemically. Five-year disease-free survival (DFS) and overall survival (OS) rates were estimated by Kaplan Meier method. Cox proportional hazards models were used to estimate hazard ratios (HRs) and 95% confidence intervals (95% CIs) with adjustment for other prognostic variables and further stratified by body mass index (BMI) or physical activity. Results: Breast cancer cells exhibited either a membranous or a cytoplasmic expression pattern of TGFβRII. Compared with the membranous pattern, the cytoplasmic pattern was more prevalent among patients with low BMI (P&amp;lt;0.01) and was associated with lower five-year DFS (91% vs. 83%, P=0.04; adjusted HR=1.66, 95% CI: 1.03-2.68). Compared with lower nuclear pSmad2 expression intensity, higher expression intensity of nuclear pSmad2 in breast cancer cells was associated with lower five-year DFS (87% vs. 82%, P=0.02; adjusted HR=1.31, 95%CI: 0.96-1.79). This association was predominantly seen among participants with moderate physical activity levels (&amp;lt;9.4 MET-hr/wk; adjusted HR=2.05, 95% CI: 1.11-3.79 for five-year DFS) and lower BMI (&amp;lt;25; adjusted HR=1.80, 95% CI: 1.17-2.77 for five-year DFS), although the test for multiplicative interaction was only significant for BMI (Pinteraction=0.02). Analyses of OS showed similar patterns of association with a significant interaction between BMI and pSmad2 (adjusted HR=2.09, 95% CI: 1.38-3.15 for patients with low BMI, Pinteraction&amp;lt;0.001). The expression of TGFβRII and nuclear pSmad2 in stromal cells was unrelated to breast cancer outcomes. Conclusion: Expression levels of TGFβRII and nuclear pSmad2 in cancer cells were associated with breast cancer outcomes. These associations may be modified by BMI. Citation Format: Yinghao Su, Wei Zheng, Hui Cai, Ying Zheng, Qingchao Qiu, Wei Lu, Xiao-Ou Shu, Qiuyin Cai. TGFβ pathway alteration, body mass index, physical activity and breast cancer outcomes: The Shanghai Breast Cancer Survival Study. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3281. doi:10.1158/1538-7445.AM2014-3281

Decreased expression of C10orf10 and its prognostic significance in human breast cancer.

Breast cancer is a common malignant tumor, which severely threatens the health of women with an increasing incidence in many countries. Here, we identified C10orf10 as a novel differentially expression gene using expression microarray screening. The expression analysis indicated that C10orf10 was frequently decreased in human breast cancers compared to noncancerous breast tissues (81/95, P = 0.0063). Kaplan-Meier analysis indicated that patients with low C10orf10 expression showed a poorer prognosis both in mRNA (n = 1115, P = 0.0013) and protein (n = 100, P = 0.003) levels. Univariate and multivariate analysis showed that the C10orf10 expression was an independent prognostic factor for overall survival of breast cancer patients. Further analysis revealed that low expression of C10orf10 was an unfavorable factor for the prognosis of the patients who were luminal A, luminal B, Her2+ subtypes, at histological grade 2, lymph node negative and ER positive. Our data provided the first evidence that C10orf10 expression was frequently decreased in breast cancer tissues, and low expression of C10orf10 may be an important prognostic factor for poorer survival time of breast cancer patients.

Abstract A27: Proteomic study of the of posttranslational modifications (phosphorylation and glycosylation) and its significance in human breast cancer.

