microRNA expression profile in a large series of bladder tumors: identification of a 3-miRNA signature associated with aggressiveness of muscle-invasive bladder cancer.

The aim of this study was to evaluate the expression levels of microRNAs (miRNAs) in bladder tumors in order to identify miRNAs involved in bladder carcinogenesis with potential prognostic implications. Expression levels of miRNAs were assessed by quantitative real-time RT-PCR in 11 human normal bladder and 166 bladder tumor samples (86 non-muscle-invasive bladder cancer (NMIBC) and 80 muscle-invasive bladder cancer (MIBC)). The expression level of 804 miRNAs was initially measured in a well-defined series of seven NMIBC, MIBC and normal bladder samples (screening set). The most strongly deregulated miRNAs in tumor samples compared to normal bladder tissue were then selected for RT-PCR validation in a well-characterized independent series of 152 bladder tumors (validation set), and in six bladder cancer cell lines. Expression levels of these miRNAs were tested for their association with clinical outcome. A robust group of 15 miRNAs was found to be significantly deregulated in bladder cancer. Except for two miRNAs, miR-146b and miR-9, which were specifically upregulated in MIBC, the majority of miRNAs (n = 13) were deregulated in the same way in the two types of bladder tumors, irrespective of pathological stage : three miRNAs were upregulated (miR-200b, miR-182 and miR-138) and the other 10 miRNAs were downregulated (miR-1, miR-133a, miR-133b, miR-145, miR-143, miR-204, miR-921, miR-1281, miR-199a and miR-199b). A 3-miRNA signature (miR-9, miR-182 and miR-200b) was found to be related to MIBC tumor aggressiveness and was associated with both recurrence-free and overall survival in univariate analysis with a trend to significance in the multivariate analysis (p = 0.05). Our results suggested a promising individual prognostic value of these new markers.

Expression analysis of DNA methyltransferases 1, 3A, and 3B in sporadic breast carcinomas.

PURPOSE: Three genes, namely DNA methyltransferase (DNMT) 1, DNMT3A, and DNMT3B, coding for DNMTs that affect promoter methylation status are thought to play an important role in the development of cancers. Little is known of the biological and clinical significance of these genes in human breast cancer. EXPERIMENTAL DESIGN: We used real-time reverse transcription-PCR assays to quantify the mRNA expression of the three DNMT genes in a series of 130 breast cancer patients. We also sought relationships between mRNA levels of the DNMTs and those of 20 target genes involved in the DNMT pathway (subgroup of 46 breast tumors). RESULTS: The DNMT3B gene showed the highest range of expression (81.8 compared with 16.6 and 14 for DNMT1 and DNMT3A, respectively). DNMT3B was overexpressed in 30% of the patients (5.4 and 3.1% for DNMT1 and DNMT3A, respectively). DNMT3B overexpression was significantly related to Scarff, Bloom, and Richardson histopathological grade III (P = 0.002), ERalpha negativity (P = 0.0015), and strong MKI67 expression (P = 3 x 10(-6)). In univariate analysis, DNMT3B overexpression was associated with poor relapse-free survival in the subgroup of patients who received adjuvant hormone therapy (with or without chemotherapy; P = 0.0064). Although the poor prognosis associated with DNMT3B overexpression was confirmed by univariate analysis in an independent series of 98 postmenopausal women exclusively treated with adjuvant tamoxifen therapy (P = 0.0036), DNMT3B expression status did not persist as an independent prognostic factor in multivariate analysis. CONCLUSIONS: Although we failed to identify underexpression of specific target genes associated with DNMT increasing expression, the frequent overexpression of DNMT3B in this breast tumor series points to DNMT3B as a potential new therapeutic target in breast cancer.

Molecular profiling of inflammatory breast cancer: identification of a poor-prognosis gene expression signature.

