The transcription factor Ets-1 regulates the expression of several angiogenic and extracellular matrix remodeling factors, and might be implicated in disease progression of breast cancer. In the present study, the prognostic value of Ets-1 expression was assessed by quantitative realtime fluorescence RT -PCR in 123 sporadic primary breast cancer samples of patients with a median followup time of 62 months. Ets-1 expression levels correlated significantly with VEGF and PAI-1 in the same tissue. In univariate (P=0.0011) and multivariate (P=0.005) analyses, Ets-1 expression showed significant prognostic value for relapse-free survival. Ets-1 is a strong, independent predictor of poor prognosis in breast cancer. This seems -at least in part -to be attributable to its role in transcriptional regulation of factors involved in angiogenesis (VEGF), and extracellular matrix remodeling (PAI-1).

uPA and PAI-1 are becoming established as amongst the most effective markers of poor prognosis for patients with node-negative breast cancer; tPA is an index of longer survival. This paper describes a sensitive ELISA for the measurement of uPA, tPA and PAI-1 in breast cancer cytosols. The structure of the assay involves coating Ab (sheep alpha-Chicken IgY), catching Ab (chicken alpha-analyte), tagging Ab (rabbit alpha-analyte) and detecting Ab (goat alpha-rabbit IgG) labelled with HRP. The assay has a high degree of accuracy and specificity. Comparison with the American Diagnostic kits shows the results' equivalence for PAI-1 and tPA. For uPA the results of the assay were twice as high. The assay is sensitive and relatively inexpensive. It is the first published assay to yield strictly comparative values for uPA, tPA and PAI-1 in tissue extracts and is readily subject to external quality control.

IntroductionHypoxia in solid tumors is associated with treatment resistance, resulting in poor prognosis. Tribbles homolog 3 (TRIB3) is induced during hypoxia and is involved in multiple cellular pathways involved in cell survival. Here, we investigated the role of TRIB3 in breast cancer.MethodsTRIB3 mRNA expression was measured in breast tumor tissue from 247 patients and correlated with clinicopathological parameters and clinical outcome. Furthermore, we studied TRIB3 expression regulation in cell lines, xenografts tissues and human breast cancer material using Reverse transcriptase, quantitative polymerase chain reaction (RT-qPCR) and immunohistochemical staining. Finally, the effect of small interfering RNA (siRNA) mediated TRIB3 knockdown on hypoxia tolerance was assessed.ResultsBreast cancer patients with low, intermediate or high TRIB3 expression exhibited a mean disease free survival (DFS) of 80 (95% confidence interval [CI] = 74 to 86), 74 (CI = 67 to 81), and 63 (CI = 55 to 71) months respectively (P = .002, Mantel-Cox log-rank). The prognostic value of TRIB3 was limited to those patients that had received radiotherapy as part of their primary treatment (n = 179, P = .005) and remained statistically significant after correction for other clinicopathological parameters (DFS, Hazard Ratio = 1.90, CI = 1.17 to 3.08, P = .009). In breast cell lines TRIB3 expression was induced by hypoxia, nutrient starvation, and endoplasmic reticulum stress in an hypoxia inducible factor 1 (HIF-1) independent manner. TRIB3 induction after hypoxia did not increase with decreasing oxygen levels. In breast tumor xenografts and human breast cancer tissues TRIB3 co-localized with the hypoxic cell marker pimonidazole. The induction of TRIB3 by hypoxia was shown to be regulated via the PERK/ATF4/CHOP pathway of the unfolded protein response and knockdown of TRIB3 resulted in a dose-dependent increase in hypoxia sensitivity.ConclusionsTRIB3 is independently associated with poor prognosis of breast cancer patients, possibly through its association with tumor cell hypoxia.

These results demonstrate that the assessment of the tumor mRNA expression level of the breast-specific protein mammaglobin could be useful to stratify patients for individual adjuvant treatment strategies.

Summary High levels of urokinase-type plasminogen activator (uPA) and plasminogen activator inhibitor type 1 (PAI-1) in breast cancer tissue extracts have been associated with rapid disease progression. In these studies, different enzyme-linked immunosorbent assay (ELISA) kits have been applied for the quantification, and consequently the ranges of uPA and PAI-1 levels reported differ considerably. Therefore, the Receptor and Biomarker Study Group (RBSG) of the European Organization for Research and Treatment of Cancer (EORTC) and a consortium of the BIOMED-1 project 'Clinical Relevance of Proteases in Tumor Invasion and Metastasis' initiated three collaborative between-laboratory assessment trials aimed at controlling uPA and PAI-1 antigen analyses. For this purpose, two control preparations were produced from different sources: pooled human breast cancer specimens (QC-240893) and human breast cancer xenografts raised in nude mice (QC-1 01094). The lyophilized preparations were stable for prolonged times (at least 3 and 27 months respectively) at 40C. Furthermore, a good parallelism following dilution was found for uPA and PAI-1. The data from QC trial no. 1 clearly indicated that acceptable betweenlaboratory coefficients of variation (CVs) for uPA (<8.2%) and PAI-1 (<16.6%) in QC-240893 could be achieved when the same type of ELISA kit (American Diagnostica) was used. From the second trial, in which ten EORTC laboratories each received five identical lyophilized QC-101094 samples, it appeared that the within-laboratory variations for uPA and PAI-1 determinations obtained by 'experienced' laboratories were lower (<12.9%) than those from non-experienced laboratories (<36.4%). In a third QC trial, five BIOMED-1 laboratories, all of which employed ELISA procedures for uPA and PAI-1, participated in six subsequent quality assessment rounds receiving five samples of QC-101094. Although for each laboratory the within-run CVs for uPA as well as for PAI-1 were low (<7.8%), the between-run CVs were found to be considerably higher (up to 56.2% for uPA and to 27.6% for PAI-1). Consequently, because of the different ELISA formats used, the absolute analyte values measured in the different laboratories varied substantially. The use of 'common external standards' in the different ELISAs resulted in a significant reduction of the between-laboratory CVs from 61.3% to 15.7% (uPA) and from 42.1% to 19.1% (PAI-1). The present data demonstrate that in multicentre studies the same ELISA kit should be used, and that external quality assurance (QA) is mandatory. Furthermore, it appears from the present study that standardization of the protein assay as a tissular parameter is imperative.Keywords: uPA; PAI-1; enzyme-linked immunosorbent assay; breast cancer; quality assessment; EORTC During the past decade, convincing evidence has accumulated progression and early death (Duffy et al, 1988;Janicke et al, 1990; suggesting that the urokinase-type plasminogen activator (uPA) Foekens et al, 1992, Spyratos et al, 1992Duffy, 1996). plays a k...