Abstract Post translational modifications are the biochemical modifications introduced into the protein after translation (or during translation in case of co-translational modification). Various changes that occur are in the form of chemical groups added to various residues of the target protein by specific enzymes like glycosyltransferase, kinase, methyltransferase, etc. Various studies have shown that cancer cells differ from non-neoplastic cells not only in expression of proteins and their related function, but they also have major differences in the pattern of post-translational modification. These changes may be direct result of difference in expression levels of PTM-responsible enzymes, or differences in the functioning of the same. In the same manner, differences in PTM pattern of cancer proteins leads to many structural and functional changes in the target proteins, many of which may be responsible in furthering the neoplastic characteristics and survival of the cancer cell. Since PTM leads to change in functionality in target proteins, this study aims to find a direct relation between change in function of both pro-apoptotic and anti-apoptotic proteins and cancer cell immortality. Theoretically, pro-apoptotic proteins need to be non-functional for the cell to skip apoptosis, as is often seen in cancer cells. Also, the activity of anti-apoptotic proteins needs to be subdued by PTM to reach the same results. Differential PTM of various proteins in cancer cells with respect to non-neoplastic cells has been seen to be of great significance. This upregulation or downregulation of certain type of specific PTM can be a factor for the overall progression and development of cancer. Present study was desined with an aim to identifying the proteome exclusive to breast cancer cells, and also to study the differential PTM pattern of the same and determine the significance of this difference in various aspects of cancer occurrence, pathogenesis and progression. We found that the there is a significant change in the expression profiling of phosphorylation and glycosylation patterns of various proteins associated with breast cancer as assessed by using various bioinformatic based tools. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):A27. Citation Format: Navkiran Kaur, Parul Jauhari. Proteomic study of the of posttranslational modifications (phosphorylation and glycosylation) and its significance in human breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr A27.

RETRACTED ARTICLE: Expression of microRNA-497 and its prognostic significance in human breast cancer

ObjectiveDysregulation of microRNAs (miRNAs) plays critical roles in tumor progression. The aim of this study was to investigate the clinicopathologic and prognostic significance of miR-497 expression in human breast cancer (BC).MethodsTaqman qRT-PCR assay was performed to detect the expression of microRNA (miR)-497 in 30 pairs of BC tissues and corresponding noncancerous breast tissues. Additionally, the expression of this miRNA was detected in another 128 BC tissues and its correlations with clinicopathologic features of patients were analyzed. Kaplan-Meier analyses were used to assess survival of patients. Univariate and multivariate analyses were performed using the Cox proportional hazards model to analyze the prognostic significance of miR-497 expression.ResultsOur data indicated that the relative level of miR-497 expression in BC tissues was significantly lower than that in corresponding noncancerous breast tissues (P = 0.0046). Of 128 BC patients, 74 (57.8%) were placed in the high-miR-497 group and 54 (42.2%) were placed in the low-miR-497 group. By statistical analyses, low miR-497 expression was observed to be closely correlated with higher differentiation grade, positive HER-2 expression, higher incidence of lymph node metastasis and advanced clinical stage. Moreover, patients with high miR-497 expression had better 5-year disease-free and overall survival compared with the low miR-497 group (P = 0.0124 and 0.0018, respectively). Univariate and multivariate analyses indicated that low miR-497 expression was an independent poor prognostic factor for BC patients.ConclusionsOur data provided the first evidence that downregulation of miR-497 was correlated with BC progression, and miR-497 might be a potential molecular biomarker for predicting the prognosis of patients.Virtual slidesThe virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/2025828761093488

PCR-SSCP-DNA Sequencing Method in Detecting PTEN Gene Mutation and its Significance in Human Breast Cancer in Turkish Populations

ABSTRACTThe aim of this study was to analyze the possible effect of PTEN gene mutations on the occurrence and development of human breast cancer. PCR-SSCP-DNA sequencing indicated that two kinds of mutation sites were found in 4 out of 80 breast cancer samples. One kind of mutation was found in exons. AA-TCC mutation was located 40 bp upstream of the 3'lateral exon 2 (115946 AA-TCC). Such mutations led to terminator formation at codon 267 of the PTEN gene. The other mutation was found in an intron, including a C-T point mutation 91 bp upstream of 2' lateral exon 2 (1903858 C-T). The PTEN protein expression in tissues with breast cancer indicated that the total positive rate of PTEN protein expression was 68% in breast cancer tissue, which was significantly lower than that (100%) in paracancerous tissues (P < 0.005). PTEN gene mutation may play an important role in the occurrence and development of breast cancer.