PURPOSE: Inflammatory breast cancer (IBC) is a rare but particularly aggressive form of primary breast cancer. The molecular mechanisms responsible for IBC are largely unknown. EXPERIMENTAL DESIGN: To obtain further insight into the molecular pathogenesis of IBC, we used real-time quantitative reverse transcription (RT)-PCR to quantify the mRNA expression of 538 selected genes in IBC relative to non-IBC. RESULTS: Twenty-seven (5.0%) of the 538 genes were significantly up-regulated in IBC compared with non-IBC. None were down-regulated. The 27 up-regulated genes mainly encoded transcription factors (JUN, EGR1, JUNB, FOS, FOSB, MYCN, and SNAIL1), growth factors (VEGF, DTR/HB-EGF, IGFBP7, IL6, ANGPT2, EREG, CCL3/MIP1A, and CCL5/RANTES) and growth factor receptors (TBXA2R, TNFRSF10A/TRAILR1, and ROBO2). We also identified a gene expression profile, based on MYCN, EREG, and SHH, which discriminated subgroups of IBC patients with good, intermediate, and poor outcome. CONCLUSION: Our study has identified a limited number of signaling pathways that require inappropriate activation for IBC development. Some of the up-regulated genes identified here could offer useful diagnostic or prognostic markers and could form the basis of novel therapeutic strategies.

Expression analysis of estrogen receptor alpha coregulators in breast carcinoma: evidence that NCOR1 expression is predictive of the response to tamoxifen.

PURPOSE: Dysregulated expression of steroid receptor transcriptional coactivators and corepressors has been implicated in tamoxifen resistance, especially in estrogen receptor (ER) alpha-positive breast cancer patients. Therefore, expression analysis of these ERalpha coregulators may identify new predictors of the response to tamoxifen treatment. EXPERIMENTAL DESIGN: We measured mRNA levels of 16 coactivator and 11 corepressor genes with a real-time quantitative reverse transcription-PCR method in 14 ERalpha-positive breast tumors. Three selected coactivator genes (TIF2, AIB1, and GCN5L2) and two corepressor genes (NCOR1 and MTA1L1) were additionally investigated in a well-characterized series of ERalpha-positive unilateral invasive primary breast tumors from 99 postmenopausal patients who only received tamoxifen as adjuvant hormone therapy after primary surgery. We sought relationships between mRNA levels of the coregulators and those of molecular markers, including ERalpha, ERbeta, CCND1, and ERBB2. RESULTS: ERalpha coregulator expression was unrelated to age, histological grade, lymph node status, and macroscopic tumor size. The relationship between mRNA expression of the coregulators, and ERalpha and beta only showed a significant positive correlation between GCN5L2 and ERalpha (P = 0.015). mRNA levels of CCND1 correlated with those of all of the coregulators studied (P < 0.05 or trend), whereas ERBB2 mRNA levels only correlated with AIB1 mRNA levels (P = 0.011). Low NCOR1 expression (versus intermediate and high) was associated with significantly shorter relapse-free survival (log-rank test; P = 0.0076). The prognostic significance of low NCOR1 expression persisted in Cox multivariate regression analysis (P = 0.043). CONCLUSIONS: These findings point to NCOR1 as a promising independent predictor of tamoxifen resistance in patients with ERalpha-positive breast tumors.

Relationship between intratumoral expression of genes coding for xenobiotic-metabolizing enzymes and benefit from adjuvant tamoxifen in estrogen receptor alpha-positive postmenopausal breast carcinoma.

INTRODUCTION: Little is known of the function and clinical significance of intratumoral dysregulation of xenobiotic-metabolizing enzyme expression in breast cancer. One molecular mechanism proposed to explain tamoxifen resistance is altered tamoxifen metabolism and bioavailability. METHODS: To test this hypothesis, we used real-time quantitative RT-PCR to quantify the mRNA expression of a large panel of genes coding for the major xenobiotic-metabolizing enzymes (12 phase I enzymes, 12 phase II enzymes and three members of the ABC transporter family) in a small series of normal breast (and liver) tissues, and in estrogen receptor alpha (ERalpha)-negative and ERalpha-positive breast tumors. Relevant genes were further investigated in a well-defined cohort of 97 ERalpha-positive postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone. RESULTS: Seven of the 27 genes showed very weak or undetectable expression in both normal and tumoral breast tissues. Among the 20 remaining genes, seven genes (CYP2A6, CYP2B6, FMO5, NAT1, SULT2B1, GSTM3 and ABCC11) showed significantly higher mRNA levels in ERalpha-positive breast tumors than in normal breast tissue, or showed higher mRNA levels in ERalpha-positive breast tumors than in ERalpha-negative breast tumors. In the 97 ERalpha-positive breast tumor series, most alterations of these seven genes corresponded to upregulations as compared with normal breast tissue, with an incidence ranging from 25% (CYP2A6) to 79% (NAT1). Downregulation was rare. CYP2A6, CYP2B6, FMO5 and NAT1 emerged as new putative ERalpha-responsive genes in human breast cancer. Relapse-free survival was longer among patients with FMO5-overexpressing tumors or NAT1-overexpressing tumors (P = 0.0066 and P = 0.000052, respectively), but only NAT1 status retained prognostic significance in Cox multivariate regression analysis (P = 0.0013). CONCLUSIONS: Taken together, these data point to a role of genes coding for xenobiotic-metabolizing enzymes in breast tumorigenesis, NAT1 being an attractive candidate molecular predictor of antiestrogen responsiveness.

