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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.

It is now clearly established that proteolytic enzymes, including plasminogen activator (uPA), play an important role in breaking down the extracellular matrix, which is considered to be a step in metastasis formation. Plasminogen activators are controlled at various levels. Two inhibitors, PAI-1 and PAI-2, have been identified, the latter being more specific for uPA. In attempts to determine their prognostic value, it is essential to investigate the relative importance of these parameters and their interactions. We used an immunoenzymatic method to assay uPA, PAI-1 and PAI-2 antigens in cytosols prepared from 314 primary breast tumours. The patients were followed up for a minimum of 6 years and all relevant clinical and laboratory findings were recorded. Univariate analysis confirmed the poor outcome of patients whose tumours contained large amounts of uPA and PAI-1. In addition, low levels of PAI-2 correlated with shorter disease-free survival in the overall population (P = 0.02), post-menopausal women (P = 0.02) and women without lymph node involvement (P = 0.02). Multivariate analysis in the 'main effects' Cox model identified node involvement, macroscopic tumour size and PAI-2 as significant variables. The 'interactive' model, taking into account interactions between uPA and its two inhibitors, identified a first subgroup with a very poor prognosis associating either high levels of PAI-1 with low levels of PAI-2 in the overall population and the women with no node involvement or high levels of uPA with low levels of PAI-2 in the group of menopausal women. We conclude that PAI-1 provides the same prognostic information as uPA, and does not appear to play a role as an inhibitor. In contrast, PAI-2 increases the prognostic value of uPA, particularly in post-menopausal women, and PAI-1 in patients with no node involvement.

The concentrations of cathepsin D (Cath D), urokinase (uPA) and two plasminogen activator inhibitors (PAI-1 and PAI-2) were analysed in the cytosols of 130 human mammary tumours (43 benign tumours and 87 primary and unilateral breast carcinomas). uPA, PAI-1 and PAI-2 levels were measured by antigenic immunoassays and Cath D by immunoradiometric assay. The median levels of the four parameters were significantly higher in the malignant tumours than in the benign ones. Cath D and uPA increases were 4-fold and 5-fold respectively. PAI-1 and PAI-2 increases were much more important, 74-fold and 29-fold respectively. In malignant tumours, median levels of Cath D and uPA did not vary according to classical prognostic factors (histologic grade, presence or absence of axillary lymph nodes, steroid receptors, UICC stage, tumour size, age, and menopausal status). However, PAI-1 decreased in ER+ and PR+ tumours and PAI-2 increased in menopausal women's tumours. When Cath D, uPA, PAI-1 and PAI-2 levels in malignant tumours were compared, positive correlations were found for all combinations. The implication of plasminogen activator inhibitors in the phenomenon was surprising and merits further investigation using tools other than global antigen measurements in tumours.

Cancer invasion is dependent on a combined action of several proteolytic enzymes, such as collagenases, other metalloproteases, and serine proteases. Cathepsin-D was the first example of a protease whose immunoassay in the cytosol of breast tumors was available and could be readily standardized with quality control. Most studies indicated a worse prognosis in high Cathepsin-D tumors. Urokinase-type plasminogen activator (UPA), urokinase receptor (UPAR), and 2 plasminogen activator inhibitors, PAI1 and PAI-2, are all involved in regulation of plasmine generation. UPA and PAI1 levels in breast cancer tissue are independant and significant prognostic markers and high levels of each of the parameters are associated with poor prognostic. In tumors with high UPA or PAI1 content, a high level of PAI-2 appeared to be an independant marker of favorable prognostic. However, components of proteolytic enzyme systems are often expressed in non malignant stromal cells in invasive areas of cancer tissue. The cellular expression pattern of these molecules appears to be characteristic for each type of cancer and is not yet fully clarified. The measurements of proteases could be helpful for classifying breast tumors for understanding processes of breast tumor invasion and metastasis, and for the development of future treatment strategies based on interference in the proteolytic cascade which lead to tumor dissemination.

Plasminogen activator is a group of proteases which catalyses the conversion of the inactive plasminogen to the active plasmin. Most transformed cell lines and solid tumors produce higher levels of plasminogen activator than the non transformed counterparts. Plasminogen activator was determined in the cytosol of 77 breast carcinomas and 10 benign tumors. The results show a strong correlation between the level of protease activity and the hormone receptor status. Plasminogen activator activity appeared as a criterium of differentiation.

The activities of selected glycolytic enzymes in breast tumor tissues were examined, based on earlier studies showing a relationship between therapy and/or prognosis of breast cancer and tissue enzyme. In this study, five glycolytic enzymatic activities, pyruvate kinase (PK), glucose6-phosphate dehydrogenase (G6PD), phosphohexoseisomerase (PHI), lactate dehydrogenase (LDH), 6phospho-gluconate dehydrogenase (6PGD) were measured in the cytosol (105,000 x g) of 57 breast carcinomas and 44 benign breast lesions. Nucleic acids and DNA were also determined. The results were related to the wet weight of the tissue, to total and tissue cytosol proteins and to DNA. These various means of expressing the results were compared. The correlations were satisfactory excepted for PHI and LDH. In these cases, this might have been due to blood contamination. The enzyme activities content was lower in the benign breast lesions than in the breast carcinomas irrespective of the way the results were expressed.