Prognostic significance of cytoplasmic SOX9 in invasive ductal carcinoma and metastatic breast cancer

SOX9, a high mobility group (HMG) box transcription factor, is required for development, differentiation and lineage commitment. It is known to exert its effects through nuclear translocation, such as cell cycle changes in response to retinoic acid treatment in breast cancer cells. However, it is not known whether SOX9 has prognostic significance in human breast cancer. Over-expression and cytoplasmic sequestration of nuclear proteins are implicated in tumor progression. To determine whether SOX9 has any prognostic significance in human breast cancer, its expression and subcellular localization were analyzed in more than 200 human breast carcinomas (BCs). SOX9 mRNA expression data for human BCs were computed from microarray studies available in public databases and correlated with known poor prognostic parameters of BCs. SOX9 protein expression and its correlation with Ki-67 staining in human BCs were assessed using immunohistochemistry. Higher SOX9 mRNA levels were significantly associated with estrogen receptor negative (P ≤ 0.001) and higher grade (P ≤ 0.01) human breast tumors. Patients with higher SOX9 mRNA level had significantly shorter overall survival (P ≤ 0.0001). SOX9 protein, which is normally nuclear, was instead localized in the cytoplasm of 25-30% invasive ductal carcinomas (IDCs) and lymph node metastases. Its cytoplasmic accumulation significantly correlated with enhanced proliferation in breast tumors (Kendall's tau = 0.337 with a P value < 0.0001). Cytoplasmic SOX9 can serve as a valuable prognostic marker for IDCs and metastatic breast cancer. Its significant correlation with breast tumor cell proliferation implies that SOX9 directly contributes to the poor clinical outcomes associated with invasive breast cancer.

Association between polymorphisms of XRCC1, p53 and MDR1 genes, the expression of their protein products and prognostic significance in human breast cancer.

SummaryBackgroundThis study aimed to examine the relationship between XRCC1, p53 and MDR1 protein, along with polymorphisms of their genes and their prognostic values in breast cancer. The following clinical and pathological parameters were evaluated: histopathological type of tumor, grade, stage, Her2/neu expression, ER, PR positivity and involvement of regional lymph nodes.Material/MethodsExpression of proteins was determined in 39 samples of breast cancer by immunohistochemistry. Nucleotide polymorphisms were analyzed by PCR-RFLP. For statistical analysis, chi-square test (Yates), Fisher’s exact test, and correlation test were used to analyze the data.ResultsThe highest protein expression was immunohistochemically found in MDR1 protein, with 54% of samples testing positive. In addition, the evaluation of MDR1 expression revealed higher positive immunoreactivity in lobular (LIC) and other types of tumor in comparison to ductal (DIC) type. The expression of p53 and XRCC1 protein was equal, but lower compared to MDR1, both testing positive in 36% of all tissue samples. Comparison of XRCC1 protein and histopathological type of tumor revealed that DIC and LIC types were mostly XRCC1-negative, while other types, papillary and mucinous were more likely to be XRCC1-positive. Interestingly, when evaluating LIC samples separately, a negative correlation between the Her2/neu and expression of XRCC1 was detected. Apparently, all Her2/neu-positive samples were XRCC1-negative (6/86%). The correlation test indicated a negative correlation between Her2/neu-positive samples and XRCC1-negative specimens (r=1, p<0.05). Statistical analysis did not reveal a correlation of p53 expression with clinical and pathological parameters. Similarly, no statistically significant difference was found between the tested polymorphisms and protein expression.ConclusionsWe did not find statistically significant correlation between tested polymorphisms and their protein expression.

Annexin A1 expression and its prognostic significance in human breast cancer

Annexin A1 (ANXA1) is a calcium- and phospholipid-binding protein and is considered to play an important role in tumorigenesis. However, the correlation between ANXA1 expression and tumor clinicopathological features in patients with breast cancer remains unclear. This study investigated the prognostic value of ANXA1 protein as breast cancer marker. Tissue microarray blocks, containing 20 cases of non-tumor breast tissue, 20 cases of benign breast lesion and 135 cases of breast cancer (107 with lymph node metastasis), were constructed. Expression of ANXA1 in these specimens was analyzed using immunohistochemistry. In non-tumor tissue and benign breast lesions, myoepithelial cells showed strong expression of ANXA1. Negative ANXA1 expression was significantly associated with advanced disease stage (P<0.05), especially pathological-N stage (P<0.01). The patients with loss of ANXA1 expression in tumor tissues showed a significantly worse overall survival compared with positive ones (P<0.05). ANXA1 did not correlate well with estrogen receptor (ER), progesterone receptor (PR) and HER2/neu status. Moreover, the level of ANXA1 expression in lymph node metastases was higher than corresponding primary breast cancer. These results suggest that ANXA1 may play a multifaceted role in breast cancer development, progression, and metastases.

