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Heparin affin regulatory peptide (HARP) and midkine (MK) are growth factors, expressed in carcinomas, neuroblastomas and gliomas. In this study, we measured the levels of HARP and MK in plasma samples from 77 cancer patients. The patients had advanced tumors with loco-regional (n=18) or metastatic (n=49) diseases and 10 patients have their diseases limited to the primary site. HARP and MK plasma concentrations were significantly higher in all of these different subgroups of cancer patients (P<0.05 in all cases), when compared to healthy controls (n=30). Neither HARP nor MK levels were significantly different between patients with loco-regional and metastatic tumors (P=0.203 and 0.242, respectively). Moreover, a strong correlation between the elevations of the plasma levels of these two proteins (r2=0.546) in these cancer patients was found. Measurements of these secreted angiogenic growth factors may be useful for evaluation of cancer diagnosis.

Among the proteases involved in the tumor invasion process, components of the plasminogen activator system (plasminogen activator type-urokinase uPA, its membrane receptor uPAR and its two inhibitors PAI-1 and PAI-2) appear to define high risk patients in primary breast cancer. As individual analysis of each component of the plasminogen activator system does not reflect the complex interactions between the different components, we studied the prognostic impact of a dissemination risk index combining the four variables. We found that this index was the most powerful prognostic factor, particularly in node-negative patients.

Purpose: To study interactions between disease-free survival (DFS) and four components of the plasminogen activator system: urokinase-type plasminogen activator (uPA), its two inhibitors (PAI-1 and PAI-2), and its membrane receptor uPAR., Patients and Methods: We conducted a retrospective study of 499 primary breast cancer patients (median follow-up, 6 years). uPA, PAI-1, and PAI-2 were determined on cytosols and uPAR on solubilized pellets, using enzyme-linked immunoadsorbent assay kits (American Diagnostica, Greenwich, CT). Classical univariate and multivariate statistical methods were used together with multiple correspondence analysis to graphically examine interactions between the variables and outcome., Results: By univariate analysis, higher uPA and PAI-1 values were significantly related to shorter DFS (P =.002; P <.00002). PAI-2 was not significantly related to DFS, although patients with high and very low PAI-2 values had a longer DFS. Multiple correspondence analysis showed the parallel impact of uPA and PAI-1 on outcome, and the clearly different behavior of PAI-2 compared with PAI-1. The prognostic contribution of uPAR seemed weak by both methods. A dissemination risk index [uPA x PAI-1/(PAI-2 + 1)], taking into account the modulation of uPA proteolytic activity by the ratio of its two inhibitors, was then tested. Dissemination risk index was selected as an independent variable in the Cox model in the overall population (P <.000001) and in node-positive patients (P <.00001). It was the only variable selected in node-negative patients (P =. 003)., Conclusion: A dissemination risk index determined on primary tumor and taking into account the different effects of PAI-1 and PAI-2 on uPA can be of major help in clinical management of breast cancer, particularly in node-negative patients.

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.

The levels of uPA, its inhibitors PAI-1 and PAI-2, and the uPA receptor (uPAR) have prognostic value in breast cancer. However, different extraction methods and assays kits are used in different laboratories and may directly influence the levels observed. To define a buffer suitable for both PAI-2 and uPAR extraction from breast cancer tissue compatible with hormone receptors and other cytosolic prognosticator assays, we compared PAI-2 and uPAR values obtained by immunoenzymatic assays (American Diagnostica, Greenwhich, USA) in several extraction conditions: 1) cytosol obtained with the standard hormone receptor buffer; 2) solubilized pellets obtained by Triton X100 extraction of the pelleted membranes obtained with standard hormone receptor buffer; 3) cytosol obtained by direct extraction in the buffer (containing Triton X100) recommended by the manufacturer, after 2 hours or 12 hours of incubation. Cytosol extracts prepared using the standard procedure recommended for hormone receptors gave the highest PAI-2 values. The highest uPAR values were obtained in the subsequent detergent extraction of the pelleted membranes. PAI-2 levels obtained with the kit manufacturer's method after 12 hours of incubation were lower than those obtained after 2 hours of incubation, whereas uPAR levels were similar. We conclude that the most suitable extraction protocol employs standard hormone receptor extraction buffer to obtain a supernatant cytosol fraction for PAI-2 assay, and subsequent detergent extraction of the pelleted membranes to obtain an extract suitable for uPAR.