Identification of a three-gene expression signature of poor-prognosis breast carcinoma.

BACKGROUND: The clinical course of breast cancer is difficult to predict on the basis of established clinical and pathological prognostic criteria. Given the genetic complexity of breast carcinomas, it is not surprising that correlations with individual genetic abnormalities have also been disappointing. The use of gene expression profiles could result in more accurate and objective prognostication. RESULTS: To this end, we used real-time quantitative RT-PCR assays to quantify the mRNA expression of a large panel (n = 47) of genes previously identified as candidate prognostic molecular markers in a series of 100 ERalpha-positive breast tumor samples from patients with known long-term follow-up. We identified a three-gene expression signature (BRCA2, DNMT3B and CCNE1) as an independent prognostic marker (P = 0.007 by univariate analysis; P = 0.006 by multivariate analysis). This "poor prognosis" signature was then tested on an independent panel of ERalpha-positive breast tumors from a well-defined cohort of 104 postmenopausal breast cancer patients treated with primary surgery followed by adjuvant tamoxifen alone: although this "poor prognosis" signature was associated with shorter relapse-free survival in univariate analysis (P = 0.029), it did not persist as an independent prognostic factor in multivariate analysis (P = 0.27). CONCLUSION: Our results confirm the value of gene expression signatures in predicting the outcome of breast cancer.

Altered expression pattern of alternatively spliced estrogen receptor beta transcripts in breast carcinoma.

Dysregulation of total estrogen receptor beta (ERbeta) expression has been implicated in breast tumorigenesis. The ERbeta gene yields five exon 8 alternatively spliced transcripts (ERbeta1-5), which encode proteins with different C-terminal amino acids. Individual expression analysis of these transcripts may provide new insights into estrogen signaling in breast cancer. We measured mRNA levels of total ERbeta and its five isoforms in normal tissues, breast carcinomas from post-menopausal patients, and breast cancer cell lines by means of real-time reverse transcription-polymerase chain reaction and fluorescent fragment analysis. In various normal human tissues, ERbeta1-5 isoforms displayed different qualitative and quantitative expression patterns that were consistent with previous reports. Total ERbeta mRNA levels were significantly lower in breast tumors than in normal breast tissues (38-fold lower, P < 0.001), mainly due to lower expression of ERbeta1 and ERbeta2 (ERbeta5 expression was similar in the two tissue types). This altered expression pattern of ERbeta isoforms in breast cancer should be taken into account in future ERbeta-based clinical applications.

Prognostic value of uPA, PAI-1 and PAI-2 mRNA expression in primary breast cancer.

BACKGROUND: The prognostic value of uPA, PAI-1 and PAI-2 mRNA expression was studied in a retrospective series of 130 primary breast cancer patients (median follow-up 8.1 years). MATERIALS AND METHODS: UPA, PAI-1 and PAI-2 mRNA were quantified by means of real-time quantitative RT-PCR. Comparison with the corresponding protein levels determined by ELISA was performed in 21 cases. RESULTS: Higher uPA protein values were found in cases with high mRNA values, but the relationship was of borderline significance. PAI-1 and PAI-2 mRNA were positively correlated to protein values. The mRNA expression of uPA, PAI-1 and PAI-2 was significantly correlated with one another. Higher uPA and PAI-1 mRNA values were significantly associated with shorter disease-free survival (DFS) (p = 0.002; p = 0.03, respectively). Low and very high PAI-2 mRNA values tended to be associated with longer DFS. In Cox multivariate analyses, higher uPA and PAI-1 mRNA values were independently associated with shorter DFS. PAI-2 was not retained as a significant variable in the Cox model. CONCLUSION: These preliminary results indicate that, in breast cancer, uPA, PAI-1 and PAI-2 mRNA analysis by quantitative RT-PCR give results comparable to those obtained at the protein level.