Estrogen Receptor-Positive and Estrogen Receptor-Negative Human Breast Cancer Cells: Regulation of Expression of Cancer-Related Genes by Estradiol and Tamoxifen

Breast cancer is one of the most prevalent forms of carcinomas among women world-wide. The oncogenes, tumor suppressor genes and antioxidant enzyme genes are cancer-related genes which play an important role in the initiation, metastasis and malignancy of many cancers, including breast cancer. Estrogen which is a key steroid hormone in the regulation and differentiation of the normal breast also appears to be involved in the carcinogenesis of this tissue. Antiestrogen therapy, such as the use of tamoxifen, targets the estrogen receptors (ER) through which estrogen exerts both its normal biological function as well as the molecular processes leading to cancer formation. The objectives of the present study were to characterize and quantify the expression of genes related to human breast carcinogenesis, using ER- positive (ZR-75-1) and ER-negative (MDA-MB-231) cells as in vitro cellular models of breast cancer. We have examined the expression of a number of genes which have an important clinical significance in human breast cancer, when expressed at levels below or above the normal range. Among these are the receptor oncogenes (EGFR, c-erbB2), other oncogenes (pS2, hMAM, MUC1 and CK-19), the tumor suppressor gene, p53, as well as four of the major antioxidant enzyme genes. These are superoxide dismutase-1 (SOD1), superoxide dismutase-2 (SOD2), Se-glutathione peroxidase (GPx1) and catalase (CAT), all of which have been well characterized in human cells, and whose activities have been localized to different cellular compartments, such as the cytoplasm, mitochondria and peroxisomes. Gene expressions were determined using real-time PCR, in control untreated cells as well as when these two cell types were exposed to 17β-estradiol (E), or tamoxifen (TAM) or a combination of E and TAM. Our results show that basal level expressions of receptor oncogenes (EGFR and c-erbB2) as well as two of the four antioxidant enzyme genes (SOD2 and GPx1) were several folds lower in ER-positive breast cancer cells (ZR-75-1) as compared to ER-negative cells (MDAMB- 231). However, treatment with E and TAM, either individually or in combination, produced much greater effects on the ER-positive cells than on the ER-negative cells, with the result that these genes were greatly overexpressed in the ER-positive cells as compared to the ER-negative cells. Of these, the receptor oncogene, c-erbB2, and the antioxidant enzyme gene, SOD2, were the most affected, resulting in striking upregulations ranging over several hundred folds. This, and other observations, would suggest that the carcinogenic effects of estradiol and the anticarcinogenic effects of tamoxifen are not only mediated by the presence of estrogen receptors, but may also be regulated, either directly or indirectly, through other mechanisms, including the mitochondrial antioxidant system. Keywords: Breast cancer, estrogen and antiestrogens, tamoxifen, oncogenes, tumor suppressor genes, antioxidant enzyme genes, oxidative stress and antioxidants, quantitative real-time PCR

Tissue microarray analysis of connexin expression and its prognostic significance in human breast cancer