The urokinase type plasminogen activator (u-PA) and the plasminogen activator inhibitor-1 (PAI-1) are among the best second-generation prognostic tissue factors in breast cancer. However, different extraction procedures and assay kits are used in different laboratories. A total of 79 breast tumour tissues stored in liquid nitrogen were analysed in this study. We compared u-PA and PAI-1 levels determined with the American Diagnostics (AD) kit after various extraction procedures. The median cytosolic extraction yield in the presence of 0.4 mol/l KCl, calculated relative to extraction in the presence of 10 ml/l Triton X100 when adapted to standard laboratory working hours (incubation for 2 h instead of 12 h) was 74.4% for u-PA and 85.8% for PAI-1. In addition, the correlations were acceptable. Cytosolic extracts prepared with KCl could permit optimal u-PA and PAI-1 assays while also enabling hormone receptors to be determined with the same specimens. Further studies with clinical data are now necessary to determine the prognostic relevance of this extraction procedure.

It is now clearly established that proteolytic enzymes, and in particular 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 tumors. The patients were followed up for a minimum of six years and all relevant clinical and laboratory findings had been recorded. Univariate analysis confirmed the poor outcome of patients whose tumors 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 using the "Main Effects" Cox model identified node involvement, macroscopic tumor size and PAI-2 as significant variables. The "interactive" Cox 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 as well as following stratification for axillary node negative disease, 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 informations as uPA, and does not appear to play its role as an inhibitor. In contrast, PAI-2 increased the prognostic value of both uPA, particularly in post-menopausal women, as well as PAI-1 in a subgroup of axillary node negative patients.

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.

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 the present study, five glycolytic enzymatic activities (pyruvate kinase [PK], glucose-6-phosphate dehydrogenase [G6PD], phosphohexose-isomerase [PHI], lactate dehydrogenase [LDH], and 6-phospho-glucocate dehydrogenase [6PGD]) were measured in the cytosol (105,000 g) of 57 breast carcinomas and 22 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. The various means of expressing the results were compared. The correlations were satisfactory except 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.

Fourteen clinical, pathologic, and pretreatment bioimmunologic variables were evaluated for their significance in predicting the survival or the length of disease-free interval of 55 patients with primary breast cancer. The variables studied were: patient age; clinical stage of disease according to the International Union Against Cancer TNM classification; number of involved nodes; sedimentation rate; peripheral lymphocyte, leucocyte, and monocyte counts; serum levels of immunoglobulins IgG, IgA, and IgM; percentages of E-, "active" E-, and EAC-rosettes; and finally, the lymphoblastic transformation test value (PHA-LTT). A multivariate analysis using the Cox proportional hazards regression model was carried out, in a stepwise manner, to identify those variables most highly related to survival or to the length of disease-free interval. The Cox analysis showed that clinical stage, number of involved nodes, percentage of EAC-rosettes, sedimentation rate, and T-lymphocyte reactivity, (i.e., the T-lymphocyte sensitivity to PHA, expressed as the ratio between the PHA-LTT in counts per minute and the percentage of E-rosettes) were the significant prognostic factors for survival, whereas the number of involved nodes and the sedimentation rate were independent of importance in predicting the length of disease-free interval. The results obtained from this analysis proved the importance of some immunologic parameters in the estimation of prognosis. In addition, a prognostic score for summarizing multiple factors with potential use in stratification was derived from the multivariate analysis.

Primary breast cancers from 85 patients undergoing post-surgical adjuvant chemotherapy were analyzed for five glycolytic enzymes: lactate dehydrogenase (LDH); phosphohexose isomerase (PHI); glucose-6-phosphate dehydrogenase (G-6PD); pyruvate-kinase (PK); and 6-phospho-gluconate dehydrogenase (6-PGD). The purpose of this study was to determine whether biochemical parameters could offer a prognostic index to determine outcome of therapy. The patients were followed up to a maximum of 54 months; during this period 30 of them developed recurrent or metastatic disease. The enzyme activities were expressed by the three following reference parameters: units/g proteins, units/g tissue weight and units/mg DNA. Two methods of analysis were compared: firstly, univariate analysis using life tables; and secondly, multivariate analysis using the Cox's model, where enzyme levels were tested for each mode of expression in addition to node status, histological features, receptor and menopausal status. Life table analyses appear limited when subsets of patients were studied because the sample size tends to become too small to warrant firm conclusions. Using the Cox's model, a prognostic index 1 was proposed, including the number of involved nodes and the product of logarithms of G-6PD and 6-PGD expressed as units/mg DNA. Compared to the number of involved nodes, this index gives a slightly better discrimination of the patients at 2 yr after mastectomy.