Gene expression profiling of ERBB receptors and ligands in human transitional cell carcinoma of the bladder.

PURPOSE: The ErbB driven growth pathway has been implicated in most human epithelial malignancies. Therefore, its blockade is a promising therapeutic strategy and several candidate drugs are currently undergoing clinical trials. Paradoxically little is known of the expression pattern or clinical significance of the 4 ErbB receptors and their 11 ligands in TCC of the bladder. MATERIALS AND METHODS: To obtain further insight into the molecular pathogenesis of TCC we used quantitative real-time reverse transcriptase-polymerase chain reaction assay to quantify mRNA expression of the 4 ERBB and their 11 known ligand genes, including recently described EPGN/epigen, in 73 tumor samples. RESULTS: The level of mRNA of 4 ligand genes (EGF, NRG1, NRG2 and NRG3) was extremely low, that is detectable but not quantifiable. Six genes were over expressed (ERBB2, TGFA, HB-EGF, AREG, EREG and EPGN), 3 were under expressed (ERBB1, ERBB4 and NRG4) and 2 were over or under expressed (ERBB3 and BTC). ERBB2 and AREG expression differed between early stage tumors (pTa grade 1) and normal samples. The most marked differences in expression were ERBB3, EREG and NRG4 between superficial and muscle invasive tumors (p = 0.0069, 0.00007 and 0.0000001, respectively), and TGFA and NRG4 between low and high grade superficial tumors, and between pT1 or greater and pTa tumors. CONCLUSIONS: This study shows the involvement of the ERBB family and ligand genes in TCC. Most receptor and ligand genes are deregulated at different stages of carcinogenesis, implying that they should be studied simultaneously. Quantitative real-time reverse transcriptase-polymerase chain reaction could be used to determine ErbB signaling pathway status in individuals with a view to tailored therapy.

Is quantitative real-time RT-PCR an adjunct to immunohistochemistry for the evaluation of ErbB2 status in transitional carcinoma of the bladder?

OBJECTIVE: To test different approaches of evaluation of the ErbB2 status in a large series of human transitional cell carcinoma (TCC) of the bladder with the prospect of finding targeted therapies. METHODS: ErbB2 status of 73 human TCC samples was analyzed by both immunohistochemistry (IHC) and by quantification of mRNA levels of expression using real-time reverse transcription-polymerase chain reaction (RT-PCR). Additionally, 18 bladder samples were studied for ERBB2 gene amplification by real-time quantitative PCR. RESULTS: Twenty-five tumors (34.2%) overexpressed ERBB2 mRNA compared to normal bladder samples; this alteration appeared in low-grade and low-stage tumors (pTaG1). Twenty-four (32.9%) tumors showed moderate (++) or strong (+++) immunostaining. A very strong agreement was found between the two methods (kappa = 0.97, 95% confidence interval, 0.90-1). ErbB2 status was not associated with tumor stage. Of the 18 bladder samples tested for ERBB2 gene amplification, only one showed ERBB2 DNA amplification. CONCLUSIONS: ErbB2 overexpression occurs in about one third of bladder TCCs. This overexpression can be detected by RT-PCR with a very good correlation with IHC. RT-PCR can therefore be used for cases considered doubtful on IHC rather than gene amplification studies because, in TCC, gene amplification is not the predominant mechanism of both mRNA and protein overexpression. Accurate quantification of ErbB2 status is mandatory for the use of anti-ErbB2-targeted therapies in bladder TCC.

Prognostic value of ERBB family mRNA expression in breast carcinomas.