Breast cancer accounts for approximately 15% of all cancer deaths. Currently, axillary nodal status is the most reliable prognostic indicator for breast cancer. Tumor size and histological grade are used to stage breast cancer. Estrogen receptor/progesterone receptor (ER/PR) and HER-2/neu status are useful in predicting patient survival and relapse. Ki67, an indicator of proliferative activity, also correlates well with prognosis. Connexin proteins form gap junction channels, permitting intercellular exchange of ions and small molecules. Reduced connexin protein levels and impaired gap junctional intercellular communication are associated with tumor phenotypes. This study investigated the prognostic value of connexin proteins as breast cancer markers. Tissue microarrays, containing 438 cases of invasive breast carcinoma, were stained with Cx26, Cx32, and Cx43 antibodies. The degree of connexin immunoreactivity was determined and then correlated with patient outcome, tumor grade, tumor size, lymph node status, and immunohistochemical markers, such as p53, ER/PR status, Ki67 and c-erbB-2 expression. Cx26, Cx32, or Cx43 did not correlate well with tumor grade, tumor size, p53 or c-erbB-2 status. There was an inverse correlation between Cx32 and lymph node status ( P < 0.05) and a positive correlation between Cx43 and PR status ( P < 0.01). Cx32 and Cx43 correlated positively with ER status ( P < 0.01). Cx43 correlated negatively with Ki67 expression ( P < 0.01). Cx26, Cx32, and Cx43 did not correlate with patient outcome. Based on our observations in this study, connexin proteins do not appear to be reliable indicators of breast cancer prognosis.

Expression of mucins (MUC1, MUC2, MUC3, MUC4, MUC5AC and MUC6) and their prognostic significance in human breast cancer

Mucins are a large family of glycoproteins expressed by many epithelial cells and their malignant counterparts. Much interest has been focused on expression of its members in breast cancer because of their potential role as prognostic indicators and their involvement in cancer therapy. We have examined 1447 cases of invasive breast carcinoma with a long-term follow-up, using tissue microarray (TMA) technology and immunohistochemistry to evaluate the expression profiles of several mucins (MUC1, MUC2, MUC3, MUC4, MUC5AC and MUC6) and to assess their prognostic value. We detected MUC1 expression in 91% of tumours. MUC1 overexpression was associated with a lower grade, smaller tumour size, a higher oestrogen receptor (ER)-positive phenotype and absence of both regional recurrence and distance metastasis. The subcellular localization but not the level of expression had a prognostic value in predicting outcome. The aberrant cytoplasmic and membranous localization of MUC1 was associated with poor outcome compared with apical localization, which is the normal physiological site of expression. MUC2 expression was noticed in only 8.3% of all cases and was restricted to the cytoplasm of the tumour cells. An inverse trend was identified between MUC2 expression and lymph node stage and vascular invasion status. On excluding cases of mucinous carcinoma from the analysis, the inverse association with vascular invasion was still defined and in addition an inverse association with ER status emerged. MUC3 expression was detected in 91% of cases and its expression was associated with increased local recurrence, and lymph node stage. The membranous expression of MUC3 was found to be a potentially poor prognostic feature, with higher grade and poorer Nottingham Prognostic Index (NPI), and negative ER expression. MUC4, MUC5AC and MUC6 were expressed in 95, 37 and 20% of cases, respectively. Apart from an association between MUC4 expression and tumour grade and between MUC6 and ER-negative tumours, no other associations with any clinicopathological variables were found. Apart from the higher expression of MUC2 and MUC6 in mucinous carcinomas, no association was found between the expression of different mucins and tumour type. No association between the level of expression of any of the studied mucins and patient outcomes has been identified. In conclusion, most breast carcinomas express MUC1, MUC3 and MUC4. Among the various mucins expressed in breast cancer, MUC1 and MUC3 are potential prognostic indicators, MUC1 having the strongest relationship with patient outcome.

Expression of the Transcription Factors Snail, Slug, and Twist and Their Clinical Significance in Human Breast Cancer

