Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): Report of an EORTC/BIOMED-1 Workshop

Benraad, Th. J.; Geurts-Moespot, J.; Grøndahl-Hansen, J.; Schmitt, Manfred; Heuvel, J. J. T. M.; Witte, J; Foekens, John A.; Leake, R E; Brünner, Nils; Sweep, Fred C.G.J.

doi:10.1016/0959-8049(96)00118-9

Cited by 61 publications

(30 citation statements)

References 19 publications

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“…It is probable that two or more markers will have to be combined to obtain a reliable prognostic score in breast cancer. Assay techniques for markers of metastatic disease must be perfectly standardized Janicke et al, 1994;Romain et al, 1995;Benraad et al, 1996;Bouchet et al, 1996;R0nne et al, 1995) if they are to be of use for diagnosis and the decision to give adjuvant therapy.…”

Section: Discussionmentioning

confidence: 99%

Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods

Bouchet

Spyratos²,

Hacène³

et al. 1998

Br J Cancer

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Summary Urokinase-type plasminogen activator (uPA) is a potentially important prognostic factor in breast cancer for identifying patients at high risk of recurrence. This retrospective study assessed two enzyme-linked immunosorbent assay (ELISA) methods measuring uPA antigen levels in 499 primary breast cancer cytosols. Both uPA methods were applied to cytosols used routinely for oestrogen (ER) and progesterone (PgR) receptor assays. uPA was determined using a classical ELISA method (Imubind; American Diagnostica) and a novel automatic immunoluminometric assay (Lia; Sangtec Medical). The uPA Imubind method revealed about twice as much uPA antigen (median 0.75 ng mg-1 protein) as the uPA Lia method (median 0.38 ng mg-1 protein). The correlation coefficient between the two methods was acceptable (r = 0.81), but the two techniques are not interchangeable. Univariate analyses confirmed the poor outcome of patients whose tumours contained large amounts of uPA, regardless of the technique used. Multivariate analyses showed that uPA Imubind and uPA Lia values were both strong independent prognostic factors.Keywords: urokinase plasminogen activator; luminometric immunoassay; prognosis; breast cancer Evidence has accumulated that invasion and metastasis by solid tumours require the action of tumour-associated proteases, which promote the dissolution of the surrounding tumour matrix and basement membranes. In several independent studies of a variety of cancer types, i.e. breast (Dano et al, 1985;Duffy et al, 1988), colorectal (Ganesh et al, 1994;Skelly et al, 1995), lung (Oka et al, 1991), ovary (Kuhn et al, 1994), gastric (Nekarda et al, 1994;Cho et al, 1997) and bladder cancer (Hasui et al, 1992), high levels of the serine protease urokinase-type plasminogen activator (uPA) antigen in tumour extracts were associated with rapid disease progression and poor prognosis. In this study of 499 primary breast cancer tumours, we used two different assay methods for uPA antigen, in order to compare an enzyme-linked immunoassay (uPA Imubind; American Diagnostica) with a new automatic immunoluminometric assay (uPA Lia; Sangtec Medical), to assess the relationships between uPA values and clinical and histological factors and to evaluate the prognostic value of the two methods in multivariate analyses. MATERIALS AND METHODS PatientsThe study group consisted of 499 breast cancer patients treated at the Centre Rene Huguenin (CRH) between 1981 and 1989. The median age was 58 years (range 24-84 years). Patients were selected according to the following criteria: (1) primary, unilateral breast tumour; (2) full follow-up at CRH; (3) previously untreated, Correspondence to: C Bouchet without evidence of metastatic disease or any other malignant tumour at the time of diagnosis; (4) surgery as the first treatment; and (5) complete clinical, histological and biological information, especially concerning hormone receptors and antigen levels of uPA measured in cytosols by the two methods. All tumours were graded by a method based on the criteri...

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Section: Discussionmentioning

confidence: 99%

Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods

Bouchet

Spyratos²,

Hacène³

et al. 1998

Br J Cancer

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“…The lower limit of detection was 2 pg/ml. In each run, a reference preparation was run to check for interassay variability and to monitor overall performance (41,42). IL-1 total protein levels were detected using an ELISA as previously described (43).…”

Section: Methodsmentioning

confidence: 99%

Increased expression of Fcγ receptors II and III on macrophages of rheumatoid arthritis patients results in higher production of tumor necrosis factor α and matrix metalloproteinase