The ErbB-driven autocrine growth pathway has been implicated in the development and progression of most common human epithelial malignancies; its blockade is therefore a promising therapeutic strategy, and several candidate drugs are currently undergoing clinical trials. Paradoxically, little is known of the expression pattern of these 4 genes in human tumors, and the clinical significance of the 2 most recently discovered ERBB genes, ERBB3 and ERBB4, is unclear. We used a real-time quantitative RT-PCR assay to quantify ERBB family mRNA copy numbers in a large series of breast tumors from patients with known long-term outcome. ERBB gene expression varied widely, by more than 2 orders of magnitude for ERBB1 and ERBB3, more than 3 orders for ERBB2 and more than 4 orders for ERBB4. We found a positive correlation between ERBB3 and ERBB4 mRNA levels, and a negative correlation between the expression of these 2 latter genes and that of ERBB1. Compared to normal breast tissue, ERBB1 was underexpressed (82.3% of tumors), ERBB2 (16.9%) and ERBB3 (46.2%) were overexpressed and ERBB4 was both underexpressed (24.6%) and overexpressed (29.2%). Links were also found between ERBB status on the one hand and Scarff-Bloom-Richardson (SBR) histopathological grade and estrogen receptor alpha (ERa) status on the other hand. Relapse-free survival (RFS) was shorter among patients with ERBB3-overexpressing tumors (p=0.0092) and longer among those with ERBB4-underexpressing tumors (p=0.0085) relative to patients with normal expression of the respective genes; in contrast, RFS was not significantly influenced by ERBB1 or ERBB2 mRNA status. Only ERBB4 status retained prognostic significance in Cox multivariate regression analysis (p=0.015). Our results point to the involvement of several ErbB-specific ligands (amphiregulin and neuregulin 1) and enzymes or adaptor molecules (PI3K, Src, Shc and Grb7) in the ErbB pathway dysregulation associated with breast cancer. These findings reveal a complex expression pattern of ERBB gene family members in breast tumors and suggest that it is this pattern of expression, rather than the expression of individual family members, that should be taken into account when evaluating antitumoral drugs designed to target these receptors.

Expression of PEA3/E1AF/ETV4, an Ets-related transcription factor, in breast tumors: positive links to MMP2, NRG1 and CGB expression.

The PEA3/E1AF/ETV4 gene encodes an Ets-related transcription factor that is expressed in the epithelial cells of the mammary gland. Previous reports have shown that PEA3 can up-regulate promoter activities of many genes associated with tumorigenesis. A significant fraction of those encode matrix metalloproteinases (MMP genes) required for degradation of the extracellular matrix. To better obtain a molecular characterization of PEA3 expression in sporadic breast cancer, we quantified PEA3 mRNA by means of real-time reverse transcriptase-polymerase chain reaction assay in a large series of human primary breast tumors. PEA3 expression showed wide variations in tumor tissues, being under-expressed in 30 of 130 (23.1%) and over-expressed in 18 of 130 (13.8%) compared with normal breast tissues. High PEA3 mRNA levels correlated significantly with Scarff-Bloom-Richardson histopathological grade III (P = 0.018) but not with poor prognosis, suggesting that PEA3 is a marker of tumor aggressiveness rather than a prognostic factor in human breast cancer. We also observed positive links between the expression of PEA3 and those of MKI67 and ERBB2 (P = 0.034 and P = 0.045, respectively) and an inverse relationship with ERalpha (P = 0.0016). Our results do not support recent findings suggesting that PEA3 could be a tumor-suppressor gene that can act therapeutically in ERBB2 over-expressed tumors. Our results also suggest major roles of the MMP2, NRG1 and CGB genes (which encode type I gelatinase, heregulin and human chorionic gonadotropin beta subunit, respectively) in the PEA3 pathway dysregulation observed in breast cancer. Taken together, the data confirm the role of the PEA3 gene in breast tumorigenesis, and suggest the existence of numerous other still unknown genes transactivated by the PEA3 transcription factor.

Evaluation of mRNA expression of estrogen receptor beta and its isoforms in human normal and neoplastic endometrium.

Endometrial cancer is well known to be estrogen-dependent. Two estrogen receptor types, ERalpha and ERbeta, are major mediators of a diversity of biologic functions of estrogen and play an important role in estrogen-dependent tissues and cancers. Cloning of ERbeta was followed by the discovery of a variety of its isoforms. Using real-time RT-PCR, the relative expression levels of ERbeta1, ERbeta2 (ERbetacx), ERbeta3, ERbeta4 and ERbeta5 were studied. We observed coexpression of ERbeta isoforms in the endometrium and upregulation of the ERbeta5 transcript in malignant endometrium. We also observed downregulation of ERbeta2Delta5 transcript in neoplastic endometrium, using a semiquantitative method. Our results suggest that analyzing the changes in ERbeta and its isoforms may be important in the diagnosis, prognosis and treatment of endometrial cancer.