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.

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...

SummaryThe prognostic value of tissue-type plasminogen activator (tPA) measured in samples derived from 865 patients with primary breast cancer using a recently developed enzyme-linked immunosorbent assay (ELISA) was evaluated. Since the assay could easily be adapted to the assessment of the complex of tPA with its type-1 inhibitor (PAI-1), it was investigated whether the tPA:PAI-1 complex also provides prognostic information. To this end, cytosolic extracts and corresponding detergent extracts of 100 000 g pellets obtained after ultracentrifugation when preparing the cytosolic fractions for routine steroid hormone receptor determination were assayed. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r s = 0.75, P < 0.001 for tPA and r = 0.50, P < 0.001 for tPA:PAI-1 complex). In both Cox univariate and multivariate analysis elevated levels of (total) tPA determined in the pellet extracts, but not in cytosols, were associated with prolonged relapse-free (RFS) and overall survival (OS). In contrast, high levels of the tPA:PAI-1 complex measured in cytosols, but not in the pellet extracts, were associated with a poor RFS and OS. The prognostic information provided by the cytosolic tPA:PAI-1 complex was comparable to that provided by cytosolic (total) PAI-1. Furthermore, the estimated levels of free, uncomplexed tPA and PAI-1, in cytosols and in pellet extracts, were related to patient prognosis in a similar way as the (total) levels of tPA and PAI-1 respectively. Determination of specific forms of components of the plasminogen activation system, i.e. tPA:PAI-1 complex and free, uncomplexed tPA and/or PAI-1, may be considered a useful adjunct to the analyses of the separate components (tPA and/or PAI-1) and provide valuable additional prognostic information with respect to survival of breast cancer patients.

SummaryTo evaluate the clinical relevance of urokinase-type plasminogen activator (uPA) and its type-1 inhibitor (PAI-1) measured by a recently developed enzyme-linked immunosorbent assay (ELISA), we analysed both components in samples derived from 892 patients with primary breast cancer (median follow-up 99 months). The assays were performed in cytosolic extracts as well as in corresponding detergent extracts of pellets obtained after ultracentrifugation, which was carried out when preparing the cytosolic fractions for routine steroid hormone receptor determination. Statistically significant correlations were found between the cytosolic levels and those determined in the pellet extracts (Spearman correlation coefficient r s = 0.60, P < 0.0001 for uPA and r s = 0.65, P < 0.0001 for PAI-1). Furthermore, strong correlations were found between the levels of both uPA (r s = 0.85, P < 0.0001) and PAI-1 (r s = 0.90, P < 0.0001) in the cytosols and their levels previously measured with ELISAs based on commercial reagents. In both Cox univariate and multivariate analysis, high cytosolic levels of uPA or PAI-1 were significantly associated with increased rates of relapse and death. The levels of uPA and PAI-1 in the pellet extracts also provided prognostic information, although to a lesser extent compared with the cytosolic extracts. The prediction of prognosis on the basis of uPA and PAI-1 assessed by an alternative ELISA once again emphasizes the established prognostic role and usefulness of these parameters in selection of breast cancer patients at high or low risk of recurrence.Keywords: urokinase-type plasminogen activator; plasminogen activator inhibitor; enzyme-linked immunosorbent assay; cytosol; breast cancer; prognostic impact 1190British Journal of Cancer (1999) 79(7/8), 1190-1198 © 1999 Cancer Research Campaign Article no. bjoc.1998 Received of uPA and PAI-1 were correlated to those obtained earlier with ELISAs based on commercial reagents. MATERIALS AND METHODS Patients and tumour characteristicsIn the present study, 892 patients with primary operable breast cancer (modified mastectomy, 446 patients; breast-conserving lumpectomy, 446 patients) were included. Inclusion criteria for patients from whom tumour biopsies or cytosol samples were stored in the tumour bank of the Rotterdam Cancer Centre (Dr Daniel den Hoed Kliniek) were: (i) primary diagnosis of breast cancer between 1979 and 1989; (ii) no signs of distant metastasis at diagnosis; (iii) no previous diagnosis of carcinoma, with the exception of basal cell skin cancer and cervical cancer stage Ia; (iv) no evidence of disease within 1 month after primary surgery. In cases of mastectomy after an initial lumpectomy for residual disease, the mastectomy is considered as (part of) primary treatment. The median number of lymph nodes removed surgically was 11. Patients without primary surgery or patients who received neoadjuvant treatment before primary surgery were excluded. Median age of the patients at the time of surgery was 56 years (range 25Ð89 years)....

Complexes between urokinase‐type plasminogen activator (uPA) and its receptor (uPAR) were assessed in plasma and serum from 39 breast cancer patients and from 20 healthy individuals, applying a recently developed enzyme‐linked immunosorbent assay (ELISA) for the analysis of these complexes in tumor tissue extracts. The assay is based on a combination of rabbit polyclonal anti‐uPA antibodies for catching and a mouse anti‐uPAR monoclonal antibody (MAb) for detection. The specificity of the assessment of uPA:uPAR complexes was verified by simultaneous analysis of the individual blood samples in corresponding non‐sense ELISA formats, in which either the anti‐uPA catching antibody or the anti‐uPAR detecting antibody was substituted with an irrelevant antibody. Assessment of native uPA:uPAR complexes was ascertained by demonstrating the absence of any de novo formation of uPA:uPAR complexes in plasma and serum during the sample incubation step in the ELISA, as verified by the use of a peptide antagonist for uPAR. Plasma and serum samples contained almost identical levels of uPA:uPAR complexes. The levels of uPA:uPAR complexes were found to be significantly lower in serum from breast cancer patients compared to the serum of healthy donors, while the levels of (total) uPAR in plasma from breast cancer patients were significantly higher than in plasma from the healthy controls. In addition, the free, uncomplexed uPAR levels, estimated by subtraction of uPA:uPAR complex levels from (total) uPAR levels, were significantly elevated in plasma as well as in serum from breast cancer patients compared to healthy individuals. The uPA:uPAR complex levels were highly comparable to the uPA levels analyzed in the same plasma and serum samples, indicating that most if not all of the uPA present in these samples is complexed with uPAR. Int. J. Cancer 77:236–242, 1998.© 1998 Wiley‐Liss, Inc.