Slug, Snail, and Twist are transcription factors that regulate the expression of tumor suppressors such as E-cadherin. We examined the distribution and expression of these three molecules together with the methylation of the Twist gene promoter in human breast cancer to elucidate their clinical significance. Frozen sections from breast cancer primary tumors (tumor, n = 114; background, n = 30) were immunostained with Slug, Snail, and Twist antibodies. RNA was reverse-transcribed, quantified, and analyzed by quantitative polymerase chain reaction (Q-PCR). Results were expressed as copy number of transcript per 50 ng of RNA (standardized against beta-actin). Immunohistochemistry revealed that all three molecules were stained in mammary tissues, with an increase in Twist within tumor tissues; this was supported by Q-PCR analysis. Q-PCR analysis showed that Slug was elevated with increasing tumor grade and prognostic indices. Twist was elevated with increasing nodal involvement (tumor-node-metastasis status). Conversely, Snail was reduced in expression corresponding with prognostic indices and tumor grade. Increased levels of Slug were associated with tumors from patients with metastatic disease or disease recurrence, and increased expression of Twist was associated with tumors from patients who had died from breast cancer. It is interesting to note that Snail expression was significantly reduced in patients with a poor outcome and those who had node-positive tumors. In addition, tumors exhibited methylation of the Twist promoter. These data demonstrate that all three transcription factors have inappropriate expression in breast cancer and that this may play a part in the progression of human breast tumors.

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 ERα-positive and 10 ERα-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 ERα-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.

The expression of ERβ protein correlates with vascular endothelial growth factor and its prognostic significance in human breast cancer

Although cloning of ER beta has prompted a reevaluation of the role of ERs in human breast cancer and there have been many studies focusing on the clinical value of ER beta detection, however, few reports evaluated the prognostic significance of ER beta based on follow-up data. The VEGF gene transcription may be mediated by different ER subtypes directly. The aim of this study was to evaluate the relationship of angiogenesis factor VEGF with different ER subtypes and the prognostic value of ER beta and VEGF in 116 human breast cancer patients. Of these patients, 40 (34.5%) were ER beta protein high expressed and 76 (65.5%) were ER beta protein low expressed. When correlated the ER beta protein levels with other clinical characteristics, statistical significance (p<0.05) was found between ER beta protein expression and menopausal status, and tumor grade. No significance was found between ER beta protein level and node status, stage, or tumor size. Inverse relationship was found between ER beta protein expression and PR status (p<0.05). When comparing the VEGF levels with different ER subtypes a significant difference between ERs and VEGF was found. In ER beta protein high expression group, the VEGF protein was highly expressed (p<0.01), inverse relationship was also found between ER alpha and VEGF. In univariate analysis ER alpha, ER beta and VEGF levels had prognostic value for both relapse-free survival and overall survival (p<0.05). However, in a multivariate study, ER beta and ER alpha protein levels lost the prognostic value either to relapse-free survival or to overall survival. Only VEGF levels acted as an independent prognostic factor to disease-free survival. The result suggested that ER beta protein may have important prognostic value in human breast cancer patients. VEGF expression may be mediated through different ER subtypes.

Messenger ribonucleic acid in situ hybridization analysis of estrogen receptors alpha and beta in human breast carcinoma.

We examined the expression of a recently characterized novel estrogen receptor (ER) beta in 25 cases of invasive ductal carcinoma of the breast, using messenger RNA (mRNA) in situ hybridization, and compared the findings with those of ERalpha, to study its localization and its possible biological significance in human breast cancer. ERalpha and ERbeta hybridization signals were both detected, predominantly in carcinoma cells and in some stromal cells, in 18 of 25 (72%) and 11 of 25 (44%) cases, respectively. The cases in which more than 25% of carcinoma cells demonstrated mRNA hybridization signals were 13 of 25 (52%) and 2 of 25 (8%) cases for ERalpha and ERbeta, respectively. Among the cases expressing ERbeta, 10 of 11 (91%) also expressed ERalpha mRNA; and in these 10 cases, coexpressing both ERalpha and beta, the number of carcinoma cells expressing ERalpha was greater than that expressing ERbeta in 9 cases. Eight cases demonstrated only ERalpha mRNA hybridization signals in carcinoma cells. These results indicate that ERbeta is coexpressed with ERalpha in most ERbeta-positive breast carcinoma cells, which suggests that the expression of ERbeta depends on the presence of ERalpha in the great majority of human breast cancer. In addition, the number of carcinoma cases and/or the ratio of carcinoma cells expressing ERalpha was much greater than those expressing ERbeta. The relative ratio of ERalpha and ERbeta expression in carcinoma cells may be related to various estrogen-dependent biological features of human breast cancer.