Blom¹,

Radstake²,

Holthuysen³

et al. 2003

Arthritis & Rheumatism

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Objective. To evaluate Fc␥ receptor (Fc␥R) expression on synovial macrophages from rheumatoid arthritis (RA) patients and to determine whether this expression correlates with the production of the proinflammatory cytokines tumor necrosis factor ␣ (TNF␣), interleukin-1␤ (IL-1␤), IL-12, and matrix metalloproteinase 1 (MMP-1). We also sought to determine whether mature macrophages from RA patients express aberrant levels of Fc␥RI, Fc␥RII, and Fc␥RIII, and to determine the production of inflammatory mediators after immune complex (IC) stimulation.Methods. Immunohistochemistry was performed on cryostat sections of synovial biopsy specimens obtained from 27 RA patients and 5 controls. Fc␥R I, II, and III were detected, as well as the proinflammatory mediators IL-1, TNF␣, IL-12, and MMP-1. Monocytes were isolated from the blood of 10 RA patients and 10 healthy controls and cultured for 7 days with macrophage colony-stimulating factor to obtain macrophages. Using fluorescence-activated cell sorting, the expression of Fc␥RI, Fc␥RII, and Fc␥RIII was determined. On day 7, macrophages were stimulated with heat-aggregated gamma globulins (HAGGs) for 24 hours. Production of cytokines was measured using enzyme-linked immunosorbent assay, and production of gelatinases/ collagenases was measured by degradation of fluorescent gelatin.Results. Immunohistochemistry showed higher Fc␥RII and Fc␥RIII expression in RA synovium than in controls. Fc␥RII and Fc␥RIII, but not Fc␥RI, were highly correlated with the number of synovial macrophages. Consistent with this, TNF␣ expression correlated positively with Fc␥RIII expression. Moreover, MMP-1 expression strongly correlated with Fc␥R I, II, and III expression. Mature macrophages from RA patients showed significantly enhanced expression of Fc␥RII and Fc␥RIII compared with controls. Twentyfour hours after stimulation of RA macrophages with HAGGs, significantly higher production of TNF␣ and gelatinase/collagenase was measured.Conclusion. RA synovium and mature RA macrophages express significantly elevated levels of Fc␥RII and Fc␥RIII, resulting in much higher production of TNF␣ and gelatinase/collagenase after IC stimulation. These data suggest that disturbed expression of Fc␥R on mature synovial macrophages is involved in the pathology of RA.

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“…The protein content of the tissue extracts (cytosolic plus secreted proteins) was measured as modified by Lowry, using BSA as the standard. uPA and PAI-1 antigen contents were determined in the high-speed supernatant by published in-house ELISA formats (20)(21)(22).…”

Section: Methodsmentioning

confidence: 99%

“…uPA and PAI-1 antigen levels were quantified by published in-house ELISA formats from cytosolic extracts (20)(21)(22) -…”

Section: ------------------------------------------------------------mentioning

confidence: 99%

Quantitative RT-PCR assays for the determination of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 mRNA in primary tumor tissue of breast cancer patients: Comparison to antigen quantification by ELISA

Biermann

Holzscheiter²,

Kotzsch³

et al. 2008

Int J Mol Med

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Abstract. Urokinase-type plasminogen activator (uPA) and its inhibitor plasminogen activator inhibitor type 1 (PAI-1) play a key role in tumor-associated processes such as the degradation of extracellular matrix proteins, tissue remodeling, cell adhesion, migration, and invasion. High antigen levels of uPA and PAI-1 in tumor tissue of various solid malignant tumors, including breast cancer, are associated with poor patient prognosis. In the present study, we examined whether analysis of uPA and PAI-1 mRNA expression represents an alternative to the measurement of the respective antigen levels in breast cancer. Highly sensitive quantitative real-time PCR (QPCR) assays, based on the LightCycler technology, were established to quantify uPA and PAI-1 mRNA expression in breast cancer cell lines as well as in tumor tissue of breast cancer patients. mRNA concentrations were normalized to the expression level of the housekeeping gene h-G6PDH. The respective uPA and PAI-1 antigen concentrations were determined by established ELISA formats. QPCR mean interassay variation coefficients were 11% (uPA) and 8% (PAI-1). In breast cancer cell lines, mRNA and antigen values were highly correlated for both uPA and PAI-1 (each: r s =0.95; p<0.001). In contrast, correlations between uPA/PAI-1 mRNA and protein in the breast cancer samples were found to be distinctly weaker or not significant. Thus, quantitative determination of mRNA expression for both factors does not mirror antigen levels in breast cancer tissue, possibly due to posttranscriptional regulation. Except for nodal status being inversely correlated with uPA mRNA levels, no significant interrelations were observed between uPA or PAI-1 mRNA expression and clinicopathological parameters. On the protein level, elevated uPA and PAI-1 values were associated with a negative steroid hormone receptor status. In conclusion, the implementation of mRNA quantification of uPA and PAI-1 in breast tumors is unable to serve as a one-to-one substitution for antigen determination by ELISA.

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Immunoassays (ELISA) of urokinase-type plasminogen activator (uPA): Report of an EORTC/BIOMED-1 Workshop

Cited by 61 publications

References 19 publications

Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods

Prognostic value of urokinase plasminogen activator in primary breast carcinoma: comparison of two immunoassay methods

Increased expression of Fcγ receptors II and III on macrophages of rheumatoid arthritis patients results in higher production of tumor necrosis factor α and matrix metalloproteinase

Quantitative RT-PCR assays for the determination of urokinase-type plasminogen activator and plasminogen activator inhibitor type 1 mRNA in primary tumor tissue of breast cancer patients: Comparison to antigen quantification by ELISA

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